A Multiplex Immunoassay Using the Guthrie Specimen to Detect T-Cell Deficiencies Including Severe Combined Immunodeficiency Disease

PubMed Central

Janik, David K.; Lindau-Shepard, Barbara; Comeau, Anne Marie; Pass, Kenneth A.

2011-01-01

BACKGROUND Severe combined immunodeficiency (SCID) fulfills many of the requirements for addition to a newborn screening panel. Two newborn screening SCID pilot studies are now underway using the T-cell receptor excision circle (TREC) assay, a molecular technique. Here we describe an immunoassay with CD3 as a marker for T cells and CD45 as a marker for total leukocytes that can be used with the Guthrie specimen. METHODS The multiplexing capabilities of the Luminex platform were used. Antibody pairs were used to capture and detect CD3 and CD45 from a single 3-mm punch of the Guthrie specimen. The assay for each bio-marker was developed separately in identical buffers and then combined to create a multiplex assay. RESULTS Using calibrators made from known amounts of leukocytes, a detection limit of 0.25 × 106 cells/mL for CD3 and 0.125 × 106 cells/mL for CD45 was obtained. Affinity tests showed no cross-reactivity between the antibodies to CD3 and CD45. The multiplex assay was validated against 8 coded specimens of known clinical status and linked to results from the TREC assay that had identified them. All were correctly identified by the CD345 assay. CONCLUSIONS The performance parameters of the CD345 assay met the performance characteristics generally accepted for immunoassays. Our assay classifications of positive specimens concur with previous TREC results. This CD345 assay warrants evaluation as a viable alternative or complement to the TREC assay as a primary screening tool for detecting T-cell immunodeficiencies, including SCID, in Guthrie specimens. PMID:20660143

Comparative evaluation of uniplex, nested, semi-nested, multiplex and nested multiplex PCR methods in the identification of microbial etiology of clinically suspected infectious endophthalmitis.

PubMed

Bharathi, Madasamy Jayahar; Murugan, Nandagopal; Rameshkumar, Gunasekaran; Ramakrishnan, Rengappa; Venugopal Reddy, Yerahaia Chinna; Shivkumar, Chandrasekar; Ramesh, Srinivasan

2013-05-01

This study is aimed to determine the utility of various polymerase chain reaction (PCR) methods in vitreous fluids (VFs) for detecting the infectious genomes in the diagnosis of infectious endophthalmitis in terms of sensitivity and specificity. This prospective and consecutive analysis included a total of 66 VFs that were submitted for the microbiological evaluation, which were obtained from 66 clinically diagnosed endophthalmitis patients presented between November 2010 and October 2011 at the tertiary eye care referral centre in South India. Part of the collected VFs were subjected to cultures and smears, and the remaining parts were utilized for five PCR methods: uniplex, nested, semi-nested, multiplex and nested multiplex after extracting DNA, using universal eubacterial and Propionibacterium acnes species-specific primer sets targeting 16S rRNA gene in all bacteria and P. acnes, and panfungal primers, targeting 28S rRNA gene in all fungi. Of the 66 VFs, five (7.5%) showed positive results in smears, 16 (24%) in cultures and 43 (65%) showed positive results in PCRs. Among the 43 positively amplified VFs, 10 (15%) were positive for P. acnes genome, one for panfungal genome and 42 (62%) for eubacterial genome (including 10 P. acnes positives). Among 42 eubacterial-positive VFs, 36 were positive by both uniplex (first round) and multiplex (first round) PCRs, while nested (second round) and nested multiplex (second round) PCRs produced positive results in 42 and 41 VFs, respectively. Of the 43 PCR-positive specimens, 16 (37%) had positive growth (15 bacterial and one fungal) in culture. Of 50 culture-negative specimens, 27 (54%) were showed positive amplification, of which 10 were amplified for both P. acnes and eubacterial genomes and the remaining 17 were for eubacterial genome alone. Nested PCRs are superior than uniplex and multiplex PCR. PCRs proved to be a powerful tool in the diagnosis of endophthalmitis, especially for detecting uncultured microbes.

Next Generation Molecular Histology Using Highly Multiplexed Ion Beam Imaging (MIBI) of Breast Cancer Tissue Specimens for Enhanced Clinical Guidance

DTIC Science & Technology

2016-07-01

AWARD NUMBER: W81XWH- 14-1-0192 TITLE: Next-Generation Molecular Histology Using Highly Multiplexed Ion Beam Imaging (MIBI) of Breast Cancer...DATES COVERED 4. TITLE AND SUBTITLE Next-Generation Molecular Histology Using Highly Multiplexed Ion Beam Imaging (MIBI) of Breast Cancer Tissue

Quantitative polarized light microscopy using spectral multiplexing interferometry.

PubMed

Li, Chengshuai; Zhu, Yizheng

2015-06-01

We propose an interferometric spectral multiplexing method for measuring birefringent specimens with simple configuration and high sensitivity. The retardation and orientation of sample birefringence are simultaneously encoded onto two spectral carrier waves, generated interferometrically by a birefringent crystal through polarization mixing. A single interference spectrum hence contains sufficient information for birefringence determination, eliminating the need for mechanical rotation or electrical modulation. The technique is analyzed theoretically and validated experimentally on cellulose film. System simplicity permits the possibility of mitigating system birefringence background. Further analysis demonstrates the technique's exquisite sensitivity as high as ∼20  pm for retardation measurement.

Rapid and simultaneous detection of Mycobacterium tuberculosis complex and Beijing/W genotype in sputum by an optimized DNA extraction protocol and a novel multiplex real-time PCR.

PubMed

Leung, Eric T Y; Zheng, L; Wong, Rity Y K; Chan, Edward W C; Au, T K; Chan, Raphael C Y; Lui, Grace; Lee, Nelson; Ip, Margaret

2011-07-01

Rapid diagnosis and genotyping of Mycobacterium tuberculosis by molecular methods are often limited by the amount and purity of DNA extracted from body fluids. In this study, we evaluated 12 DNA extraction methods and developed a highly sensitive protocol for mycobacterial DNA extraction directly from sputa using surface-coated magnetic particles. We have also developed a novel multiplex real-time PCR for simultaneous identification of M. tuberculosis complex and the Beijing/W genotype (a hypervirulent sublineage of M. tuberculosis) by using multiple fluorogenic probes targeting both the M. tuberculosis IS6110 and the Rv0927c-pstS3 intergenic region. With reference strains and clinical isolates, our real-time PCR accurately identified 20 non-Beijing/W and 20 Beijing/W M. tuberculosis strains from 17 different species of nontuberculosis Mycobacterium (NTM). Further assessment of our DNA extraction protocol and real-time PCR with 335 nonduplicate sputum specimens correctly identified all 74 M. tuberculosis culture-positive specimens. In addition, 15 culture-negative specimens from patients with confirmed tuberculosis were also identified. No cross-reactivity was detected with NTM specimens (n = 31). The detection limit of the assay is 10 M. tuberculosis bacilli, as determined by endpoint dilution analysis. In conclusion, an optimized DNA extraction protocol coupled with a novel multiprobe multiplex real-time PCR for the direct detection of M. tuberculosis, including Beijing/W M. tuberculosis, was found to confer high sensitivity and specificity. The combined procedure has the potential to compensate for the drawbacks of conventional mycobacterial culture in routine clinical laboratory setting, such as the lengthy incubation period and the limitation to viable organisms.

Novel application of digital microfluidics for the detection of biotinidase deficiency in newborns.

PubMed

Graham, Carrie; Sista, Ramakrishna S; Kleinert, Jairus; Wu, Ning; Eckhardt, Allen; Bali, Deeksha; Millington, David S; Pamula, Vamsee K

2013-12-01

Newborn screening for biotinidase deficiency can be performed using a fluorometric enzyme assay on dried blood spot specimens. As a pre-requisite to the consolidation of different enzymatic assays onto a single platform, we describe here a novel analytical method for detecting biotinidase deficiency using the same digital microfluidic cartridge that has already been demonstrated to screen for five lysosomal storage diseases (Pompe, Fabry, Gaucher, Hurler and Hunter) in a multiplex format. A novel assay to quantify biotinidase concentration in dried blood spots (DBS) was developed and optimized on the digital microfluidic platform using proficiency testing samples from the Centers for Disease Control and Prevention. The enzymatic assay uses 4-methylumbelliferyl biotin as the fluorogenic substrate. Biotinidase deficiency assays were performed on normal (n=200) and deficient (n=7) newborn DBS specimens. Enzymatic activity analysis of biotinidase deficiency revealed distinct separation between normal and affected DBS specimens using digital microfluidics and these results matched the expected activity. This study has demonstrated performance of biotinidase deficiency assays by measurement of 4-methylumbelliferyl product on a digital microfluidic platform. Due to the inherent ease in multiplexing on such a platform, consolidation of other fluorometric assays onto a single cartridge may be realized. © 2013.

Comparison of nested-multiplex, Taqman & SYBR Green real-time PCR in diagnosis of amoebic liver abscess in a tertiary health care institute in India.

PubMed

Dinoop, K P; Parija, Subhash Chandra; Mandal, Jharna; Swaminathan, R P; Narayanan, P

2016-01-01

Amoebiasis is a common parasitic infection caused by Entamoeba histolytica and amoebic liver abscess (ALA) is the most common extraintestinal manifestation of amoebiasis. The aim of this study was to standardise real-time PCR assays (Taqman and SYBR Green) to detect E. histolytica from liver abscess pus and stool samples and compare its results with nested-multiplex PCR. Liver abscess pus specimens were subjected to DNA extraction. The extracted DNA samples were subjected to amplification by nested-multiplex PCR, Taqman (18S rRNA) and SYBR Green real-time PCR (16S-like rRNA assays to detect E. histolytica/E. dispar/E. moshkovskii). The amplification products were further confirmed by DNA sequence analysis. Receiver operator characteristic (ROC) curve analysis was done for nested-multiplex and SYBR Green real-time PCR and the area under the curve was calculated for evaluating the accuracy of the tests to dignose ALA. In all, 17, 19 and 25 liver abscess samples were positive for E. histolytica by nested-multiplex PCR, SYBR Green and Taqman real-time PCR assays, respectively. Significant differences in detection of E. histolytica were noted in the real-time PCR assays evaluated ( P<0.0001). The nested-multiplex PCR, SYBR Green real-time PCR and Taqman real-time PCR evaluated showed a positivity rate of 34, 38 and 50 per cent, respectively. Based on ROC curve analysis (considering Taqman real-time PCR as the gold standard), it was observed that SYBR Green real-time PCR was better than conventional nested-multiplex PCR for the diagnosis of ALA. Taqman real-time PCR targeting the 18S rRNA had the highest positivity rate evaluated in this study. Both nested multiplex and SYBR Green real-time PCR assays utilized were evaluated to give accurate results. Real-time PCR assays can be used as the gold standard in rapid and reliable diagnosis, and appropriate management of amoebiasis, replacing the conventional molecular methods.

Real-time nested multiplex PCR for the detection of herpes simplex virus types 1 and 2 and varicella zoster virus.

PubMed

O'Neill, Hugh J; Wyatt, Dorothy E; Coyle, Peter V; McCaughey, Conall; Mitchell, Frederick

2003-12-01

One hundred forty-nine specimens were tested in a LightCycler nested multiplex polymerase chain reaction (LCnmPCR) for Herpes simplex virus (HSV)1, HSV2, and VZV. Eighty-one were from genitourinary medicine (GUM) patients and the other 68 specimens were from other patients with skin lesions. The results were compared to a conventional multiplex nested PCR (nmPCR) using agarose gel electrophoresis. Twenty-five specimens were positive in both assays for HSV1 and 29 were positive for VZV. For HSV2 there were 27 positive in the LCnmPCR and 26 positive in the nmPCR assay. The melting temperatures (Tms) of each target were different with a mean of 84.75 degrees C for HSV1, 88.57 degrees C for HSV2, and 83.62 degrees C for VZV. The melting curves of positive specimens directly overlaid the melting curves of the positive controls in the assay. The LCnmPCR assay is a convenient alternative to conventional PCR using agarose gel electrophoresis. It improves specimen turnaround time by eliminating the need for gel electrophoresis, transillumination, and gel photography. It also shows increased sensitivity for HSV2 over our standard assay. This LCnmPCR reduces further the possibility of amplicon contamination with nested PCR protocols. Copyright 2003 Wiley-Liss, Inc.

Multiplex PCR reveals that viruses are more frequent than bacteria in children with cystic fibrosis.

PubMed

Miró-Cañís, Sílvia; Capilla-Rubio, Sílvia; Marzo-Checa, Laura; Fontanals-Aymerich, Dionisia; Sanfeliu-Sala, Isabel; Espasa-Soley, Mateu; Asensio-de-la-Cruz, Oscar

2017-01-01

Cystic fibrosis is a degenerative disease characterized by progressive epithelial secretory gland dysfunction associated with repeated respiratory infections. Bacterial infections are very frequent in children with cystic fibrosis, but because rapid METHODS: for screening for the wide variety of potentially involved viruses were unavailable until recently, the frequency of viral presence is unknown. Multiplex PCR enables screening for many viruses involved in respiratory infections. This study aimed to evaluate the frequency of viruses and bacteria in respiratory specimens from children with cystic fibrosis and to clarify the incidence and characteristics (seasonality and age of patients) of different viruses detected in children with cystic fibrosis. In this 2-year prospective study, we obtained paired nasopharyngeal-swab and sputum specimens from children with cystic fibrosis during clinical respiratory examinations separated by at least 14days. We analyzed viruses in nasopharyngeal-swab specimens with multiplex PCR and bacteria in sputum with standard methods. We analyzed 368 paired specimens from 33 children. We detected viruses in 154 (41.8%) and bacteria in 132 (35.9%). Bacteria were commoner in spring and summer; viruses were commoner in autumn and winter. In every season, Staphylococcus aureus was the commonest bacteria and rhinovirus was the commonest virus. Nearly all infections with Haemophilus influenzae occurred in autumn and winter. Viruses were more prevalent in children <5 years old, and bacteria were more prevalent in children ≥12 years old. Multiplex PCR screening for respiratory viruses is feasible in children with cystic fibrosis; the clinical implications of screening warrant further study. Copyright © 2016 Elsevier B.V. All rights reserved.

Multiplex real-time quantitative PCR, microscopy and rapid diagnostic immuno-chromatographic tests for the detection of Plasmodium spp: performance, limit of detection analysis and quality assurance.

PubMed

Khairnar, Krishna; Martin, Donald; Lau, Rachel; Ralevski, Filip; Pillai, Dylan R

2009-12-09

Accurate laboratory diagnosis of malaria species in returning travelers is paramount in the treatment of this potentially fatal infectious disease. A total of 466 blood specimens from returning travelers to Africa, Asia, and South/Central America with suspected malaria infection were collected between 2007 and 2009 at the reference public health laboratory. These specimens were assessed by reference microscopy, multipex real-time quantitative polymerase chain reaction (QPCR), and two rapid diagnostic immuno-chromatographic tests (ICT) in a blinded manner. Key clinical laboratory parameters such as limit of detection (LOD) analysis on clinical specimens by parasite stage, inter-reader variability of ICTs, staffing implications, quality assurance and cost analysis were evaluated. QPCR is the most analytically sensitive method (sensitivity 99.41%), followed by CARESTART (sensitivity 88.24%), and BINAXNOW (sensitivity 86.47%) for the diagnosis of malaria in returning travelers when compared to reference microscopy. However, microscopy was unable to specifically identify Plasmodia spp. in 18 out of 170 positive samples by QPCR. Moreover, the 17 samples that were negative by microscopy and positive by QPCR were also positive by ICTs. Quality assurance was achieved for QPCR by exchanging a blinded proficiency panel with another reference laboratory. The Kappa value of inter-reader variability among three readers for BINAXNOW and CARESTART was calculated to be 0.872 and 0.898 respectively. Serial dilution studies demonstrated that the QPCR cycle threshold correlates linearly with parasitemia (R(2) = 0.9746) in a clinically relevant dynamic range and retains a LOD of 11 rDNA copies/microl for P. falciparum, which was several log lower than reference microscopy and ICTs. LOD for QPCR is affected not only by parasitemia but the parasite stage distribution of each clinical specimen. QPCR was approximately 6-fold more costly than reference microscopy. These data suggest that multiplex QPCR although more costly confers a significant diagnostic advantage in terms of LOD compared to reference microscopy and ICTs for all four species. Quality assurance of QPCR is essential to the maintenance of proficiency in the clinical laboratory. ICTs showed good concordance between readers however lacked sensitivity for non-falciparum species due to antigenic differences and low parasitemia. Multiplex QPCR but not ICTs is an essential adjunct to microscopy in the reference laboratory detection of malaria species specifically due to the superior LOD. ICTs are better suited to the non-reference laboratory where lower specimen volumes challenge microscopy proficiency in the non-endemic setting.

Two Multiplex Real-Time PCR Assays to Detect and Differentiate Acinetobacter baumannii and Non- baumannii Acinetobacter spp. Carrying blaNDM, blaOXA-23-Like, blaOXA-40-Like, blaOXA-51-Like, and blaOXA-58-Like Genes

PubMed Central

Yang, Qiu; Rui, Yongyu

2016-01-01

Nosocomial infections caused by Acinetobacter spp. resistant to carbapenems are increasingly reported worldwide. Carbapenem-resistant Acinetobacter (CRA) is becoming a serious concern with increasing patient morbidity, mortality, and lengths of hospital stay. Therefore, the rapid detection of CRA is essential for epidemiological surveillance. Polymerase chain reaction (PCR) has been extensively used for the rapid identification of most pathogens. In this study, we have developed two multiplex real-time PCR assays to detect and differentiate A. baumannii and non-A. baumannii Acinetobacter spp, and common carbapenemase genes, including blaNDM, blaOXA-23-like, blaOXA-40-like, blaOXA-51-like, and blaOXA-58-like. We demonstrate the potential utility of these assays for the direct detection of blaNDM-, blaOXA-23-like-, blaOXA-40-like-, blaOXA-51-like-, and blaOXA-58-like-positive CRA in clinical specimens. Primers were specifically designed, and two multiplex real-time PCR assays were developed: multiplex real-time PCR assay1 for the detection of Acinetobacter baumannii 16S–23S rRNA internal transcribed spacer sequence, the Acinetobacter recA gene, and class-B-metalloenzyme-encoding gene blaNDM; and multiplex real-time PCR assay2 to detect class-D-oxacillinase-encoding genes (blaOXA-23-like, blaOXA-40-like, blaOXA-51-like,and blaOXA-58-like). The assays were performed on an ABI Prism 7500 FAST Real-Time PCR System. CRA isolates were used to compare the assays with conventional PCR and sequencing. Known amounts of CRA cells were added to sputum and fecal specimens and used to test the multiplex real-time PCR assays. The results for target and nontarget amplification showed that the multiplex real-time PCR assays were specific, the limit of detection for each target was 10 copies per 20 μL reaction volume, the assays were linear over six log dilutions of the target genes (r2 > 0.99), and the Ct values of the coefficients of variation for intra- and interassay reproducibility were less than 5%. The multiplex real-time PCR assays showed 100% concordance with conventional PCR when tested against 400 CRA isolates and their sensitivity for the target DNA in sputum and fecal specimens was 102 CFU/mL. Therefore, these novel multiplex real-time PCR assays allow the sensitive and specific characterization and differentiation of blaNDM-, blaOXA-23-like-, blaOXA-40-like-, blaOXA-51-like-, and blaOXA-58-like-positive CRA, making them potential tools for the direct detection of CRA in clinical specimens and the surveillance of nosocomial infections. PMID:27391234

Multiplex Real-Time PCR Assay for Rapid Detection of Methicillin-Resistant Staphylococci Directly from Positive Blood Cultures

PubMed Central

Wang, Hye-young; Kim, Sunghyun; Kim, Jungho; Park, Soon-Deok

2014-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent cause of bloodstream infections (BSIs) and is recognized as a major nosocomial pathogen. This study aimed to evaluate a newly designed multiplex real-time PCR assay capable of the simultaneous detection of mecA, S. aureus, and coagulase-negative staphylococci (CoNS) in blood culture specimens. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays (M&D, Republic of Korea) use the TaqMan probes 16S rRNA for Staphylococcus spp., the nuc gene for S. aureus, and the mecA gene for methicillin resistance. The detection limit of the multiplex real-time PCR assay was 103 CFU/ml per PCR for each gene target. The multiplex real-time PCR assay was evaluated using 118 clinical isolates from various specimen types and a total of 350 positive blood cultures from a continuous monitoring blood culture system. The results obtained with the multiplex real-time PCR assay for the three targets were in agreement with those of conventional identification and susceptibility testing methods except for one organism. Of 350 positive bottle cultures, the sensitivities of the multiplex real-time PCR kit were 100% (166/166 cultures), 97.2% (35/36 cultures), and 99.2% (117/118 cultures) for the 16S rRNA, nuc, and mecA genes, respectively, and the specificities for all three targets were 100%. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays are very useful for the rapid accurate diagnosis of staphylococcal BSIs. In addition, the Real-MRSA and Real-MRCoNS multiplex real-time PCR assays could have an important impact on the choice of appropriate antimicrobial therapy, based on detection of the mecA gene. PMID:24648566

Multiplex Amplification Coupled with COLD-PCR and High Resolution Melting Enables Identification of Low-Abundance Mutations in Cancer Samples with Low DNA Content

PubMed Central

Milbury, Coren A.; Chen, Clark C.; Mamon, Harvey; Liu, Pingfang; Santagata, Sandro; Makrigiorgos, G. Mike

2011-01-01

Thorough screening of cancer-specific biomarkers, such as DNA mutations, can require large amounts of genomic material; however, the amount of genomic material obtained from some specimens (such as biopsies, fine-needle aspirations, circulating-DNA or tumor cells, and histological slides) may limit the analyses that can be performed. Furthermore, mutant alleles may be at low-abundance relative to wild-type DNA, reducing detection ability. We present a multiplex-PCR approach tailored to amplify targets of interest from small amounts of precious specimens, for extensive downstream detection of low-abundance alleles. Using 3 ng of DNA (1000 genome-equivalents), we amplified the 1 coding exons (2-11) of TP53 via multiplex-PCR. Following multiplex-PCR, we performed COLD-PCR (co-amplification of major and minor alleles at lower denaturation temperature) to enrich low-abundance variants and high resolution melting (HRM) to screen for aberrant melting profiles. Mutation-positive samples were sequenced. Evaluation of mutation-containing dilutions revealed improved sensitivities after COLD-PCR over conventional-PCR. COLD-PCR improved HRM sensitivity by approximately threefold to sixfold. Similarly, COLD-PCR improved mutation identification in sequence-chromatograms over conventional PCR. In clinical specimens, eight mutations were detected via conventional-PCR-HRM, whereas 12 were detected by COLD-PCR-HRM, yielding a 33% improvement in mutation detection. In summary, we demonstrate an efficient approach to increase screening capabilities from limited DNA material via multiplex-PCR and improve mutation detection sensitivity via COLD-PCR amplification. PMID:21354058

Rapid identification of 11 human intestinal Lactobacillus species by multiplex PCR assays using group- and species-specific primers derived from the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA.

PubMed

Song, Y; Kato, N; Liu, C; Matsumiya, Y; Kato, H; Watanabe, K

2000-06-15

Rapid and reliable two-step multiplex polymerase chain reaction (PCR) assays were established to identify human intestinal lactobacilli; a multiplex PCR was used for grouping of lactobacilli with a mixture of group-specific primers followed by four multiplex PCR assays with four sorts of species-specific primer mixtures for identification at the species level. Primers used were designed from nucleotide sequences of the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA gene of members of the genus Lactobacillus which are commonly isolated from human stool specimens: Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii (ssp. bulgaricus and ssp. lactis), Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus paracasei (ssp. paracasei and ssp. tolerans), Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus and Lactobacillus salivarius (ssp. salicinius and ssp. salivarius). The established two-step multiplex PCR assays were applied to the identification of 84 Lactobacillus strains isolated from human stool specimens and the PCR results were consistent with the results from the DNA-DNA hybridization assay. These results suggest that the multiplex PCR system established in this study is a simple, rapid and reliable method for the identification of common Lactobacillus isolates from human stool samples.

A Robust Protocol for Using Multiplexed Droplet Digital PCR to Quantify Somatic Copy Number Alterations in Clinical Tissue Specimens.

PubMed

Hughesman, Curtis B; Lu, X J David; Liu, Kelly Y P; Zhu, Yuqi; Poh, Catherine F; Haynes, Charles

2016-01-01

The ability of droplet digital PCR (ddPCR) to accurately determine the concentrations of amplifiable targets makes it a promising platform for measuring copy number alterations (CNAs) in genomic biomarkers. However, its application to clinical samples, particularly formalin-fixed paraffin-embedded specimens, will require strategies to reliably determine CNAs in DNA of limited quantity and quality. When applied to cancerous tissue, those methods must also account for global genetic instability and the associated probability that the abundance(s) of one or more chosen reference loci do not represent the average ploidy of cells comprising the specimen. Here we present an experimental design strategy and associated data analysis tool that enables accurate determination of CNAs in a panel of biomarkers using multiplexed ddPCR. The method includes strategies to optimize primer and probes design to cleanly segregate droplets in the data output from reaction wells amplifying multiple independent templates, and to correct for bias from artifacts such as DNA fragmentation. We demonstrate how a panel of reference loci can be used to determine a stable CNA-neutral benchmark. These innovations, when taken together, provide a comprehensive strategy that can be used to reliably detect biomarker CNAs in DNA extracted from either frozen or FFPE tissue biopsies.

Digital Transplantation Pathology: Combining Whole Slide Imaging, Multiplex Staining, and Automated Image Analysis

PubMed Central

Isse, Kumiko; Lesniak, Andrew; Grama, Kedar; Roysam, Badrinath; Minervini, Martha I.; Demetris, Anthony J

2013-01-01

Conventional histopathology is the gold standard for allograft monitoring, but its value proposition is increasingly questioned. “-Omics” analysis of tissues, peripheral blood and fluids and targeted serologic studies provide mechanistic insights into allograft injury not currently provided by conventional histology. Microscopic biopsy analysis, however, provides valuable and unique information: a) spatial-temporal relationships; b) rare events/cells; c) complex structural context; and d) integration into a “systems” model. Nevertheless, except for immunostaining, no transformative advancements have “modernized” routine microscopy in over 100 years. Pathologists now team with hardware and software engineers to exploit remarkable developments in digital imaging, nanoparticle multiplex staining, and computational image analysis software to bridge the traditional histology - global “–omic” analyses gap. Included are side-by-side comparisons, objective biopsy finding quantification, multiplexing, automated image analysis, and electronic data and resource sharing. Current utilization for teaching, quality assurance, conferencing, consultations, research and clinical trials is evolving toward implementation for low-volume, high-complexity clinical services like transplantation pathology. Cost, complexities of implementation, fluid/evolving standards, and unsettled medical/legal and regulatory issues remain as challenges. Regardless, challenges will be overcome and these technologies will enable transplant pathologists to increase information extraction from tissue specimens and contribute to cross-platform biomarker discovery for improved outcomes. PMID:22053785

ACVP-14: Next-Generation Multiplex vRNA and vDNA Lineage Specific In Situ Hybridization Detection With Immunohisto-Fluorescence or Chromogen in the Same Tissue Section with Quantitative Image Analysis in Fixed Tissues from Virally Infected Specimens | Frederick National Laboratory for Cancer Research

Cancer.gov

The Tissue Analysis Core within the AIDS and Cancer Virus Program will process, embed and perform microtomy on fixed tissue samples presented in ethanol. HIV/SIVin situhybridization for detection of vRNA and vDNA will be performed using the next-gene

Development of a Multiplex Single Base Extension Assay for Mitochondrial DNA Haplogroup Typing

PubMed Central

Nelson, Tahnee M.; Just, Rebecca S.; Loreille, Odile; Schanfield, Moses S.; Podini, Daniele

2007-01-01

Aim To provide a screening tool to reduce time and sample consumption when attempting mtDNA haplogroup typing. Methods A single base primer extension assay was developed to enable typing, in a single reaction, of twelve mtDNA haplogroup specific polymorphisms. For validation purposes a total of 147 samples were tested including 73 samples successfully haplogroup typed using mtDNA control region (CR) sequence data, 21 samples inconclusively haplogroup typed by CR data, 20 samples previously haplogroup typed using restriction fragment length polymorphism (RFLP) analysis, and 31 samples of known ancestral origin without previous haplogroup typing. Additionally, two highly degraded human bones embalmed and buried in the early 1950s were analyzed using the single nucleotide polymorphisms (SNP) multiplex. Results When the SNP multiplex was used to type the 96 previously CR sequenced specimens, an increase in haplogroup or macrohaplogroup assignment relative to conventional CR sequence analysis was observed. The single base extension assay was also successfully used to assign a haplogroup to decades-old, embalmed skeletal remains dating to World War II. Conclusion The SNP multiplex was successfully used to obtain haplogroup status of highly degraded human bones, and demonstrated the ability to eliminate possible contributors. The SNP multiplex provides a low-cost, high throughput method for typing of mtDNA haplogroups A, B, C, D, E, F, G, H, L1/L2, L3, M, and N that could be useful for screening purposes for human identification efforts and anthropological studies. PMID:17696300

Detection of respiratory bacterial pathogens causing atypical pneumonia by multiplex Lightmix® RT-PCR.

PubMed

Wagner, Karoline; Springer, Burkard; Imkamp, Frank; Opota, Onya; Greub, Gilbert; Keller, Peter M

2018-04-01

Pneumonia is a severe infectious disease. In addition to common viruses and bacterial pathogens (e.g. Streptococcus pneumoniae), fastidious respiratory pathogens like Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella spp. can cause severe atypical pneumonia. They do not respond to penicillin derivatives, which may cause failure of antibiotic empirical therapy. The same applies for infections with B. pertussis and B. parapertussis, the cause of pertussis disease, that may present atypically and need to be treated with macrolides. Moreover, these fastidious bacteria are difficult to identify by culture or serology, and therefore often remain undetected. Thus, rapid and accurate identification of bacterial pathogens causing atypical pneumonia is crucial. We performed a retrospective method evaluation study to evaluate the diagnostic performance of the new, commercially available Lightmix ® multiplex RT-PCR assay that detects these fastidious bacterial pathogens causing atypical pneumonia. In this retrospective study, 368 clinical respiratory specimens, obtained from patients suffering from atypical pneumonia that have been tested negative for the presence of common agents of pneumonia by culture and viral PCR, were investigated. These clinical specimens have been previously characterized by singleplex RT-PCR assays in our diagnostic laboratory and were used to evaluate the diagnostic performance of the respiratory multiplex Lightmix ® RT-PCR. The multiplex RT-PCR displayed a limit of detection between 5 and 10 DNA copies for different in-panel organisms and showed identical performance characteristics with respect to specificity and sensitivity as in-house singleplex RT-PCRs for pathogen detection. The Lightmix ® multiplex RT-PCR assay represents a low-cost, time-saving and accurate diagnostic tool with high throughput potential. The time-to-result using an automated DNA extraction device for respiratory specimens followed by multiplex RT-PCR detection was below 4 h, which is expected to significantly improve diagnostics for atypical pneumonia-associated bacterial pathogens. Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.

Comparison of nested-multiplex, Taqman & SYBR Green real-time PCR in diagnosis of amoebic liver abscess in a tertiary health care institute in India

PubMed Central

Dinoop, K.P.; Parija, Subhash Chandra; Mandal, Jharna; Swaminathan, R.P.; Narayanan, P.

2016-01-01

Background & objectives: Amoebiasis is a common parasitic infection caused by Entamoeba histolytica and amoebic liver abscess (ALA) is the most common extraintestinal manifestation of amoebiasis. The aim of this study was to standardise real-time PCR assays (Taqman and SYBR Green) to detect E. histolytica from liver abscess pus and stool samples and compare its results with nested-multiplex PCR. Methods: Liver abscess pus specimens were subjected to DNA extraction. The extracted DNA samples were subjected to amplification by nested-multiplex PCR, Taqman (18S rRNA) and SYBR Green real-time PCR (16S-like rRNA assays to detect E. histolytica/E. dispar/E. moshkovskii). The amplification products were further confirmed by DNA sequence analysis. Receiver operator characteristic (ROC) curve analysis was done for nested-multiplex and SYBR Green real-time PCR and the area under the curve was calculated for evaluating the accuracy of the tests to dignose ALA. Results: In all, 17, 19 and 25 liver abscess samples were positive for E. histolytica by nested-multiplex PCR, SYBR Green and Taqman real-time PCR assays, respectively. Significant differences in detection of E. histolytica were noted in the real-time PCR assays evaluated (P<0.0001). The nested-multiplex PCR, SYBR Green real-time PCR and Taqman real-time PCR evaluated showed a positivity rate of 34, 38 and 50 per cent, respectively. Based on ROC curve analysis (considering Taqman real-time PCR as the gold standard), it was observed that SYBR Green real-time PCR was better than conventional nested-multiplex PCR for the diagnosis of ALA. Interpretation & conclusions: Taqman real-time PCR targeting the 18S rRNA had the highest positivity rate evaluated in this study. Both nested multiplex and SYBR Green real-time PCR assays utilized were evaluated to give accurate results. Real-time PCR assays can be used as the gold standard in rapid and reliable diagnosis, and appropriate management of amoebiasis, replacing the conventional molecular methods. PMID:26997014

Enterovirus D68 detection in respiratory specimens: Association with severe disease.

PubMed

Engelmann, Ilka; Fatoux, Marie; Lazrek, Mouna; Alidjinou, Enagnon K; Mirand, Audrey; Henquell, Cécile; Dewilde, Anny; Hober, Didier

2017-07-01

Molecular techniques increased the number of documented respiratory infections. In a substantial number of cases the causative agent remains undetected. Since August 2014, an increase in Enterovirus(EV)-D68 infections was reported. We aimed to investigate epidemiology and clinical relevance of EV-D68. From June to December 2014 and from September to December 2015, 803 and 847 respiratory specimens, respectively, were tested for respiratory viruses with a multiplex RT-PCR. This multiplex RT-PCR does not detect EV-D68. Therefore, 457 (2014) and 343 (2015) specimens with negative results were submitted to an EV-specific-RT-PCR. EV-positive specimens were tested with an EV-D68-specific-RT-PCR and genotyped. Eleven specimens of 2014 tested positive in the EV-specific-RT-PCR and of these seven were positive in the EV-D68-specific-RT-PCR. Typing confirmed these as EV-D68. Median age of EV-D68-positive patients was 3 years (1 month-91 years). Common symptoms included fever (n = 6, 86%), respiratory distress (n = 5, 71%), and cough (n = 4, 57%). All EV-D68-positive patients were admitted to hospital, 4 (57%) were admitted to intensive care units and 6 (86%) received oxygen. One patient suffered from acute flaccid paralysis. Seven specimens of 2015 were positive in the EV-specific-RT-PCR but negative in the EV-D68-specific-RT-PCR. In conclusion, use of an EV-specific-RT-PCR allowed us to detect EV-D68 circulation in autumn 2014 that was not detected by the multiplex RT-PCR and was associated with severe disease. © 2017 Wiley Periodicals, Inc.

Multiplex KRASG12/G13 mutation testing of unamplified cell-free DNA from the plasma of patients with advanced cancers using droplet digital polymerase chain reaction.

PubMed

Janku, F; Huang, H J; Fujii, T; Shelton, D N; Madwani, K; Fu, S; Tsimberidou, A M; Piha-Paul, S A; Wheler, J J; Zinner, R G; Naing, A; Hong, D S; Karp, D D; Cabrilo, G; Kopetz, E S; Subbiah, V; Luthra, R; Kee, B K; Eng, C; Morris, V K; Karlin-Neumann, G A; Meric-Bernstam, F

2017-03-01

Cell-free DNA (cfDNA) from plasma offers easily obtainable material for KRAS mutation analysis. Novel, multiplex, and accurate diagnostic systems using small amounts of DNA are needed to further the use of plasma cfDNA testing in personalized therapy. Samples of 16 ng of unamplified plasma cfDNA from 121 patients with diverse progressing advanced cancers were tested with a KRASG12/G13 multiplex assay to detect the seven most common mutations in the hotspot of exon 2 using droplet digital polymerase chain reaction (ddPCR). The results were retrospectively compared to mutation analysis of archival primary or metastatic tumor tissue obtained at different points of clinical care. Eighty-eight patients (73%) had KRASG12/G13 mutations in archival tumor specimens collected on average 18.5 months before plasma analysis, and 78 patients (64%) had KRASG12/G13 mutations in plasma cfDNA samples. The two methods had initial overall agreement in 103 (85%) patients (kappa, 0.66; ddPCR sensitivity, 84%; ddPCR specificity, 88%). Of the 18 discordant cases, 12 (67%) were resolved by increasing the amount of cfDNA, using mutation-specific probes, or re-testing the tumor tissue, yielding overall agreement in 115 patients (95%; kappa 0.87; ddPCR sensitivity, 96%; ddPCR specificity, 94%). The presence of ≥ 6.2% of KRASG12/G13 cfDNA in the wild-type background was associated with shorter survival (P = 0.001). Multiplex detection of KRASG12/G13 mutations in a small amount of unamplified plasma cfDNA using ddPCR has good sensitivity and specificity and good concordance with conventional clinical mutation testing of archival specimens. A higher percentage of mutant KRASG12/G13 in cfDNA corresponded with shorter survival. © The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Novel Multiplex MethyLight Protocol for Detection of DNA Methylation in Patient Tissues and Bodily Fluids

PubMed Central

Olkhov-Mitsel, Ekaterina; Zdravic, Darko; Kron, Ken; van der Kwast, Theodorus; Fleshner, Neil; Bapat, Bharati

2014-01-01

Aberrant DNA methylation is a hallmark of cancer and is an important potential biomarker. Particularly, combined analysis of a panel of hypermethylated genes shows the most promising clinical performance. Herein, we developed, optimized and standardized a multiplex MethyLight assay to simultaneously detect hypermethylation of APC, HOXD3 and TGFB2 in DNA extracted from prostate cancer (PCa) cell lines, archival tissue specimens, and urine samples. We established that the assay is capable of discriminating between fully methylated and unmethylated alleles with 100% specificity and demonstrated the assay as highly accurate and reproducible as the singleplex approach. For proof of principle, we analyzed the methylation status of these genes in tissue and urine samples of PCa patients as well as PCa-free controls. These data show that the multiplex MethyLight assay offers a significant advantage when working with limited quantities of DNA and has potential applications in research and clinical settings. PMID:24651255

Optimization and Standardization of Fluorescent Cell Barcoding for Multiplexed Flow Cytometric Phenotyping

PubMed Central

Giudice, Valentina; Feng, Xingmin; Kajigaya, Sachiko; Young, Neal S.; Biancotto, Angélique

2017-01-01

Fluorescent cell barcoding (FCB) is a cell-based multiplexing technique for high-throughput flow cytometry. Barcoded samples can be stained and acquired collectively, minimizing staining variability and antibody consumption, and decreasing required sample volumes. Combined with functional measurements, FCB can be used for drug screening, signaling profiling, and cytokine detection, but technical issues are present. We optimized the FCB technique for routine utilization using DyLight 350, DyLight 800, Pacific Orange, and CBD500 for barcoding six, nine, or 36 human peripheral blood specimens. Working concentrations of FCB dyes ranging from 0 to 500 μg/ml were tested, and viability dye staining was optimized to increase robustness of data. A five-color staining with surface markers for Vβ usage analysis in CD4+ and CD8+ T cells was achieved in combination with nine sample barcoding. We provide improvements of the FCB technique that should be useful for multiplex drug screening and for lymphocyte characterization and perturbations in the diagnosis and during the course of disease. PMID:28692789

Diagnostic multiplex PCR for toxin genotyping of Clostridium perfringens isolates.

PubMed

Baums, Christoph G; Schotte, Ulrich; Amtsberg, Gunter; Goethe, Ralph

2004-05-20

In this study we provide a protocol for genotyping Clostridium perfringens with a new multiplex PCR. This PCR enables reliable and specific detection of the toxin genes cpa, cpb, etx, iap, cpe and cpb2 from heat lysed bacterial suspensions. The efficiency of the protocol was demonstrated by typing C. perfringens reference strains and isolates from veterinary bacteriological routine diagnostic specimens.

Digital transplantation pathology: combining whole slide imaging, multiplex staining and automated image analysis.

PubMed

Isse, K; Lesniak, A; Grama, K; Roysam, B; Minervini, M I; Demetris, A J

2012-01-01

Conventional histopathology is the gold standard for allograft monitoring, but its value proposition is increasingly questioned. "-Omics" analysis of tissues, peripheral blood and fluids and targeted serologic studies provide mechanistic insights into allograft injury not currently provided by conventional histology. Microscopic biopsy analysis, however, provides valuable and unique information: (a) spatial-temporal relationships; (b) rare events/cells; (c) complex structural context; and (d) integration into a "systems" model. Nevertheless, except for immunostaining, no transformative advancements have "modernized" routine microscopy in over 100 years. Pathologists now team with hardware and software engineers to exploit remarkable developments in digital imaging, nanoparticle multiplex staining, and computational image analysis software to bridge the traditional histology-global "-omic" analyses gap. Included are side-by-side comparisons, objective biopsy finding quantification, multiplexing, automated image analysis, and electronic data and resource sharing. Current utilization for teaching, quality assurance, conferencing, consultations, research and clinical trials is evolving toward implementation for low-volume, high-complexity clinical services like transplantation pathology. Cost, complexities of implementation, fluid/evolving standards, and unsettled medical/legal and regulatory issues remain as challenges. Regardless, challenges will be overcome and these technologies will enable transplant pathologists to increase information extraction from tissue specimens and contribute to cross-platform biomarker discovery for improved outcomes. ©Copyright 2011 The American Society of Transplantation and the American Society of Transplant Surgeons.

Development of Multiplex Reverse Transcription-Polymerase Chain Reaction for Simultaneous Detection of Influenza A, B and Adenoviruses

PubMed Central

Nakhaie, Mohsen; Soleimanjahi, Hoorieh; Mollaie, Hamid Reza; Arabzadeh, Seyed Mohamad Ali

2018-01-01

Background and objective: Millions of people in developing countries lose their lives due to acute respiratory infections, such as Influenza A & B and Adeno viruses. Given the importance of rapid identification of the virus, in this study the researchers attempted to design a method that enables detection of influenza A, B, and adenoviruses, quickly and simultaneously. The Multiplex RT PCR method was the preferred method for the detection of influenza A, B, and adenoviruses in clinical specimens because it is rapid, sensitive, specific, and more cost-effective than alternative methods Methods: After collecting samples from patients with respiratory disease, virus genome was extracted, then Monoplex PCR was used on positive samples and Multiplex RT-PCR on clinical specimens. Finally, by comparing the bands of these samples, the type of virus in the clinical samples was determined. Results: Performing Multiplex RT-PCR on 50 samples of respiratory tract led to following results; flu A: 12.5%, fluB: 50%, adeno: 27.5%, negative: 7.5%, and 2.5% contamination. Conclusion: Reverse transcription-multiplex Polymerase Chain Reaction (PCR) technique, a rapid diagnostic tool, has potential for high-throughput testing. This method has a significant advantage, which provides simultaneous amplification of numerous viruses in a single reaction. This study concentrates on multiplex molecular technologies and their clinical application for the detection and quantification of respiratory pathogens. The improvement in diagnostic testing for viral respiratory pathogens effects patient management, and leads to more cost-effective delivery of care. It limits unnecessary antibiotic use and improves clinical management by use of suitable treatment. PMID:29731796

A color-corrected strategy for information multiplexed Fourier ptychographic imaging

NASA Astrophysics Data System (ADS)

Wang, Mingqun; Zhang, Yuzhen; Chen, Qian; Sun, Jiasong; Fan, Yao; Zuo, Chao

2017-12-01

Fourier ptychography (FP) is a novel computational imaging technique that provides both wide field of view (FoV) and high-resolution (HR) imaging capacity for biomedical imaging. Combined with information multiplexing technology, wavelength multiplexed (or color multiplexed) FP imaging can be implemented by lighting up R/G/B LED units simultaneously. Furthermore, a HR image can be recovered at each wavelength from the multiplexed dataset. This enhances the efficiency of data acquisition. However, since the same dataset of intensity measurement is used to recover the HR image at each wavelength, the mean value in each channel would converge to the same value. In this paper, a color correction strategy embedded in the multiplexing FP scheme is demonstrated, which is termed as color corrected wavelength multiplexed Fourier ptychography (CWMFP). Three images captured by turning on a LED array in R/G/B are required as priori knowledge to improve the accuracy of reconstruction in the recovery process. Using the reported technique, the redundancy requirement of information multiplexed FP is reduced. Moreover, the accuracy of reconstruction at each channel is improved with correct color reproduction of the specimen.

Detection of hepatitis C virus subtypes 6a, 6n, 6w and mixed infections using a modified multiplex real-time polymerase chain reaction protocol.

PubMed

Lee, Yuan-Ming; Chen, Yen-Ju; Lee, Cheng-Ming; Kuo, Lou-Hui; Wong, Wing-Wai; Chen, Yi-Ming Arthur

2011-12-01

In the past few years, many new subtypes in hepatitis C virus (HCV) genotype 6 have been identified. The aim of this study was to modify the multiplex real-time polymerase chain reaction (RT-PCR) protocol and use it to determine the HCV subtypes of a group of Taiwanese injection drug users (IDUs). We used 76 serum specimens collected in northern Taiwan in 2008. Multiplex RT-PCR was used for HCV subtyping among those serum samples having anti-HCV antibodies. Twenty cases were randomly selected for comparison with subtyping results from Inno-LiPa II tests and phylogenetic tree analysis using NS5B sequences. Multiplex RT-PCR assays showed that 60.5% (46/76) of IDUs had single HCV infection. Three out of 76 (3.9%) had double HCV infection (1b/6a, 2a/2b and 2b/6a). Besides this, 27.6% (21/76) had no HCV signal. One IDU had subtype 6n and two had subtype 6w infection. Inno-LiPa II tests misclassified all 6n and 6w cases as 1b subtype. Our modified multiplex RT-PCR protocol can be used to support molecular epidemiological studies and laboratory diagnoses of different HCV subtypes including genotype 6. Copyright © 2011. Published by Elsevier B.V.

Paradigm for diagnosing mycobacterial disease: direct detection and differentiation of Mycobacterium tuberculosis complex and non-tuberculous mycobacteria in clinical specimens using multiplex real-time PCR.

PubMed

Kim, Jeong-Uk; Ryu, Dae-Shick; Cha, Choong-Hwan; Park, Seon-Hee

2018-03-20

Mycobacterium tuberculosis and non-tuberculous mycobacteria (NTM) are clinically different, and the rapid detection and differentiation of M. tuberculosis complex (MTBC) and NTM is crucial for patient management and infection control. Given the slow growth of most pathogenic mycobacteria, nucleic acid amplification assays are excellent tools for direct identification of mycobacteria in clinical specimens. Recently, a multiplex real-time PCR assay was developed that can directly detect 20 mycobacterial species in clinical specimens. Here, we evaluated the diagnostic performance of the assay for diagnosing mycobacterial disease under routine laboratory conditions. A total of 3334 specimens collected from 1437 patients suspected of tuberculosis infection were subjected to acid-fast bacilli staining, conventional culture and the multiplex real-time PCR assay. To evaluate the sensitivity and specificity of the assay, the overall diagnosis of tuberculosis was defined by positive culture plus medical history, and the 2007 American Thoracic Society and Infectious Disease Society of America diagnostic criteria for NTM disease were applied. The sensitivity, specificity, positive predictive value and negative predictive value were 87.5%, 99.6%, 96.1% and 98.5%, respectively, for the detection of MTBC isolates and 53.3%, 99.9%, 95.2%, and 98.9%, respectively, for detecting NTM isolates. Thus, the assay can correctly differentiate between MTBC and NTM isolates in clinical specimens and would be a useful tool for the rapid differentiation of tuberculosis and NTM disease, despite its limited sensitivity for the diagnosis of NTM disease. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Comparison of a new transport medium with universal transport medium at a tropical field site.

PubMed

Schlaudecker, Elizabeth P; Heck, Joan P; MacIntyre, Elizabeth T; Martinez, Ruben; Dodd, Caitlin N; McNeal, Monica M; Staat, Mary A; Heck, Jeffery E; Steinhoff, Mark C

2014-10-01

Limited data are available in rural Honduran settings describing the etiology of respiratory infections, partially due to limited specimen transport. A new molecular transport media (MTM) preserves released nucleic acid at ambient temperature for later detection. Prospective surveillance was conducted in a Honduran clinic to identify 233 children less than 5 years of age presenting with respiratory symptoms. We obtained 2 nasopharyngeal samples and stored 1 in PrimeStore® MTM at room temperature and 1 in universal transport media (UTM) at -80 °C. The specimens were then transported to Cincinnati Children's Hospital and tested for 16 respiratory viruses using a multiplex PCR panel. The 2 specimen collection systems were similar for detecting the 4 most common viruses: influenza (Kappa = 0.7676, P < 0.0001), human metapneumovirus (Kappa = 0.8770, P < 0.0001), respiratory syncytial virus (Kappa = 0.6849, P < 0.0001), and parainfluenza (Kappa = 0.8796, P < 0.0001). These results suggest that clinical specimens transported via PrimeStore® MTM and UTM yield similar viral multiplex PCR results. Copyright © 2014 Elsevier Inc. All rights reserved.

Lutzomyia (Pintomyia) fischeri (Diptera: Psychodidae: Phlebotominae), a probable vector of American cutaneous leishmaniasis: detection of natural infection by Leishmania (Viannia) DNA in specimens from the municipality of Porto Alegre (RS), Brazil, using multiplex PCR assay.

PubMed

Pita-Pereira, Daniela de; Souza, Getúlio D; Pereira, Thaís de Araújo; Zwetsch, Adriana; Britto, Constança; Rangel, Elizabeth F

2011-12-01

In order to determine natural Leishmania (Viannia) infection in Lutzomyia (Pintomyia) fischeri, a multiplex PCR methodology coupled to non-isotopic hybridization was adopted for the analysis of sand fly samples collected by CDC light traps in an endemic area of American Cutaneous Leishmaniasis (ACL) in the periurban region of the municipality of Porto Alegre, Rio Grande do Sul State, Brazil. We analyzed by PCR methodology 560 specimens of Lutzomyia (Pintomyia) fischeri (520 females and 40 males). The wild sand flies were grouped into 56 pools (52 females and 4 males) of 10 each, and positive results were detected in 2 of the 52 female pools, representing a minimum infection rate of 0.38% based on the presence of at least 1 infected insect in the pool. This result associated with some local evidence such as anthopophily, spatial distribution in accordance with the transmission area and human case incidence, suggests that L. (P.)fischeri may be considered as a secondary vector of ACL in the studied locality. Copyright © 2011 Elsevier B.V. All rights reserved.

Multiplexed Molecular Diagnostics for Respiratory, Gastrointestinal, and Central Nervous System Infections.

PubMed

Hanson, Kimberly E; Couturier, Marc Roger

2016-11-15

The development and implementation of highly multiplexed molecular diagnostic tests have allowed clinical microbiology laboratories to more rapidly and sensitively detect a variety of pathogens directly in clinical specimens. Current US Food and Drug Administration-approved multiplex panels target multiple different organisms simultaneously and can identify the most common pathogens implicated in respiratory viral, gastrointestinal, or central nervous system infections. This review summarizes the test characteristics of available assays, highlights the advantages and limitations of multiplex technology for infectious diseases, and discusses potential utilization of these new tests in clinical practice. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

EZH2 and CD79B mutational status over time in B-cell non-Hodgkin lymphomas detected by high-throughput sequencing using minimal samples

PubMed Central

Saieg, Mauro Ajaj; Geddie, William R; Boerner, Scott L; Bailey, Denis; Crump, Michael; da Cunha Santos, Gilda

2013-01-01

BACKGROUND: Numerous genomic abnormalities in B-cell non-Hodgkin lymphomas (NHLs) have been revealed by novel high-throughput technologies, including recurrent mutations in EZH2 (enhancer of zeste homolog 2) and CD79B (B cell antigen receptor complex-associated protein beta chain) genes. This study sought to determine the evolution of the mutational status of EZH2 and CD79B over time in different samples from the same patient in a cohort of B-cell NHLs, through use of a customized multiplex mutation assay. METHODS: DNA that was extracted from cytological material stored on FTA cards as well as from additional specimens, including archived frozen and formalin-fixed histological specimens, archived stained smears, and cytospin preparations, were submitted to a multiplex mutation assay specifically designed for the detection of point mutations involving EZH2 and CD79B, using MassARRAY spectrometry followed by Sanger sequencing. RESULTS: All 121 samples from 80 B-cell NHL cases were successfully analyzed. Mutations in EZH2 (Y646) and CD79B (Y196) were detected in 13.2% and 8% of the samples, respectively, almost exclusively in follicular lymphomas and diffuse large B-cell lymphomas. In one-third of the positive cases, a wild type was detected in a different sample from the same patient during follow-up. CONCLUSIONS: Testing multiple minimal tissue samples using a high-throughput multiplex platform exponentially increases tissue availability for molecular analysis and might facilitate future studies of tumor progression and the related molecular events. Mutational status of EZH2 and CD79B may vary in B-cell NHL samples over time and support the concept that individualized therapy should be based on molecular findings at the time of treatment, rather than on results obtained from previous specimens. Cancer (Cancer Cytopathol) 2013;121:377–386. © 2013 American Cancer Society. PMID:23361872

Current Status of Human Taeniasis in Lao People's Democratic Republic

PubMed Central

Jeon, Hyeong-Kyu; Yong, Tai-Soon; Sohn, Woon-Mok; Chai, Jong-Yil; Min, Duk-Young; Yun, Cheong-Ha; Rim, Han-Jong; Pongvongsa, Tiengkham; Banouvong, Virasack; Insisiengmay, Bounnaloth; Phommasack, Bounlay

2013-01-01

Human taeniasis was investigated in Lao People's Democratic Republic (Lao PDR) between 2000 and 2011 as part of the nation's helminthiasis survey. A total of 55,038 inhabitants, including 29,846 school children, were examined using the Kato-Katz and scotch-tape anal swab method, and morphological observation of adult worms. Molecular identification of Taenia tapeworms was performed by multiplex PCR or DNA sequence analysis of the mitochondrial cox1 gene. Taenia eggs were present at a rate of 1.5% (845/55,038) in the subject population. Adult tapeworms were identified as T. solium or T. saginata by analyzing the collectable stool specimens (n=126). Three specimens identified as T. solium were found in Luang Prabang, while the remaining 123 specimens, which were T. saginata, were found in Bokeo, Bolikhamxay, Champasak, Houaphan, Khammouane, Luang Namta, Luang Prabang, Oudomxay, Phongsaly, Saysomboune, Saravane, Savannakhet, Xayaboury, Xekong, Xieng Khouang Province, and Vientiane Municipality. PMID:23710098

Current status of human taeniasis in Lao People's Democratic Republic.

PubMed

Jeon, Hyeong-Kyu; Yong, Tai-Soon; Sohn, Woon-Mok; Chai, Jong-Yil; Min, Duk-Young; Yun, Cheong-Ha; Rim, Han-Jong; Pongvongsa, Tiengkham; Banouvong, Virasack; Insisiengmay, Bounnaloth; Phommasack, Bounlay; Eom, Keeseon S

2013-04-01

Human taeniasis was investigated in Lao People's Democratic Republic (Lao PDR) between 2000 and 2011 as part of the nation's helminthiasis survey. A total of 55,038 inhabitants, including 29,846 school children, were examined using the Kato-Katz and scotch-tape anal swab method, and morphological observation of adult worms. Molecular identification of Taenia tapeworms was performed by multiplex PCR or DNA sequence analysis of the mitochondrial cox1 gene. Taenia eggs were present at a rate of 1.5% (845/55,038) in the subject population. Adult tapeworms were identified as T. solium or T. saginata by analyzing the collectable stool specimens (n=126). Three specimens identified as T. solium were found in Luang Prabang, while the remaining 123 specimens, which were T. saginata, were found in Bokeo, Bolikhamxay, Champasak, Houaphan, Khammouane, Luang Namta, Luang Prabang, Oudomxay, Phongsaly, Saysomboune, Saravane, Savannakhet, Xayaboury, Xekong, Xieng Khouang Province, and Vientiane Municipality.

A novel RT-multiplex PCR for enteroviruses, hepatitis A and E viruses and influenza A virus among infants and children with diarrhea in Vietnam.

PubMed

Phan, T G; Nguyen, T A; Yan, H; Okitsu, S; Ushijima, H

2005-06-01

A novel reverse transcription-multiplex polymerase chain reaction (RT-multiplex PCR) assay that can detect enteroviruses, hepatitis A and E viruses and influenza A virus from various hosts (avian species, human, swine and horse) was developed. The identification of that group of viruses was performed with the mixture of four pairs of published specific primers (F1 and R1, P3 and P4, 2s and 2as, MMU42 and MMU43) for amplifying viral genomes and specifically generated four different amplicon sizes of 440, 267, 146 and 219 bp for enteroviruses, hepatitis A and E viruses and influenza A virus, respectively. A total of 276 fecal specimens (previously screened for rotavirus, adenovirus, norovirus, sapovirus and astrovirus-negative) from infants and children admitted into hospital with acute gastroenteritis in Ho Chi Minh city, Vietnam during October 2002 and September 2003 were collected and further tested for the presence of those viruses by RT-multiplex PCR. Enteroviruses were identified in 27 specimens and this represented 9.8%. No hepatitis A and E viruses and influenza A virus was found among these subjects. The sensitivity and specificity of RT-multiplex PCR were also assessed and demonstrated the strong validation against RT-monoplex PCR. Taken together, the findings clearly indicated that this novel RT-multiplex PCR is a simple and potential assay for rapid, sensitive, specific and cost-effective laboratory diagnosis to investigate molecular epidemiology of acute gastroenteritis caused by enteroviruses, hepatitis A and E viruses and influenza A virus. This report is the first, to our knowledge, detecting these kinds of viruses in diarrheal feces from infants and children in Vietnam.

A multiplex PCR method for detection of Aspergillus spp. and Mycobacterium tuberculosis in BAL specimens.

PubMed

Amini, F; Kachuei, R; Noorbakhsh, F; Imani Fooladi, A A

2015-06-01

The aim of this study was the detection of Aspergillus species and Mycobacterium tuberculosis together in bronchoalveolar lavage (BAL) using of multiplex PCR. In this study, from September 2012 until June 2013, 100 bronchoalveolar lavage (BAL) specimens were collected from patients suspected of tuberculosis (TB). After the direct and culture test, multiplex PCR were utilized in order to diagnose Aspergillus species and M. tuberculosis. Phenol-chloroform manual method was used in order to extract DNA from these microorganisms. Aspergillus specific primers, M. tuberculosis designed primers and beta actin primers were used for multiplex PCR. In this study, by multiplex PCR method, Aspergillus species were identified in 12 samples (12%), positive samples in direct and culture test were respectively 11% and 10%. Sensitivity and specificity of this method in comparison to direct test were respectively 100% and 98.8%, also sensitivity and specificity of this method in comparison to culture test were respectively 100% and 97.7%. In this assay, M. tuberculosis was identified in 8 samples (8%). Mycobacterium-positive samples in molecular method, direct and culture test were respectively 6%, 5% and 7%. Sensitivity and specificity of PCR method in comparison to direct test were 80% and 97.8% also sensitivity and specificity of this method in comparison to culture test was 71.4% and 98.9%. In the present study, multiplex PCR method had higher sensitivity than direct and culture test in order to identify and detect Aspergillus, also this method had lower sensitivity for identification of M. tuberculosis, suggesting that the method of DNA extraction was not suitable. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Tissue Microarray Analysis Applied to Bone Diagenesis

PubMed Central

Mello, Rafael Barrios; Silva, Maria Regina Regis; Alves, Maria Teresa Seixas; Evison, Martin Paul; Guimarães, Marco Aurelio; Francisco, Rafaella Arrabaca; Astolphi, Rafael Dias; Iwamura, Edna Sadayo Miazato

2017-01-01

Taphonomic processes affecting bone post mortem are important in forensic, archaeological and palaeontological investigations. In this study, the application of tissue microarray (TMA) analysis to a sample of femoral bone specimens from 20 exhumed individuals of known period of burial and age at death is described. TMA allows multiplexing of subsamples, permitting standardized comparative analysis of adjacent sections in 3-D and of representative cross-sections of a large number of specimens. Standard hematoxylin and eosin, periodic acid-Schiff and silver methenamine, and picrosirius red staining, and CD31 and CD34 immunohistochemistry were applied to TMA sections. Osteocyte and osteocyte lacuna counts, percent bone matrix loss, and fungal spheroid element counts could be measured and collagen fibre bundles observed in all specimens. Decalcification with 7% nitric acid proceeded more rapidly than with 0.5 M EDTA and may offer better preservation of histological and cellular structure. No endothelial cells could be detected using CD31 and CD34 immunohistochemistry. Correlation between osteocytes per lacuna and age at death may reflect reported age-related responses to microdamage. Methodological limitations and caveats, and results of the TMA analysis of post mortem diagenesis in bone are discussed, and implications for DNA survival and recovery considered. PMID:28051148

Rapid Detection of Chlamydia trachomatis and Typing of the Lymphogranuloma venereum associated L-Serovars by TaqMan PCR

PubMed Central

Schaeffer, Anke; Henrich, Birgit

2008-01-01

Background Infection due to Chlamydia trachomatis is the most common sexually transmitted bacterial disease of global health significance, and especially the L-serovars causing lymphogranuloma venereum are increasingly being found in Europe in men who have sex with men. Results The design and evaluation of a rapid, multiplex, real-time PCR targeting the major outer membrane protein (omp-1) -gene and a L-serovar-specific region of the polymorphic protein H (pmp-H) -gene for the detection of Chlamydia trachomatis is reported here. The PCR takes place as a single reaction with an internal control. For L1-, L2- and L3-serovar differentiation a second set of real-time PCRs was evaluated based on the amplification of serovar-specific omp-1-regions. The detection limit of each real-time PCR, multiplexed or not, was 50 genome copies per reaction with an efficiency ranging from 90,5–95,2%. In a retrospective analysis of 50 ocular, rectal and urogenital specimens formerly tested to be positive for C. trachomatis we identified six L2-serovars in rectal specimens of HIV-positive men, one in a double-infection with L3, and one L2 in a urethral specimen of an HIV-negative male. Conclusion This unique real-time PCR is specific and convenient for the rapid routine-diagnostic detection of lymphogranuloma venereum-associated L-serovars and enables the subsequent differentiation of L1, L2 and L3 for epidemiologic studies. PMID:18447917

Multiplex picoliter-droplet digital PCR for quantitative assessment of EGFR mutations in circulating cell-free DNA derived from advanced non-small cell lung cancer patients.

PubMed

Yu, Qian; Huang, Fei; Zhang, Meilin; Ji, Haiying; Wu, Shenchao; Zhao, Ying; Zhang, Chunyan; Wu, Jiong; Wang, Beili; Pan, Baisheng; Zhang, Xin; Guo, Wei

2017-08-01

To explore the possible diagnostic value of liquid biopsy, two multiplex panels using picoliter-droplet digital polymerase chain reaction (ddPCR) were established to quantitatively assess the epidermal growth factor receptor (EGFR) mutations in cell‑free DNA (cfDNA) extracted from the plasma of advanced non‑small cell lung cancer (NSCLC) patients. Plasma samples derived from 22 patients with stage IIIB/IV NSCLC harboring EGFR mutations in matched tumor tissues confirmed by amplification refractory mutation system (ARMS) analysis were subjected to two multiplex ddPCR panels to assess the abundance of tyrosine kinase inhibitor (TKI) ‑sensitive (19DEL, L858R) and TKI‑resistant (T790 M) mutations. Fluctuations in EGFR mutant abundance were monitored by either of the multiplex ddPCR panels for three patients undergoing EGFR‑TKI treatment, with serial plasma sample collections over 2 months. The multiplex ddPCR panels applied to plasma cfDNA from advanced NSCLC patients achieved a total concordance rate of 80% with the EGFR mutation profiles obtained by ARMS from matched biopsy tumor specimens (90% for 19DEL, 95% for L858R, 95% for T790M, respectively) and revealed additional mutant alleles in two subjects. The respective sensitivity and specificity were 90.9 and 88.9% for 19DEL, 87.5 and 100% for L858R, 100 and 93.8% for T790M. The fluctuations of EGFR mutant abundance in serial plasma cfDNA were in accordance with the changes in tumor size as assessed by imaging scans. The authors demonstrated the utility of multiplex ddPCR panels with ultra‑sensitivity for quantitative analysis of EGFR mutations in plasma cfDNA and obtained promising usefulness in EGFR‑TKI decision‑making for advanced NSCLC patients.

Multiplex picoliter-droplet digital PCR for quantitative assessment of EGFR mutations in circulating cell-free DNA derived from advanced non-small cell lung cancer patients

PubMed Central

Yu, Qian; Huang, Fei; Zhang, Meilin; Ji, Haiying; Wu, Shenchao; Zhao, Ying; Zhang, Chunyan; Wu, Jiong; Wang, Beili; Pan, Baisheng; Zhang, Xin; Guo, Wei

2017-01-01

To explore the possible diagnostic value of liquid biopsy, two multiplex panels using picoliter-droplet digital polymerase chain reaction (ddPCR) were established to quantitatively assess the epidermal growth factor receptor (EGFR) mutations in cell-free DNA (cfDNA) extracted from the plasma of advanced non-small cell lung cancer (NSCLC) patients. Plasma samples derived from 22 patients with stage IIIB/IV NSCLC harboring EGFR mutations in matched tumor tissues confirmed by amplification refractory mutation system (ARMS) analysis were subjected to two multiplex ddPCR panels to assess the abundance of tyrosine kinase inhibitor (TKI) -sensitive (19DEL, L858R) and TKI-resistant (T790 M) mutations. Fluctuations in EGFR mutant abundance were monitored by either of the multiplex ddPCR panels for three patients undergoing EGFR-TKI treatment, with serial plasma sample collections over 2 months. The multiplex ddPCR panels applied to plasma cfDNA from advanced NSCLC patients achieved a total concordance rate of 80% with the EGFR mutation profiles obtained by ARMS from matched biopsy tumor specimens (90% for 19DEL, 95% for L858R, 95% for T790M, respectively) and revealed additional mutant alleles in two subjects. The respective sensitivity and specificity were 90.9 and 88.9% for 19DEL, 87.5 and 100% for L858R, 100 and 93.8% for T790M. The fluctuations of EGFR mutant abundance in serial plasma cfDNA were in accordance with the changes in tumor size as assessed by imaging scans. The authors demonstrated the utility of multiplex ddPCR panels with ultra-sensitivity for quantitative analysis of EGFR mutations in plasma cfDNA and obtained promising usefulness in EGFR-TKI decision-making for advanced NSCLC patients. PMID:29067441

High resolution multiplexed functional imaging in live embryos (Conference Presentation)

NASA Astrophysics Data System (ADS)

Xu, Dongli; Zhou, Weibin; Peng, Leilei

2017-02-01

Fourier multiplexed fluorescence lifetime imaging (FmFLIM) scanning laser optical tomography (FmFLIM-SLOT) combines FmFLIM and Scanning laser optical tomography (SLOT) to perform multiplexed 3D FLIM imaging of live embryos. The system had demonstrate multiplexed functional imaging of zebrafish embryos genetically express Foster Resonant Energy Transfer (FRET) sensors. However, previous system has a 20 micron resolution because the focused Gaussian beam diverges quickly from the focused plane, makes it difficult to achieve high resolution imaging over a long projection depth. Here, we present a high-resolution FmFLIM-SLOT system with achromatic Bessel beam, which achieves 3 micron resolution in 3D deep tissue imaging. In Bessel-FmFLIM-SLOT, multiple laser excitation lines are firstly intensity modulated by a Michelson interferometer with a spinning polygon mirror optical delay line, which enables Fourier multiplexed multi-channel lifetime measurements. Then, a spatial light modulator and a prism are used to transform the modulated Gaussian laser beam to an achromatic Bessel beam. The achromatic Bessel beam scans across the whole specimen with equal angular intervals as sample rotated. After tomography reconstruction and the frequency domain lifetime analysis method, both the 3D intensity and lifetime image of multiple excitation-emission can be obtained. Using Bessel-FmFLIM-SLOT system, we performed cellular-resolution FLIM tomography imaging of live zebrafish embryo. Genetically expressed FRET sensors in these embryo will allow non-invasive observation of multiple biochemical processes in vivo.

Multiplex mass spectrometry imaging for latent fingerprints.

PubMed

Yagnik, Gargey B; Korte, Andrew R; Lee, Young Jin

2013-01-01

We have previously developed in-parallel data acquisition of orbitrap mass spectrometry (MS) and ion trap MS and/or MS/MS scans for matrix-assisted laser desorption/ionization MS imaging (MSI) to obtain rich chemical information in less data acquisition time. In the present study, we demonstrate a novel application of this multiplex MSI methodology for latent fingerprints. In a single imaging experiment, we could obtain chemical images of various endogenous and exogenous compounds, along with simultaneous MS/MS images of a few selected compounds. This work confirms the usefulness of multiplex MSI to explore chemical markers when the sample specimen is very limited. Copyright © 2013 John Wiley & Sons, Ltd.

Snapshot Hyperspectral Volumetric Microscopy

NASA Astrophysics Data System (ADS)

Wu, Jiamin; Xiong, Bo; Lin, Xing; He, Jijun; Suo, Jinli; Dai, Qionghai

2016-04-01

The comprehensive analysis of biological specimens brings about the demand for capturing the spatial, temporal and spectral dimensions of visual information together. However, such high-dimensional video acquisition faces major challenges in developing large data throughput and effective multiplexing techniques. Here, we report the snapshot hyperspectral volumetric microscopy that computationally reconstructs hyperspectral profiles for high-resolution volumes of ~1000 μm × 1000 μm × 500 μm at video rate by a novel four-dimensional (4D) deconvolution algorithm. We validated the proposed approach with both numerical simulations for quantitative evaluation and various real experimental results on the prototype system. Different applications such as biological component analysis in bright field and spectral unmixing of multiple fluorescence are demonstrated. The experiments on moving fluorescent beads and GFP labelled drosophila larvae indicate the great potential of our method for observing multiple fluorescent markers in dynamic specimens.

Pathogen Identification by Multiplex LightMix Real-Time PCR Assay in Patients with Meningitis and Culture-Negative Cerebrospinal Fluid Specimens

PubMed Central

Wagner, Karoline; Springer, Burkard; Pires, Valeria P.

2017-01-01

ABSTRACT Acute bacterial meningitis is a medical emergency, and delays in initiating effective antimicrobial therapy result in increased morbidity and mortality. Culture-based methods, thus far considered the “gold standard” for identifying bacterial microorganisms, require 24 to 48 h to provide a diagnosis. In addition, antimicrobial therapy is often started prior to clinical sample collection, thereby decreasing the probability of confirming the bacterial pathogen by culture-based methods. To enable a fast and accurate detection of the most important bacterial pathogens causing meningitis, namely, Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Streptococcus agalactiae, and Listeria monocytogenes, we evaluated a commercially available multiplex LightMix real-time PCR (RT-PCR) in 220 cerebrospinal fluid (CSF) specimens. The majority of CSF samples were collected by lumbar puncture, but we also included some CSF samples from patients with symptoms of meningitis from the neurology department that were recovered from shunts. CSF samples were analyzed by multiplex RT-PCR enabling a first diagnosis within a few hours after sample arrival at our institute. In contrast, bacterial identification took between 24 and 48 h by culture. Overall, a high agreement of bacterial identification between culture and multiplex RT-PCR was observed (99%). Moreover, multiplex RT-PCR enabled the detection of pathogens, S. pneumoniae (n = 2), S. agalactiae (n = 1), and N. meningitidis (n = 1), in four culture-negative samples. As a complement to classical bacteriological CSF culture, the LightMix RT-PCR assay proved to be valuable by improving the rapidity and accuracy of the diagnosis of bacterial meningitis. PMID:29237781

How Well Does Physician Selection of Microbiologic Tests Identify Clostridium difficile and other Pathogens in Paediatric Diarrhoea? Insights Using Multiplex PCR-Based Detection

PubMed Central

Stockmann, Chris; Rogatcheva, Margarita; Harrel, Brian; Vaughn, Mike; Crisp, Rob; Poritz, Mark; Thatcher, Stephanie; Korgenski, Ernest K; Barney, Trenda; Daly, Judy; Pavia, Andrew T

2014-01-01

The objective of this study was to compare the aetiologic yield of standard of care microbiologic testing ordered by physicians with that of a multiplex PCR platform. Stool specimens obtained from children and young adults with gastrointestinal illness were evaluated by standard laboratory methods and a developmental version of the FilmArray Gastrointestinal Diagnostic System (FilmArray GI Panel), a rapid multiplex PCR platform that detects 23 bacterial, viral, and protozoal agents. Results were classified according to the microbiologic tests requested by the treating physician. A median of 3 (range 1-10) microbiologic tests were performed by the clinical laboratory during 378 unique diarrhoeal episodes. A potential aetiologic agent was identified in 46% of stool specimens by standard laboratory methods and in 65% of specimens tested using the FilmArray GI Panel (P<0.001). For those patients who only had Clostridium difficile testing requested, an alternative pathogen was identified in 29% of cases with the FilmArray GI Panel. Notably, 11 (12%) cases of norovirus were identified among children who only had testing for C. difficile ordered. Among those who had C. difficile testing ordered in combination with other tests, an additional pathogen was identified in 57% of stool specimens with the FilmArray GI Panel. For patients who had no C. difficile testing performed, the FilmArray GI Panel identified a pathogen in 63% of cases, including C. difficile in 8%. Physician-specified laboratory testing may miss important diarrhoeal pathogens. Additionally, standard laboratory testing is likely to underestimate co-infections with multiple infectious diarrhoeagenic agents. PMID:25599941

Rapid targeted somatic mutation analysis of solid tumors in routine clinical diagnostics.

PubMed

Magliacane, Gilda; Grassini, Greta; Bartocci, Paola; Francaviglia, Ilaria; Dal Cin, Elena; Barbieri, Gianluca; Arrigoni, Gianluigi; Pecciarini, Lorenza; Doglioni, Claudio; Cangi, Maria Giulia

2015-10-13

Tumor genotyping is an essential step in routine clinical practice and pathology laboratories face a major challenge in being able to provide rapid, sensitive and updated molecular tests. We developed a novel mass spectrometry multiplexed genotyping platform named PentaPanel to concurrently assess single nucleotide polymorphisms in 56 hotspots of the 5 most clinically relevant cancer genes, KRAS, NRAS, BRAF, EGFR and PIK3CA for a total of 221 detectable mutations. To both evaluate and validate the PentaPanel performance, we investigated 1025 tumor specimens of 6 different cancer types (carcinomas of colon, lung, breast, pancreas, and biliary tract, and melanomas), systematically addressing sensitivity, specificity, and reproducibility of our platform. Sanger sequencing was also performed for all the study samples. Our data showed that PentaPanel is a high throughput and robust tool, allowing genotyping for targeted therapy selection of 10 patients in the same run, with a practical turnaround time of 2 working days. Importantly, it was successfully used to interrogate different DNAs isolated from routinely processed specimens (formalin-fixed paraffin embedded, frozen, and cytological samples), covering all the requirements of clinical tests. In conclusion, the PentaPanel platform can provide an immediate, accurate and cost effective multiplex approach for clinically relevant gene mutation analysis in many solid tumors and its utility across many diseases can be particularly relevant in multiple clinical trials, including the new basket trial approach, aiming to identify appropriate targeted drug combination strategies.

Next-Generation Molecular Histology Using Highly Multiplexed Ion Beam Imaging (MIBI) of Breast Cancer Tissue Specimens for Enhanced Clinical Guidance

DTIC Science & Technology

2017-07-01

panels of MIBI multiplexed in situ detection reagents, and compare the quantitative data to the conventional clinically derived “one at a time” and...Measure standard curves for each analyte against western blots using cell lines and tumor samples. Compare quantitation dynamic ranges to...GSTM1, CD68, BAG1, ER, PGR, BCL2, SCUBE2, ACTB, GAPDH, RPLPO, GUS, TFRC) IIa. Compare hybridization results for mass tagged probe designs from both

Next Generation Molecular Histology Using Highly Multiplexed Ion Beam Imaging (MIBI) of Breast Cancer Tissue Specimens for Enhanced Clinical Guidance

DTIC Science & Technology

2016-07-01

AWARD NUMBER: W81XWH-14-1-0180 TITLE: Next-Generation Molecular Histology Using Highly Multiplexed Ion Beam Imaging (MIBI) of Breast Cancer Tissue...1212 REPORT DATE: July 2016 TYPE OF REPORT: Annual PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012...SPONSOR/MONITOR’S ACRONYM(S) U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 11. SPONSOR/MONITOR’S REPORT NUMBER

T-cell clonality analysis in biopsy specimens from two different skin sites shows high specificity in the diagnosis of patients with suggested mycosis fungoides.

PubMed

Thurber, Stacy E; Zhang, Bing; Kim, Youn H; Schrijver, Iris; Zehnder, James; Kohler, Sabine

2007-11-01

The diagnosis of mycosis fungoides (MF) is often difficult because of significant clinical and histopathologic overlap with inflammatory dermatoses. T-cell receptor (TCR)gamma chain rearrangement by polymerase chain reaction (PCR) (TCR-PCR) is a helpful adjuvant tool in this setting, but several of the inflammatory dermatoses in the differential diagnosis of MF may contain a clonal T-cell proliferation. We examined whether analysis for T-cell clonality and comparison of the clones with the standardized BIOMED-2 PCR multiplex primers for the TCRgamma chain from two anatomically distinct skin sites improves diagnostic accuracy. We examined two biopsy specimens each from 10 patients with unequivocal MF, from 18 patients with inflammatory dermatoses, and from 18 patients who could initially not be definitively given a diagnosis based on clinical and histopathologic criteria. Eight of 10 patients with unequivocal MF had an identical clone in both biopsy specimens. Two of 18 patients with inflammatory dermatoses were found to have a clone in one of the biopsy specimens. On further follow-up of the 18 patients with morphologically nondiagnostic biopsy specimens, 13 of 18 were later confirmed to have MF and 5 of 18 had inflammatory dermatoses. Eleven of 13 patients with MF had an identical clone in both biopsy specimens; two of 13 had a polyclonal amplification pattern in both biopsy specimens. Four of 5 patients with inflammatory dermatoses had no clone in either biopsy specimen. One patient with an inflammatory dermatosis had an identical clone in both specimens. The sensitivity of TCR-PCR analysis to evaluate for an identical clone at different anatomic skin sites (dual TCR-PCR) is 82.6% and the specificity is 95.7%. The number of patients in the study group was limited. These data suggest that dual TCR-PCR is a very promising technique with high specificity in distinguishing MF from inflammatory dermatoses.

Forensic SNP Genotyping with SNaPshot: Development of a Novel In-house SBE Multiplex SNP Assay.

PubMed

Zar, Mian Sahib; Shahid, Ahmad Ali; Shahzad, Muhammad Saqib; Shin, Kyoung-Jin; Lee, Hwan Young; Lee, Sang-Seob; Israr, Muhammad; Wiegand, Peter; Kulstein, Galina

2018-04-10

This study introduces a newly developed in-house SNaPshot single-base extension (SBE) multiplex assay for forensic single nucleotide polymorphism (SNP) genotyping of fresh and degraded samples. The assay was validated with fresh blood samples from four different populations. In addition, altogether 24 samples from skeletal remains were analyzed with the multiplex. Full SNP profiles could be obtained from 14 specimens, while ten remains showed partial SNP profiles. Minor allele frequencies (MAF) of bone samples and different populations were compared and used for association of skeletal remains with a certain population. The results reveal that the SNPs of the bone samples are genetically close to the Pathan population. The findings show that the new multiplex system can be utilized for SNP genotyping of degraded and forensic relevant skeletal material, enabling to provide additional investigative leads in criminal cases. © 2018 American Academy of Forensic Sciences.

Newborn screening for autism: in search of candidate biomarkers

PubMed Central

Mizejewski, Gerald J; Lindau-Shepard, Barbara; Pass, Kenneth A

2013-01-01

Background Autism spectrum disorder (ASD) represents a wide range of neurodevelopmental disorders characterized by impairments in social interaction, language, communication and range of interests. Autism is usually diagnosed in children 3–5 years of age using behavioral characteristics; thus, diagnosis shortly after birth would be beneficial for early initiation of treatment. Aim This retrospective study sought to identify newborns at risk for ASD utilizing bloodspot specimens in an immunoassay. Materials & methods The present study utilized stored frozen specimens from ASD children already diagnosed at 15–36 months of age. The newborn specimens and controls were analyzed by immunoassay in a multiplex system that included 90 serum biomarkers and subjected to statisical analysis. Results Three sets of five biomarkers associated with ASD were found that differed from control groups. The 15 candidate biomarkers were then discussed regarding their association with ASD. Conclusion This study determined that a statistically selected panel of 15 biomarkers successfully discriminated presumptive newborns at risk for ASD from those of nonaffected controls. PMID:23547820

Mapping a multiplexed zoo of mRNA expression.

PubMed

Choi, Harry M T; Calvert, Colby R; Husain, Naeem; Huss, David; Barsi, Julius C; Deverman, Benjamin E; Hunter, Ryan C; Kato, Mihoko; Lee, S Melanie; Abelin, Anna C T; Rosenthal, Adam Z; Akbari, Omar S; Li, Yuwei; Hay, Bruce A; Sternberg, Paul W; Patterson, Paul H; Davidson, Eric H; Mazmanian, Sarkis K; Prober, David A; van de Rijn, Matt; Leadbetter, Jared R; Newman, Dianne K; Readhead, Carol; Bronner, Marianne E; Wold, Barbara; Lansford, Rusty; Sauka-Spengler, Tatjana; Fraser, Scott E; Pierce, Niles A

2016-10-01

In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin-embedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences. © 2016. Published by The Company of Biologists Ltd.

Mapping a multiplexed zoo of mRNA expression

PubMed Central

Choi, Harry M. T.; Calvert, Colby R.; Husain, Naeem; Huss, David; Barsi, Julius C.; Deverman, Benjamin E.; Hunter, Ryan C.; Kato, Mihoko; Lee, S. Melanie; Abelin, Anna C. T.; Rosenthal, Adam Z.; Akbari, Omar S.; Li, Yuwei; Hay, Bruce A.; Sternberg, Paul W.; Patterson, Paul H.; Davidson, Eric H.; Mazmanian, Sarkis K.; Prober, David A.; van de Rijn, Matt; Leadbetter, Jared R.; Newman, Dianne K.; Readhead, Carol; Bronner, Marianne E.; Wold, Barbara; Lansford, Rusty; Sauka-Spengler, Tatjana; Fraser, Scott E.

2016-01-01

In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin-embedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences. PMID:27702788

Multiplex PCR detection of Cryptosporidium sp, Giardia lamblia and Entamoeba histolytica directly from dried stool samples from Guinea-Bissauan children with diarrhoea.

PubMed

Mero, Sointu; Kirveskari, Juha; Antikainen, Jenni; Ursing, Johan; Rombo, Lars; Kofoed, Poul-Erik; Kantele, Anu

2017-09-01

In developing countries, diarrhoea is the most common cause of death for children under five years of age, with Giardia lamblia, Cryptosporidium and Entamoeba histolytica as the most frequent pathogenic parasites. Traditional microscopy for stool parasites has poor sensitivity and specificity, while new molecular methods may provide more accurate diagnostics. In poor regions with sample storage hampered by uncertain electricity supply, research would benefit from a method capable of analysing dried stools. A real-time multiplex PCR method with internal inhibition control was developed for detecting Giardia lamblia, Cryptosporidium hominis/parvum and Entamoeba histolytica directly from stool specimens. Applicability to dried samples was checked by comparing with fresh ones in a small test material. Finally, the assay was applied to dried specimens collected from Guinea-Bissauan children with diarrhoea. The PCR's analytical sensitivity limit was 0.1 ng/ml for G. lamblia DNA, 0.01 ng/ml for E. histolytica DNA and 0.1 ng/ml for Cryptosporidium sp. In the test material, the assay performed similarly with fresh and dried stools. Of the 52 Guinea-Bissauan samples, local microscopy revealed a parasite in 15%, while PCR detected 62% positive for at least one parasite: 44% of the dried samples had Giardia, 23% Cryptosporidium and 0% E. histolytica. Our new multiplex real-time PCR for protozoa presents a sensitive method applicable to dried samples. As proof of concept, it worked well on stools collected from Guinea-Bissauan children with diarrhoea. It provides an epidemiological tool for analysing dried specimens from regions poor in resources.

Next-Generation Molecular Histology Using Highly Multiplexed Ion Beam Imaging (MIBI) of Breast Cancer Tissue Specimens for Enhanced Clinical Guidance

DTIC Science & Technology

2015-07-01

SLC7A5, NRDG1, HTF9C, CEACAM5). Gene-expression assays using qRT-PCR, array hybridization, and RNA sequence assays have also been developed. The...and RNA sequence assays have also been developed. The OncotypeDX, for example, uses a panel of 21 genes (16 analytical, 5 controls: Ki67, STK15...Provide a brief list of keywords (limit to 20 words). Breast Cancer Diagnosis Pathology Immunophenotype Multiplex Morphology RNA In Situ

The ichnogenus Curvolithus revisited

USGS Publications Warehouse

Buatois, L.A.; Mangano, M.G.; Mikulas, R.; Maples, C.G.

1998-01-01

The ichnogenus Curvolithus Fritsch, 1908, originally described from the Ordovician of the Prague Basin, typically comprises ribbonlike or tonguelike, flattened, endostratal traces with three rounded lobes on the upper surface. However, considerable confusion persists regarding the ichnotaxonomic status and diagnostic features of its ichnospecies. The type specimens of this ichnotaxon, overlooked in most subsequent reports, are redescribed herein. Curvolithus multiplex Fritsch, 1908, the type species, is retained for specimens with a trilobate upper surface and a quadralobate lower surface, in contrast to the criteria adopted by subsequent authors. The other ichnospecies originally proposed from the type locality, C. gregarius Fritsch, 1908, actually consists of a series of grouped parallel scratch marks forming ridges and should be removed from Curvolithus. Subsequently, four ichnospecies were defined: C.? davidis Webby 1970; C. annulatus Badve and Ghare 1978; C. aequus Walter et al. 1989; and C. manitouensis Maples and Suttner 1990. Curvolithus? davidis shows the typical trilobation of Curvolithus apparently in its lower surface, but the morphology of the upper surface is uncertain. Accordingly, it does not warrant ichnospecific assessment, and is regarded as a nomen dubium. The nature of the annulations on the trilobate upper surface of C. annulatus is unclear, and this ichnospecies is also best considered as a nomen dubium. Curvolithus aequus has a bilobate lower surface and probably represents washed out specimens of Didymaulichnus. Finally, C. manitouensis comprises specimens with a smooth, trilobate upper surface and a smooth, quadralobate lower surface, and is best regarded as a junior synonym of C. multiplex. Curvolithus multiplex has been used incorrectly for Curvolithus with a trilobate upper surface and a trilobate to unilobate lower surface. The new ichnospecies, Curvolithus simplex, is proposed herein for such traces. Curvolithus is interpreted as a locomotion trace (Repichnia) of endostratal carnivores, possibly gastropods, flatworms, or nemerteans. Curvolithus is a component of the Cruziana ichnofacies in shallow-marine facies, either of normal salinity or slightly brackish, in the latter case typically associated with fan deltas.

Development of a multiplex real-time PCR for the simultaneous detection of herpes simplex and varicella zoster viruses in cerebrospinal fluid and lesion swab specimens.

PubMed

Wong, Anita A; Pabbaraju, Kanti; Wong, Sallene; Tellier, Raymond

2016-03-01

Herpes simplex viruses (HSV) and varicella zoster virus (VZV) can have very similar and wide-ranging clinical presentations. Rapid identification is necessary for timely antiviral therapy, especially with infections involving the central nervous system, neonates, and immunocompromised individuals. Detection of HSV-1, HSV-2 and VZV was combined into one real-time PCR reaction utilizing hydrolysis probes. The assay was validated on the LightCycler(®) (Roche) and Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific Inc.) to detect alphaherpesviruses in cerebral spinal fluid (CSF) and lesion swab specimens, respectively. Validation data on blood and tissue samples are also presented. The multiplex assay showed excellent sensitivity, specificity and reproducibility when compared to two singleplex real-time PCR assays for CSF samples and direct fluorescent antigen/culture for lesion swab samples. Implementation of the multiplex assay has facilitated improved sensitivity and accuracy as well as reduced turn-around-times and costs. The results from a large data set of 16,622 prospective samples tested between August 16, 2012 to February 1, 2014 at the Provincial Laboratory for Public Health (Alberta, Canada) are presented here. Copyright © 2015 Elsevier B.V. All rights reserved.

Simultaneous detection of eight avian influenza A virus subtypes by multiplex reverse transcription-PCR using a GeXP analyser.

PubMed

Li, Meng; Xie, Zhixun; Xie, Zhiqin; Liu, Jiabo; Xie, Liji; Deng, Xianwen; Luo, Sisi; Fan, Qing; Huang, Li; Huang, Jiaoling; Zhang, Yanfang; Zeng, Tingting; Wang, Sheng

2018-04-18

Recent studies have demonstrated that at least eight subtypes of avian influenza virus (AIV) can infect humans, including H1, H2, H3, H5, H6, H7, H9 and H10. A GeXP analyser-based multiplex reverse transcription (RT)-PCR (GeXP-multiplex RT-PCR) assay was developed in our recent studies to simultaneously detect these eight AIV subtypes using the haemagglutinin (HA) gene. The assay consists of chimeric primer-based PCR amplification with fluorescent labelling and capillary electrophoresis separation. RNA was extracted from chick embryo allantoic fluid or liquid cultures of viral isolates. In addition, RNA synthesised via in vitro transcription was used to determine the specificity and sensitivity of the assay. After selecting the primer pairs, their concentrations and GeXP-multiplex RT-PCR conditions were optimised. The established GeXP-multiplex RT-PCR assay can detect as few as 100 copies of premixed RNA templates. In the present study, 120 clinical specimens collected from domestic poultry at live bird markets and from wild birds were used to evaluate the performance of the assay. The GeXP-multiplex RT-PCR assay specificity was the same as that of conventional RT-PCR. Thus, the GeXP-multiplex RT-PCR assay is a rapid and relatively high-throughput method for detecting and identifying eight AIV subtypes that may infect humans.

Single tube multiplex real-time PCR for the rapid detection of herpesvirus infections of the central nervous system.

PubMed

Sankuntaw, Nipaporn; Sukprasert, Saovaluk; Engchanil, Chulapan; Kaewkes, Wanlop; Chantratita, Wasun; Pairoj, Vantanit; Lulitanond, Viraphong

2011-01-01

Human herpesvirus infection of immunocompromised hosts may lead to central nervous system (CNS) infection and diseases. In this study, a single tube multiplex real-time PCR was developed for the detection of five herpesviruses (HSV-1, HSV-2, VZV, EBV and CMV) in clinical cerebrospinal fluid (CSF) specimens. Two primer pairs specific for the herpesvirus polymerase gene and five hybridization probe pairs for the specific identification of the herpesvirus types were used in a LightCycler multiplex real-time PCR. A singleplex real-time PCR was first optimized and then applied to the multiplex real-time PCR. The singleplex and multiplex real-time PCRs showed no cross-reactivity. The sensitivity of the singleplex real-time PCR was 1 copy per reaction for each herpesvirus, while that of the multiplex real-time PCR was 1 copy per reaction for HSV-1 and VZV and 10 copies per reaction for HSV-2, EBV and CMV. Intra and inter-assay variations of the single tube multiplex assay were in the range of 0.02%-3.67% and 0.79%-4.35%, respectively. The assay was evaluated by testing 62 clinical CSF samples and was found to have equivalent sensitivity, specificity and agreement as the routine real-time PCR, but reducing time, cost and amount of used sample. Copyright © 2011 Elsevier Ltd. All rights reserved.

Using next-generation sequencing for high resolution multiplex analysis of copy number variation from nanogram quantities of DNA from formalin-fixed paraffin-embedded specimens.

PubMed

Wood, Henry M; Belvedere, Ornella; Conway, Caroline; Daly, Catherine; Chalkley, Rebecca; Bickerdike, Melissa; McKinley, Claire; Egan, Phil; Ross, Lisa; Hayward, Bruce; Morgan, Joanne; Davidson, Leslie; MacLennan, Ken; Ong, Thian K; Papagiannopoulos, Kostas; Cook, Ian; Adams, David J; Taylor, Graham R; Rabbitts, Pamela

2010-08-01

The use of next-generation sequencing technologies to produce genomic copy number data has recently been described. Most approaches, however, reply on optimal starting DNA, and are therefore unsuitable for the analysis of formalin-fixed paraffin-embedded (FFPE) samples, which largely precludes the analysis of many tumour series. We have sought to challenge the limits of this technique with regards to quality and quantity of starting material and the depth of sequencing required. We confirm that the technique can be used to interrogate DNA from cell lines, fresh frozen material and FFPE samples to assess copy number variation. We show that as little as 5 ng of DNA is needed to generate a copy number karyogram, and follow this up with data from a series of FFPE biopsies and surgical samples. We have used various levels of sample multiplexing to demonstrate the adjustable resolution of the methodology, depending on the number of samples and available resources. We also demonstrate reproducibility by use of replicate samples and comparison with microarray-based comparative genomic hybridization (aCGH) and digital PCR. This technique can be valuable in both the analysis of routine diagnostic samples and in examining large repositories of fixed archival material.

Multiplex qPCR for serodetection and serotyping of hepatitis viruses: A brief review.

PubMed

Irshad, Mohammad; Gupta, Priyanka; Mankotia, Dhananjay Singh; Ansari, Mohammad Ahmad

2016-05-28

The present review describes the current status of multiplex quantitative real time polymerase chain reaction (qPCR) assays developed and used globally for detection and subtyping of hepatitis viruses in body fluids. Several studies have reported the use of multiplex qPCR for the detection of hepatitis viruses, including hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV). In addition, multiplex qPCR has also been developed for genotyping HBV, HCV, and HEV subtypes. Although a single step multiplex qPCR assay for all six hepatitis viruses, i.e., A to G viruses, is not yet reported, it may be available in the near future as the technologies continue to advance. All studies use a conserved region of the viral genome as the basis of amplification and hydrolysis probes as the preferred chemistries for improved detection. Based on a standard plot prepared using varying concentrations of template and the observed threshold cycle value, it is possible to determine the linear dynamic range and to calculate an exact copy number of virus in the specimen. Advantages of multiplex qPCR assay over singleplex or other molecular techniques in samples from patients with co-infection include fast results, low cost, and a single step investigation process.

Multiplex qPCR for serodetection and serotyping of hepatitis viruses: A brief review

PubMed Central

Irshad, Mohammad; Gupta, Priyanka; Mankotia, Dhananjay Singh; Ansari, Mohammad Ahmad

2016-01-01

The present review describes the current status of multiplex quantitative real time polymerase chain reaction (qPCR) assays developed and used globally for detection and subtyping of hepatitis viruses in body fluids. Several studies have reported the use of multiplex qPCR for the detection of hepatitis viruses, including hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV). In addition, multiplex qPCR has also been developed for genotyping HBV, HCV, and HEV subtypes. Although a single step multiplex qPCR assay for all six hepatitis viruses, i.e., A to G viruses, is not yet reported, it may be available in the near future as the technologies continue to advance. All studies use a conserved region of the viral genome as the basis of amplification and hydrolysis probes as the preferred chemistries for improved detection. Based on a standard plot prepared using varying concentrations of template and the observed threshold cycle value, it is possible to determine the linear dynamic range and to calculate an exact copy number of virus in the specimen. Advantages of multiplex qPCR assay over singleplex or other molecular techniques in samples from patients with co-infection include fast results, low cost, and a single step investigation process. PMID:27239109

Thermally multiplexed polymerase chain reaction.

PubMed

Phaneuf, Christopher R; Pak, Nikita; Saunders, D Curtis; Holst, Gregory L; Birjiniuk, Joav; Nagpal, Nikita; Culpepper, Stephen; Popler, Emily; Shane, Andi L; Jerris, Robert; Forest, Craig R

2015-07-01

Amplification of multiple unique genetic targets using the polymerase chain reaction (PCR) is commonly required in molecular biology laboratories. Such reactions are typically performed either serially or by multiplex PCR. Serial reactions are time consuming, and multiplex PCR, while powerful and widely used, can be prone to amplification bias, PCR drift, and primer-primer interactions. We present a new thermocycling method, termed thermal multiplexing, in which a single heat source is uniformly distributed and selectively modulated for independent temperature control of an array of PCR reactions. Thermal multiplexing allows amplification of multiple targets simultaneously-each reaction segregated and performed at optimal conditions. We demonstrate the method using a microfluidic system consisting of an infrared laser thermocycler, a polymer microchip featuring 1 μl, oil-encapsulated reactions, and closed-loop pulse-width modulation control. Heat transfer modeling is used to characterize thermal performance limitations of the system. We validate the model and perform two reactions simultaneously with widely varying annealing temperatures (48 °C and 68 °C), demonstrating excellent amplification. In addition, to demonstrate microfluidic infrared PCR using clinical specimens, we successfully amplified and detected both influenza A and B from human nasopharyngeal swabs. Thermal multiplexing is scalable and applicable to challenges such as pathogen detection where patients presenting non-specific symptoms need to be efficiently screened across a viral or bacterial panel.

A multiplex PCR/LDR assay for simultaneous detection and identification of the NIAID category B bacterial food and water-borne pathogens.

PubMed

Rundell, Mark S; Pingle, Maneesh; Das, Sanchita; Hussain, Aashiq; Ocheretina, Oksana; Charles, Macarthur; Larone, Davise H; Spitzer, Eric D; Golightly, Linnie; Barany, Francis

2014-06-01

Enteric pathogens that cause gastroenteritis remain a major global health concern. The goal of this study was to develop a multiplex PCR/ligation detection reaction (LDR) assay for the detection of all NIAID category B bacterial food and water-borne pathogens directly from stool specimens. To validate the PCR/LDR assay, clinical isolates of Campylobacter spp., Vibrio spp., Shigella spp., Salmonella spp., Listeria monocytogenes, Yersinia enterocolitica, and diarrheagenic Escherichia coli were tested. The sensitivity and specificity of the assay were assessed using a large number of seeded culture-negative stool specimens and a smaller set of clinical specimens from Haiti. The overall sensitivity ranged from 91% to 100% (median 100%) depending on the species. For the majority of organisms, the sensitivity was 100%. The overall specificity based on initial testing ranged from 98% to 100% depending on the species. After additional testing of discordant samples, the lowest specificity was 99.4%. PCR/LDR detected additional category B agents (particularly diarrheagenic E. coli) in 11/40 specimens from Haiti that were culture-positive for V. cholerae and in approximately 1% of routine culture-negative stool specimens from a hospital in New York. This study demonstrated the ability of the PCR/LDR assay to detect a large comprehensive panel of category B enteric bacterial pathogens as well as mixed infections. This type of assay has the potential to provide earlier warnings of possible public health threats and more accurate surveillance of food and water-borne pathogens. Copyright © 2014 Elsevier Inc. All rights reserved.

A Multiplex PCR/LDR Assay for Simultaneous Detection and Identification of the NIAID Category B Bacterial Food and Water-borne Pathogens

PubMed Central

Rundell, Mark S.; Pingle, Maneesh; Das, Sanchita; Hussain, Aashiq; Ocheretina, Oksana; Charles, Macarthur; Larone, Davise H.; Spitzer, Eric D.; Golightly, Linnie; Barany, Francis

2014-01-01

Enteric pathogens that cause gastroenteritis remain a major global health concern. The goal of this study was to develop a multiplex PCR/LDR assay for the detection of all NIAID category B bacterial food and water-borne pathogens directly from stool specimens. To validate the PCR/LDR assay, clinical isolates of Campylobacter spp., Vibrio spp., Shigella spp., Salmonella spp., Listeria monocytogenes, Yersinia enterocolitica, and diarrheagenic Escherichia coli were tested. The sensitivity and specificity of the assay was assessed using a large number of seeded culture-negative stool specimens and a smaller set of clinical specimens from Haiti. The overall sensitivity ranged from 91 to 100% (median 100%) depending on the species. For the majority of organisms the sensitivity was 100%. The overall specificity based on initial testing ranged from 98% to 100% depending on the species. After additional testing of discordant samples the lowest specificity was 99.4%. PCR/LDR detected additional category B agents (particularly diarrheagenic E. coli) in 11/40 specimens from Haiti that were culture-positive for V. cholerae and in approximately 1% of routine culture-negative stool specimens from a hospital in New York. This study demonstrated the ability of the PCR/LDR assay to detect a large comprehensive panel of category B enteric bacterial pathogens as well as mixed infections. This type of assay has the potential to provide earlier warnings of possible public health threats and more accurate surveillance of food and water-borne pathogens. PMID:24709368

Molecular profiling of single cancer cells and clinical tissue specimens with semiconductor quantum dots

PubMed Central

Xing, Yun; Smith, Andrew M; Agrawal, Amit; Ruan, Gang; Nie, Shuming

2006-01-01

Semiconductor quantum dots (QDs) are a new class of fluorescent labels with broad applications in biomedical imaging, disease diagnostics, and molecular and cell biology. In comparison with organic dyes and fluorescent proteins, quantum dots have unique optical and electronic properties such as size-tunable light emission, improved signal brightness, resistance against photobleaching, and simultaneous excitation of multiple fluorescence colors. Recent advances have led to multifunctional nanoparticle probes that are highly bright and stable under complex in vitro and in vivo conditions. New designs involve encapsulating luminescent QDs with amphiphilic block copolymers, and linking the polymer coating to tumor-targeting ligands and drug-delivery functionalities. These improved QDs have opened new possibilities for real-time imaging and tracking of molecular targets in living cells, for multiplexed analysis of biomolecular markers in clinical tissue specimens, and for ultrasensitive imaging of malignant tumors in living animal models. In this article, we briefly discuss recent developments in bioaffinity QD probes and their applications in molecular profiling of individual cancer cells and clinical tissue specimens. PMID:17722280

Development and preliminary evaluation of a multiplexed amplification and next generation sequencing method for viral hemorrhagic fever diagnostics

PubMed Central

Radonić, Aleksandar; Kocak Tufan, Zeliha; Domingo, Cristina

2017-01-01

Background We describe the development and evaluation of a novel method for targeted amplification and Next Generation Sequencing (NGS)-based identification of viral hemorrhagic fever (VHF) agents and assess the feasibility of this approach in diagnostics. Methodology An ultrahigh-multiplex panel was designed with primers to amplify all known variants of VHF-associated viruses and relevant controls. The performance of the panel was evaluated via serially quantified nucleic acids from Yellow fever virus, Rift Valley fever virus, Crimean-Congo hemorrhagic fever (CCHF) virus, Ebola virus, Junin virus and Chikungunya virus in a semiconductor-based sequencing platform. A comparison of direct NGS and targeted amplification-NGS was performed. The panel was further tested via a real-time nanopore sequencing-based platform, using clinical specimens from CCHF patients. Principal findings The multiplex primer panel comprises two pools of 285 and 256 primer pairs for the identification of 46 virus species causing hemorrhagic fevers, encompassing 6,130 genetic variants of the strains involved. In silico validation revealed that the panel detected over 97% of all known genetic variants of the targeted virus species. High levels of specificity and sensitivity were observed for the tested virus strains. Targeted amplification ensured viral read detection in specimens with the lowest virus concentration (1–10 genome equivalents) and enabled significant increases in specific reads over background for all viruses investigated. In clinical specimens, the panel enabled detection of the causative agent and its characterization within 10 minutes of sequencing, with sample-to-result time of less than 3.5 hours. Conclusions Virus enrichment via targeted amplification followed by NGS is an applicable strategy for the diagnosis of VHFs which can be adapted for high-throughput or nanopore sequencing platforms and employed for surveillance or outbreak monitoring. PMID:29155823

Development and preliminary evaluation of a multiplexed amplification and next generation sequencing method for viral hemorrhagic fever diagnostics.

PubMed

Brinkmann, Annika; Ergünay, Koray; Radonić, Aleksandar; Kocak Tufan, Zeliha; Domingo, Cristina; Nitsche, Andreas

2017-11-01

We describe the development and evaluation of a novel method for targeted amplification and Next Generation Sequencing (NGS)-based identification of viral hemorrhagic fever (VHF) agents and assess the feasibility of this approach in diagnostics. An ultrahigh-multiplex panel was designed with primers to amplify all known variants of VHF-associated viruses and relevant controls. The performance of the panel was evaluated via serially quantified nucleic acids from Yellow fever virus, Rift Valley fever virus, Crimean-Congo hemorrhagic fever (CCHF) virus, Ebola virus, Junin virus and Chikungunya virus in a semiconductor-based sequencing platform. A comparison of direct NGS and targeted amplification-NGS was performed. The panel was further tested via a real-time nanopore sequencing-based platform, using clinical specimens from CCHF patients. The multiplex primer panel comprises two pools of 285 and 256 primer pairs for the identification of 46 virus species causing hemorrhagic fevers, encompassing 6,130 genetic variants of the strains involved. In silico validation revealed that the panel detected over 97% of all known genetic variants of the targeted virus species. High levels of specificity and sensitivity were observed for the tested virus strains. Targeted amplification ensured viral read detection in specimens with the lowest virus concentration (1-10 genome equivalents) and enabled significant increases in specific reads over background for all viruses investigated. In clinical specimens, the panel enabled detection of the causative agent and its characterization within 10 minutes of sequencing, with sample-to-result time of less than 3.5 hours. Virus enrichment via targeted amplification followed by NGS is an applicable strategy for the diagnosis of VHFs which can be adapted for high-throughput or nanopore sequencing platforms and employed for surveillance or outbreak monitoring.

High Throughput Multiplex PCR and Probe-based Detection with Luminex Beads for Seven Intestinal Parasites

PubMed Central

Taniuchi, Mami; Verweij, Jaco J.; Noor, Zannatun; Sobuz, Shihab U.; van Lieshout, Lisette; Petri, William A.; Haque, Rashidul; Houpt, Eric R.

2011-01-01

Polymerase chain reaction (PCR) assays for intestinal parasites are increasingly being used on fecal DNA samples for enhanced specificity and sensitivity of detection. Comparison of these tests against microscopy and copro-antigen detection has been favorable, and substitution of PCR-based assays for the ova and parasite stool examination is a foreseeable goal for the near future. One challenge is the diverse list of protozoan and helminth parasites. Several existing real-time PCR assays for the major intestinal parasites—Cryptosporidium spp., Giardia intestinalis, Entamoeba histolytica, Ancylostoma duodenale, Ascaris lumbricoides, Necator americanus, and Strongyloides stercoralis—were adapted into a high throughput protocol. The assay involves two multiplex PCR reactions, one with specific primers for the protozoa and one with specific primers for the helminths, after which PCR products are hybridized to beads linked to internal oligonucleotide probes and detected on a Luminex platform. When compared with the parent multiplex real-time PCR assays, this multiplex PCR-bead assay afforded between 83% and 100% sensitivity and specificity on a total of 319 clinical specimens. In conclusion, this multiplex PCR-bead protocol provides a sensitive diagnostic screen for a large panel of intestinal parasites. PMID:21292910

A Multiplexed Amplicon Approach for Detecting Gene Fusions by Next-Generation Sequencing.

PubMed

Beadling, Carol; Wald, Abigail I; Warrick, Andrea; Neff, Tanaya L; Zhong, Shan; Nikiforov, Yuri E; Corless, Christopher L; Nikiforova, Marina N

2016-03-01

Chromosomal rearrangements that result in oncogenic gene fusions are clinically important drivers of many cancer types. Rapid and sensitive methods are therefore needed to detect a broad range of gene fusions in clinical specimens that are often of limited quantity and quality. We describe a next-generation sequencing approach that uses a multiplex PCR-based amplicon panel to interrogate fusion transcripts that involve 19 driver genes and 94 partners implicated in solid tumors. The panel also includes control assays that evaluate the 3'/5' expression ratios of 12 oncogenic kinases, which might be used to infer gene fusion events when the partner is unknown or not included on the panel. There was good concordance between the solid tumor fusion gene panel and other methods, including fluorescence in situ hybridization, real-time PCR, Sanger sequencing, and other next-generation sequencing panels, because 40 specimens known to harbor gene fusions were correctly identified. No specific fusion reads were observed in 59 fusion-negative specimens. The 3'/5' expression ratio was informative for fusions that involved ALK, RET, and NTRK1 but not for BRAF or ROS1 fusions. However, among 37 ALK or RET fusion-negative specimens, four exhibited elevated 3'/5' expression ratios, indicating that fusions predicted solely by 3'/5' read ratios require confirmatory testing. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Multiplex PCR for rapid diagnosis and differentiation of pox and pox-like diseases in dromedary Camels.

PubMed

Khalafalla, Abdelmalik I; Al-Busada, Khalid A; El-Sabagh, Ibrahim M

2015-07-07

Pox and pox-like diseases of camels are a group of exanthematous skin conditions that have become increasingly important economically. Three distinct viruses may cause them: camelpox virus (CMLV), camel parapox virus (CPPV) and camelus dromedary papilloma virus (CdPV). These diseases are often difficult to differentiate based on clinical presentation in disease outbreaks. Molecular methods such as PCR targeting species-specific genes have been developed and used to identify these diseases, but not simultaneously in a single tube. Recently, multiplex PCR has gained reputation as a convenient diagnostic method with cost-and timesaving benefits. In the present communication, we describe the development, optimization and validation of a multiplex PCR assay able to detect simultaneously the genome of the three viruses in one single test allowing for rapid and efficient molecular diagnosis. The assay was developed based on the evaluation and combination of published and new primer sets and was validated with viral genomic DNA extracted from known virus strains (n = 14) and DNA extracted from homogenized clinical skin specimens (n = 86). The assay detects correctly the target pathogens by amplification of targeted genes, even in case of co-infection. The method showed high sensitivity, and the specificity was confirmed by PCR-product sequencing. This assay provide rapid, sensitive and specific method for identifying three important viruses in specimens collected from dromedary camels with varying clinical presentations.

The development and application of a multiplex short tandem repeat (STR) system for identifying subspecies, individuals and sex in tigers.

PubMed

Zou, Zheng-Ting; Uphyrkina, Olga V; Fomenko, Pavel; Luo, Shu-Jin

2015-07-01

Poaching and trans-boundary trafficking of tigers and body parts are threatening the world's last remaining wild tigers. Development of an efficient molecular genetic assay for tracing the origins of confiscated specimens will assist in law enforcement and wildlife forensics for this iconic flagship species. We developed a multiplex genotyping system "tigrisPlex" to simultaneously assess 22 short tandem repeat (STR, or microsatellite) loci and a gender-identifying SRY gene, all amplified in 4 reactions using as little as 1 ng of template DNA. With DNA samples used for between-run calibration, the system generates STR genotypes that are directly compatible with voucher tiger subspecies genetic profiles, hence making it possible to identify subspecies via bi-parentally inherited markers. We applied "tigrisPlex" to 12 confiscated specimens from Russia and identified 6 individuals (3 females and 3 males), each represented by duplicated samples and all designated as Amur tigers (Panthera tigris altaica) with high confidence. This STR multiplex system can serve as an effective and versatile approach for genetic profiling of both wild and captive tigers as well as confiscated tiger products, fulfilling various conservation needs for identifying the origins of tiger samples. © 2015 International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and Wiley Publishing Asia Pty Ltd.

Usefulness of rapid urease test samples for molecular analysis of clarithromycin resistance in Helicobacter pylori.

PubMed

Baroni, María R; Bucci, Pamela; Giani, Rita N; Giusti, Antonela; Tedeschi, Fabian A; Salvatierra, Emiliano; Barbaglia, Yanina; Jimenez, Félix; Zalazar, Fabian E

2018-03-27

Helicobacter pylori is a gastric pathogen that is widely recognized as a causative agent of gastric disease. Its eradication is variable, mainly due to increased resistance to clarithromycin. Our objective was: to evaluate (i) if the biopsy specimen used for the rapid urease test is a useful sample to detect resistance to clarithromycin by PCR-RFLP and (ii) the distribution of A2142G and A2143G point mutations in the 23S rRNA gene, in relation to virulence factors in our region. Gastric specimens were collected from adult dyspeptic patients (n=141) and H. pylori was investigated by the rapid urease test, histopathological analysis and PCR for the hsp60 gene. Clarithromycin resistance was detected by PCR-RFLP in 62 H. pylori (+) paired biopsy specimens submitted to molecular analysis and the rapid urease test. H. pylori virulence factors were analyzed by multiplex PCR using specific primers for the cagA, vacA and babA2 genes. Thirteen out of 62 strains (20.9%) were resistant to clarithromycin: 6/13 (46.2%) harbored the A2143G mutation whereas 7/13 (53.8%) carried the A2142G point mutation. vacA m1s1 was the most frequent genotype among the resistant strains. In conclusion, the biopsy specimens used for the rapid urease test were suitable samples for clarithromycin resistance detection in patients infected with H. pylori, which became especially useful in cases where the number or size of the biopsies is limited. In addition, this is the first report of a molecular analysis for clarithromycin resistance performed directly from gastric biopsies in our region. Copyright © 2018 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Application of Multiplexed FBG and PZT Impedance Sensors for Health Monitoring of Rocks

PubMed Central

Yang, Yaowen; Annamdas, Venu Gopal Madhav; Wang, Chao; Zhou, Yingxin

2008-01-01

Reliable structural health monitoring (SHM) including nondestructive evaluation (NDE) is essential for safe operation of infrastructure systems. Effective monitoring of the rock components of civil infrastructures such as tunnels and caverns remains challenging. The feasibility of employing smart optical fibre sensor (OFS) and piezoelectric impedance sensor made up of lead zirconate titanate (PZT) for comprehensive health monitoring of rocks, covering load history monitoring/retrieval as well as damage assessment is presented in this paper. The rock specimens are subjected to cyclic loading and their conditions are continuously monitored using OFS and PZT sensors. OFS based multiplexed fibre Bragg grating (FBG) sensors are surface bonded on the rock specimens. Their strain sensing performance is compared with the conventional electric strain gauges (ESGs). In addition, PZT patches are also bonded on the specimens to study the damage pattern during different loading cycles. Unlike the FBGs or ESGs, PZT patches are used as bi-functional sensors and actuators, enabling them to be efficient detectors of incipient damages using the principle of electromechanical impedance. The experimental study demonstrated superior performance of these smart FBG and PZT impedance sensors. This work is expected to be useful for SHM based NDE application of rock structures such as caverns and tunnels. PMID:27879708

Rapid detection of avian influenza virus a and subtype H5N1 by single step multiplex reverse transcription-polymerase chain reaction.

PubMed

Wei, Hui-Ling; Bai, Gui-Rong; Mweene, Aaron S; Zhou, Ying-Chun; Cong, Yan-Long; Pu, Juan; Wang, Shuai; Kida, Hiroshi; Liu, Jin-Hua

2006-06-01

Outbreaks of H5N1 highly pathogenic avian influenza (HPAI) virus caused great economic losses to the poultry industry and resulted in human deaths in Thailand and Viet Nam in 2004. Rapid typing and subtyping of H5N1 viruses, especially from clinical specimens, are desirable for taking prompt control measures to prevent the spread of the disease. Here, we developed a set of oligonucleotide primers able to detect, type and subtype H5 and N1 influenza viruses in a single step multiplex reverse transcription-polymerase chain reaction (RT-PCR). RNA was extracted from allantoic fluid or from specimens with guanidinium isothiocyanate reagent. Reverse transcription and PCR were carried out with a mixture of primers specific for influenza viruses of type A, subtype H5 and N1 in a single reaction system under identical conditions. The amplified DNA fragments were analyzed by agarose gel electrophoresis. All the H5N1 viruses tested in the study and the experimental specimens presented three specific bands by the method established here. The results presented here suggest that the method described below is rapid and specific and, therefore, could be valuable in the rapid detection of H5N1 influenza viruses in clinics.

Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue

PubMed Central

Gerdes, Michael J.; Sevinsky, Christopher J.; Sood, Anup; Adak, Sudeshna; Bello, Musodiq O.; Bordwell, Alexander; Can, Ali; Corwin, Alex; Dinn, Sean; Filkins, Robert J.; Hollman, Denise; Kamath, Vidya; Kaanumalle, Sireesha; Kenny, Kevin; Larsen, Melinda; Lazare, Michael; Lowes, Christina; McCulloch, Colin C.; McDonough, Elizabeth; Pang, Zhengyu; Rittscher, Jens; Santamaria-Pang, Alberto; Sarachan, Brion D.; Seel, Maximilian L.; Seppo, Antti; Shaikh, Kashan; Sui, Yunxia; Zhang, Jingyu; Ginty, Fiona

2013-01-01

Limitations on the number of unique protein and DNA molecules that can be characterized microscopically in a single tissue specimen impede advances in understanding the biological basis of health and disease. Here we present a multiplexed fluorescence microscopy method (MxIF) for quantitative, single-cell, and subcellular characterization of multiple analytes in formalin-fixed paraffin-embedded tissue. Chemical inactivation of fluorescent dyes after each image acquisition round allows reuse of common dyes in iterative staining and imaging cycles. The mild inactivation chemistry is compatible with total and phosphoprotein detection, as well as DNA FISH. Accurate computational registration of sequential images is achieved by aligning nuclear counterstain-derived fiducial points. Individual cells, plasma membrane, cytoplasm, nucleus, tumor, and stromal regions are segmented to achieve cellular and subcellular quantification of multiplexed targets. In a comparison of pathologist scoring of diaminobenzidine staining of serial sections and automated MxIF scoring of a single section, human epidermal growth factor receptor 2, estrogen receptor, p53, and androgen receptor staining by diaminobenzidine and MxIF methods yielded similar results. Single-cell staining patterns of 61 protein antigens by MxIF in 747 colorectal cancer subjects reveals extensive tumor heterogeneity, and cluster analysis of divergent signaling through ERK1/2, S6 kinase 1, and 4E binding protein 1 provides insights into the spatial organization of mechanistic target of rapamycin and MAPK signal transduction. Our results suggest MxIF should be broadly applicable to problems in the fields of basic biological research, drug discovery and development, and clinical diagnostics. PMID:23818604

Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue.

PubMed

Gerdes, Michael J; Sevinsky, Christopher J; Sood, Anup; Adak, Sudeshna; Bello, Musodiq O; Bordwell, Alexander; Can, Ali; Corwin, Alex; Dinn, Sean; Filkins, Robert J; Hollman, Denise; Kamath, Vidya; Kaanumalle, Sireesha; Kenny, Kevin; Larsen, Melinda; Lazare, Michael; Li, Qing; Lowes, Christina; McCulloch, Colin C; McDonough, Elizabeth; Montalto, Michael C; Pang, Zhengyu; Rittscher, Jens; Santamaria-Pang, Alberto; Sarachan, Brion D; Seel, Maximilian L; Seppo, Antti; Shaikh, Kashan; Sui, Yunxia; Zhang, Jingyu; Ginty, Fiona

2013-07-16

Limitations on the number of unique protein and DNA molecules that can be characterized microscopically in a single tissue specimen impede advances in understanding the biological basis of health and disease. Here we present a multiplexed fluorescence microscopy method (MxIF) for quantitative, single-cell, and subcellular characterization of multiple analytes in formalin-fixed paraffin-embedded tissue. Chemical inactivation of fluorescent dyes after each image acquisition round allows reuse of common dyes in iterative staining and imaging cycles. The mild inactivation chemistry is compatible with total and phosphoprotein detection, as well as DNA FISH. Accurate computational registration of sequential images is achieved by aligning nuclear counterstain-derived fiducial points. Individual cells, plasma membrane, cytoplasm, nucleus, tumor, and stromal regions are segmented to achieve cellular and subcellular quantification of multiplexed targets. In a comparison of pathologist scoring of diaminobenzidine staining of serial sections and automated MxIF scoring of a single section, human epidermal growth factor receptor 2, estrogen receptor, p53, and androgen receptor staining by diaminobenzidine and MxIF methods yielded similar results. Single-cell staining patterns of 61 protein antigens by MxIF in 747 colorectal cancer subjects reveals extensive tumor heterogeneity, and cluster analysis of divergent signaling through ERK1/2, S6 kinase 1, and 4E binding protein 1 provides insights into the spatial organization of mechanistic target of rapamycin and MAPK signal transduction. Our results suggest MxIF should be broadly applicable to problems in the fields of basic biological research, drug discovery and development, and clinical diagnostics.

Single-band upconversion nanoprobes for multiplexed simultaneous in situ molecular mapping of cancer biomarkers.

PubMed

Zhou, Lei; Wang, Rui; Yao, Chi; Li, Xiaomin; Wang, Chengli; Zhang, Xiaoyan; Xu, Congjian; Zeng, Aijun; Zhao, Dongyuan; Zhang, Fan

2015-04-24

The identification of potential diagnostic markers and target molecules among the plethora of tumour oncoproteins for cancer diagnosis requires facile technology that is capable of quantitatively analysing multiple biomarkers in tumour cells and tissues. Diagnostic and prognostic classifications of human tumours are currently based on the western blotting and single-colour immunohistochemical methods that are not suitable for multiplexed detection. Herein, we report a general and novel method to prepare single-band upconversion nanoparticles with different colours. The expression levels of three biomarkers in breast cancer cells were determined using single-band upconversion nanoparticles, western blotting and immunohistochemical technologies with excellent correlation. Significantly, the application of antibody-conjugated single-band upconversion nanoparticle molecular profiling technology can achieve the multiplexed simultaneous in situ biodetection of biomarkers in breast cancer cells and tissue specimens and produce more accurate results for the simultaneous quantification of proteins present at low levels compared with classical immunohistochemical technology.

Differentiation of Campylobacter jejuni and Campylobacter coli Using Multiplex-PCR and High Resolution Melt Curve Analysis

PubMed Central

Banowary, Banya; Dang, Van Tuan; Sarker, Subir; Connolly, Joanne H.; Chenu, Jeremy; Groves, Peter; Ayton, Michelle; Raidal, Shane; Devi, Aruna; Vanniasinkam, Thiru; Ghorashi, Seyed A.

2015-01-01

Campylobacter spp. are important causes of bacterial gastroenteritis in humans in developed countries. Among Campylobacter spp. Campylobacter jejuni (C. jejuni) and C. coli are the most common causes of human infection. In this study, a multiplex PCR (mPCR) and high resolution melt (HRM) curve analysis were optimized for simultaneous detection and differentiation of C. jejuni and C. coli isolates. A segment of the hippuricase gene (hipO) of C. jejuni and putative aspartokinase (asp) gene of C. coli were amplified from 26 Campylobacter isolates and amplicons were subjected to HRM curve analysis. The mPCR-HRM was able to differentiate between C. jejuni and C. coli species. All DNA amplicons generated by mPCR were sequenced. Analysis of the nucleotide sequences from each isolate revealed that the HRM curves were correlated with the nucleotide sequences of the amplicons. Minor variation in melting point temperatures of C. coli or C. jejuni isolates was also observed and enabled some intraspecies differentiation between C. coli and/or C. jejuni isolates. The potential of PCR-HRM curve analysis for the detection and speciation of Campylobacter in additional human clinical specimens and chicken swab samples was also confirmed. The sensitivity and specificity of the test were found to be 100% and 92%, respectively. The results indicated that mPCR followed by HRM curve analysis provides a rapid (8 hours) technique for differentiation between C. jejuni and C. coli isolates. PMID:26394042

Resin embedded multicycle imaging (REMI): a tool to evaluate protein domains.

PubMed

Busse, B L; Bezrukov, L; Blank, P S; Zimmerberg, J

2016-08-08

Protein complexes associated with cellular processes comprise a significant fraction of all biology, but our understanding of their heterogeneous organization remains inadequate, particularly for physiological densities of multiple protein species. Towards resolving this limitation, we here present a new technique based on resin-embedded multicycle imaging (REMI) of proteins in-situ. By stabilizing protein structure and antigenicity in acrylic resins, affinity labels were repeatedly applied, imaged, removed, and replaced. In principle, an arbitrarily large number of proteins of interest may be imaged on the same specimen with subsequent digital overlay. A series of novel preparative methods were developed to address the problem of imaging multiple protein species in areas of the plasma membrane or volumes of cytoplasm of individual cells. For multiplexed examination of antibody staining we used straightforward computational techniques to align sequential images, and super-resolution microscopy was used to further define membrane protein colocalization. We give one example of a fibroblast membrane with eight multiplexed proteins. A simple statistical analysis of this limited membrane proteomic dataset is sufficient to demonstrate the analytical power contributed by additional imaged proteins when studying membrane protein domains.

Resin embedded multicycle imaging (REMI): a tool to evaluate protein domains

PubMed Central

Busse, B. L.; Bezrukov, L.; Blank, P. S.; Zimmerberg, J.

2016-01-01

Protein complexes associated with cellular processes comprise a significant fraction of all biology, but our understanding of their heterogeneous organization remains inadequate, particularly for physiological densities of multiple protein species. Towards resolving this limitation, we here present a new technique based on resin-embedded multicycle imaging (REMI) of proteins in-situ. By stabilizing protein structure and antigenicity in acrylic resins, affinity labels were repeatedly applied, imaged, removed, and replaced. In principle, an arbitrarily large number of proteins of interest may be imaged on the same specimen with subsequent digital overlay. A series of novel preparative methods were developed to address the problem of imaging multiple protein species in areas of the plasma membrane or volumes of cytoplasm of individual cells. For multiplexed examination of antibody staining we used straightforward computational techniques to align sequential images, and super-resolution microscopy was used to further define membrane protein colocalization. We give one example of a fibroblast membrane with eight multiplexed proteins. A simple statistical analysis of this limited membrane proteomic dataset is sufficient to demonstrate the analytical power contributed by additional imaged proteins when studying membrane protein domains. PMID:27499335

Comparison of NxTAG Respiratory Pathogen Panel and Anyplex II RV16 Tests for Multiplex Detection of Respiratory Pathogens in Hospitalized Children

PubMed Central

Brotons, Pedro; Henares, Desiree; Latorre, Irene; Cepillo, Antonio; Launes, Cristian

2016-01-01

Multiplex molecular techniques can detect a diversity of respiratory viruses and bacteria that cause childhood acute respiratory infection rapidly and conveniently. However, currently available techniques show high variation in performance. We sought to compare the diagnostic accuracy of the novel multiplex NxTAG respiratory pathogen panel (RPP) RUO test versus a routine multiplex Anyplex II RV16 assay in respiratory specimens collected from children <18 years of age hospitalized with nonspecific symptoms of acute lower respiratory infection. Parallel testing was performed on nasopharyngeal aspirates prospectively collected at referral Children's Hospital Sant Joan de Déu (Barcelona, Spain) between June and November 2015. Agreement values between the two tests and kappa coefficients were assessed. Bidirectional sequencing was performed for the resolution of discordant results. A total of 319 samples were analyzed by both techniques. A total of 268 (84.0%) of them yielded concordant results. Positive percent agreement values ranged from 83.3 to 100%, while the negative percent agreement was more than 99% for all targets except for enterovirus/rhinovirus (EV/RV; 94.4%). Kappa coefficients ranged from 0.83 to 1.00. Discrepancy analysis confirmed 66.0% of NxTAG RPP RUO results. A total of 260 viruses were detected, with EV/RV (n = 105, 40.4%) being the most prevalent target. Viral coinfections were found in 44 (14.2%) samples. In addition, NxTAG RPP RUO detected single bacterial and mixed viral-bacterial infections in seven samples. NxTAG RPP RUO showed high positive and negative agreement with Anyplex II RV16 for main viruses that cause acute respiratory infections in children, coupled with an additional capability to detect some respiratory bacteria. PMID:27629904

A strategy to analyse activity-based profiling of tyrosine kinase substrates in OCT-embedded lung cancer tissue.

PubMed

Arni, Stephan; de Wijn, Rik; Garcia-Villegas, Refugio; Bitanihirwe, Byron K Y; Caviezel, Claudio; Weder, Walter; Hillinger, Sven

2018-04-15

The use of optimal cutting temperature (OCT) medium has served to improve the long-term preservation of surgical tissue specimens. Unfortunately, the presence of polymers in OCT has been found to generate signal interference in proteomic-based techniques. Indeed the presence of OCT medium in tissue lysates precludes the analysis of activity based proteomic profiles obtained from lung adenocarcinoma (LuAdCa) resection specimens. In order to probe this question further tissue lysates were prepared from 47 lung non-neoplastic and tumour, node, metastasis (TNM) stage 1 LuAdCa resection specimens embedded with or without OCT, and data of activity based multiplex profiles of protein tyrosine kinase peptide substrates were obtained. We found that changes in overall phosphorylation level coincided with the use of OCT and subsequently developed an OCT per peptide median correcting strategy by performing median centering on the values of each peptide. Application of this post-analytical strategy not only can identify changes in kinase activity but can also assist in identifying novel targets for therapeutic intervention against LuAdCa. Copyright © 2018 Elsevier Inc. All rights reserved.

Detection of Borrelia miyamotoi and other tick-borne pathogens in human clinical specimens and Ixodes scapularis ticks in New York State, 2012-2015.

PubMed

Wroblewski, Danielle; Gebhardt, Linda; Prusinski, Melissa A; Meehan, Lisa J; Halse, Tanya A; Musser, Kimberlee A

2017-03-01

Borrelia miyamotoi (Bm) is a recently emerging bacterial agent transmitted by several species of ixodid ticks. Diagnosis of Bm infection can be challenging, as the organism is not easily cultivable. We have developed and validated a multiplex real-time PCR to simultaneously identify Bm infection and the agents causing human granulocytic anaplasmosis and human monocytic ehrlichiosis, Anaplasma phagocytophilum and Ehrlichia chaffeensis, respectively. The assay is 100% specific; highly sensitive, detecting 11 gene copies of Bm DNA in both whole blood and cerebral spinal fluid; and provides rapid results in less than two hours. A retrospective study of 796 clinical specimens collected between the years 2012 and 2014 and a prospective study of 366 clinical specimens were performed utilizing this novel assay to evaluate the frequency of Bm infection in New York State (NYS). Eight clinical specimens (1%) were found to be positive for Bm, 216 were positive for A. phagocytophilum, and 10 were positive for E. chaffeensis. Additionally, we tested 411 I. scapularis ticks collected in NYS during 2013 and 2014 in a separate multiplex real-time PCR to determine the prevalence of Bm, A. phagocytophilum, Borrelia burgdorferi s.s., and Borrelia species. Our results indicated rates of 1.5%, 27%, 19.7%, and 8.8% respectively. The ability to monitor both the frequency and geographic distribution of Bm cases and the prevalence and geographic distribution of Bm in ticks will help create a better understanding of this emerging tick-borne pathogen. Published by Elsevier GmbH.

Multiplexed immunofluorescence delineates proteomic cancer cell states associated with metabolism

PubMed Central

Sood, Anup; Miller, Alexandra M.; Brogi, Edi; Sui, Yunxia; Armenia, Joshua; McDonough, Elizabeth; Santamaria-Pang, Alberto; Stamper, Aleksandra; Campos, Carl; Pang, Zhengyu; Li, Qing; Port, Elisa; Graeber, Thomas G.; Schultz, Nikolaus; Ginty, Fiona; Larson, Steven M.

2016-01-01

The phenotypic diversity of cancer results from genetic and nongenetic factors. Most studies of cancer heterogeneity have focused on DNA alterations, as technologies for proteomic measurements in clinical specimen are currently less advanced. Here, we used a multiplexed immunofluorescence staining platform to measure the expression of 27 proteins at the single-cell level in formalin-fixed and paraffin-embedded samples from treatment-naive stage II/III human breast cancer. Unsupervised clustering of protein expression data from 638,577 tumor cells in 26 breast cancers identified 8 clusters of protein coexpression. In about one-third of breast cancers, over 95% of all neoplastic cells expressed a single protein coexpression cluster. The remaining tumors harbored tumor cells representing multiple protein coexpression clusters, either in a regional distribution or intermingled throughout the tumor. Tumor uptake of the radiotracer 18F-fluorodeoxyglucose was associated with protein expression clusters characterized by hormone receptor loss, PTEN alteration, and HER2 gene amplification. Our study demonstrates an approach to generate cellular heterogeneity metrics in routinely collected solid tumor specimens and integrate them with in vivo cancer phenotypes. PMID:27182557

Human Papillomavirus Genotyping Using an Automated Film-Based Chip Array

PubMed Central

Erali, Maria; Pattison, David C.; Wittwer, Carl T.; Petti, Cathy A.

2009-01-01

The INFINITI HPV-QUAD assay is a commercially available genotyping platform for human papillomavirus (HPV) that uses multiplex PCR, followed by automated processing for primer extension, hybridization, and detection. The analytical performance of the HPV-QUAD assay was evaluated using liquid cervical cytology specimens, and the results were compared with those results obtained using the digene High-Risk HPV hc2 Test (HC2). The specimen types included Surepath and PreservCyt transport media, as well as residual SurePath and HC2 transport media from the HC2 assay. The overall concordance of positive and negative results following the resolution of indeterminate and intermediate results was 83% among the 197 specimens tested. HC2 positive (+) and HPV-QUAD negative (−) results were noted in 24 specimens that were shown by real-time PCR and sequence analysis to contain no HPV, HPV types that were cross-reactive in the HC2 assay, or low virus levels. Conversely, HC2 (−) and HPV-QUAD (+) results were noted in four specimens and were subsequently attributed to cross-contamination. The most common HPV types to be identified in this study were HPV16, HPV18, HPV52/58, and HPV39/56. We show that the HPV-QUAD assay is a user friendly, automated system for the identification of distinct HPV genotypes. Based on its analytical performance, future studies with this platform are warranted to assess its clinical utility for HPV detection and genotyping. PMID:19644025

Human papillomavirus genotyping using an automated film-based chip array.

PubMed

Erali, Maria; Pattison, David C; Wittwer, Carl T; Petti, Cathy A

2009-09-01

The INFINITI HPV-QUAD assay is a commercially available genotyping platform for human papillomavirus (HPV) that uses multiplex PCR, followed by automated processing for primer extension, hybridization, and detection. The analytical performance of the HPV-QUAD assay was evaluated using liquid cervical cytology specimens, and the results were compared with those results obtained using the digene High-Risk HPV hc2 Test (HC2). The specimen types included Surepath and PreservCyt transport media, as well as residual SurePath and HC2 transport media from the HC2 assay. The overall concordance of positive and negative results following the resolution of indeterminate and intermediate results was 83% among the 197 specimens tested. HC2 positive (+) and HPV-QUAD negative (-) results were noted in 24 specimens that were shown by real-time PCR and sequence analysis to contain no HPV, HPV types that were cross-reactive in the HC2 assay, or low virus levels. Conversely, HC2 (-) and HPV-QUAD (+) results were noted in four specimens and were subsequently attributed to cross-contamination. The most common HPV types to be identified in this study were HPV16, HPV18, HPV52/58, and HPV39/56. We show that the HPV-QUAD assay is a user friendly, automated system for the identification of distinct HPV genotypes. Based on its analytical performance, future studies with this platform are warranted to assess its clinical utility for HPV detection and genotyping.

Multiplexed transcriptome analysis to detect ALK, ROS1 and RET rearrangements in lung cancer

PubMed Central

Rogers, Toni-Maree; Arnau, Gisela Mir; Ryland, Georgina L.; Huang, Stephen; Lira, Maruja E.; Emmanuel, Yvette; Perez, Omar D.; Irwin, Darryl; Fellowes, Andrew P.; Wong, Stephen Q.; Fox, Stephen B.

2017-01-01

ALK, ROS1 and RET gene fusions are important predictive biomarkers for tyrosine kinase inhibitors in lung cancer. Currently, the gold standard method for gene fusion detection is Fluorescence In Situ Hybridization (FISH) and while highly sensitive and specific, it is also labour intensive, subjective in analysis, and unable to screen a large numbers of gene fusions. Recent developments in high-throughput transcriptome-based methods may provide a suitable alternative to FISH as they are compatible with multiplexing and diagnostic workflows. However, the concordance between these different methods compared with FISH has not been evaluated. In this study we compared the results from three transcriptome-based platforms (Nanostring Elements, Agena LungFusion panel and ThermoFisher NGS fusion panel) to those obtained from ALK, ROS1 and RET FISH on 51 clinical specimens. Overall agreement of results ranged from 86–96% depending on the platform used. While all platforms were highly sensitive, both the Agena panel and Thermo Fisher NGS fusion panel reported minor fusions that were not detectable by FISH. Our proof–of–principle study illustrates that transcriptome-based analyses are sensitive and robust methods for detecting actionable gene fusions in lung cancer and could provide a robust alternative to FISH testing in the diagnostic setting. PMID:28181564

Hydrogel microstructure live-cell array for multiplexed analyses of cancer stem cells, tumor heterogeneity and differential drug response at single-element resolution.

PubMed

Afrimzon, E; Botchkina, G; Zurgil, N; Shafran, Y; Sobolev, M; Moshkov, S; Ravid-Hermesh, O; Ojima, I; Deutsch, M

2016-03-21

Specific phenotypic subpopulations of cancer stem cells (CSCs) are responsible for tumor development, production of heterogeneous differentiated tumor mass, metastasis, and resistance to therapies. The development of therapeutic approaches based on targeting rare CSCs has been limited partially due to the lack of appropriate experimental models and measurement approaches. The current study presents new tools and methodologies based on a hydrogel microstructure array (HMA) for identification and multiplex analyses of CSCs. Low-melt agarose integrated with type I collagen, a major component of the extracellular matrix (ECM), was used to form a solid hydrogel array with natural non-adhesive characteristics and high optical quality. The array contained thousands of individual pyramidal shaped, nanoliter-volume micro-chambers (MCs), allowing concomitant generation and measurement of large populations of free-floating CSC spheroids from single cells, each in an individual micro-chamber (MC). The optical live cell platform, based on an imaging plate patterned with HMA, was validated using CSC-enriched prostate and colon cancer cell lines. The HMA methodology and quantitative image analysis at single-element resolution clearly demonstrates several levels of tumor cell heterogeneity, including morphological and phenotypic variability, differences in proliferation capacity and in drug response. Moreover, the system facilitates real-time examination of single stem cell (SC) fate, as well as drug-induced alteration in expression of stemness markers. The technology may be applicable in personalized cancer treatment, including multiplex ex vivo analysis of heterogeneous patient-derived tumor specimens, precise detection and characterization of potentially dangerous cell phenotypes, and for representative evaluation of drug sensitivity of CSCs and other types of tumor cells.

Quantum Dot Platform for Single-Cell Molecular Profiling

NASA Astrophysics Data System (ADS)

Zrazhevskiy, Pavel S.

In-depth understanding of the nature of cell physiology and ability to diagnose and control the progression of pathological processes heavily rely on untangling the complexity of intracellular molecular mechanisms and pathways. Therefore, comprehensive molecular profiling of individual cells within the context of their natural tissue or cell culture microenvironment is essential. In principle, this goal can be achieved by tagging each molecular target with a unique reporter probe and detecting its localization with high sensitivity at sub-cellular resolution, primarily via microscopy-based imaging. Yet, neither widely used conventional methods nor more advanced nanoparticle-based techniques have been able to address this task up to date. High multiplexing potential of fluorescent probes is heavily restrained by the inability to uniquely match probes with corresponding molecular targets. This issue is especially relevant for quantum dot probes---while simultaneous spectral imaging of up to 10 different probes is possible, only few can be used concurrently for staining with existing methods. To fully utilize multiplexing potential of quantum dots, it is necessary to design a new staining platform featuring unique assignment of each target to a corresponding quantum dot probe. This dissertation presents two complementary versatile approaches towards achieving comprehensive single-cell molecular profiling and describes engineering of quantum dot probes specifically tailored for each staining method. Analysis of expanded molecular profiles is achieved through augmenting parallel multiplexing capacity with performing several staining cycles on the same specimen in sequential manner. In contrast to other methods utilizing quantum dots or other nanoparticles, which often involve sophisticated probe synthesis, the platform technology presented here takes advantage of simple covalent bioconjugation and non-covalent self-assembly mechanisms for straightforward probe preparation and specimen labeling, requiring no advanced technical skills and being directly applicable for a wide range of molecular profiling studies. Utilization of quantum dot platform for single-cell molecular profiling promises to greatly benefit both biomedical research and clinical diagnostics by providing a tool for addressing phenotypic heterogeneity within large cell populations, opening access to studying low-abundance events often masked or completely erased by batch processing, and elucidating biomarker signatures of diseases critical for accurate diagnostics and targeted therapy.

Evaluation of the Quantamatrix Multiplexed Assay Platform system for simultaneous detection of Mycobacterium tuberculosis and the rifampicin resistance gene using cultured mycobacteria.

PubMed

Wang, Hye-Young; Uh, Young; Kim, Seoyong; Shim, Tae-Sun; Lee, Hyeyoung

2017-08-01

The differentiation of Mycobacterium tuberculosis complex (MTBC) from non-tuberculous mycobacteria (NTM) is of primary importance for infection control and the selection of anti-tuberculosis drugs. Up to date data on rifampicin (RIF)-resistant tuberculosis (TB) is essential for the early management of multidrug-resistant TB. The aim of this study was to evaluate the usefulness of a newly developed multiplexed, bead-based bioassay (Quantamatrix Multiplexed Assay Platform, QMAP) for the rapid differentiation of 23 Mycobacterium species including MTBC and RIF-resistant strains. A total of 314 clinical Mycobacterium isolates cultured from respiratory specimens were used in this study. The sensitivity and specificity of the QMAP system for Mycobacterium species were 100% (95% CI 99.15-100%, p<0.0001) and 97.8% (95% CI 91.86-99.87%, p<0.0001), respectively. The results of conventional drug susceptibility testing and the QMAP Dual-ID assay were completely concordant for all clinical isolates (100%, 95% CI 98.56-100%). Out of 223 M. tuberculosis (MTB) isolates, 196 were pan-susceptible and 27 were resistant to RIF according to QMAP results. All of the mutations in the RIF resistance-determining region detected by the QMAP system were confirmed by rpoB sequence analysis and a REBA MTB-Rifa reverse blot hybridization assay. The majority of the mutations (n=26, 96.3%), including those missing wild-type probe signals, were located in three codons (529-534, 524-529, and 514-520), and 17 (65.4%) of these mutations were detected by three mutation probes (531TTG, 526TAC, and 516GTC). The entire QMAP system assay takes about 3h to complete, while results from the culture-based conventional method can take up to 48-72h. Although improvements to the QMAP system are needed for direct respiratory specimens, it may be useful for rapid screening, not only to identify and accurately discriminate MTBC from NTM, but also to identify RIF-resistant MTB strains in positive culture samples. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Comparison between culture and a multiplex quantitative real-time polymerase chain reaction assay detecting Ureaplasma urealyticum and U. parvum.

PubMed

Frølund, Maria; Björnelius, Eva; Lidbrink, Peter; Ahrens, Peter; Jensen, Jørgen Skov

2014-01-01

A novel multiplex quantitative real-time polymerase chain reaction (qPCR) for simultaneous detection of U. urealyticum and U. parvum was developed and compared with quantitative culture in Shepard's 10 C medium for ureaplasmas in urethral swabs from 129 men and 66 women, and cervical swabs from 61 women. Using culture as the gold standard, the sensitivity of the qPCR was 96% and 95% for female urethral and cervical swabs, respectively. In male urethral swabs the sensitivity was 89%. The corresponding specificities were 100%, 87% and 99%. The qPCR showed a linear increasing DNA copy number with increasing colour-changing units. Although slightly less sensitive than culture, this multiplex qPCR assay detecting U. urealyticum and U. parvum constitutes a simple and fast alternative to the traditional methods for identification of ureaplasmas and allows simultaneous species differentiation and quantitation in clinical samples. Furthermore, specimens overgrown by other bacteria using the culture method can be evaluated in the qPCR.

Impact of 6-month frozen storage of cervical specimens in alkaline buffer conditions on human papillomavirus genotyping.

PubMed

LaMere, Brandon J; Howell, Renee; Fetterman, Barbara; Shieh, Jen; Castle, Philip E

2008-08-01

The impact of 6-month storage of cervical specimens under alkaline conditions that occurs as the result of Hybrid Capture 2 testing on human papillomavirus (HPV) genotyping is not well documented. To examine this issue, 143 frozen hc2-positive specimens in specimen transport medium were selected at random from each of the following groups: specimens stored for 6 months, 4 months, and 2.5 months under alkaline pH (pH 12-13) and specimens stored 1 month at neutral pH (pH 6-7) as controls. Specimens were tested in a masked fashion for 20 HPV genotypes (HPV6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82) using a prototype, research-use-only GP5+/6+ L1 consensus PCR method and multiplex hybridization using Luminex xMAP for detection of specific HPV genotypes One control specimen had missing test results. There were no statistical differences in the number of HPV genotypes detected, number of carcinogenic HPV genotypes detected, or in the signal strength among HPV-positive results across groups. Six-month frozen storage of cervical specimens at alkaline pH had little impact on testing for HPV genotypes among hc2-positive women using this HPV genotyping method.

Evaluation of five enzyme immunoassays compared with the cytotoxicity assay for diagnosis of Clostridium difficile-associated diarrhea in dogs.

PubMed

Chouicha, Nadira; Marks, Stanley L

2006-03-01

Clostridium difficile-associated-diarrhea (CDAD) is a nosocomial infection in dogs. Diagnosis of this infection is dependent on clinical signs of disease supported by laboratory detection of C. difficile toxins A or B, or both, in fecal specimens via enzyme-linked immunosorbent assay (ELISA). Unfortunately, to the authors' knowledge, commercially available ELISAs have not been validated in dogs to date. We evaluated 5 ELISAs done on 143 canine fecal specimens (100 diarrheic and 43 nondiarrheic dogs) and on 29 C. difficile isolates. The results of each ELISA were compared with the cytotoxin B tissue culture assay (CTA). Clostridium difficile was isolated from 23% of the fecal specimens. Eighteen of the 143 fecal specimens were toxin positive (15 diarrheic and 3 nondiarrheic dogs). On the basis of multiplex polymerase chain reaction (PCR) analysis for toxin-A and -B genes, 72% of the isolates were toxigenic. The carriage rate of toxigenic isolates in diarrheic dogs was higher than that in the nondiarrheic dogs; however, these differences were not statistically significant. A good correlation was found between CTA, PCR, and culture results. The ELISAs done on fecal specimens collected from diarrheic dogs had low sensitivity (7-33%). In contrast, ELISA for toxin A or B, or both, performed on toxigenic isolates had high sensitivity (93%). These results suggest that commercially available human ELISAs are inadequate for the diagnosis of canine C. difficile-associated diarrhea when tested on fecal specimens. In contrast, the Premier ToxinA/B and Techlab ToxinA/B ELISAs may be useful for the diagnosis of canine CDAD when used on toxigenic isolates.

Molecular barcoding of venomous snakes and species-specific multiplex PCR assay to identify snake groups for which antivenom is available in Thailand.

PubMed

Supikamolseni, A; Ngaoburanawit, N; Sumontha, M; Chanhome, L; Suntrarachun, S; Peyachoknagul, S; Srikulnath, K

2015-10-30

DNA barcodes of mitochondrial COI and Cytb genes were constructed from 54 specimens of 16 species for species identification. Intra- and interspecific sequence divergence of the COI gene (10 times) was greater than that of the Cytb gene (4 times), which suggests that the former gene may be a better marker than the latter for species delimitation in snakes. The COI barcode cut-off scores differed by more than 3% between most species, and the minimum interspecific divergence was greater than the maximum intraspecific divergence. Clustering analysis indicated that most species fell into monophyletic clades. These results suggest that these species could be reliably differentiated using COI DNA barcodes. Moreover, a novel species-specific multiplex PCR assay was developed to distinguish between Naja spp, Ophiophagus hannah, Trimeresurus spp, Hydrophiinae, Daboia siamensis, Bungarus fasciatus, and Calloselasma rhodostoma. Antivenom for these species is produced and kept by the Thai Red Cross for clinical use. Our novel PCR assay could easily be applied to venom and saliva samples and could be used effectively for the rapid and accurate identification of species during forensic work, conservation study, and medical research.

Recent Situation of Taeniasis in Mongolia (2002-2012)

PubMed Central

Dorjsuren, Temuulen; Yanagida, Tetsuya; Sako, Yasuhito; Nakaya, Kazuhiro; Davaajav, Abmed; Agvaandaram, Gurbadam; Enkhbat, Tsatsral; Gonchigoo, Battsetseg; Dulmaa, Nyamkhuu; Chuluunbaatar, Gantigmaa; Ito, Akira

2014-01-01

Epidemiological situation of taeniasis in Mongolia was assessed based on mitochondrial DNA identification of the parasite species. Multiplex PCR was used on a total of 194 proglottid specimens of Taenia species and copro-PCR and loop-mediated isothermal amplification (LAMP) assays were utilized for detection of copro-DNA of 37 fecal samples from taeniasis patients submitted to the Mongolian National Center for Communicable Diseases (NCCD) from 2002 to 2012. In addition, 4 out of 44 calcified cysts in beef kept in formalin since 2003 were evaluated for histopathological confirmation of cattle cysticercosis. All proglottid specimens and stool samples were confirmed to be Taenia saginata by multiplex PCR and by copro-PCR and LAMP, respectively. Cysts collected from cattle were morphologically confirmed to be metacestodes of Taenia species. T. saginata taeniasis was identified from almost all ages from a 2-year-old boy up to a 88-year-old woman and most prominently in 15-29 age group (37%, 74/198) followed by 30-44 age group (34.8%, 69/198 ) from 15 of Mongolia's 21 provinces, while cattle cysticerci were found from 12 provinces. The highest proportion of taeniasis patients was in Ulaanbaatar, the capital of Mongolia. PMID:24850968

Recent situation of taeniasis in Mongolia (2002-2012).

PubMed

Davaasuren, Anu; Dorjsuren, Temuulen; Yanagida, Tetsuya; Sako, Yasuhito; Nakaya, Kazuhiro; Davaajav, Abmed; Agvaandaram, Gurbadam; Enkhbat, Tsatsral; Gonchigoo, Battsetseg; Dulmaa, Nyamkhuu; Chuluunbaatar, Gantigmaa; Ito, Akira

2014-04-01

Epidemiological situation of taeniasis in Mongolia was assessed based on mitochondrial DNA identification of the parasite species. Multiplex PCR was used on a total of 194 proglottid specimens of Taenia species and copro-PCR and loop-mediated isothermal amplification (LAMP) assays were utilized for detection of copro-DNA of 37 fecal samples from taeniasis patients submitted to the Mongolian National Center for Communicable Diseases (NCCD) from 2002 to 2012. In addition, 4 out of 44 calcified cysts in beef kept in formalin since 2003 were evaluated for histopathological confirmation of cattle cysticercosis. All proglottid specimens and stool samples were confirmed to be Taenia saginata by multiplex PCR and by copro-PCR and LAMP, respectively. Cysts collected from cattle were morphologically confirmed to be metacestodes of Taenia species. T. saginata taeniasis was identified from almost all ages from a 2-year-old boy up to a 88-year-old woman and most prominently in 15-29 age group (37%, 74/198) followed by 30-44 age group (34.8%, 69/198 ) from 15 of Mongolia's 21 provinces, while cattle cysticerci were found from 12 provinces. The highest proportion of taeniasis patients was in Ulaanbaatar, the capital of Mongolia.

Application of Multiplexed FBG and PZT Impedance Sensors for Health Monitoring of Rocks.

PubMed

Yang, Yaowen; Annamdas, Venu Gopal Madhav; Wang, Chao; Zhou, Yingxin

2008-01-21

Reliable structural health monitoring (SHM) including nondestructiveevaluation (NDE) is essential for safe operation of infrastructure systems. Effectivemonitoring of the rock components of civil infrastructures such as tunnels and cavernsremains challenging. The feasibility of employing smart optical fibre sensor (OFS) andpiezoelectric impedance sensor made up of lead zirconate titanate (PZT) forcomprehensive health monitoring of rocks, covering load history monitoring/retrieval aswell as damage assessment is presented in this paper. The rock specimens are subjected tocyclic loading and their conditions are continuously monitored using OFS and PZTsensors. OFS based multiplexed fibre Bragg grating (FBG) sensors are surface bonded onthe rock specimens. Their strain sensing performance is compared with the conventionalelectric strain gauges (ESGs). In addition, PZT patches are also bonded on the specimensto study the damage pattern during different loading cycles. Unlike the FBGs or ESGs,PZT patches are used as bi-functional sensors and actuators, enabling them to be efficientdetectors of incipient damages using the principle of electromechanical impedance. Theexperimental study demonstrated superior performance of these smart FBG and PZTimpedance sensors. This work is expected to be useful for SHM based NDE application ofrock structures such as caverns and tunnels.

Multiplex Real-Time PCR for Detecting and Typing Clostridium botulinum Group III Organisms and Their Mosaic Variants

PubMed Central

Auricchio, Bruna; Woudstra, Cédric; Fach, Patrick; Fiore, Alfonsina; Skarin, Hanna; Bano, Luca; Segerman, Bo; Knutsson, Rickard; De Medici, Dario

2013-01-01

Botulism is a neuroparalytic disease that can occur in all warm-blooded animals, birds, and fishes. The disease in animals is mainly caused by toxins produced by Clostridium botulinum strains belonging to group III, although outbreaks due to toxins produced by group I and II organisms have been recognized. Group III strains are capable of producing botulinum toxins of type C, D, and C/D and D/C mosaic variants. Definitive diagnosis of animal botulism is made by combining clinical findings with laboratory investigations. Detection of toxins in clinical specimens and feed is the gold standard for laboratory diagnosis. Since toxins may be degraded by organisms contained in the gastrointestinal tract or may be present at levels below the detection limit, the recovery of C. botulinum from sick animal specimens is consistent for laboratory confirmation. In this article we report the development and in-house validation of a new multiplex real-time PCR for detecting and typing the neurotoxin genes found in C. botulinum group III organisms. Validation procedures have been carried out according to ISO 16140, using strains and samples recovered from cases of animal botulism in Italy and France. PMID:23971808

Evaluation of the Cepheid Xpert Flu Assay for rapid identification and differentiation of influenza A, influenza A 2009 H1N1, and influenza B viruses.

PubMed

Novak-Weekley, S M; Marlowe, E M; Poulter, M; Dwyer, D; Speers, D; Rawlinson, W; Baleriola, C; Robinson, C C

2012-05-01

The Xpert Flu Assay cartridge is a next-generation nucleic acid amplification system that provides multiplexed PCR detection of the influenza A, influenza A 2009 H1N1, and influenza B viruses in approximately 70 min with minimal hands-on time. Six laboratories participated in a clinical trial comparing the results of the new Cepheid Xpert Flu Assay to those of culture or real-time PCR with archived and prospectively collected nasal aspirate-wash (NA-W) specimens and nasopharyngeal (NP) swabs from children and adults. Discrepant results were resolved by DNA sequence analysis. After discrepant-result analysis, the sensitivities of the Xpert Flu Assay for prospective NA-W specimens containing the influenza A, influenza A 2009 H1N1, and influenza B viruses compared to those of culture were 90.0%, 100%, and 100%, respectively, while the sensitivities of the assay for prospective NP swabs compared to those of culture were 100%, 100%, and 100%, respectively. The sensitivities of the Xpert Flu Assay for archived NA-W specimens compared to those of Gen-Probe ProFlu+ PCR for the influenza A, influenza A 2009 H1N1, and influenza B viruses were 99.4%, 98.4%, and 100%, respectively, while the sensitivities of the Xpert Flu Assay for archived NP swabs compared to those of ProFlu+ were 98.1%, 100%, and 93.8%, respectively. The sensitivities of the Xpert Flu Assay with archived NP specimens compared to those of culture for the three targets were 97.5%, 100%, and 93.8%, respectively. We conclude that the Cepheid Xpert Flu Assay is an accurate and rapid method that is suitable for on-demand testing for influenza viral infection.

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens.

PubMed

Loudig, Olivier; Liu, Christina; Rohan, Thomas; Ben-Dov, Iddo Z

2018-05-05

-Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of cancer development. By using non-invasive or pre-malignant lesions from patients who later develop invasive disease, gene expression analyses may help identify early molecular alterations that predispose to cancer risk. It has been well described that nucleic acids recovered from FFPE tissues have undergone severe physical damage and chemical modifications, which make their analysis difficult and generally requires adapted assays. MicroRNAs (miRNAs), however, which represent a small class of RNA molecules spanning only up to ~18-24 nucleotides, have been shown to withstand long-term storage and have been successfully analyzed in FFPE samples. Here we present a 3' barcoded complementary DNA (cDNA) library preparation protocol specifically optimized for the analysis of small RNAs extracted from archived tissues, which was recently demonstrated to be robust and highly reproducible when using archived clinical specimens stored for up to 35 years. This library preparation is well adapted to the multiplex analysis of compromised/degraded material where RNA samples (up to 18) are ligated with individual 3' barcoded adapters and then pooled together for subsequent enzymatic and biochemical preparations prior to analysis. All purifications are performed by polyacrylamide gel electrophoresis (PAGE), which allows size-specific selections and enrichments of barcoded small RNA species. This cDNA library preparation is well adapted to minute RNA inputs, as a pilot polymerase chain reaction (PCR) allows determination of a specific amplification cycle to produce optimal amounts of material for next-generation sequencing (NGS). This approach was optimized for the use of degraded FFPE RNA from specimens archived for up to 35 years and provides highly reproducible NGS data.

Large-Scale and Deep Quantitative Proteome Profiling Using Isobaric Labeling Coupled with Two-Dimensional LC-MS/MS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gritsenko, Marina A.; Xu, Zhe; Liu, Tao

Comprehensive, quantitative information on abundances of proteins and their post-translational modifications (PTMs) can potentially provide novel biological insights into diseases pathogenesis and therapeutic intervention. Herein, we introduce a quantitative strategy utilizing isobaric stable isotope-labelling techniques combined with two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) for large-scale, deep quantitative proteome profiling of biological samples or clinical specimens such as tumor tissues. The workflow includes isobaric labeling of tryptic peptides for multiplexed and accurate quantitative analysis, basic reversed-phase LC fractionation and concatenation for reduced sample complexity, and nano-LC coupled to high resolution and high mass accuracy MS analysis for high confidence identification andmore » quantification of proteins. This proteomic analysis strategy has been successfully applied for in-depth quantitative proteomic analysis of tumor samples, and can also be used for integrated proteome and PTM characterization, as well as comprehensive quantitative proteomic analysis across samples from large clinical cohorts.« less

Large-Scale and Deep Quantitative Proteome Profiling Using Isobaric Labeling Coupled with Two-Dimensional LC-MS/MS.

PubMed

Gritsenko, Marina A; Xu, Zhe; Liu, Tao; Smith, Richard D

2016-01-01

Comprehensive, quantitative information on abundances of proteins and their posttranslational modifications (PTMs) can potentially provide novel biological insights into diseases pathogenesis and therapeutic intervention. Herein, we introduce a quantitative strategy utilizing isobaric stable isotope-labeling techniques combined with two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) for large-scale, deep quantitative proteome profiling of biological samples or clinical specimens such as tumor tissues. The workflow includes isobaric labeling of tryptic peptides for multiplexed and accurate quantitative analysis, basic reversed-phase LC fractionation and concatenation for reduced sample complexity, and nano-LC coupled to high resolution and high mass accuracy MS analysis for high confidence identification and quantification of proteins. This proteomic analysis strategy has been successfully applied for in-depth quantitative proteomic analysis of tumor samples and can also be used for integrated proteome and PTM characterization, as well as comprehensive quantitative proteomic analysis across samples from large clinical cohorts.

Next-Generation Molecular Testing of Newborn Dried Blood Spots for Cystic Fibrosis.

PubMed

Lefterova, Martina I; Shen, Peidong; Odegaard, Justin I; Fung, Eula; Chiang, Tsoyu; Peng, Gang; Davis, Ronald W; Wang, Wenyi; Kharrazi, Martin; Schrijver, Iris; Scharfe, Curt

2016-03-01

Newborn screening for cystic fibrosis enables early detection and management of this debilitating genetic disease. Implementing comprehensive CFTR analysis using Sanger sequencing as a component of confirmatory testing of all screen-positive newborns has remained impractical due to relatively lengthy turnaround times and high cost. Here, we describe CFseq, a highly sensitive, specific, rapid (<3 days), and cost-effective assay for comprehensive CFTR gene analysis from dried blood spots, the common newborn screening specimen. The unique design of CFseq integrates optimized dried blood spot sample processing, a novel multiplex amplification method from as little as 1 ng of genomic DNA, and multiplex next-generation sequencing of 96 samples in a single run to detect all relevant CFTR mutation types. Sequence data analysis utilizes publicly available software supplemented by an expert-curated compendium of >2000 CFTR variants. Validation studies across 190 dried blood spots demonstrated 100% sensitivity and a positive predictive value of 100% for single-nucleotide variants and insertions and deletions and complete concordance across the polymorphic poly-TG and consecutive poly-T tracts. Additionally, we accurately detected both a known exon 2,3 deletion and a previously undetected exon 22,23 deletion. CFseq is thus able to replace all existing CFTR molecular assays with a single robust, definitive assay at significant cost and time savings and could be adapted to high-throughput screening of other inherited conditions. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Rapid Identification of Mycobacteria and Drug-Resistant Mycobacterium tuberculosis by Use of a Single Multiplex PCR and DNA Sequencing

PubMed Central

Pérez-Osorio, Ailyn C.; Boyle, David S.; Ingham, Zachary K.; Ostash, Alla; Gautom, Romesh K.; Colombel, Craig; Houze, Yolanda

2012-01-01

Tuberculosis (TB) remains a significant global health problem for which rapid diagnosis is critical to both treatment and control. This report describes a multiplex PCR method, the Mycobacterial IDentification and Drug Resistance Screen (MID-DRS) assay, which allows identification of members of the Mycobacterium tuberculosis complex (MTBC) and the simultaneous amplification of targets for sequencing-based drug resistance screening of rifampin-resistant (rifampinr), isoniazidr, and pyrazinamider TB. Additionally, the same multiplex reaction amplifies a specific 16S rRNA gene target for rapid identification of M. avium complex (MAC) and a region of the heat shock protein 65 gene (hsp65) for further DNA sequencing-based confirmation or identification of other mycobacterial species. Comparison of preliminary results generated with MID-DRS versus culture-based methods for a total of 188 bacterial isolates demonstrated MID-DRS sensitivity and specificity as 100% and 96.8% for MTBC identification; 100% and 98.3% for MAC identification; 97.4% and 98.7% for rifampinr TB identification; 60.6% and 100% for isoniazidr TB identification; and 75.0% and 98.1% for pyrazinamider TB identification. The performance of the MID-DRS was also tested on acid-fast-bacterium (AFB)-positive clinical specimens, resulting in sensitivity and specificity of 100% and 78.6% for detection of MTBC and 100% and 97.8% for detection of MAC. In conclusion, use of the MID-DRS reduces the time necessary for initial identification and drug resistance screening of TB specimens to as little as 2 days. Since all targets needed for completing the assay are included in a single PCR amplification step, assay costs, preparation time, and risks due to user errors are also reduced. PMID:22162548

Bessel beam fluorescence lifetime tomography of live embryos (Conference Presentation)

NASA Astrophysics Data System (ADS)

Xu, Dongli; Peng, Leilei

2016-03-01

Optical tomography allows isotropic 3D imaging of embryos. Scanning-laser optical tomography (SLOT) has superior light collecting efficiency than wide-field optical tomography, making it ideal for fluorescence imaging of live embryos. We previously reported an imaging system that combines SLOT with a novel Fourier-multiplexed fluorescence lifetime imaging (FmFLIM) technique named FmFLIM-SLOT. FmFLIM-SLOT performs multiplexed FLIM-FRET readout of multiple FRET sensors in live embryos. Here we report a recent effort on improving the spatial resolution of the FmFLIM-SLOT system in order to image complex biochemical processes in live embryos at the cellular level. Optical tomography has to compromise between resolution and the depth of view. In SLOT, the commonly-used focused Gaussian beam diverges quickly from the focal plane, making it impossible to achieve high resolution imaging in a large volume specimen. We thus introduce Bessel beam laser-scanning tomography, which illuminates the sample with a spatial-light-modulator-generated Bessel beam that has an extended focal depth. The Bessel beam is scanned across the whole specimen. Fluorescence projection images are acquired at equal angular intervals as the sample rotates. Reconstruction artifacts due to annular-rings of the Bessel beam are removed by a modified 3D filtered back projection algorithm. Furthermore, in combination of Fourier-multiplexing fluorescence lifetime imaging (FmFLIM) method, the Bessel FmFLIM-SLOT system is capable of perform 3D lifetime imaging of live embryos at cellular resolution. The system is applied to in-vivo imaging of transgenic Zebrafish embryos. Results prove that Bessel FmFLIM-SLOT is a promising imaging method in development biology research.

Implementation and Evaluation of a Fully Automated Multiplex Real-Time PCR Assay on the BD Max Platform to Detect and Differentiate Herpesviridae from Cerebrospinal Fluids

PubMed Central

Köller, Thomas; Kurze, Daniel; Lange, Mirjam; Scherdin, Martin; Podbielski, Andreas; Warnke, Philipp

2016-01-01

A fully automated multiplex real-time PCR assay—including a sample process control and a plasmid based positive control—for the detection and differentiation of herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2) and varicella-zoster virus (VZV) from cerebrospinal fluids (CSF) was developed on the BD Max platform. Performance was compared to an established accredited multiplex real time PCR protocol utilizing the easyMAG and the LightCycler 480/II, both very common devices in viral molecular diagnostics. For clinical validation, 123 CSF specimens and 40 reference samples from national interlaboratory comparisons were examined with both methods, resulting in 97.6% and 100% concordance for CSF and reference samples, respectively. Utilizing the BD Max platform revealed sensitivities of 173 (CI 95%, 88–258) copies/ml for HSV1, 171 (CI 95%, 148–194) copies/ml for HSV2 and 84 (CI 95%, 5–163) copies/ml for VZV. Cross reactivity could be excluded by checking 25 common viral, bacterial and fungal human pathogens. Workflow analyses displayed shorter test duration as well as remarkable fewer and easier preparation steps with the potential to reduce error rates occurring when manually assessing patient samples. This protocol allows for a fully automated PCR assay on the BD Max platform for the simultaneously detection of herpesviridae from CSF specimens. Singular or multiple infections due to HSV1, HSV2 and VZV can reliably be differentiated with good sensitivities. Control parameters are included within the assay, thereby rendering its suitability for current quality management requirements. PMID:27092772

A single-electrode electrochemical system for multiplex electrochemiluminescence analysis based on a resistance induced potential difference.

PubMed

Gao, Wenyue; Muzyka, Kateryna; Ma, Xiangui; Lou, Baohua; Xu, Guobao

2018-04-28

Developing low-cost and simple electrochemical systems is becoming increasingly important but still challenged for multiplex experiments. Here we report a single-electrode electrochemical system (SEES) using only one electrode not only for a single experiment but also for multiplex experiments based on a resistance induced potential difference. SEESs for a single experiment and multiplex experiments are fabricated by attaching a self-adhesive label with a hole and multiple holes onto an ITO electrode, respectively. This enables multiplex electrochemiluminescence analysis with high sensitivity at a very low safe voltage using a smartphone as a detector. For the multiplex analysis, the SEES using a single electrode is much simpler, cheaper and more user-friendly than conventional electrochemical systems and bipolar electrochemical systems using electrode arrays. Moreover, SEESs are free from the electrochemiluminescent background problem from driving electrodes in bipolar electrochemical systems. Since numerous electrodes and cover materials can be used to fabricate SEESs readily and electrochemistry is being extensively used, SEESs are very promising for broad applications, such as drug screening and high throughput analysis.

Can we trust intraoperative culture results in nonunions?

PubMed

Palmer, Michael P; Altman, Daniel T; Altman, Gregory T; Sewecke, Jeffrey J; Ehrlich, Garth D; Hu, Fen Z; Nistico, Laura; Melton-Kreft, Rachel; Gause, Trent M; Costerton, John W

2014-07-01

To identify the presence of bacterial biofilms in nonunions comparing molecular techniques (multiplex polymerase chain reaction and mass spectrometry, fluorescent in situ hybridization) with routine intraoperative cultures. Thirty-four patients with nonunions were scheduled for surgery and enrolled in this ongoing prospective study. Intraoperative specimens were collected from removed implants, surrounding tissue membrane, and local soft tissue followed by standard culture analysis, Ibis's second generation molecular diagnostics (Ibis Biosystems), and bacterial 16S rRNA-based fluorescence in situ hybridization (FISH). Confocal microscopy was used to visualize the tissue specimens reacted with the FISH probes, which were chosen based on the Ibis analysis. Thirty-four patient encounters were analyzed. Eight were diagnosed as infected nonunions by positive intraoperative culture results. Ibis confirmed the presence of bacteria in all 8 samples. Ibis identified bacteria in a total of 30 of 34 encounters, and these data were confirmed by FISH. Twenty-two of 30 Ibis-positive samples were culture-negative. Four samples were negative by all methods of analysis. No samples were positive by culture, but negative by molecular techniques. Our preliminary data indicate that molecular diagnostics are more sensitive for identifying bacteria than cultures in cases of bony nonunion. This is likely because of the inability of cultures to detect biofilms and bacteria previously exposed to antibiotic therapy. Diagnostic Level I. See Instructions for Authors for a complete description of levels of evidence.

The suitability of small biopsy and cytology specimens for EGFR and other mutation testing in non-small cell lung cancer

PubMed Central

Wang, Shu; Yu, Bing; Ng, Chiu Chin; Mercorella, Belinda; Selinger, Christina I.; O’Toole, Sandra A.

2015-01-01

Background Patients with advanced non-small cell lung cancer (NSCLC) benefit from treatment with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) when their tumor harbors an activating EGFR mutation. As the majority of NSCLC patients present with advanced disease, cytology and small biopsy specimens are frequently the only tissue available for mutation testing, but can pose challenges due to low tumor content. We aim to better define the suitability of these specimens for mutation testing. Methods NSCLC cases referred to our institution for mutation testing over a 15-month period were retrospectively reviewed. Specimens were tested for mutations including EGFR, KRAS, and BRAF, using a multiplex PCR assay (OncoCarta Panel v1.0) and analyzed on the Agena Bioscience MassARRAY platform. Results A total of 146 specimens were tested, comprising 53 (36.3%) resection specimens (including 28 lung resection specimens), 55 (37.7%) small biopsy specimens and 38 (26%) cytology specimens. Of 142 cases with sufficient DNA for mutation testing, EGFR mutations were detected in 31 specimens (21.8%), KRAS mutations in 31 specimens (21.8%) and BRAF mutations in three specimens (2.1%). There was no significant difference in the EGFR mutation rate between lung resection (10 of 28 cases; 35.7%), small biopsy (9 of 53 cases; 17%), and cytology specimens (8 of 36 cases; 22.2%). Conclusions Our results support the utility of small biopsy and cytology specimens for mutation testing. Careful evaluation of the adequacy of small specimens is required to minimize the risk of false negative or positive results. PMID:25870794

The Development and Fabrication of an Implantable, Multiplexed, Semiconductor Multielectrode Array.

DTIC Science & Technology

1983-12-01

ILt D. C. Denton . Their individual and collective efforts in designing, fabricating, and testing an implantable array resulted in a semiconductor device... contaminating sodium ions were attracted by the electrical field developed by the JFET pinch-off voltage. These sodium ions produced leakage paths...34 was implanted in a biological specimen (dog) by Hensley and Denton (Ref 5). Summary of Current Knowledge Hensley and Denton showed the feasibility

Breast Cancer Diagnosis Using a Microfluidic Multiplexed Immunohistochemistry Platform

PubMed Central

Kim, Minseok S.; Kim, Taemin; Kong, Sun-Young; Kwon, Soim; Bae, Chae Yun; Choi, Jaekyu; Kim, Chul Hwan; Lee, Eun Sook; Park, Je-Kyun

2010-01-01

Background Biomarkers play a key role in risk assessment, assessing treatment response, and detecting recurrence and the investigation of multiple biomarkers may also prove useful in accurate prediction and prognosis of cancers. Immunohistochemistry (IHC) has been a major diagnostic tool to identify therapeutic biomarkers and to subclassify breast cancer patients. However, there is no suitable IHC platform for multiplex assay toward personalized cancer therapy. Here, we report a microfluidics-based multiplexed IHC (MMIHC) platform that significantly improves IHC performance in reduction of time and tissue consumption, quantification, consistency, sensitivity, specificity and cost-effectiveness. Methodology/Principal Findings By creating a simple and robust interface between the device and human breast tissue samples, we not only applied conventional thin-section tissues into on-chip without any additional modification process, but also attained perfect fluid control for various solutions, without any leakage, bubble formation, or cross-contamination. Four biomarkers, estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), progesterone receptor (PR) and Ki-67, were examined simultaneously on breast cancer cells and human breast cancer tissues. The MMIHC method improved immunoreaction, reducing time and reagent consumption. Moreover, it showed the availability of semi-quantitative analysis by comparing Western blot. Concordance study proved strong consensus between conventional whole-section analysis and MMIHC (n = 105, lowest Kendall's coefficient of concordance, 0.90). To demonstrate the suitability of MMIHC for scarce samples, it was also applied successfully to tissues from needle biopsies. Conclusions/Significance The microfluidic system, for the first time, was successfully applied to human clinical tissue samples and histopathological diagnosis was realized for breast cancers. Our results showing substantial agreement indicate that several cancer-related proteins can be simultaneously investigated on a single tumor section, giving clear advantages and technical advances over standard immunohistochemical method. This novel concept will enable histopathological diagnosis using numerous specific biomarkers at a time even for small-sized specimens, thus facilitating the individualization of cancer therapy. PMID:20454672

Development of an improved PCR-ICT hybrid assay for direct detection of Legionellae and Legionella pneumophila from cooling tower water specimens.

PubMed

Horng, Yu-Tze; Soo, Po-Chi; Shen, Bin-Jon; Hung, Yu-Li; Lo, Kai-Yin; Su, Hsun-Pi; Wei, Jun-Rong; Hsieh, Shang-Chen; Hsueh, Po-Ren; Lai, Hsin-Chih

2006-06-01

A novelly improved polymerase chian reaction and immunochromatography test (PCR-ICT) hybrid assay comprising traditional multiplex-nested PCR and ICT, (a lateral-flow device) was developed for direct detection of Legionella bacteria from environmental cooling tower samples. The partial 16S rDNA (specific for Legionella spp.) and dnaJ (specific for Legionella pneumophila) genes from Legionella chromosome were first specifically amplified by multiplex-nested PCR, respectively, followed by detection using ICT strip. Reading of results was based on presence or absence of the two test lines on the strips. Presence of test line 1 indicated existence of Legionella spp. specific 16S rDNA and identified Legionella spp. Presence of test line 2 further indicated existence of dnaJ and thus specifically identified L. pneumophila. In contrast, for non-Legionellae bacteria no test line formation was observed. Results of direct detection of Legionella bacteria and L. pneumophila from water tower specimens by this assay showed 100% sensitivity, and 96.6% and 100% specificity, respectively compared with traditional culture, biochemical and serological identification methods. The PCR-ICT hybrid assay does not require sophisticated equipment and was proved to be practically useful in rapid and direct Legionellae detection from environmental water samples.

FIMic: design for ultimate 3D-integral microscopy of in-vivo biological samples

PubMed Central

Scrofani, G.; Sola-Pikabea, J.; Llavador, A.; Sanchez-Ortiga, E.; Barreiro, J. C.; Saavedra, G.; Garcia-Sucerquia, J.; Martínez-Corral, M.

2017-01-01

In this work, Fourier integral microscope (FIMic), an ultimate design of 3D-integral microscopy, is presented. By placing a multiplexing microlens array at the aperture stop of the microscope objective of the host microscope, FIMic shows extended depth of field and enhanced lateral resolution in comparison with regular integral microscopy. As FIMic directly produces a set of orthographic views of the 3D-micrometer-sized sample, it is suitable for real-time imaging. Following regular integral-imaging reconstruction algorithms, a 2.75-fold enhanced depth of field and 2-time better spatial resolution in comparison with conventional integral microscopy is reported. Our claims are supported by theoretical analysis and experimental images of a resolution test target, cotton fibers, and in-vivo 3D-imaging of biological specimens. PMID:29359107

Development of genomic microsatellite multiplex PCR using dye-labeled universal primer and its validation in pedigree analysis of Pacific oyster ( Crassostrea gigas)

NASA Astrophysics Data System (ADS)

Liu, Ting; Li, Qi; Song, Junlin; Yu, Hong

2017-02-01

There is an increasing requirement for traceability of aquaculture products, both for consumer protection and for food safety. There are high error rates in the conventional traceability systems depending on physical labels. Genetic traceability technique depending on DNA-based tracking system can overcome this problem. Genealogy information is essential for genetic traceability, and microsatellite DNA marker is a good choice for pedigree analysis. As increasing genotyping throughput of microsatellites, microsatellite multiplex PCR has become a fast and cost-effective technique. As a commercially important cultured aquatic species, Pacific oyster Crassostrea gigas has the highest global production. The objective of this study was to develop microsatellite multiplex PCR panels with dye-labeled universal primer for pedigree analysis in C. gigas, and these multiplex PCRs were validated using 12 full-sib families with known pedigrees. Here we developed six informative multiplex PCRs using 18 genomic microsatellites in C. gigas. Each multiplex panel contained a single universal primer M13(-21) used as a tail on each locus-specific forward primer and a single universal primer M13(-21) labeled with fluorophores. The polymorphisms of the markers were moderate, with an average of 10.3 alleles per locus and average polymorphic information content of 0.740. The observed heterozygosity per locus ranged from 0.492 to 0.822. Cervus simulations revealed that the six panels would still be of great value when massive families were analysed. Pedigree analysis of real offspring demonstrated that 100% of the offspring were unambiguously allocated to their parents when two multiplex PCRs were used. The six sets of multiplex PCRs can be an important tool for tracing cultured individuals, population genetic analysis, and selective breeding program in C. gigas.

Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection

NASA Astrophysics Data System (ADS)

Dobnik, David; Štebih, Dejan; Blejec, Andrej; Morisset, Dany; Žel, Jana

2016-10-01

The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR. Two 4-plex assays were developed for quantification of 8 different DNA targets (7 genetically modified maize events and maize endogene). Per assay, two of the targets were labelled with one fluorophore and two with another. As current analysis software does not support analysis of more than duplex, a new R- and Shiny-based web application analysis tool (http://bit.ly/ddPCRmulti) was developed that automates the analysis of 4-plex results. In conclusion, the two developed multiplex assays are suitable for quantification of GMO maize events and the same approach can be used in any other field with a need for accurate and reliable quantification of multiple DNA targets.

Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection.

PubMed

Dobnik, David; Štebih, Dejan; Blejec, Andrej; Morisset, Dany; Žel, Jana

2016-10-14

The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR. Two 4-plex assays were developed for quantification of 8 different DNA targets (7 genetically modified maize events and maize endogene). Per assay, two of the targets were labelled with one fluorophore and two with another. As current analysis software does not support analysis of more than duplex, a new R- and Shiny-based web application analysis tool (http://bit.ly/ddPCRmulti) was developed that automates the analysis of 4-plex results. In conclusion, the two developed multiplex assays are suitable for quantification of GMO maize events and the same approach can be used in any other field with a need for accurate and reliable quantification of multiple DNA targets.

Broadband plasmonic-enhanced forward and backward multiplex coherent anti-Stokes Raman scattering microscopy

NASA Astrophysics Data System (ADS)

Guo, Baoshan; Jiang, Lan; Hua, Yanhong; Li, Xin; Cui, Tianhong; Lu, Yongfeng

2018-03-01

Coherent anti-Stokes Raman scattering (CARS) microscopy is an attractive technique for label-free biochemical-specific characterization of biological specimens. However, it has very low sensitivity in monitoring and imaging molecules present in extremely low concentrations or at fast speeds. To improve this sensitivity, especially for multiplex CARS, the intensity of the pump beam and broadband Stokes beam should be enhanced simultaneously. Therefore, the gold shell particle and gold surface are demonstrated to enhance the forward and backward CARS, respectively. Results show that a signal enhancement factor of ˜25,000 can be achieved for the gold surface and an even higher enhancement factor can be achieved for the gold shell particles. Thus, we can obtain an enhanced CARS signal in a broad spectral range, which will substantially improve the detection sensitivity of hyperspectral CARS spectroscopy and imaging.

High throughput, multiplexed pathogen detection authenticates plague waves in medieval Venice, Italy.

PubMed

Tran, Thi-Nguyen-Ny; Signoli, Michel; Fozzati, Luigi; Aboudharam, Gérard; Raoult, Didier; Drancourt, Michel

2011-03-10

Historical records suggest that multiple burial sites from the 14th-16th centuries in Venice, Italy, were used during the Black Death and subsequent plague epidemics. High throughput, multiplexed real-time PCR detected DNA of seven highly transmissible pathogens in 173 dental pulp specimens collected from 46 graves. Bartonella quintana DNA was identified in five (2.9%) samples, including three from the 16th century and two from the 15th century, and Yersinia pestis DNA was detected in three (1.7%) samples, including two from the 14th century and one from the 16th century. Partial glpD gene sequencing indicated that the detected Y. pestis was the Orientalis biotype. These data document for the first time successive plague epidemics in the medieval European city where quarantine was first instituted in the 14th century.

Molecular differentiation of Opisthorchis viverrini and Clonorchis sinensis eggs by multiplex real-time PCR with high resolution melting analysis.

PubMed

Kaewkong, Worasak; Intapan, Pewpan M; Sanpool, Oranuch; Janwan, Penchom; Thanchomnang, Tongjit; Laummaunwai, Porntip; Lulitanond, Viraphong; Doanh, Pham Ngoc; Maleewong, Wanchai

2013-12-01

Opisthorchis viverrini and Clonorchis sinensis are parasites known to be carcinogenic and causative agents of cholangiocarcinoma in Asia. The standard method for diagnosis for those parasite infections is stool examination to detect parasite eggs. However, the method has low sensitivity, and eggs of O. viverrini and C. sinensis are difficult to distinguish from each other and from those of some other trematodes. Here, we report a multiplex real-time PCR coupled with high resolution melting (HRM) analysis for the differentiation of O. viverrini and C. sinensis eggs in fecal samples. Using 2 pairs of species-specific primers, DNA sequences from a portion of the mitochondrial NADH dehydrogenase subunit 2 (nad 2) gene, were amplified to generate 209 and 165 bp products for O. viverrini and C. sinensis, respectively. The distinct characteristics of HRM patterns were analyzed, and the melting temperatures peaked at 82.4±0.09℃ and 85.9±0.08℃ for O. viverrini and C. sinensis, respectively. This technique was able to detect as few as 1 egg of O. viverrini and 2 eggs of C. sinensis in a 150 mg fecal sample, which is equivalent to 7 and 14 eggs per gram of feces, respectively. The method is species-specific, rapid, simple, and does not require fluorescent probes or post-PCR processing for discrimination of eggs of the 2 species. It offers a new tool for differentiation and detection of Asian liver fluke infections in stool specimens.

Comparison of Multiplex Suspension Array Large-Panel Kits for Profiling Cytokines and Chemokines in Rheumatoid Arthritis Patients

PubMed Central

Khan, Imran H.; Krishnan, V.V.; Ziman, Melanie; Janatpour, Kim; Wun, Ted; Luciw, Paul A.; Tuscano, Joseph

2015-01-01

Background Multiplex analysis allows measurements of a large number of analytes simultaneously in each sample. Based on the Luminex multiplex technology (xMAP), kits for measuring multiple cytokines and chemokines (immunomodulators) are commercially available and are useful in investigations on inflammatory diseases. This study evaluated four multiplex kits (Bio-Plex, LINCOplex, Fluorokine, and Beadlyte) that contained 27, 29, 20 and 22 analytes each, respectively, for the analysis of immunomodulators in plasma of rheumatoid arthritis (RA) patients who underwent treatment with antibody against CD20 (rituximab), a B-cell reductive therapy. Methods Multiplex kits were tested on serial plasma samples obtained from six RA patients at baseline and multiple time points (3, 6, and 9 months) post-treatment with rituximab. The RA patients included in this study had previously failed therapy with disease modifying anti-arthritis drugs (DMARD) and treatment with anti-TNFα antibody (infliximab). Results Computer modeling and hierarchical cluster analysis of the multiplex data allowed a comparison of the performance of multiplex assay kits and revealed profiles of immunomodulators in the RA patients. Conclusions In plasma of RA patients who appeared to have benefited from rituximab treatment the profile of significantly elevated immunomodulators by at least two of the three kits (BioPlex, LINCOplex, Beadlyte), is as follows: IL-12p70, Eotaxin, IL-4, TNFα, Il-9, IL-1β, IFNγ, IL-10, IL-6, and IL-13. Immunomodulator profiling by multiplex analysis may provide useful plasma biomarkers for monitoring response to B-cell reductive therapy in RA patients. PMID:18823005

The use of FTA cards for preserving unfixed cytological material for high-throughput molecular analysis.

PubMed

Saieg, Mauro Ajaj; Geddie, William R; Boerner, Scott L; Liu, Ni; Tsao, Ming; Zhang, Tong; Kamel-Reid, Suzanne; da Cunha Santos, Gilda

2012-06-25

Novel high-throughput molecular technologies have made the collection and storage of cells and small tissue specimens a critical issue. The FTA card provides an alternative to cryopreservation for biobanking fresh unfixed cells. The current study compared the quality and integrity of the DNA obtained from 2 types of FTA cards (Classic and Elute) using 2 different extraction protocols ("Classic" and "Elute") and assessed the feasibility of performing multiplex mutational screening using fine-needle aspiration (FNA) biopsy samples. Residual material from 42 FNA biopsies was collected in the cards (21 Classic and 21 Elute cards). DNA was extracted using the Classic protocol for Classic cards and both protocols for Elute cards. Polymerase chain reaction for p53 (1.5 kilobase) and CARD11 (500 base pair) was performed to assess DNA integrity. Successful p53 amplification was achieved in 95.2% of the samples from the Classic cards and in 80.9% of the samples from the Elute cards using the Classic protocol and 28.5% using the Elute protocol (P = .001). All samples (both cards) could be amplified for CARD11. There was no significant difference in the DNA concentration or 260/280 purity ratio when the 2 types of cards were compared. Five samples were also successfully analyzed by multiplex MassARRAY spectrometry, with a mutation in KRAS found in 1 case. High molecular weight DNA was extracted from the cards in sufficient amounts and quality to perform high-throughput multiplex mutation assays. The results of the current study also suggest that FTA Classic cards preserve better DNA integrity for molecular applications compared with the FTA Elute cards. Copyright © 2012 American Cancer Society.

Persistence of Penaeus stylirostris densovirus delays mortality caused by white spot syndrome virus infection in black tiger shrimp (Penaeus monodon)

PubMed Central

2013-01-01

Background Persistent infection of Penaeus stylirostris densovirus (PstDNV) (also called IHHNV) and its non-infectious inserts in the black tiger shrimp, Penaeus monodon (P. monodon) genome are commonly found without apparent disease. Here, we introduced the method of multiplex PCR in order to differentiate shrimp with viral inserts from ones with the infectious virus. The method allowed us to study the effect of pre-infection of IHHNV, in comparison to IHHNV inserts, on WSSV resistance in P. monodon. Results A multiplex PCR system was developed to amplify the entire IHHNV genome, ensuring the accurate diagnosis. Field samples containing IHHNV DNA templates as low as 20 pg or equivalent 150 viral copies can be detected by this method. By challenging the two groups of diagnosed shrimp with WSSV, we found that shrimp with IHHNV infection and those with viral inserts responded to WSSV differently. Considering cumulative mortality, average time to death of shrimp in IHHNV-infected group (day 14) was significantly delayed relative to that (day 10) of IHHNV-inserted group. Real-time PCR analysis of WSSV copy number indicated the lower amount of WSSV in the IHHNV-infected group than the virus-inserted group. The ratio of IHHNV: WSSV copy number in all determined IHHNV-infected samples ranged from approximately 4 to 300-fold. Conclusion The multiplex PCR assay developed herein proved optimal for convenient differentiation of shrimp specimens with real IHHNV infection and those with insert types. Diagnosed shrimp were also found to exhibit different WSSV tolerance. After exposed to WSSV, the naturally pre-infected IHHNV P. monodon were less susceptible to WSSV and, consequently, survived longer than the IHHNV-inserted shrimp. PMID:23414329

Evaluation of the new AmpliSens multiplex real-time PCR assay for simultaneous detection of Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, and Trichomonas vaginalis.

PubMed

Rumyantseva, Tatiana; Golparian, Daniel; Nilsson, Christian S; Johansson, Emma; Falk, My; Fredlund, Hans; Van Dam, Alje; Guschin, Alexander; Unemo, Magnus

2015-10-01

In this study, we performed an evaluation of the new CE-marked multiplex real-time AmpliSens N.gonorrhoeae/C.trachomatis/M.genitalium/T.vaginalis-MULTIPRIME-FRT PCR assay compared to APTIMA tests, i.e., APTIMA COMBO 2 assay, APTIMA Trichomonas vaginalis assay (FDA-approved), and two different APTIMA Mycoplasma genitalium assays (research use only; one of them only used for discrepancy analysis). Vaginal swabs (n = 209) and first-void urine (FVU) specimens from females (n = 498) and males (n = 554), consecutive attendees (n = 1261) at a dermatovenerological clinic in Sweden, were examined. The sensitivity of the AmpliSens PCR assay for detection of C. trachomatis (6.3% prevalence), M. genitalium (5.7% prevalence), N. gonorrhoeae (0.3% prevalence), and T. vaginalis (0.08% prevalence) was 97.5% (95% confidence interval (CI): 91.2-99.6%), 81.9% (95% CI: 70.7-89.7%), 100% (95% CI: 40.2-100%) and 100% (95% CI: 16.5-100%), respectively. The specificity of the AmpliSens PCR assay was 100% (95% CI: 99.6-100%) for all agents. The analytical sensitivity and specificity for N. gonorrhoeae detection was excellent, i.e., 55 international gonococcal strains detected and 135 isolates of 13 non-gonococcal Neisseria species were negative. In conclusion, the multiplex real-time AmpliSens N.gonorrhoeae/C.trachomatis/M.genitalium/T.vaginalis-MULTIPRIME-FRT PCR assay demonstrated high sensitivity and excellent specificity for the detection of C. trachomatis, N. gonorrhoeae, and T. vaginalis, and excellent specificity but suboptimal sensitivity for M. genitalium detection. © 2015 APMIS. Published by John Wiley & Sons Ltd.

Simultaneous identification of 36 mutations in KRAS codons 61and 146, BRAF, NRAS, and PIK3CA in a single reaction by multiplex assay kit

PubMed Central

2013-01-01

Background Retrospective analyses in the West suggest that mutations in KRAS codons 61 and 146, BRAF, NRAS, and PIK3CA are negative predictive factors for cetuximab treatment in colorectal cancer patients. We developed a novel multiplex kit detecting 36 mutations in KRAS codons 61 and 146, BRAF, NRAS, and PIK3CA using Luminex (xMAP) assay in a single reaction. Methods Tumor samples and clinical data from Asian colorectal cancer patients treated with cetuximab were collected. We investigated KRAS, BRAF, NRAS, and PIK3CA mutations using both the multiplex kit and direct sequencing methods, and evaluated the concordance between the 2 methods. Objective response, progression-free survival (PFS), and overall survival (OS) were also evaluated according to mutational status. Results In total, 82 of 83 samples (78 surgically resected specimens and 5 biopsy specimens) were analyzed using both methods. All multiplex assays were performed using 50 ng of template DNA. The concordance rate between the methods was 100%. Overall, 49 (59.8%) patients had all wild-type tumors, 21 (25.6%) had tumors harboring KRAS codon 12 or 13 mutations, and 12 (14.6%) had tumors harboring KRAS codon 61, KRAS codon 146, BRAF, NRAS, or PIK3CA mutations. The response rates in these patient groups were 38.8%, 4.8%, and 0%, respectively. Median PFS in these groups was 6.1 months (95% confidence interval (CI): 3.1–9.2), 2.7 months (1.2–4.2), and 1.6 months (1.5–1.7); median OS was 13.8 months (9.2–18.4), 8.2 months (5.7–10.7), and 6.3 months (1.3–11.3), respectively. Statistically significant differences in both PFS and OS were found between patients with all wild-type tumors and those with KRAS codon 61, KRAS codon 146, BRAF, NRAS, or PIK3CA mutations (PFS: 95% CI, 0.11–0.44; P < 0.0001; OS: 95% CI, 0.15–0.61; P < 0.0001). Conclusions Our newly developed multiplex kit is practical and feasible for investigation of a range of sample types. Moreover, mutations in KRAS codon 61, KRAS codon 146, BRAF, NRAS, or PIK3CA detected in Asian patients were not predictive of clinical benefits from cetuximab treatment, similar to the result obtained in European studies. PMID:24006859

Standard operating procedures for serum and plasma collection: early detection research network consensus statement standard operating procedure integration working group.

PubMed

Tuck, Melissa K; Chan, Daniel W; Chia, David; Godwin, Andrew K; Grizzle, William E; Krueger, Karl E; Rom, William; Sanda, Martin; Sorbara, Lynn; Stass, Sanford; Wang, Wendy; Brenner, Dean E

2009-01-01

Specimen collection is an integral component of clinical research. Specimens from subjects with various stages of cancers or other conditions, as well as those without disease, are critical tools in the hunt for biomarkers, predictors, or tests that will detect serious diseases earlier or more readily than currently possible. Analytic methodologies evolve quickly. Access to high-quality specimens, collected and handled in standardized ways that minimize potential bias or confounding factors, is key to the "bench to bedside" aim of translational research. It is essential that standard operating procedures, "the how" of creating the repositories, be defined prospectively when designing clinical trials. Small differences in the processing or handling of a specimen can have dramatic effects in analytical reliability and reproducibility, especially when multiplex methods are used. A representative working group, Standard Operating Procedures Internal Working Group (SOPIWG), comprised of members from across Early Detection Research Network (EDRN) was formed to develop standard operating procedures (SOPs) for various types of specimens collected and managed for our biomarker discovery and validation work. This report presents our consensus on SOPs for the collection, processing, handling, and storage of serum and plasma for biomarker discovery and validation.

Standardized Methods to Generate Mock (Spiked) Clinical Specimens by Spiking Blood or Plasma with Cultured Pathogens

PubMed Central

Dong, Ming; Fisher, Carolyn; Añez, Germán; Rios, Maria; Nakhasi, Hira L.; Hobson, J. Peyton; Beanan, Maureen; Hockman, Donna; Grigorenko, Elena; Duncan, Robert

2016-01-01

Aims To demonstrate standardized methods for spiking pathogens into human matrices for evaluation and comparison among diagnostic platforms. Methods and Results This study presents detailed methods for spiking bacteria or protozoan parasites into whole blood and virus into plasma. Proper methods must start with a documented, reproducible pathogen source followed by steps that include standardized culture, preparation of cryopreserved aliquots, quantification of the aliquots by molecular methods, production of sufficient numbers of individual specimens and testing of the platform with multiple mock specimens. Results are presented following the described procedures that showed acceptable reproducibility comparing in-house real-time PCR assays to a commercially available multiplex molecular assay. Conclusions A step by step procedure has been described that can be followed by assay developers who are targeting low prevalence pathogens. Significance and Impact of Study The development of diagnostic platforms for detection of low prevalence pathogens such as biothreat or emerging agents is challenged by the lack of clinical specimens for performance evaluation. This deficit can be overcome using mock clinical specimens made by spiking cultured pathogens into human matrices. To facilitate evaluation and comparison among platforms, standardized methods must be followed in the preparation and application of spiked specimens. PMID:26835651

Available number of multiplexed holograms based on signal-to-noise ratio analysis in reflection-type holographic memory using three-dimensional speckle-shift multiplexing.

PubMed

Nishizaki, Tatsuya; Matoba, Osamu; Nitta, Kouichi

2014-09-01

The recording properties of three-dimensional speckle-shift multiplexing in reflection-type holographic memory are analyzed numerically. Three-dimensional recording can increase the number of multiplexed holograms by suppressing the cross-talk noise from adjacent holograms by using depth-direction multiplexing rather than in-plane multiplexing. Numerical results indicate that the number of multiplexed holograms in three-layer recording can be increased by 1.44 times as large as that of a single-layer recording when an acceptable signal-to-noise ratio is set to be 2 when NA=0.43 and the thickness of the recording medium is 0.5 mm.

A new design approach to MMI-based (de)multiplexers

NASA Astrophysics Data System (ADS)

Yueyu, Xiao; Sailing, He

2004-09-01

A novel design method of the wavelength (de)multiplexer is presented. The output spectral response of a (de)multiplexer is designed from the view of FIR filters. Avoiding laborious mathematic analysis, the (de)multiplexer is analyzed and designed in this explicit and simple method. A four channel (de)multiplexer based on multimode interference (MMI) is designed as an example. The result obtained agrees with that of the commonly used method, and is verified by a finite difference beam propagation method (FDBPM) simulation.

Gut Microbiota Dysbiosis and Diarrhea in Kidney Transplant Recipients.

PubMed

Lee, John Richard; Magruder, Matthew; Zhang, Lisa; Westblade, Lars F; Satlin, Michael J; Robertson, Amy; Edusei, Emmanuel; Crawford, Carl; Ling, Lilan; Taur, Ying; Schlueter, Jonas; Lubetzky, Michelle; Dadhania, Darshana; Pamer, Eric; Suthanthiran, Manikkam

2018-06-19

Post-transplant diarrhea is associated with kidney allograft failure and death but its etiology remains unknown in the majority of cases. Because altered gut microbial ecology is a potential basis for diarrhea, we investigated whether post-transplant diarrhea is associated with gut dysbiosis. We enrolled 71 kidney allograft recipients for serial fecal specimen collections in the first 3 months of transplantation and profiled the gut microbiota using 16S rRNA gene V4-V5 deep sequencing. The Shannon diversity index was significantly lower in 28 diarrheal fecal specimens from 25 recipients with post-transplant diarrhea than in 112 fecal specimens from 46 recipients without post-transplant diarrhea. We found lower relative abundance of 13 commensal genera (Benjamini-Hochberg adjusted p value ≤ 0.15) in the diarrheal fecal specimens including the same 4 genera identified in our prior study. The 28 diarrheal fecal specimens were also evaluated by a multiplexed PCR assay for 22 bacterial, viral, and protozoan gastrointestinal pathogens, and 26 specimens were negative for infectious etiologies. Using PICRUSt to predict metagenomic functions, we found diarrheal fecal specimens had a lower abundance of metabolic genes. Our findings suggest that post-transplant diarrhea is not associated with common infectious diarrheal pathogens but with a gut dysbiosis. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Next-Generation Molecular Histology Using Highly Multiplexed Ion Beam Imaging (MIBI) of Breast Cancer Tissue Specimens for Enhanced Clinical Guidance

DTIC Science & Technology

2015-07-01

describe: 1) major activities ; 2) specific objectives; 3) significant results or key outcomes, including major findings, developments, or conclusions...project progresses to completion, the emphasis in reporting in this section should shift from reporting activities to reporting accomplishments. B...high-resolution axial (depth) imaging as well. Of course, both techniques are under active development, and their properties may change sub

The unexpected detection of varicella-zoster virus in genital specimens using the Lyra™ Direct HSV 1+2/VZV Assay.

PubMed

Granato, Paul A; DeGilio, Marcia A; Wilson, Elsie M

2016-11-01

The Lyra™ Direct HSV 1+2/VZV Assay is a moderately complex, multiplex PCR assay that qualitatively detects the presence of HSV 1, HSV 2, and VZV DNA in cutaneous and mucocutaneous specimens with a time-to-result of less than 60min. To report a one-year laboratory experience using Lyra assay for testing cutaneous and mucocutaneous specimens for HSV and VZV that resulted in the unexpected detection of VZV in 14 male and female genital specimens. Over a one-year period, 2113 cutaneous and mucocutaneous specimens from male and female patients were submitted for testing using the Lyra assay. An unexpected 14 genital specimens were positive for the presence VZV DNA. Eleven of the 14 specimens were available for confirmatory testing using two alternative molecular methods and Sanger sequencing. Fourteen male and female genital specimens were positive for the presence of VZV DNA. All of the 11 specimens (9 female and 2 male) that were available for confirmatory testing by the alternative molecular method and Sanger sequencing were confirmed as containing VZV DNA. Using of the Lyra assay over a one-year time period, VZV DNA was detected in 126 specimens of which 14 (11.1%) were from male and female genital sites. This rare and unexpected finding suggests that the appearance of zoster lesions in the genital area may not be as uncommon as previously thought and that this finding would have considerable impact on patient counseling and public health considerations. Copyright © 2016 Elsevier B.V. All rights reserved.

Comprehensive Multiplex One-Step Real-Time TaqMan qRT-PCR Assays for Detection and Quantification of Hemorrhagic Fever Viruses

PubMed Central

Li, Jiandong; Qu, Jing; He, Chengcheng; Zhang, Shuo; Li, Chuan; Zhang, Quanfu; Liang, Mifang; Li, Dexin

2014-01-01

Background Viral hemorrhagic fevers (VHFs) are a group of animal and human illnesses that are mostly caused by several distinct families of viruses including bunyaviruses, flaviviruses, filoviruses and arenaviruses. Although specific signs and symptoms vary by the type of VHF, initial signs and symptoms are very similar. Therefore rapid immunologic and molecular tools for differential diagnosis of hemorrhagic fever viruses (HFVs) are important for effective case management and control of the spread of VHFs. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay is one of the reliable and desirable methods for specific detection and quantification of virus load. Multiplex PCR assay has the potential to produce considerable savings in time and resources in the laboratory detection. Results Primers/probe sets were designed based on appropriate specific genes for each of 28 HFVs which nearly covered all the HFVs, and identified with good specificity and sensitivity using monoplex assays. Seven groups of multiplex one-step real-time qRT-PCR assays in a universal experimental system were then developed by combining all primers/probe sets into 4-plex reactions and evaluated with serial dilutions of synthesized viral RNAs. For all the multiplex assays, no cross-reactivity with other HFVs was observed, and the limits of detection were mainly between 45 and 150 copies/PCR. The reproducibility was satisfactory, since the coefficient of variation of Ct values were all less than 5% in each dilution of synthesized viral RNAs for both intra-assays and inter-assays. Evaluation of the method with available clinical serum samples collected from HFRS patients, SFTS patients and Dengue fever patients showed high sensitivity and specificity of the related multiplex assays on the clinical specimens. Conclusions Overall, the comprehensive multiplex one-step real-time qRT-PCR assays were established in this study, and proved to be specific, sensitive, stable and easy to serve as a useful tool for rapid detection of HFVs. PMID:24752452

A single-step polymerase chain reaction for simultaneous detection and differentiation of nontypeable and serotypeable Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae.

PubMed

Kunthalert, Duangkamol; Henghiranyawong, Kritsada; Sistayanarain, Anchalee; Khoothiam, Krissana

2013-02-01

The critically high prevalence of bacterial otitis media worldwide has prompted a proper disease management. While vaccine development for otitis media is promising, the reliable and effective methods for diagnosis of such etiologic agents are of importance. We developed a multiplex polymerase chain reaction assay for simultaneous detection and differentiation of nontypeable and serotypeable Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae. Five primer pairs targeting genes fumarate reductase (H. influenzae), outer membrane protein B (M. catarrhalis), major autolysin (S. pneumoniae), capsulation-associated BexA protein (all encapsulated H. influenzae) and 16S rRNA were incorporated in this single-step PCR. Validation of the multiplex PCR was also performed on clinical isolates. The developed multiplex PCR was highly specific, enabling the detection of the target pathogens in a specific manner, either individually or as a mixture of all target organisms. The assay was also found to be sensitive with the lowest detection limit of 1 ng of bacterial DNA. When applied to clinical isolates from diverse specimen sources, the multiplex PCR developed in this study correctly identified each microorganism individually or in a combination of two or more target organisms. All results matched with conventional culture identification. In addition, the ability of such assay to differentiate H. influenzae encapsulation from the study clinical isolates was 100%. Our multiplex PCR provides a rapid and accurate diagnostic tool for detection of the 4 target organisms. Such assay would serve as a useful tool for clinicians and epidemiologists in their efforts to the proper treatment and disease management caused by these organisms. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

The Relevance of a Novel Quantitative Assay to Detect up to 40 Major Streptococcus pneumoniae Serotypes Directly in Clinical Nasopharyngeal and Blood Specimens

PubMed Central

Albrich, Werner C.; van der Linden, Mark P. G.; Bénet, Thomas; Chou, Monidarin; Sylla, Mariam; Barreto Costa, Patricia; Richard, Nathalie; Klugman, Keith P.; Endtz, Hubert P.; Paranhos-Baccalà, Gláucia; Telles, Jean-Noël

2016-01-01

For epidemiological and surveillance purposes, it is relevant to monitor the distribution and dynamics of Streptococcus pneumoniae serotypes. Conventional serotyping methods do not provide rapid or quantitative information on serotype loads. Quantitative serotyping may enable prediction of the invasiveness of a specific serotype compared to other serotypes carried. Here, we describe a novel, rapid multiplex real-time PCR assay for identification and quantification of the 40 most prevalent pneumococcal serotypes and the assay impacts in pneumonia specimens from emerging and developing countries. Eleven multiplex PCR to detect 40 serotypes or serogroups were optimized. Quantification was enabled by reference to standard dilutions of known bacterial load. Performance of the assay was evaluated to specifically type and quantify S. pneumoniae in nasopharyngeal and blood samples from adult and pediatric patients hospitalized with pneumonia (n = 664) from five different countries. Serogroup 6 was widely represented in nasopharyngeal specimens from all five cohorts. The most frequent serotypes in the French, South African, and Brazilian cohorts were 1 and 7A/F, 3 and 19F, and 14, respectively. When both samples were available, the serotype in blood was always present as carriage with other serotypes in the nasopharynx. Moreover, the ability of a serotype to invade the bloodstream may be linked to its nasopharyngeal load. The mean nasopharyngeal concentration of the serotypes that moved to the blood was 3 log-fold higher than the ones only found in the nasopharynx. This novel, rapid, quantitative assay may potentially predict some of the S. pneumoniae serotypes invasiveness and assessment of pneumococcal serotype distribution. PMID:26986831

Development of two real-time multiplex PCR assays for the detection and quantification of eight key bacterial pathogens in lower respiratory tract infections

PubMed Central

Gadsby, N.J.; McHugh, M.P.; Russell, C.D.; Mark, H.; Conway Morris, A.; Laurenson, I.F.; Hill, A.T.; Templeton, K.E.

2015-01-01

The frequent lack of a positive and timely microbiological diagnosis in patients with lower respiratory tract infection (LRTI) is an important obstacle to antimicrobial stewardship. Patients are typically prescribed broad-spectrum empirical antibiotics while microbiology results are awaited, but, because these are often slow, negative, or inconclusive, de-escalation to narrow-spectrum agents rarely occurs in clinical practice. The aim of this study was to develop and evaluate two multiplex real-time PCR assays for the sensitive detection and accurate quantification of Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Moraxella catarrhalis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. We found that all eight bacterial targets could be reliably quantified from sputum specimens down to a concentration of 100 CFUs/reaction (8333 CFUs/mL). Furthermore, all 249 positive control isolates were correctly detected with our assay, demonstrating effectiveness on both reference strains and local clinical isolates. The specificity was 98% on a panel of nearly 100 negative control isolates. Bacterial load was quantified accurately when three bacterial targets were present in mixtures of varying concentrations, mimicking likely clinical scenarios in LRTI. Concordance with culture was 100% for culture-positive sputum specimens, and 90% for bronchoalveolar lavage fluid specimens, and additional culture-negative bacterial infections were detected and quantified. In conclusion, a quantitative molecular test for eight key bacterial causes of LRTI has the potential to provide a more sensitive decision-making tool, closer to the time-point of patient admission than current standard methods. This should facilitate de-escalation from broad-spectrum to narrow-spectrum antibiotics, substantially improving patient management and supporting efforts to curtail inappropriate antibiotic use. PMID:25980353

Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection

PubMed Central

Dobnik, David; Štebih, Dejan; Blejec, Andrej; Morisset, Dany; Žel, Jana

2016-01-01

The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR. Two 4-plex assays were developed for quantification of 8 different DNA targets (7 genetically modified maize events and maize endogene). Per assay, two of the targets were labelled with one fluorophore and two with another. As current analysis software does not support analysis of more than duplex, a new R- and Shiny-based web application analysis tool (http://bit.ly/ddPCRmulti) was developed that automates the analysis of 4-plex results. In conclusion, the two developed multiplex assays are suitable for quantification of GMO maize events and the same approach can be used in any other field with a need for accurate and reliable quantification of multiple DNA targets. PMID:27739510

Multiplexed Liquid Chromatography-Multiple Reaction Monitoring Mass Spectrometry Quantification of Cancer Signaling Proteins

PubMed Central

Chen, Yi; Fisher, Kate J.; Lloyd, Mark; Wood, Elizabeth R.; Coppola, Domenico; Siegel, Erin; Shibata, David; Chen, Yian A.; Koomen, John M.

2017-01-01

Quantitative evaluation of protein expression across multiple cancer-related signaling pathways (e.g. Wnt/β-catenin, TGF-β, receptor tyrosine kinases (RTK), MAP kinases, NF-κB, and apoptosis) in tumor tissues may enable the development of a molecular profile for each individual tumor that can aid in the selection of appropriate targeted cancer therapies. Here, we describe the development of a broadly applicable protocol to develop and implement quantitative mass spectrometry assays using cell line models and frozen tissue specimens from colon cancer patients. Cell lines are used to develop peptide-based assays for protein quantification, which are incorporated into a method based on SDS-PAGE protein fractionation, in-gel digestion, and liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM/MS). This analytical platform is then applied to frozen tumor tissues. This protocol can be broadly applied to the study of human disease using multiplexed LC-MRM assays. PMID:28808993

Molecular diagnosis of Salmonella typhi and its virulence in suspected typhoid blood samples through nested multiplex PCR.

PubMed

Prabagaran, Solai Ramatchandirane; Kalaiselvi, Vellingiri; Chandramouleeswaran, Naganathan; Deepthi, Krishnan Nair Geetha; Brahmadathan, Kootallur Narayanan; Mani, Mariappa

2017-08-01

A nested multiplex polymerase chain reaction (PCR) based diagnosis was developed for the detection of virulent Salmonella typhi in the blood specimens from patients suspected for typhoid fever. After the Widal test, two pairs of primers were used for the detection of flagellin gene (fliC) of S. typhi. Among them, those positive for fliC alone were subjected to identification of genes in Via B operon of Salmonella Pathogenesity Island (SPI-7) where four primer pairs were used to detect tviA and tviB genes. Among 250 blood samples tested, 115 were positive by fliC PCR; 22 of these were negative for tviA and tviB. Hence, the method described here can be used to diagnose the incidence of Vi-negative serovar typhi especially in endemic regions where the Vi vaccine is administered. Copyright © 2017 Elsevier B.V. All rights reserved.

Multiplex PCR-Based Next-Generation Sequencing and Global Diversity of Seoul Virus in Humans and Rats.

PubMed

Kim, Won-Keun; No, Jin Sun; Lee, Seung-Ho; Song, Dong Hyun; Lee, Daesang; Kim, Jeong-Ah; Gu, Se Hun; Park, Sunhye; Jeong, Seong Tae; Kim, Heung-Chul; Klein, Terry A; Wiley, Michael R; Palacios, Gustavo; Song, Jin-Won

2018-02-01

Seoul virus (SEOV) poses a worldwide public health threat. This virus, which is harbored by Rattus norvegicus and R. rattus rats, is the causative agent of hemorrhagic fever with renal syndrome (HFRS) in humans, which has been reported in Asia, Europe, the Americas, and Africa. Defining SEOV genome sequences plays a critical role in development of preventive and therapeutic strategies against the unique worldwide hantavirus. We applied multiplex PCR-based next-generation sequencing to obtain SEOV genome sequences from clinical and reservoir host specimens. Epidemiologic surveillance of R. norvegicus rats in South Korea during 2000-2016 demonstrated that the serologic prevalence of enzootic SEOV infections was not significant on the basis of sex, weight (age), and season. Viral loads of SEOV in rats showed wide dissemination in tissues and dynamic circulation among populations. Phylogenetic analyses showed the global diversity of SEOV and possible genomic configuration of genetic exchanges.

Multiplex Reverse Transcription-PCR for Simultaneous Surveillance of Influenza A and B Viruses

PubMed Central

Zhou, Bin; Barnes, John R.; Sessions, October M.; Chou, Tsui-Wen; Wilson, Malania; Stark, Thomas J.; Volk, Michelle; Spirason, Natalie; Halpin, Rebecca A.; Kamaraj, Uma Sangumathi; Ding, Tao; Stockwell, Timothy B.; Ghedin, Elodie; Barr, Ian G.

2017-01-01

ABSTRACT Influenza A and B viruses are the causative agents of annual influenza epidemics that can be severe, and influenza A viruses intermittently cause pandemics. Sequence information from influenza virus genomes is instrumental in determining mechanisms underpinning antigenic evolution and antiviral resistance. However, due to sequence diversity and the dynamics of influenza virus evolution, rapid and high-throughput sequencing of influenza viruses remains a challenge. We developed a single-reaction influenza A/B virus (FluA/B) multiplex reverse transcription-PCR (RT-PCR) method that amplifies the most critical genomic segments (hemagglutinin [HA], neuraminidase [NA], and matrix [M]) of seasonal influenza A and B viruses for next-generation sequencing, regardless of viral type, subtype, or lineage. Herein, we demonstrate that the strategy is highly sensitive and robust. The strategy was validated on thousands of seasonal influenza A and B virus-positive specimens using multiple next-generation sequencing platforms. PMID:28978683

High Throughput, Multiplexed Pathogen Detection Authenticates Plague Waves in Medieval Venice, Italy

PubMed Central

Tran, Thi-Nguyen-Ny; Signoli, Michel; Fozzati, Luigi; Aboudharam, Gérard; Raoult, Didier; Drancourt, Michel

2011-01-01

Background Historical records suggest that multiple burial sites from the 14th–16th centuries in Venice, Italy, were used during the Black Death and subsequent plague epidemics. Methodology/Principal Findings High throughput, multiplexed real-time PCR detected DNA of seven highly transmissible pathogens in 173 dental pulp specimens collected from 46 graves. Bartonella quintana DNA was identified in five (2.9%) samples, including three from the 16th century and two from the 15th century, and Yersinia pestis DNA was detected in three (1.7%) samples, including two from the 14th century and one from the 16th century. Partial glpD gene sequencing indicated that the detected Y. pestis was the Orientalis biotype. Conclusions These data document for the first time successive plague epidemics in the medieval European city where quarantine was first instituted in the 14th century. PMID:21423736

Multiplex pyrosequencing of InDel markers for forensic DNA analysis.

PubMed

Bus, Magdalena M; Karas, Ognjen; Allen, Marie

2016-12-01

The capillary electrophoresis (CE) technology is commonly used for fragment length separation of markers in forensic DNA analysis. In this study, pyrosequencing technology was used as an alternative and rapid tool for the analysis of biallelic InDel (insertion/deletion) markers for individual identification. The DNA typing is based on a subset of the InDel markers that are included in the Investigator ® DIPplex Kit, which are sequenced in a multiplex pyrosequencing analysis. To facilitate the analysis of degraded DNA, the polymerase chain reaction (PCR) fragments were kept short in the primer design. Samples from individuals of Swedish origin were genotyped using the pyrosequencing strategy and analysis of the Investigator ® DIPplex markers with CE. A comparison between the pyrosequencing and CE data revealed concordant results demonstrating a robust and correct genotyping by pyrosequencing. Using optimal marker combination and a directed dispensation strategy, five markers could be multiplexed and analyzed simultaneously. In this proof-of-principle study, we demonstrate that multiplex InDel pyrosequencing analysis is possible. However, further studies on degraded samples, lower DNA quantities, and mixtures will be required to fully optimize InDel analysis by pyrosequencing for forensic applications. Overall, although CE analysis is implemented in most forensic laboratories, multiplex InDel pyrosequencing offers a cost-effective alternative for some applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Photocleavable DNA barcode-antibody conjugates allow sensitive and multiplexed protein analysis in single cells.

PubMed

Agasti, Sarit S; Liong, Monty; Peterson, Vanessa M; Lee, Hakho; Weissleder, Ralph

2012-11-14

DNA barcoding is an attractive technology, as it allows sensitive and multiplexed target analysis. However, DNA barcoding of cellular proteins remains challenging, primarily because barcode amplification and readout techniques are often incompatible with the cellular microenvironment. Here we describe the development and validation of a photocleavable DNA barcode-antibody conjugate method for rapid, quantitative, and multiplexed detection of proteins in single live cells. Following target binding, this method allows DNA barcodes to be photoreleased in solution, enabling easy isolation, amplification, and readout. As a proof of principle, we demonstrate sensitive and multiplexed detection of protein biomarkers in a variety of cancer cells.

Simultaneous detection of antibodies to five Actinobacillus pleuropneumoniae serovars using bead-based multiplex analysis.

PubMed

Berger, Sanne Schou; Lauritsen, Klara Tølbøll; Boas, Ulrik; Lind, Peter; Andresen, Lars Ole

2017-11-01

We developed and made a preliminary validation of a bead-based multiplexed immunoassay for simultaneous detection of porcine serum antibodies to Actinobacillus pleuropneumoniae serovars 1, 2, 6, 7, and 12. Magnetic fluorescent beads were coupled with A. pleuropneumoniae antigens and tested with a panel of serum samples from experimentally infected pigs and with serum samples from uninfected and naturally infected pigs. The multiplex assay was compared to in-house ELISAs and complement fixation (CF) tests, which have been used for decades as tools for herd classification in the Danish Specific Pathogen Free system. Assay specificities and sensitivities as well as the corresponding cutoff values were determined using receiver operating characteristic (ROC) curve analysis, and the A. pleuropneumoniae multiplex assay showed good correlation with the in-house ELISAs and CF tests with areas under ROC curves ≥ 0.988. Benefits of multiplexed assays compared to ELISAs and CF tests include reduced serum sample volumes needed for analysis, less labor, and shorter assay time.

Modified midi- and mini-multiplex PCR systems for mitochondrial DNA control region sequence analysis in degraded samples.

PubMed

Kim, Na Young; Lee, Hwan Young; Park, Sun Joo; Yang, Woo Ick; Shin, Kyoung-Jin

2013-05-01

Two multiplex polymerase chain reaction (PCR) systems (Midiplex and Miniplex) were developed for the amplification of the mitochondrial DNA (mtDNA) control region, and the efficiencies of the multiplexes for amplifying degraded DNA were validated using old skeletal remains. The Midiplex system consisted of two multiplex PCRs to amplify six overlapping amplicons ranging in length from 227 to 267 bp. The Miniplex system consisted of three multiplex PCRs to amplify 10 overlapping short amplicons ranging in length from 142 to 185 bp. Most mtDNA control region sequences of several 60-year-old and 400-500-year-old skeletal remains were successfully obtained using both PCR systems and consistent with those previously obtained by monoplex amplification. The multiplex system consisting of smaller amplicons is effective for mtDNA sequence analyses of ancient and forensic degraded samples, saving time, cost, and the amount of DNA sample consumed during analysis. © 2013 American Academy of Forensic Sciences.

Simultaneous detection of viruses and Toxoplasma gondii in cerebrospinal fluid specimens by multiplex polymerase chain reaction-based reverse hybridization assay.

PubMed

Del Prete, Raffaele; Di Taranto, Anna Maria; Lipsi, Maria Rosaria; Natalicchio, Maria Iole; Antonetti, Raffaele; Miragliotta, Giuseppe

2009-04-01

The lack of rapidity and the low sensitivity and specificity of traditional laboratory methods limits their usefulness in the laboratory diagnosis of viral central nervous system (CNS) infections. This study describes the use of a commercially available multiplex polymerase chain reaction (mPCR)-based reverse hybridization assay (RHA) for the simultaneous detection of the genomes of 8 viruses and Toxoplasma gondii in cerebrospinal fluids (CSF) from 181 patients suspected of having viral meningitis. Twenty-two/181 (12.15%) CSF samples resulted positive by mPCR. Eighteen/22 were positive for 1 viral pathogen, whereas a dual infection was detected in 4/22 samples. Epstein-Barr virus (EBV) was the most commonly detected virus (6/22), followed by herpes simplex virus type-1 (HSV-1) (5/22) and -2 (HSV-2) (4/22). Cytomegalovirus (CMV), human herpesvirus-6 (HHV-6), and Epstein-Barr virus (EBV) were detected in 1 specimen each. Two CSF samples were co-infected by HSV-1/HSV-2, 1 sample by HHV-6/T. gondii, and 1 sample by EBV/EV, respectively. Our data support the usefulness of mPCR as a rapid molecular method for the simultaneous detection of major viral pathogens and T. gondii in aseptic meningitis also to allow the earlier application of specific antiviral therapy.

Advanced Code-Division Multiplexers for Superconducting Detector Arrays

NASA Astrophysics Data System (ADS)

Irwin, K. D.; Cho, H. M.; Doriese, W. B.; Fowler, J. W.; Hilton, G. C.; Niemack, M. D.; Reintsema, C. D.; Schmidt, D. R.; Ullom, J. N.; Vale, L. R.

2012-06-01

Multiplexers based on the modulation of superconducting quantum interference devices are now regularly used in multi-kilopixel arrays of superconducting detectors for astrophysics, cosmology, and materials analysis. Over the next decade, much larger arrays will be needed. These larger arrays require new modulation techniques and compact multiplexer elements that fit within each pixel. We present a new in-focal-plane code-division multiplexer that provides multiplexing elements with the required scalability. This code-division multiplexer uses compact lithographic modulation elements that simultaneously multiplex both signal outputs and superconducting transition-edge sensor (TES) detector bias voltages. It eliminates the shunt resistor used to voltage bias TES detectors, greatly reduces power dissipation, allows different dc bias voltages for each TES, and makes all elements sufficiently compact to fit inside the detector pixel area. These in-focal plane code-division multiplexers can be combined with multi-GHz readout based on superconducting microresonators to scale to even larger arrays.

Final Report Nucleic Acid System - Hybrid PCR and Multiplex Assay Project Phase 2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koopman, R P; Langlois, R G; Nasarabadi, S

2002-04-17

This report covers phase 2 (year 2) of the Nucleic Acid System--Hybrid PCR and Multiplex Assay project. The objective of the project is to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition technique of PCR (polymerase chain reaction) in a multiplex mode using flow cytometry. The Hybrid instrument consists of a flow-through PCR module capable of handling a multiplexed PCR assay, a hybridizing module capable of hybridizing multiplexed PCR amplicons and beads, and a flow cytometer module for bead-based identification, all controlled by a single computer. Multiplex immunoassay using bead-based Luminex flowmore » cytometry is available, allowing rapid screening for many agents. PCR is highly specific and complements and verifies immunoassay. It can also be multiplexed and detection provided using the bead-based Luminex flow cytometer. This approach allows full access to the speed and 100-fold multiplex capability of flow cytometry for rapid screening as well as the accuracy and specificity of PCR. This project has two principal activities: (1) Design, build and test a prototype hybrid PCR/flow cytometer with the basic capabilities for rapid, broad spectrum detection and identification, and (2) Develop and evaluate multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products. This project requires not only building operationally functional instrumentation but also developing the chemical assays for detection of priority pathogens. This involves development and evaluation of multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products.« less

Multiplex Profiling Identifies Distinct Local and Systemic Alterations during Intraperitoneal Chemotherapy for Ovarian Cancer: an NRG Oncology/Gynecologic Oncology Group Study

PubMed Central

Grabosch, Shannon; Tseng, George; Edwards, Robert P; Lankes, Heather A.; Moore, Kathleen; Odunsi, Kunle; Vlad, Anda; Ma, Tianzhou; Strange, Mary; Brozick, Joan; Lugade, Amit; Omilian, Angela; Bshara, Wiam; Stuckey, Ashley R.; Walker, Joan L.; Birrer, Michael

2017-01-01

OBJECTIVES Ovarian cancer leads to abdominal carcinomatosis and late stage (III/IV) diagnosis in 75% of patients. Three randomized phase III trials have demonstrated that intraperitoneal (IP) chemotherapy improves outcomes in epithelial ovarian cancer. While IP treatment is validated by clinical trials, there is a poor understanding of the mechanism(s) leading to the survival advantage other than the increased concentration of cytotoxic drugs within the tumor microenvironment. A better understanding of this process through analysis of dynamic biomarkers should promote novel approaches that may enhance tumor clearance. We propose this pilot study to confirm the feasibility of collecting serial peritoneal samples from implanted catheters in women receiving IP chemotherapy. We believe these specimens may be used for multiplex analysis to reveal unique biomarker fluctuations when compared to peripheral blood. METHODS From 13 women participating on GOG 252, 30 whole blood, 12 peritoneal fluid (PF), and 20 peritoneal wash (PW) with 30 mL saline were obtained. Samples were requested prior to the first three chemotherapy cycles. Samples were assessed for volume, cell populations, protein, RNA, and miRNA content changes. RESULTS Median volume for PF was 1.6 mL and 3.1 mL for PW. PW is a dilution of PF capable of capturing measurable biomarkers. Peritoneal aspirates contain a unique profile of biomarkers distinct from blood. miRNA undergo earlier alteration with chemotherapy than genes. Flow cytometry does not adequately capture biomarker fluctuations. CONCLUSIONS As a proof of principle study, this trial provides evidence that sampling the peritoneal cavity can be adapted for biomarker analysis. PMID:28483269

[Molecular diagnosis of spinal muscular atrophy by multiplex ligation-dependent probe amplification].

PubMed

Zeng, Jian; Ke, Long-feng; Deng, Xiao-jun; Cai, Mei-ying; Tu, Xiang-dong; Lan, Feng-hua

2008-12-16

To investigate the effect of multiplex ligation-dependent probe amplification (MLPA) in molecular diagnosis of spinal muscular atrophy (SMA). Peripheral blood samples were collected from 13 SMA patients, 31 parents of SMA patients, 50 healthy individuals without family history of SMA, and 10 specimens of amniotic fluid from these families were collected too. Genomic DNA was analyzed by MLPA, conventional PCR-RFLP, and allele-specific PCR. In complete agreement with the results of conventional PCR-RFLP and allele-specific PCR, MLPA analysis showed that all of the 13 patients had homozygous deletion of the survival of motor neuron 1 (SMN1) gene, and there was significant difference between the SMA severity (type I to type III) and SMN2 copy number (P < 0.05). Of the 31 parents 29 (93.5%) had 1 copy of SMN1, 2 (6.5%) had 2 copies of SMN1. Of the 50 healthy individuals, 1 (2.0%) had 1 copy of SMN1, 48 (96.0%) had 2 copies of SMN1, and 1 (2.0%) had 3 copies. The SMN1 copy number of the parents was significantly higher than that of the healthy individuals (P < 0.01). Two of the 10 fetuses had homozygous deletion of SMN1. The MLPA technique has proved to be an accurate and reliable tool for the molecular diagnosis of SMA, both in patients and in healthy carriers.

Assessment of HER2 status in breast cancer biopsies is not affected by accelerated tissue processing.

PubMed

Bulte, Joris P; Halilovic, Altuna; Kalkman, Shona; van Cleef, Patricia H J; van Diest, Paul J; Strobbe, Luc J A; de Wilt, Johannes H W; Bult, Peter

2018-03-01

To establish whether core needle biopsy (CNB) specimens processed with an accelerated processing method with short fixation time can be used to determine accurately the human epidermal growth factor receptor 2 (HER2) status of breast cancer. A consecutive case-series from two high-volume breast clinics was created. We compared routine HER2 immunohistochemistry (IHC) assessment between accelerated processing CNB specimens and routinely processed postoperative excision specimens. Additional amplification-based testing was performed in cases with equivocal results. The formalin fixation time was less than 2 h and between 6 and 72 h, respectively. Fluorescence in-situ hybridisation and multiplex ligation-dependent probe amplification were used for amplification testing. One hundred and forty-four cases were included, 15 of which were HER2-positive on the routinely processed excision specimens. On the CNB specimens, 44 were equivocal on IHC and required an amplification-based test. Correlation between the CNB specimens and the corresponding excision specimens was high for final HER2 status, with an accuracy of 97% and a kappa of 0.85. HER2 status can be determined reliably on CNB specimens with accelerated processing time using standard clinical testing methods. Using this accelerated technology the minimum 6 h of formalin fixation, which current guidelines consider necessary, can be decreased safely. This allows for a complete and expedited histology-based diagnosis of breast lesions in the setting of a one-stop-shop, same-day breast clinic. © 2018 The Authors. Histopathology Published by John Wiley & Sons Ltd.

Limited Utility of Polymerase Chain Reaction in Induced Sputum Specimens for Determining the Causes of Childhood Pneumonia in Resource-Poor Settings: Findings From the Pneumonia Etiology Research for Child Health (PERCH) Study

PubMed Central

Seidenberg, Phil; Park, Daniel E.; Mwananyanda, Lawrence; Fu, Wei; Shi, Qiyuan; Baggett, Henry C.; Brooks, W. Abdullah; Feikin, Daniel R.; Howie, Stephen R.C.; Knoll, Maria Deloria; Kotloff, Karen L.; Levine, Orin S.; Madhi, Shabir A.; O’Brien, Katherine L.; Scott, J. Anthony G.; Antonio, Martin; Awori, Juliet O.; Baillie, Vicky L.; DeLuca, Andrea N.; Driscoll, Amanda J.; Higdon, Melissa M.; Hossain, Lokman; Jahan, Yasmin; Karron, Ruth A.; Kazungu, Sidi; Li, Mengying; Moore, David P.; Morpeth, Susan C.; Ofordile, Ogochukwu; Prosperi, Christine; Sangwichian, Ornuma; Sawatwong, Pongpun; Sylla, Mamadou; Tapia, Milagritos D.; Zeger, Scott L.; Murdoch, David R.; Hammitt, Laura L.; O., K. L.; L., O. S.; K., M. D.; F., D. R.; D., A. N.; D., A. J.; Fancourt, Nicholas; F., W.; H., L. L.; H., M. M.; Wangeci Kagucia, E.; K., R. A.; L., M.; P., D. E.; P., C.; Wu, Zhenke; Z., S. L.; Watson, Nora L.; Crawley, Jane; M., D. R.; W. A., B.; Endtz, Hubert P.; Khalequ, Zaman; Goswami, Doli; H., L.; J., Y.; Ashraf, Hasan; H., S. R. C.; Ebruke, Bernard E.; A., M.; McLellan, Jessica; Machuka, Eunice; Shamsul, Arifin; Zaman, Syed M. A.; Mackenzie, Grant; S., J. A. G.; A., J. O.; M., S. C.; Kamau, Alice; K., S.; Ominde, Micah Silaba; K., K. L.; T., M. D.; Sow, Samba O.; S., M.; Tamboura, Boubou; Onwuchekwa, Uma; Kourouma, Nana; Toure, Aliou; M., S. A.; M., D. P.; Adrian, Peter V.; B., V. L.; Kuwanda, Locadiah; Mudau, Azwifarwi; Groome, Michelle J.; Mahomed, Nasreen; B., H. C.; Thamthitiwat, Somsak; Maloney, Susan A.; Bunthi, Charatdao; Rhodes, Julia; S., P.; Akarasewi, Pasakorn; T., D. M.; M., L.; Chipeta, James; S., P.; Mwansa, James; wa Somwe, Somwe; Kwenda, Geoffrey; Anderson, Trevor P.; Mitchell, Joanne

2017-01-01

Abstract Background. Sputum examination can be useful in diagnosing the cause of pneumonia in adults but is less well established in children. We sought to assess the diagnostic utility of polymerase chain reaction (PCR) for detection of respiratory viruses and bacteria in induced sputum (IS) specimens from children hospitalized with severe or very severe pneumonia. Methods. Among children aged 1–59 months, we compared organism detection by multiplex PCR in IS and nasopharyngeal/oropharyngeal (NP/OP) specimens. To assess whether organism presence or density in IS specimens was associated with chest radiographic evidence of pneumonia (radiographic pneumonia), we compared prevalence and density in IS specimens from children with radiographic pneumonia and children with suspected pneumonia but without chest radiographic changes or clinical or laboratory findings suggestive of pneumonia (nonpneumonia group). Results. Among 4232 cases with World Health Organization–defined severe or very severe pneumonia, we identified 1935 (45.7%) with radiographic pneumonia and 573 (13.5%) with nonpneumonia. The organism detection yield was marginally improved with IS specimens (96.2% vs 92.4% for NP/OP specimens for all viruses combined [P = .41]; 96.9% vs 93.3% for all bacteria combined [P = .01]). After accounting for presence in NP/OP specimens, no organism was detected more frequently in the IS specimens from the radiographic pneumonia compared with the nonpneumonia cases. Among high-quality IS specimens, there were no statistically significant differences in organism density, except with cytomegalovirus, for which there was a higher quantity in the IS specimens from cases with radiographic pneumonia compared with the nonpneumonia cases (median cycle threshold value, 27.9 vs 28.5, respectively; P = .01). Conclusions. Using advanced molecular methods with IS specimens provided little additional diagnostic information beyond that obtained with NP/OP swab specimens. PMID:28575363

Limited Utility of Polymerase Chain Reaction in Induced Sputum Specimens for Determining the Causes of Childhood Pneumonia in Resource-Poor Settings: Findings From the Pneumonia Etiology Research for Child Health (PERCH) Study.

PubMed

Thea, Donald M; Seidenberg, Phil; Park, Daniel E; Mwananyanda, Lawrence; Fu, Wei; Shi, Qiyuan; Baggett, Henry C; Brooks, W Abdullah; Feikin, Daniel R; Howie, Stephen R C; Knoll, Maria Deloria; Kotloff, Karen L; Levine, Orin S; Madhi, Shabir A; O'Brien, Katherine L; Scott, J Anthony G; Antonio, Martin; Awori, Juliet O; Baillie, Vicky L; DeLuca, Andrea N; Driscoll, Amanda J; Higdon, Melissa M; Hossain, Lokman; Jahan, Yasmin; Karron, Ruth A; Kazungu, Sidi; Li, Mengying; Moore, David P; Morpeth, Susan C; Ofordile, Ogochukwu; Prosperi, Christine; Sangwichian, Ornuma; Sawatwong, Pongpun; Sylla, Mamadou; Tapia, Milagritos D; Zeger, Scott L; Murdoch, David R; Hammitt, Laura L

2017-06-15

Sputum examination can be useful in diagnosing the cause of pneumonia in adults but is less well established in children. We sought to assess the diagnostic utility of polymerase chain reaction (PCR) for detection of respiratory viruses and bacteria in induced sputum (IS) specimens from children hospitalized with severe or very severe pneumonia. Among children aged 1-59 months, we compared organism detection by multiplex PCR in IS and nasopharyngeal/oropharyngeal (NP/OP) specimens. To assess whether organism presence or density in IS specimens was associated with chest radiographic evidence of pneumonia (radiographic pneumonia), we compared prevalence and density in IS specimens from children with radiographic pneumonia and children with suspected pneumonia but without chest radiographic changes or clinical or laboratory findings suggestive of pneumonia (nonpneumonia group). Among 4232 cases with World Health Organization-defined severe or very severe pneumonia, we identified 1935 (45.7%) with radiographic pneumonia and 573 (13.5%) with nonpneumonia. The organism detection yield was marginally improved with IS specimens (96.2% vs 92.4% for NP/OP specimens for all viruses combined [P = .41]; 96.9% vs 93.3% for all bacteria combined [P = .01]). After accounting for presence in NP/OP specimens, no organism was detected more frequently in the IS specimens from the radiographic pneumonia compared with the nonpneumonia cases. Among high-quality IS specimens, there were no statistically significant differences in organism density, except with cytomegalovirus, for which there was a higher quantity in the IS specimens from cases with radiographic pneumonia compared with the nonpneumonia cases (median cycle threshold value, 27.9 vs 28.5, respectively; P = .01). Using advanced molecular methods with IS specimens provided little additional diagnostic information beyond that obtained with NP/OP swab specimens. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

Nanowire array chips for molecular typing of rare trafficking leukocytes with application to neurodegenerative pathology

NASA Astrophysics Data System (ADS)

Kwak, Minsuk; Kim, Dong-Joo; Lee, Mi-Ri; Wu, Yu; Han, Lin; Lee, Sang-Kwon; Fan, Rong

2014-05-01

Despite the presence of the blood-brain barrier (BBB) that restricts the entry of immune cells and mediators into the central nervous system (CNS), a small number of peripheral leukocytes can traverse the BBB and infiltrate into the CNS. The cerebrospinal fluid (CSF) is one of the major routes through which trafficking leukocytes migrate into the CNS. Therefore, the number of leukocytes and their phenotypic compositions in the CSF may represent important sources to investigate immune-to-brain interactions or diagnose and monitor neurodegenerative diseases. Due to the paucity of trafficking leucocytes in the CSF, a technology capable of efficient isolation, enumeration, and molecular typing of these cells in the clinical settings has not been achieved. In this study, we report on a biofunctionalized silicon nanowire array chip for highly efficient capture and multiplexed phenotyping of rare trafficking leukocytes in small quantities (50 microliters) of clinical CSF specimens collected from neurodegenerative disease patients. The antibody coated 3D nanostructured materials exhibited vastly improved rare cell capture efficiency due to high-affinity binding and enhanced cell-substrate interactions. Moreover, our platform creates multiple cell capture interfaces, each of which can selectively isolate specific leukocyte phenotypes. A comparison with the traditional immunophenotyping using flow cytometry demonstrated that our novel silicon nanowire-based rare cell analysis platform can perform rapid detection and simultaneous molecular characterization of heterogeneous immune cells. Multiplexed molecular typing of rare leukocytes in CSF samples collected from Alzheimer's disease patients revealed the elevation of white blood cell counts and significant alterations in the distribution of major leukocyte phenotypes. Our technology represents a practical tool for potentially diagnosing and monitoring the pathogenesis of neurodegenerative diseases by allowing an effective hematological analysis of the CSF from patients.Despite the presence of the blood-brain barrier (BBB) that restricts the entry of immune cells and mediators into the central nervous system (CNS), a small number of peripheral leukocytes can traverse the BBB and infiltrate into the CNS. The cerebrospinal fluid (CSF) is one of the major routes through which trafficking leukocytes migrate into the CNS. Therefore, the number of leukocytes and their phenotypic compositions in the CSF may represent important sources to investigate immune-to-brain interactions or diagnose and monitor neurodegenerative diseases. Due to the paucity of trafficking leucocytes in the CSF, a technology capable of efficient isolation, enumeration, and molecular typing of these cells in the clinical settings has not been achieved. In this study, we report on a biofunctionalized silicon nanowire array chip for highly efficient capture and multiplexed phenotyping of rare trafficking leukocytes in small quantities (50 microliters) of clinical CSF specimens collected from neurodegenerative disease patients. The antibody coated 3D nanostructured materials exhibited vastly improved rare cell capture efficiency due to high-affinity binding and enhanced cell-substrate interactions. Moreover, our platform creates multiple cell capture interfaces, each of which can selectively isolate specific leukocyte phenotypes. A comparison with the traditional immunophenotyping using flow cytometry demonstrated that our novel silicon nanowire-based rare cell analysis platform can perform rapid detection and simultaneous molecular characterization of heterogeneous immune cells. Multiplexed molecular typing of rare leukocytes in CSF samples collected from Alzheimer's disease patients revealed the elevation of white blood cell counts and significant alterations in the distribution of major leukocyte phenotypes. Our technology represents a practical tool for potentially diagnosing and monitoring the pathogenesis of neurodegenerative diseases by allowing an effective hematological analysis of the CSF from patients. Electronic supplementary information (ESI) available: Additional data are available in the supplementary tables and supplementary figures. See DOI: 10.1039/c3nr06465d

An investigation of the diversity of strains of enteroaggregative Escherichia coli isolated from cases associated with a large multi-pathogen foodborne outbreak in the UK.

PubMed

Dallman, Timothy J; Chattaway, Marie A; Cowley, Lauren A; Doumith, Michel; Tewolde, Rediat; Wooldridge, David J; Underwood, Anthony; Ready, Derren; Wain, John; Foster, Kirsty; Grant, Kathie A; Jenkins, Claire

2014-01-01

Following a large outbreak of foodborne gastrointestinal (GI) disease, a multiplex PCR approach was used retrospectively to investigate faecal specimens from 88 of the 413 reported cases. Gene targets from a range of bacterial GI pathogens were detected, including Salmonella species, Shigella species and Shiga toxin-producing Escherichia coli, with the majority (75%) of faecal specimens being PCR positive for aggR associated with the Enteroaggregative E. coli (EAEC) group. The 20 isolates of EAEC recovered from the outbreak specimens exhibited a range of serotypes, the most frequent being O104:H4 and O131:H27. None of the EAEC isolates had the Shiga toxin (stx) genes. Multilocus sequence typing and single nucleotide polymorphism analysis of the core genome confirmed the diverse phylogeny of the strains. The analysis also revealed a close phylogenetic relationship between the EAEC O104:H4 strains in this outbreak and the strain of E. coli O104:H4 associated with a large outbreak of haemolytic ureamic syndrome in Germany in 2011. Further analysis of the EAEC plasmids, encoding the key enteroaggregative virulence genes, showed diversity with respect to FIB/FII type, gene content and genomic architecture. Known EAEC virulence genes, such as aggR, aat and aap, were present in all but one of the strains. A variety of fimbrial genes were observed, including genes encoding all five known fimbrial types, AAF/1 to AAF/V. The AAI operon was present in its entirety in 15 of the EAEC strains, absent in three and present, but incomplete, in two isolates. EAEC is known to be a diverse pathotype and this study demonstrates that a high level of diversity in strains recovered from cases associated with a single outbreak. Although the EAEC in this study did not carry the stx genes, this outbreak provides further evidence of the pathogenic potential of the EAEC O104:H4 serotype.

Single-Tube Multiplexed Molecular Detection of Endemic Porcine Viruses in Combination with Background Screening for Transboundary Diseases

PubMed Central

Wernike, Kerstin; Hoffmann, Bernd

2013-01-01

Detection of several pathogens with multiplexed real-time quantitative PCR (qPCR) assays in a one-step setup allows the simultaneous detection of two endemic porcine and four different selected transboundary viruses. Reverse transcription (RT)-qPCR systems for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2), two of the most economically important pathogens of swine worldwide, were combined with a screening system for diseases notifiable to the World Organization of Animal Health, namely, classical and African swine fever, foot-and-mouth disease, and Aujeszky's disease. Background screening was implemented using the identical fluorophore for all four different RT-qPCR assays. The novel multiplex RT-qPCR system was validated with a large panel of different body fluids and tissues from pigs and other animal species. Both reference samples and clinical specimens were used for a complete evaluation. It could be demonstrated that a highly sensitive and specific parallel detection of the different viruses was possible. The assays for the notifiable diseases were even not affected by the simultaneous amplification of very high loads of PRRSV- and PCV2-specific sequences. The novel broad-spectrum multiplex assay allows in a unique form the routine investigation for endemic porcine pathogens with exclusion diagnostics of the most important transboundary diseases in samples from pigs with unspecific clinical signs, such as fever or hemorrhages. The new system could significantly improve early detection of the most important notifiable diseases of swine and could lead to a new approach in syndromic surveillance. PMID:23303496

Multiplex RT-PCR and indirect immunofluorescence assays for detection and subtyping of human influenza virus in Tunisia.

PubMed

Ben M'hadheb, Manel; Harrabi, Myriam; Souii, Amira; Jrad-Battikh, Nadia; Gharbi, Jawhar

2015-03-01

Influenza viruses are negative stranded segmented RNA viruses belonging to Orthomyxoviridae family. They are classified into three types A, B, and C. Type A influenza viruses are classified into subtypes according to the antigenic characters of the surface glycoproteins: hemagglutinin (H) and neuraminidase (N). The aim of the present study is to develop a fast and reliable multiplex RT-PCR technique for detecting simultaneously the subtypes A/H1N1 and A/H3N2 of influenza virus. Our study included 398 patients (mean age 30.33 ± 19.92 years) with flu or flu-like syndromes, consulting physicians affiliated with collaborating teams. A multiplex RT-PCR detecting A/H1N1 and A/H3N2 influenza viruses and an examination by indirect immunofluorescence (IFI) were performed. In the optimized conditions, we diagnosed by IFI a viral infection in 90 patients (22.6 %): 85 cases of influenza type A, four cases of influenza type B, and only one case of coinfection with types A and B. An evaluation of the technique was performed on 19 clinical specimens positive in IFI, and we detected eight cases of A/H3N2, five cases of A/H1N1, one case of influenza virus type A which is not an H1N1 nor H3N2, and five negative cases. Multiplex RT-PCR is a sensitive technique allowing an effective and fast diagnosis of respiratory infections caused by influenza viruses in which the optimization often collides with problems of sensibility.

Detection of influenza virus types A and B and type A subtypes (H1, H3, and H5) by multiplex polymerase chain reaction.

PubMed

Boonsuk, Pitirat; Payungporn, Sunchai; Chieochansin, Thaweesak; Samransamruajkit, Rujipat; Amonsin, Alongkorn; Songserm, Thaweesak; Chaisingh, Arunee; Chamnanpood, Pornchai; Chutinimitkul, Salin; Theamboonlers, Apiradee; Poovorawan, Yong

2008-07-01

Infections with influenza virus type A and B present serious public health problems on a global scale. However, only influenza A virus has been reported to cause fatal pandemic in many species. To provide suitable clinical management and prevent further virus transmission, efficient and effective clinical diagnosis is essential. Therefore, we developed multiplex PCR assays for detecting influenza types A and B and the subtypes of influenza A virus (H1, H3 and H5). Upon performing multiplex PCR assays with type-specific primer sets, the clearly distinguishable products representing influenza A and B virus were separated by agarose gel electrophoresis. In addition, the subtypes of influenza A virus (H1, H3 and H5), which are most common in humans, can be readily distinguished by PCR with subtype-specific primer sets, yielding PCR products of different sizes depending on which subtype has been amplified. This method was tested on 46 influenza virus positive specimens of avian and mammalian (dog and human) origins collected between 2006 and 2008. The sensitivity of this method, tested against known concentrations of each type and subtype specific plasmid, was established to detect 10(3) copies/microl. The method's specificity was determined by testing against other subtypes of influenza A virus (H2, H4 and H6-H15) and respiratory pathogens commonly found in humans. None of them could be amplified, thus excluding cross reactivity. In conclusion, the multiplex PCR assays developed are advantageous as to rapidity, specificity, and cost effectiveness.

Rapid Multiplex PCR Assay To Identify Respiratory Viral Pathogens: Moving Forward Diagnosing The Common Cold

PubMed Central

Gordon, Sarah M; Elegino-Steffens, Diane U; Agee, Willie; Barnhill, Jason; Hsue, Gunther

2013-01-01

Upper respiratory tract infections (URIs) can be a serious burden to the healthcare system. The majority of URIs are viral in etiology, but definitive diagnosis can prove difficult due to frequently overlapping clinical presentations of viral and bacterial infections, and the variable sensitivity, and lengthy turn-around time of viral culture. We tested new automated nested multiplex PCR technology, the FilmArray® system, in the TAMC department of clinical investigations, to determine the feasibility of replacing the standard viral culture with a rapid turn-around system. We conducted a feasibility study using a single-blinded comparison study, comparing PCR results with archived viral culture results from a convenience sample of cryopreserved archived nasopharyngeal swabs from acutely ill ED patients who presented with complaints of URI symptoms. A total of 61 archived samples were processed. Viral culture had previously identified 31 positive specimens from these samples. The automated nested multiplex PCR detected 38 positive samples. In total, PCR was 94.5% concordant with the previously positive viral culture results. However, PCR was only 63.4% concordant with the negative viral culture results, owing to PCR detection of 11 additional viral pathogens not recovered on viral culture. The average time to process a sample was 75 minutes. We determined that an automated nested multiplex PCR is a feasible alternative to viral culture in an acute clinical setting. We were able to detect at least 94.5% as many viral pathogens as viral culture is able to identify, with a faster turn-around time. PMID:24052914

Multiplex Quantitative Histologic Analysis of Human Breast Cancer Cell Signaling and Cell Fate

DTIC Science & Technology

2010-05-01

Breast cancer, cell signaling, cell proliferation, histology, image analysis 15. NUMBER OF PAGES - 51 16. PRICE CODE 17. SECURITY CLASSIFICATION...revealed by individual stains in multiplex combinations; and (3) software (FARSIGHT) for automated multispectral image analysis that (i) segments...Task 3. Develop computational algorithms for multispectral immunohistological image analysis FARSIGHT software was developed to quantify intrinsic

Microfluidic platform for multiplexed detection in single cells and methods thereof

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Meiye; Singh, Anup K.

The present invention relates to a microfluidic device and platform configured to conduct multiplexed analysis within the device. In particular, the device allows multiple targets to be detected on a single-cell level. Also provided are methods of performing multiplexed analyses to detect one or more target nucleic acids, proteins, and post-translational modifications.

Multiplexing 200 spatial modes with a single hologram

NASA Astrophysics Data System (ADS)

Rosales-Guzmán, Carmelo; Bhebhe, Nkosiphile; Mahonisi, Nyiku; Forbes, Andrew

2017-11-01

The on-demand tailoring of light's spatial shape is of great relevance in a wide variety of research areas. Computer-controlled devices, such as spatial light modulators (SLMs) or digital micromirror devices, offer a very accurate, flexible and fast holographic means to this end. Remarkably, digital holography affords the simultaneous generation of multiple beams (multiplexing), a tool with numerous applications in many fields. Here, we provide a self-contained tutorial on light beam multiplexing. Through the use of several examples, the readers will be guided step by step in the process of light beam shaping and multiplexing. Additionally, we provide a quantitative analysis on the multiplexing capabilities of SLMs to assess the maximum number of beams that can be multiplexed on a single SLM, showing approximately 200 modes on a single hologram.

Analysis on the propagation characteristics of two multiplexed groups of coaxial OAM beams in atmospheric turbulence

NASA Astrophysics Data System (ADS)

Zheng, Yongping; Tian, Qinghua; Zhang, Wei; Zhang, Qi; Zhu, Lei; Wang, Yongjun; Liu, Bo; Xin, Xiangjun

2018-01-01

Orbital angular momentum (OAM) as a new degree of freedom, greatly improves the spectrum efficiency and channel capacity of optical communication system. It has become the research focus in the field of optical communications. Some scholars have demonstrated that the feasibility of two multiplexed groups of concentric rings of Laguerre-Gaussian (LG) beams with OAM multiplexing transmission in free space. Based on the point, this paper makes the further research on the propagation characteristics of LG beams with this spatial multiplexing structure in atmospheric turbulence. The random phase screen is established by using the modified von Karman power spectrum and the received power and crosstalk power of OAM modes of LG beams are obtained under the Rytov approximation. We investigate the characteristic parameters of LG beams with this spatial multiplexing structure for mitigating turbulence. Simulation results show that the system exists an optimum beam waist related to wavelength in which the received power of OAM modes reaches the maximum. Meanwhile, the BER and aggregate capacity of the system with two multiplexed groups of concentric rings of LG beams with OAM multiplexing are simulated and analyzed under different intensities of atmospheric turbulence. The results reveal that the system with larger mode spacing generally has lower inter-modal crosstalk and larger aggregate capacity than that with the smaller mode spacing. Finally, on the basis of above the analysis and research, some suggestions for efficient OAM multiplexing detection scheme are proposed.

In Vivo Persistence of Human Rhinoviruses in Immunosuppressed Patients

PubMed Central

Engelmann, Ilka; Dewilde, Anny; Lazrek, Mouna; Batteux, Mathilde; Hamissi, Aminati; Yakoub-Agha, Ibrahim; Hober, Didier

2017-01-01

Several species of the genus Enterovirus cause persistent infections in humans. Human rhinovirus (HRV) infections are generally self-limiting but occasionally persistent infections have been described. This study aimed to identify persistent HRV infections and investigate the clinical and virologic characteristics of patients with persistent infections. From January 2012 to March 2015, 3714 respiratory specimens from 2608 patients were tested for respiratory viruses by using a multiplex reverse transcription–polymerase chain reaction. A retrospective study was performed. Patients with at least two specimens positive for HRV/enterovirus taken 45 days or longer apart were identified and the HRV/enteroviruses were typed. Patients with persistent infection were compared to patients with reinfection and patients with cleared infection. Phylogenetic analysis of the viral protein(VP)4/VP2 region was performed. 18 patients with persistent HRV/enterovirus infection were identified. Minimum median duration of persistence was 92 days (range 50–455 days). All but one patients with persistence were immunosuppressed. Immunosuppression and hematologic disorders were more frequent in patients with persistence (n = 18) than in patients with reinfection (n = 33) and with cleared infection (n = 25) (p = 0.003 and p = 0.001, respectively). In conclusion, this retrospective study identified HRV persistence in vivo which occurred mainly in immunosuppressed patients. PMID:28151988

Multiplexed operation of a micromachined ultrasonic droplet ejector array.

PubMed

Forbes, Thomas P; Degertekin, F Levent; Fedorov, Andrei G

2007-10-01

A dual-sample ultrasonic droplet ejector array is developed for use as a soft-ionization ion source for multiplexed mass spectrometry (MS). Such a multiplexed ion source aims to reduce MS analysis time for multiple analyte streams, as well as allow for the synchronized ejection of the sample(s) and an internal standard for quantitative results and mass calibration. Multiplexing is achieved at the device level by division of the fluid reservoir and separating the active electrodes of the piezoelectric transducer for isolated application of ultrasonic wave energy to each domain. The transducer is mechanically shaped to further reduce the acoustical crosstalk between the domains. Device design is performed using finite-element analysis simulations and supported by experimental characterization. Isolated ejection of approximately 5 microm diameter water droplets from individual domains in the micromachined droplet ejector array at around 1 MHz frequency is demonstrated by experiments. The proof-of-concept demonstration using a dual-sample device also shows potential for multiplexing with larger numbers of analytes.

Multiplexed operation of a micromachined ultrasonic droplet ejector array

DOE Office of Scientific and Technical Information (OSTI.GOV)

Forbes, Thomas P.; Degertekin, F. Levent; Fedorov, Andrei G.

2007-10-15

A dual-sample ultrasonic droplet ejector array is developed for use as a soft-ionization ion source for multiplexed mass spectrometry (MS). Such a multiplexed ion source aims to reduce MS analysis time for multiple analyte streams, as well as allow for the synchronized ejection of the sample(s) and an internal standard for quantitative results and mass calibration. Multiplexing is achieved at the device level by division of the fluid reservoir and separating the active electrodes of the piezoelectric transducer for isolated application of ultrasonic wave energy to each domain. The transducer is mechanically shaped to further reduce the acoustical crosstalk betweenmore » the domains. Device design is performed using finite-element analysis simulations and supported by experimental characterization. Isolated ejection of {approx}5 {mu}m diameter water droplets from individual domains in the micromachined droplet ejector array at around 1 MHz frequency is demonstrated by experiments. The proof-of-concept demonstration using a dual-sample device also shows potential for multiplexing with larger numbers of analytes.« less

Interlaboratory study of DNA extraction from multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for individual kernel detection system of genetically modified maize.

PubMed

Akiyama, Hiroshi; Sakata, Kozue; Makiyma, Daiki; Nakamura, Kosuke; Teshima, Reiko; Nakashima, Akie; Ogawa, Asako; Yamagishi, Toru; Futo, Satoshi; Oguchi, Taichi; Mano, Junichi; Kitta, Kazumi

2011-01-01

In many countries, the labeling of grains, feed, and foodstuff is mandatory if the genetically modified (GM) organism content exceeds a certain level of approved GM varieties. We previously developed an individual kernel detection system consisting of grinding individual kernels, DNA extraction from the individually ground kernels, GM detection using multiplex real-time PCR, and GM event detection using multiplex qualitative PCR to analyze the precise commingling level and varieties of GM maize in real sample grains. We performed the interlaboratory study of the DNA extraction with multiple ground samples, multiplex real-time PCR detection, and multiplex qualitative PCR detection to evaluate its applicability, practicality, and ruggedness for the individual kernel detection system of GM maize. DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR were evaluated by five laboratories in Japan, and all results from these laboratories were consistent with the expected results in terms of the commingling level and event analysis. Thus, the DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for the individual kernel detection system is applicable and practicable in a laboratory to regulate the commingling level of GM maize grain for GM samples, including stacked GM maize.

A Quantitative Analysis of Ureaplasma urealyticum and Ureaplasma parvum Compared with Host Immune Response in Preterm Neonates at Risk of Developing Bronchopulmonary Dysplasia

PubMed Central

Payne, Matthew S.; Goss, Kevin C. W.; Connett, Gary J.; Legg, Julian P.; Bruce, Ken D.

2012-01-01

Multiplex, real-time PCR for the identification of Ureaplasma urealyticum and Ureaplasma parvum was performed on nucleic acids extracted from sequential endotracheal aspirates obtained from preterm neonates born at <29 weeks of gestation and ventilated for more than 48 h admitted to two level 3 neonatal intensive care units. Specimens were obtained shortly after birth and sequentially up until extubation. One hundred fifty-two specimens (93.8%) contained material suitable for analysis. Ureaplasma spp. were identified in 5 of 13 neonates studied. In most cases, the DNA load of the detected Ureaplasma species was low and decreased over time. In addition, changes in detectable Ureaplasma species DNA did not relate to changes in the inflammatory marker C-reactive protein (CRP) or respiratory status. All but two blood samples obtained at times of suspected sepsis were culture positive for other microorganisms; the species cultured were typically coagulase-negative staphylococci and were associated with increased levels of CRP (>10 mg/liter). This study was limited by the small number of patients examined and does not have the power to support or contradict the hypothesis that postnatal lung infection with Ureaplasma parvum is causally related to bronchopulmonary dysplasia (BPD) or adverse respiratory outcomes after preterm birth. However, in this study, increases in CRP levels were not associated with patients in whom Ureaplasma parvum was detected, in contrast to the detection of other bacterial species. PMID:22189123

A quantitative analysis of Ureaplasma urealyticum and Ureaplasma parvum compared with host immune response in preterm neonates at risk of developing bronchopulmonary dysplasia.

PubMed

Payne, Matthew S; Goss, Kevin C W; Connett, Gary J; Legg, Julian P; Bruce, Ken D; Chalker, Vicki

2012-03-01

Multiplex, real-time PCR for the identification of Ureaplasma urealyticum and Ureaplasma parvum was performed on nucleic acids extracted from sequential endotracheal aspirates obtained from preterm neonates born at <29 weeks of gestation and ventilated for more than 48 h admitted to two level 3 neonatal intensive care units. Specimens were obtained shortly after birth and sequentially up until extubation. One hundred fifty-two specimens (93.8%) contained material suitable for analysis. Ureaplasma spp. were identified in 5 of 13 neonates studied. In most cases, the DNA load of the detected Ureaplasma species was low and decreased over time. In addition, changes in detectable Ureaplasma species DNA did not relate to changes in the inflammatory marker C-reactive protein (CRP) or respiratory status. All but two blood samples obtained at times of suspected sepsis were culture positive for other microorganisms; the species cultured were typically coagulase-negative staphylococci and were associated with increased levels of CRP (>10 mg/liter). This study was limited by the small number of patients examined and does not have the power to support or contradict the hypothesis that postnatal lung infection with Ureaplasma parvum is causally related to bronchopulmonary dysplasia (BPD) or adverse respiratory outcomes after preterm birth. However, in this study, increases in CRP levels were not associated with patients in whom Ureaplasma parvum was detected, in contrast to the detection of other bacterial species.

Pair-barcode high-throughput sequencing for large-scale multiplexed sample analysis

PubMed Central

2012-01-01

Background The multiplexing becomes the major limitation of the next-generation sequencing (NGS) in application to low complexity samples. Physical space segregation allows limited multiplexing, while the existing barcode approach only permits simultaneously analysis of up to several dozen samples. Results Here we introduce pair-barcode sequencing (PBS), an economic and flexible barcoding technique that permits parallel analysis of large-scale multiplexed samples. In two pilot runs using SOLiD sequencer (Applied Biosystems Inc.), 32 independent pair-barcoded miRNA libraries were simultaneously discovered by the combination of 4 unique forward barcodes and 8 unique reverse barcodes. Over 174,000,000 reads were generated and about 64% of them are assigned to both of the barcodes. After mapping all reads to pre-miRNAs in miRBase, different miRNA expression patterns are captured from the two clinical groups. The strong correlation using different barcode pairs and the high consistency of miRNA expression in two independent runs demonstrates that PBS approach is valid. Conclusions By employing PBS approach in NGS, large-scale multiplexed pooled samples could be practically analyzed in parallel so that high-throughput sequencing economically meets the requirements of samples which are low sequencing throughput demand. PMID:22276739

Pair-barcode high-throughput sequencing for large-scale multiplexed sample analysis.

PubMed

Tu, Jing; Ge, Qinyu; Wang, Shengqin; Wang, Lei; Sun, Beili; Yang, Qi; Bai, Yunfei; Lu, Zuhong

2012-01-25

The multiplexing becomes the major limitation of the next-generation sequencing (NGS) in application to low complexity samples. Physical space segregation allows limited multiplexing, while the existing barcode approach only permits simultaneously analysis of up to several dozen samples. Here we introduce pair-barcode sequencing (PBS), an economic and flexible barcoding technique that permits parallel analysis of large-scale multiplexed samples. In two pilot runs using SOLiD sequencer (Applied Biosystems Inc.), 32 independent pair-barcoded miRNA libraries were simultaneously discovered by the combination of 4 unique forward barcodes and 8 unique reverse barcodes. Over 174,000,000 reads were generated and about 64% of them are assigned to both of the barcodes. After mapping all reads to pre-miRNAs in miRBase, different miRNA expression patterns are captured from the two clinical groups. The strong correlation using different barcode pairs and the high consistency of miRNA expression in two independent runs demonstrates that PBS approach is valid. By employing PBS approach in NGS, large-scale multiplexed pooled samples could be practically analyzed in parallel so that high-throughput sequencing economically meets the requirements of samples which are low sequencing throughput demand.

Development of a low-flow multiplexed interface for capillary electrophoresis/electrospray ion trap mass spectrometry using sequential spray.

PubMed

Chen, Chao-Jung; Li, Fu-An; Her, Guor-Rong

2008-05-01

A multiplexed CE-MS interface using four low-flow sheath liquid ESI sprayers has been developed. Because of the limited space between the low-flow sprayers and the entrance aperture of the ESI source, multichannel analysis is difficult using conventional rotating plate approaches. Instead, a multiplexed low-flow system was achieved by applying an ESI potential sequentially to the four low-flow sprayers, resulting in only one sprayer being sprayed at any given time. The synchronization of the scan event and the voltage relays was accomplished by using the data acquisition signal from the IT mass spectrometer. This synchronization resulted in the ESI voltage being sequentially applied to each of the four sprayers according to the corresponding scan event. With this design, a four-fold increase in analytical throughput was achieved. Because of the use of low-flow interfaces, this multiplexed system has superior sensitivity than a rotating plate design using conventional sheath liquid interfaces. The multiplexed design presented has the potential to be applied to other low-flow multiplexed systems, such as multiplexed capillary LC and multiplexed CEC.

Two-dimensional simulation of holographic data storage medium for multiplexed recording.

PubMed

Toishi, Mitsuru; Takeda, Takahiro; Tanaka, Kenji; Tanaka, Tomiji; Fukumoto, Atsushi; Watanabe, Kenjiro

2008-02-18

In this paper, we propose a new analysis model for photopolymer recording processes that calculate the two-dimensional refractive index distribution of multiplexed holograms. For the simulation of the photopolymer medium, time evolution of monomer diffusion and polymerization need to be calculated simultaneously. The distribution of the refractive index inside the medium is induced by these processes. By evaluating the refractive index pattern on each layer, the diffraction beams from the multiplexed hologram can be read out by beam propagation method (BPM). This is the first paper to determine the diffraction beam from a multiplexed hologram in a simulated photopolymer medium process. We analyze the time response of the multiplexed hologram recording processes in the photopolymer, and estimate the degradation of diffraction efficiency with multiplexed recording. This work can greatly contribute to understanding the process of hologram recording.

Optimal percolation on multiplex networks.

PubMed

Osat, Saeed; Faqeeh, Ali; Radicchi, Filippo

2017-11-16

Optimal percolation is the problem of finding the minimal set of nodes whose removal from a network fragments the system into non-extensive disconnected clusters. The solution to this problem is important for strategies of immunization in disease spreading, and influence maximization in opinion dynamics. Optimal percolation has received considerable attention in the context of isolated networks. However, its generalization to multiplex networks has not yet been considered. Here we show that approximating the solution of the optimal percolation problem on a multiplex network with solutions valid for single-layer networks extracted from the multiplex may have serious consequences in the characterization of the true robustness of the system. We reach this conclusion by extending many of the methods for finding approximate solutions of the optimal percolation problem from single-layer to multiplex networks, and performing a systematic analysis on synthetic and real-world multiplex networks.

Targeted Multiplex Imaging Mass Spectrometry in Transmission Geometry for Subcellular Spatial Resolution

PubMed Central

Lavenant, Gwendoline Thiery; Zavalin, Andrey I.; Caprioli, Richard M.

2013-01-01

Targeted multiplex Imaging Mass Spectrometry utilizes several different antigen-specific primary antibodies, each directly labeled with a unique photocleavable mass tag, to detect multiple antigens in a single tissue section. Each photocleavable mass tag bound to an antibody has a unique molecular weight and can be readily ionized by laser desorption ionization mass spectrometry. This manuscript describes a mass spectrometry method that allows imaging of targeted single cells within tissue using transmission geometry laser desorption ionization mass spectrometry. Transmission geometry focuses the laser beam on the back side of the tissue placed on a glass slide, providing a 2 μm diameter laser spot irradiating the biological specimen. This matrix-free method enables simultaneous localization at the sub-cellular level of multiple antigens using specific tagged antibodies. We have used this technology to visualize the co-expression of synaptophysin and two major hormones peptides, insulin and somatostatin, in duplex assays in beta and delta cells contained in a human pancreatic islet. PMID:23397138

Comparison of Two Multiplex Methods for Detection of Respiratory Viruses: FilmArray RP and xTAG RVP ▿†

PubMed Central

Rand, Kenneth H.; Rampersaud, Howard; Houck, Herbert J.

2011-01-01

We compared the FilmArray RP (Idaho Technology, Inc., Salt Lake City, UT) and the xTAG RVP (Luminex Corporation, Toronto, Canada) multiplex respiratory virus PCR methods for the detection of respiratory viruses in a set of 200 patient specimens frozen at −70°C after standard viral culture and antigen detection methods were done. Both systems detected between 40 to 50% more viruses than traditional methods, primarily rhinoviruses and human metapneumovirus. The FilmArray RP detected significantly more total viruses either alone or as part of mixed infections than the xTAG RVP, as well as an additional 21.6% more respiratory syncytial viruses. The xTAG RVP requires 5 to 6 h with 2.5 to 3 h of hands-on time, while the FilmArray RP takes about an hour with 3 to 5 min of hands-on time, making it much easier to perform. PMID:21508156

Targeted Multiplex Imaging Mass Spectrometry in Transmission Geometry for Subcellular Spatial Resolution

NASA Astrophysics Data System (ADS)

Thiery-Lavenant, Gwendoline; Zavalin, Andre I.; Caprioli, Richard M.

2013-04-01

Targeted multiplex imaging mass spectrometry utilizes several different antigen-specific primary antibodies, each directly labeled with a unique photocleavable mass tag, to detect multiple antigens in a single tissue section. Each photocleavable mass tag bound to an antibody has a unique molecular weight and can be readily ionized by laser desorption ionization mass spectrometry. This article describes a mass spectrometry method that allows imaging of targeted single cells within tissue using transmission geometry laser desorption ionization mass spectrometry. Transmission geometry focuses the laser beam on the back side of the tissue placed on a glass slide, providing a 2 μm diameter laser spot irradiating the biological specimen. This matrix-free method enables simultaneous localization at the sub-cellular level of multiple antigens using specific tagged antibodies. We have used this technology to visualize the co-expression of synaptophysin and two major hormones peptides, insulin and somatostatin, in duplex assays in beta and delta cells contained in a human pancreatic islet.

Reverse transcription multiplex PCR for differentiation between polio- and enteroviruses from clinical and environmental samples.

PubMed

Egger, D; Pasamontes, L; Ostermayer, M; Bienz, K

1995-06-01

For the rapid detection of polioviruses and their differentiation from nonpoliovirus enteroviruses, we developed a protocol in which clinical or environmental specimens are first inoculated onto cell cultures in tubes. After overnight incubation, the cultures are subjected to reverse transcription multiplex PCR with a primer pair which detects all enteroviruses (T. Hyypiä, P. Auvinen, and M. Maaronen, J. Gen. Virol. 70:3261-3268 1989) and two newly designed primer pairs specific for all 36 poliovirus strains tested. The PCR products can unequivocally be identified by their lengths in agarose gels, whereas the genetic heterogeneity of the poliovirus strains precludes identification by back-hybridization with internal probes. The proposed protocol is highly insensitive to the inhibitory effects of substances in the sample (stool, sewage). It allows for the detection of polioviruses and for polioviruses to be distinguished from nonpoliovirus enteroviruses within 24 h, and it allows for the concomitant isolation of a viable strain suitable for further typing.

Deletion detection for diagnosis of Duchenne muscular dystrophy in the Japanese population--comparison between the polymerase chain reaction and the Southern blot analysis.

PubMed

Katayama, S; Takeshita, N; Yano, T; Ubagai, T; Qiu, X J; Katagiri, Y; Kubo, H; Hirakawa, S

1993-06-01

We compared the efficacy of the multiplex PCR with that of the cDNA analysis for detection of deletions of the DMD gene in the Japanese patients. Thirty males with DMD from 27 Japanese families were studied by the multiplex PCR, and 24 of them were also investigated by Southern blot analysis. We used five dystrophin cDNA probes for deletion analysis. A total of 19 regions were amplified by the PCR to detect deletions, 9 regions by the method of Chamberlain et al. and another 10 regions by the method of Beggs et al. Deletions were detected in 14 (52%) out of 27 DMD families by the PCR. Southern blot analysis detected deletions in 14 (64%) out of 22 families. Thirteen (93%) of the 14 DMD families with deletions detected by Southern blotting were also confirmed by the multiplex PCR. Provided care is taken in cases where the deletion is limited to a single exon, the multiplex PCR appears to be an efficient and useful alternative to conventional Southern blot analysis for detecting deletions during the prenatal and postnatal diagnosis of DMD.

Development and Validation of a Laboratory-Developed Multiplex Real-Time PCR Assay on the BD Max System for Detection of Herpes Simplex Virus and Varicella-Zoster Virus DNA in Various Clinical Specimens.

PubMed

Pillet, Sylvie; Verhoeven, Paul O; Epercieux, Amélie; Bourlet, Thomas; Pozzetto, Bruno

2015-06-01

A multiplex real-time PCR (quantitative PCR [qPCR]) assay detecting herpes simplex virus (HSV) and varicella-zoster virus (VZV) DNA together with an internal control was developed on the BD Max platform combining automated DNA extraction and an open amplification procedure. Its performance was compared to those of PCR assays routinely used in the laboratory, namely, a laboratory-developed test for HSV DNA on the LightCycler instrument and a test using a commercial master mix for VZV DNA on the ABI7500fast system. Using a pool of negative cerebrospinal fluid (CSF) samples spiked with either calibrated controls for HSV-1 and VZV or dilutions of a clinical strain that was previously quantified for HSV-2, the empirical limit of detection of the BD Max assay was 195.65, 91.80, and 414.07 copies/ml for HSV-1, HSV-2, and VZV, respectively. All the samples from HSV and VZV DNA quality control panels (Quality Control for Molecular Diagnostics [QCMD], 2013, Glasgow, United Kingdom) were correctly identified by the BD Max assay. From 180 clinical specimens of various origins, 2 CSF samples were found invalid by the BD Max assay due to the absence of detection of the internal control; a concordance of 100% was observed between the BD Max assay and the corresponding routine tests. The BD Max assay detected the PCR signal 3 to 4 cycles earlier than did the routine methods. With results available within 2 h on a wide range of specimens, this sensitive and fully automated PCR assay exhibited the qualities required for detecting simultaneously HSV and VZV DNA on a routine basis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Development of analytical methods for multiplex bio-assay with inductively coupled plasma mass spectrometry.

PubMed

Ornatsky, Olga I; Kinach, Robert; Bandura, Dmitry R; Lou, Xudong; Tanner, Scott D; Baranov, Vladimir I; Nitz, Mark; Winnik, Mitchell A

2008-01-01

Advances in the development of highly multiplexed bio-analytical assays with inductively coupled plasma mass spectrometry (ICP-MS) detection are discussed. Use of novel reagents specifically designed for immunological methods utilizing elemental analysis is presented. The major steps of method development, including selection of elements for tags, validation of tagged reagents, and examples of multiplexed assays, are considered in detail. The paper further describes experimental protocols for elemental tagging of antibodies, immunostaining of live and fixed human leukemia cells, and preparation of samples for ICP-MS analysis. Quantitative analysis of surface antigens on model cell lines using a cocktail of seven lanthanide labeled antibodies demonstrated high specificity and concordance with conventional immunophenotyping.

Testing implications of varying targets for Bordetella pertussis: comparison of the FilmArray Respiratory Panel and the Focus B. pertussis PCR assay

PubMed Central

Jerris, Robert C; Williams, Sally R; MacDonald, Heather J; Ingebrigtsen, Danielle R; Westblade, Lars F; Rogers, Beverly B

2015-01-01

Background The FilmArray Respiratory Panel (RP) detects multiple pathogens, including Bordetella pertussis. The multiplex PCR system is appropriate for a core laboratory or point of care due to ease of use. The purpose of this study is to compare the analytical sensitivity of the FilmArray RP, which targets the promoter region of the B. pertussis toxin gene, with the Focus real-time PCR assay, which targets the insertion sequence IS481. Methods Seventy-one specimens from patients aged 1 month to 18 years, which had tested positive for B. pertussis using the Focus assay, were analysed using the FilmArray RP. Results Forty-six specimens were positive for B. pertussis by both the Focus and the FilmArray RP assays. Twenty-five specimens were negative for B. pertussis using the FilmArray RP assay, but positive using the Focus assay. Conclusions The FilmArray RP assays will detect approximately 1/3 less cases of B. pertussis than the Focus assay. PMID:25742911

Development of a Time Domain Fluorimeter for Fluorescent Lifetime Multiplexing Analysis

PubMed Central

Weissleder, Ralph; Mahmood, Umar

2009-01-01

We show that a portable, inexpensive USB-powered time domain fluorimeter (TDF) and analysis scheme were developed for use in evaluating a new class of fluorescent lifetime multiplexed dyes. Fluorescent proteins, organic dyes, and quantum dots allow the labeling of more and more individual features within biological systems, but the wide absorption and emission spectra of these fluorophores limit the number of distinct processes which may be simultaneously imaged using spectral separation alone. By additionally separating reporters in a second dimension, fluorescent lifetime multiplexing provides a means to multiply the number of available imaging channels. PMID:19830273

Reliable Gene Expression Measurements from Fine Needle Aspirates of Pancreatic Tumors

PubMed Central

Anderson, Michelle A.; Brenner, Dean E.; Scheiman, James M.; Simeone, Diane M.; Singh, Nalina; Sikora, Matthew J.; Zhao, Lili; Mertens, Amy N.; Rae, James M.

2010-01-01

Background and aims: Biomarker use for pancreatic cancer diagnosis has been impaired by a lack of samples suitable for reliable quantitative RT-PCR (qRT-PCR). Fine needle aspirates (FNAs) from pancreatic masses were studied to define potential causes of RNA degradation and develop methods for accurately measuring gene expression. Methods: Samples from 32 patients were studied. RNA degradation was assessed by using a multiplex PCR assay for varying lengths of glyceraldehyde-3-phosphate dehydrogenase, and effects on qRT-PCR were determined by using a 150-bp and a 80-bp amplicon for RPS6. Potential causes of and methods to circumvent RNA degradation were studied by using FNAs from a pancreatic cancer xenograft. Results: RNA extracted from pancreatic mass FNAs was extensively degraded. Fragmentation was related to needle bore diameter and could not be overcome by alterations in aspiration technique. Multiplex PCR for glyceraldehyde-3-phosphate dehydrogenase could distinguish samples that were suitable for qRT-PCR. The use of short PCR amplicons (<100 bp) provided reliable gene expression analysis from FNAs. When appropriate samples were used, the assay was highly reproducible for gene copy number with minimal (0.0003 or about 0.7% of total) variance. Conclusions: The degraded properties of endoscopic FNAs markedly affect the accuracy of gene expression measurements. Our novel approach to designate specimens “informative” for qRT-PCR allowed accurate molecular assessment for the diagnosis of pancreatic diseases. PMID:20709792

Comparison of three multiplex cytokine analysis systems: Luminex, SearchLight and FAST Quant.

PubMed

Lash, Gendie E; Scaife, Paula J; Innes, Barbara A; Otun, Harry A; Robson, Steven C; Searle, Roger F; Bulmer, Judith N

2006-02-20

Multiplex cytokine analysis technologies have become readily available in the last five years. Two main formats exist: multiplex sandwich ELISA and bead based assays. While these have each been compared to individual ELISAs, there has been no direct comparison between the two formats. We report here the comparison of two multiplex sandwich ELISA procedures (FAST Quant and SearchLight) and a bead based assay (UpState Luminex). All three kits differed from each other for different analytes and there was no clear pattern of one system giving systematically different results than another for any analyte studied. We suggest that each system has merits and several factors including range of analytes available, prospect of development of new analytes, dynamic range of the assay, sensitivity of the assay, cost of equipment, cost of consumables, ease of use and ease of data analysis need to be considered when choosing a system for use. We also suggest that results obtained from different systems cannot be combined.

Multiplex network analysis of employee performance and employee social relationships

NASA Astrophysics Data System (ADS)

Cai, Meng; Wang, Wei; Cui, Ying; Stanley, H. Eugene

2018-01-01

In human resource management, employee performance is strongly affected by both formal and informal employee networks. Most previous research on employee performance has focused on monolayer networks that can represent only single categories of employee social relationships. We study employee performance by taking into account the entire multiplex structure of underlying employee social networks. We collect three datasets consisting of five different employee relationship categories in three firms, and predict employee performance using degree centrality and eigenvector centrality in a superimposed multiplex network (SMN) and an unfolded multiplex network (UMN). We use a quadratic assignment procedure (QAP) analysis and a regression analysis to demonstrate that the different categories of relationship are mutually embedded and that the strength of their impact on employee performance differs. We also use weighted/unweighted SMN/UMN to measure the predictive accuracy of this approach and find that employees with high centrality in a weighted UMN are more likely to perform well. Our results shed new light on how social structures affect employee performance.

Genomic treasure troves: complete genome sequencing of herbarium and insect museum specimens.

PubMed

Staats, Martijn; Erkens, Roy H J; van de Vossenberg, Bart; Wieringa, Jan J; Kraaijeveld, Ken; Stielow, Benjamin; Geml, József; Richardson, James E; Bakker, Freek T

2013-01-01

Unlocking the vast genomic diversity stored in natural history collections would create unprecedented opportunities for genome-scale evolutionary, phylogenetic, domestication and population genomic studies. Many researchers have been discouraged from using historical specimens in molecular studies because of both generally limited success of DNA extraction and the challenges associated with PCR-amplifying highly degraded DNA. In today's next-generation sequencing (NGS) world, opportunities and prospects for historical DNA have changed dramatically, as most NGS methods are actually designed for taking short fragmented DNA molecules as templates. Here we show that using a standard multiplex and paired-end Illumina sequencing approach, genome-scale sequence data can be generated reliably from dry-preserved plant, fungal and insect specimens collected up to 115 years ago, and with minimal destructive sampling. Using a reference-based assembly approach, we were able to produce the entire nuclear genome of a 43-year-old Arabidopsis thaliana (Brassicaceae) herbarium specimen with high and uniform sequence coverage. Nuclear genome sequences of three fungal specimens of 22-82 years of age (Agaricus bisporus, Laccaria bicolor, Pleurotus ostreatus) were generated with 81.4-97.9% exome coverage. Complete organellar genome sequences were assembled for all specimens. Using de novo assembly we retrieved between 16.2-71.0% of coding sequence regions, and hence remain somewhat cautious about prospects for de novo genome assembly from historical specimens. Non-target sequence contaminations were observed in 2 of our insect museum specimens. We anticipate that future museum genomics projects will perhaps not generate entire genome sequences in all cases (our specimens contained relatively small and low-complexity genomes), but at least generating vital comparative genomic data for testing (phylo)genetic, demographic and genetic hypotheses, that become increasingly more horizontal. Furthermore, NGS of historical DNA enables recovering crucial genetic information from old type specimens that to date have remained mostly unutilized and, thus, opens up a new frontier for taxonomic research as well.

A Multiplex PCR for Detection of Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, and Bordetella pertussis in Clinical Specimens

DTIC Science & Technology

2005-01-24

detection of S . pneumoniae from throat swab or sputum samples may indicate colonization rather than illness, as it is often found in nonsterile sites...pertussis were considered in this paper. 1.2. Mycoplasma pneumoniae M. pneumoniae may be second only to S . pneumoniae as a causative agent of CAP, with...performed using an iCycler Thermal Cycler (Bio-Rad). Denaturation was performed for 15 min at 95°C followed by 35 cycles of denaturation at 94°C for 30 s

Hormone Receptor Expression Analyses in Neoplastic and Non-Neoplastic Canine Mammary Tissue by a Bead Based Multiplex Branched DNA Assay: A Gene Expression Study in Fresh Frozen and Formalin-Fixed, Paraffin-Embedded Samples.

PubMed

Mohr, Annika; Lüder Ripoli, Florenza; Hammer, Susanne Conradine; Willenbrock, Saskia; Hewicker-Trautwein, Marion; Kiełbowicz, Zdzisław; Murua Escobar, Hugo; Nolte, Ingo

2016-01-01

Immunohistochemistry (IHC) is currently considered the method of choice for steroid hormone receptor status evaluation in human breast cancer and, therefore, it is commonly utilized for assessing canine mammary tumors. In case of low hormone receptor expression, IHC is limited and thus is complemented by molecular analyses. In the present study, a multiplex bDNA assay was evaluated as a method for hormone receptor gene expression detection in canine mammary tissues. Estrogen receptor (ESR1), progesterone receptor (PGR), prolactin receptor (PRLR) and growth hormone receptor (GHR) gene expressions were evaluated in neoplastic and non-neoplastic canine mammary tissues. A set of 119 fresh frozen and 180 formalin-fixed, paraffin-embedded (FFPE) was comparatively analyzed and used for assay evaluation. Furthermore, a possible association between the hormone receptor expression in different histological subtypes of canine malignant mammary tumors and the castration status, breed and invasive growth of the tumor were analyzed. The multiplex bDNA assay proved to be more sensitive for fresh frozen specimens. Hormone receptor expression found was significantly decreased in malignant mammary tumors in comparison to non-neoplastic tissue and benign mammary tumors. Among the histological subtypes the lowest gene expression levels of ESR1, PGR and PRLR were found in solid, anaplastic and ductal carcinomas. In summary, the evaluation showed that the measurement of hormone receptors with the multiplex bDNA assay represents a practicable method for obtaining detailed quantitative information about gene expression in canine mammary tissue for future studies. Still, comparison with IHC or quantitative real-time PCR is needed for further validation of the present method.

Multiplexed detection of serological cancer markers with plasmon-enhanced Raman spectro-immunoassay.

PubMed

Li, Ming; Kang, Jeon Woong; Sukumar, Saraswati; Dasari, Ramachandra Rao; Barman, Ishan

2015-07-01

Circulating biomarkers have emerged as promising non-invasive, real-time surrogates for cancer diagnosis, prognostication and monitoring of therapeutic response. Emerging data, however, suggest that single markers are inadequate in describing complex pathologic transformations. Architecting assays capable of parallel measurements of multiple biomarkers can help achieve the desired clinical sensitivity and specificity while conserving patient specimen and reducing turn-around time. Here we describe a plasmon-enhanced Raman spectroscopic assay featuring nanostructured biomolecular probes and spectroscopic imaging for multiplexed detection of disseminated breast cancer markers cancer antigen (CA) 15-3, CA 27-29 and cancer embryonic antigen (CEA). In the developed SERS assay, both the assay chip and surface-enhanced Raman spectroscopy (SERS) tags are functionalized with monoclonal antibodies against CA15-3, CA27-29 and CEA, respectively. Sequential addition of biomarkers and functionalized SERS tags onto the functionalized assay chip enable the specific recognition of these biomarkers through the antibody-antigen interactions, leading to a sandwich spectro-immunoassay. In addition to offering extensive multiplexing capability, our method provides higher sensitivity than conventional immunoassays and demonstrates exquisite specificity owing to selective formation of conjugated complexes and fingerprint spectra of the Raman reporter. We envision that clinical translation of this assay may further enable asymptomatic surveillance of cancer survivors and speedy assessment of treatment benefit through a simple blood test.

Diagnostic Utility of a Clonality Test for Lymphoproliferative Diseases in Koreans Using the BIOMED-2 PCR Assay

PubMed Central

Kim, Young; Choi, Yoo Duk; Choi, Chan

2013-01-01

Background A clonality test for immunoglobulin (IG) and T cell receptor (TCR) is a useful adjunctive method for the diagnosis of lymphoproliferative diseases (LPDs). Recently, the BIOMED-2 multiplex polymerase chain reaction (PCR) assay has been established as a standard method for assessing the clonality of LPDs. We tested clonality in LPDs in Koreans using the BIOMED-2 multiplex PCR and compared the results with those obtained in European, Taiwanese, and Thai participants. We also evaluated the usefulness of the test as an ancillary method for diagnosing LPDs. Methods Two hundred and nineteen specimens embedded in paraffin, including 78 B cell lymphomas, 80 T cell lymphomas and 61 cases of reactive lymphadenitis, were used for the clonality test. Results Mature B cell malignancies showed 95.7% clonality for IG, 2.9% co-existing clonality, and 4.3% polyclonality. Mature T cell malignancies exhibited 83.8% clonality for TCR, 8.1% co-existing clonality, and 16.2% polyclonality. Reactive lymphadenitis showed 93.4% polyclonality for IG and TCR. The majority of our results were similar to those obtained in Europeans. However, the clonality for IGK of B cell malignancies and TCRG of T cell malignancies was lower in Koreans than Europeans. Conclusions The BIOMED-2 multiplex PCR assay was a useful adjunctive method for diagnosing LPDs. PMID:24255634

Simultaneous and Rapid Detection of Salmonella typhi, Bacillus anthracis, and Yersinia pestis by Using Multiplex Polymerase Chain Reaction (PCR)

PubMed Central

Safari Foroshani, Nargess; Karami, Ali; Pourali, Fatemeh

2013-01-01

Background Salmonella typhi, Bacillus anthracis, and Yersinia pestis are some serious human pathogens, which their early diagnosis is of great importance. Salmonella typhi, Bacillus anthracis, and Yersinia pestis cause typhoid fever, anthrax, and plague respectively. These bacteria can be used to make biologic weapons. Objectives In this study, we designed a new and rapid diagnostic method based on Uniplex and Multiplex PCR method. Materials and Methods Uniplex and multiplex Polymerase Chain Reaction (PCR) were conducted on virulent genes of hp and invA of Salmonella typhimurium, Pa and chr of Bacillus anthracis, and pla of Yersinia pestis. A genome from other bacteria was used to study the specificity of the primer and the PCR test. Results Standard strains used in this study showed that primers were specific. As for sensitivity, it was shown that this method can diagnose 1-10 copies of the genome, or 1-10 Colony Forming Units (CFU) for each of the bacteria. All pieces except anthrax were sequenced in PCR to validate the product. DNA fragment resulted from Bacillus anthracis was confirmed by restriction enzyme digestions. Conclusion The designed methods are accurate, rapid, and inexpensive to find and differentiate these bacteria from similar bacteria. They can be applied for rapid diagnosis of these agents in different specimens, and bioterrorism cases. PMID:24719692

The robustness of multiplex networks under layer node-based attack

PubMed Central

Zhao, Da-wei; Wang, Lian-hai; Zhi, Yong-feng; Zhang, Jun; Wang, Zhen

2016-01-01

From transportation networks to complex infrastructures, and to social and economic networks, a large variety of systems can be described in terms of multiplex networks formed by a set of nodes interacting through different network layers. Network robustness, as one of the most successful application areas of complex networks, has attracted great interest in a myriad of research realms. In this regard, how multiplex networks respond to potential attack is still an open issue. Here we study the robustness of multiplex networks under layer node-based random or targeted attack, which means that nodes just suffer attacks in a given layer yet no additional influence to their connections beyond this layer. A theoretical analysis framework is proposed to calculate the critical threshold and the size of giant component of multiplex networks when nodes are removed randomly or intentionally. Via numerous simulations, it is unveiled that the theoretical method can accurately predict the threshold and the size of giant component, irrespective of attack strategies. Moreover, we also compare the robustness of multiplex networks under multiplex node-based attack and layer node-based attack, and find that layer node-based attack makes multiplex networks more vulnerable, regardless of average degree and underlying topology. PMID:27075870

The robustness of multiplex networks under layer node-based attack.

PubMed

Zhao, Da-wei; Wang, Lian-hai; Zhi, Yong-feng; Zhang, Jun; Wang, Zhen

2016-04-14

From transportation networks to complex infrastructures, and to social and economic networks, a large variety of systems can be described in terms of multiplex networks formed by a set of nodes interacting through different network layers. Network robustness, as one of the most successful application areas of complex networks, has attracted great interest in a myriad of research realms. In this regard, how multiplex networks respond to potential attack is still an open issue. Here we study the robustness of multiplex networks under layer node-based random or targeted attack, which means that nodes just suffer attacks in a given layer yet no additional influence to their connections beyond this layer. A theoretical analysis framework is proposed to calculate the critical threshold and the size of giant component of multiplex networks when nodes are removed randomly or intentionally. Via numerous simulations, it is unveiled that the theoretical method can accurately predict the threshold and the size of giant component, irrespective of attack strategies. Moreover, we also compare the robustness of multiplex networks under multiplex node-based attack and layer node-based attack, and find that layer node-based attack makes multiplex networks more vulnerable, regardless of average degree and underlying topology.

A Novel Universal Primer-Multiplex-PCR Method with Sequencing Gel Electrophoresis Analysis

PubMed Central

Huang, Kunlun; Zhang, Nan; Yuan, Yanfang; Shang, Ying; Luo, Yunbo

2012-01-01

In this study, a novel universal primer-multiplex-PCR (UP-M-PCR) method adding a universal primer (UP) in the multiplex PCR reaction system was described. A universal adapter was designed in the 5′-end of each specific primer pairs which matched with the specific DNA sequences for each template and also used as the universal primer (UP). PCR products were analyzed on sequencing gel electrophoresis (SGE) which had the advantage of exhibiting extraordinary resolution. This method overcame the disadvantages rooted deeply in conventional multiplex PCR such as complex manipulation, lower sensitivity, self-inhibition and amplification disparity resulting from different primers, and it got a high specificity and had a low detection limit of 0.1 ng for single kind of crops when screening the presence of genetically modified (GM) crops in mixture samples. The novel developed multiplex PCR assay with sequencing gel electrophoresis analysis will be useful in many fields, such as verifying the GM status of a sample irrespective of the crop and GM trait and so on. PMID:22272223

Development of analytical methods for multiplex bio-assay with inductively coupled plasma mass spectrometry

PubMed Central

Ornatsky, Olga I.; Kinach, Robert; Bandura, Dmitry R.; Lou, Xudong; Tanner, Scott D.; Baranov, Vladimir I.; Nitz, Mark; Winnik, Mitchell A.

2008-01-01

Advances in the development of highly multiplexed bio-analytical assays with inductively coupled plasma mass spectrometry (ICP-MS) detection are discussed. Use of novel reagents specifically designed for immunological methods utilizing elemental analysis is presented. The major steps of method development, including selection of elements for tags, validation of tagged reagents, and examples of multiplexed assays, are considered in detail. The paper further describes experimental protocols for elemental tagging of antibodies, immunostaining of live and fixed human leukemia cells, and preparation of samples for ICP-MS analysis. Quantitative analysis of surface antigens on model cell lines using a cocktail of seven lanthanide labeled antibodies demonstrated high specificity and concordance with conventional immunophenotyping. PMID:19122859

Linkage analysis with multiplexed short tandem repeat polymorphisms using infrared fluorescence and M13 tailed primers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oetting, W.S.; Lee, H.K.; Flanders, D.J.

The use of short tandem repeat polymorphisms (STRPs) as marker loci for linkage analysis is becoming increasingly important due to their large numbers in the human genome and their high degree of polymorphism. Fluorescence-based detection of the STRP pattern with an automated DNA sequencer has improved the efficiency of this technique by eliminating the need for radioactivity and producing a digitized autoradiogram-like image that can be used for computer analysis. In an effort to simplify the procedure and to reduce the cost of fluorescence STRP analysis, we have developed a technique known as multiplexing STRPs with tailed primers (MSTP) usingmore » primers that have a 19-bp extension, identical to the sequence of an M13 sequencing primer, on the 5{prime} end of the forward primer in conjunction with multiplexing several primer pairs in a single polymerase chain reaction (PCR) amplification. The banding pattern is detected with the addition of the M13 primer-dye conjugate as the sole primer conjugated to the fluorescent dye, eliminating the need for direct conjugation of the infrared fluorescent dye to the STRP primers. The use of MSTP for linkage analysis greatly reduces the number of PCR reactions. Up to five primer pairs can be multiplexed together in the same reaction. At present, a set of 148 STRP markers spaced at an average genetic distance of 28 cM throughout the autosomal genome can be analyzed in 37 sets of multiplexed amplification reactions. We have automated the analysis of these patterns for linkage using software that both detects the STRP banding pattern and determines their sizes. This information can then be exported in a user-defined format from a database manager for linkage analysis. 15 refs., 2 figs., 4 tabs.« less

Multicenter Evaluation of BD Max Enteric Parasite Real-Time PCR Assay for Detection of Giardia duodenalis, Cryptosporidium hominis, Cryptosporidium parvum, and Entamoeba histolytica

PubMed Central

Relich, R. F.; Doyle, L.; Espina, N.; Fuller, D.; Karchmer, T.; Lainesse, A.; Mortensen, J. E.; Pancholi, P.; Veros, W.; Harrington, S. M.

2016-01-01

Common causes of chronic diarrhea among travelers worldwide include protozoan parasites. The majority of parasitic infections are caused by Giardia duodenalis, Entamoeba histolytica, Cryptosporidium parvum, and Cryptosporidium hominis. Similarly, these species cause the majority of parasitic diarrhea acquired in the United States. Detection of parasites by gold standard microscopic methods is time-consuming and requires considerable expertise; enzyme immunoassays and direct fluorescent-antibody (DFA) stains have lowered hands-on time for testing, but improvements in sensitivity and technical time may be possible with a PCR assay. We performed a clinical evaluation of a multiplex PCR panel, the enteric parasite panel (EPP), for the detection of these common parasites using the BD Max instrument, which performs automated extraction and amplification. A total of 2,495 compliant specimens were enrolled, including 2,104 (84%) specimens collected prospectively and 391 (16%) specimens collected retrospectively. Approximately equal numbers were received in 10% formalin (1,273 specimens) and unpreserved (1,222 specimens). The results from the EPP were compared to those from alternate PCR and bidirectional sequencing (APCR), as well as DFA (G. duodenalis and C. parvum or C. hominis) or trichrome stain (E. histolytica). The sensitivity and specificity for prospective and retrospective specimens combined were 98.2% and 99.5% for G. duodenalis, 95.5% and 99.6 for C. parvum or C. hominis, and 100% and 100% for E. histolytica, respectively. The performance of the FDA-approved BD Max EPP compared well to the reference methods and may be an appropriate substitute for microscopic examination or immunoassays. PMID:27535690

Functional protease profiling with reporter peptides in serum specimens of colorectal cancer patients: demonstration of its routine diagnostic applicability.

PubMed

Findeisen, Peter; Costina, Victor; Yepes, Diego; Hofheinz, Ralf; Neumaier, Michael

2012-06-08

The progression of many solid tumors is characterized by the release of tumor-associated proteases and the detection of tumor specific proteolytic activity in serum specimens is a promising diagnostic tool in oncology. Here we describe a mass spectrometry-based functional proteomic profiling approach that tracks the ex-vivo degradation of a synthetic endoprotease substrate in serum specimens of colorectal tumor patients. A reporter peptide (RP) with the amino acid sequence WKPYDAAD was synthesized that has a known cleavage site for the cysteine-endopeptidase cancer procoagulant (EC 3.4.22.26). The RP was added to serum specimens from colorectal cancer patients (n = 30), inflammatory controls (n = 30) and healthy controls (n = 30) and incubated under strictly standardized conditions. The proteolytic fragment of the RP was quantified with liquid chromatography / mass spectrometry (LC/MS). RP-spiking showed good intra- and inter-day reproducibility with coefficients of variation (CVs) that did not exceed a value of 10%. The calibration curve for the anchor peptide was linear in the concentration range of 0.4 - 50 μmol/L. The median concentration of the RP-fragment in serum specimens from tumor patients (TU: 17.6 μmol/L, SD 9.0) was significantly higher when compared to non-malignant inflammatory controls (IC: 11.1 μmol/L, SD 6.1) and healthy controls (HC: 10.3 μmol/L, SD 3.1). Highest area under receiver operating characteristic (AUROC) values were seen for discrimination of TU versus HC (0.89) followed by TU versus IC (0.77). IC and HC could barely be separated indicated by an AUROC value of 0.57. The proteolytic activity towards the RP was conserved in serum specimens that were kept at room temperature for up to 24 hours prior to the analysis. The proteolytic cleavage of reporter peptides is a surrogate marker for tumor associated proteolytic activity in serum specimens of cancer patients. A simple, robust and highly reproducible LC/MS method has been developed that allows the quantification of proteolytic fragments in serum specimens. The preanalytical impact of sample handling is minimal as the tumor-associated proteolytic activity towards the reporter peptide is stable for at least up to 24 h. Taken together, the functional protease profiling shows characteristics that are in line with routinely performed diagnostic assays. Further work will focus on the identification of additional reporter peptides for the construction of a multiplex assay to increase diagnostic accuracy of the functional protease profiling.

Functional protease profiling with reporter peptides in serum specimens of colorectal cancer patients: demonstration of its routine diagnostic applicability

PubMed Central

2012-01-01

Background The progression of many solid tumors is characterized by the release of tumor-associated proteases and the detection of tumor specific proteolytic activity in serum specimens is a promising diagnostic tool in oncology. Here we describe a mass spectrometry-based functional proteomic profiling approach that tracks the ex-vivo degradation of a synthetic endoprotease substrate in serum specimens of colorectal tumor patients. Methods A reporter peptide (RP) with the amino acid sequence WKPYDAAD was synthesized that has a known cleavage site for the cysteine-endopeptidase cancer procoagulant (EC 3.4.22.26). The RP was added to serum specimens from colorectal cancer patients (n = 30), inflammatory controls (n = 30) and healthy controls (n = 30) and incubated under strictly standardized conditions. The proteolytic fragment of the RP was quantified with liquid chromatography / mass spectrometry (LC/MS). Results RP-spiking showed good intra- and inter-day reproducibility with coefficients of variation (CVs) that did not exceed a value of 10%. The calibration curve for the anchor peptide was linear in the concentration range of 0.4 – 50 μmol/L. The median concentration of the RP-fragment in serum specimens from tumor patients (TU: 17.6 μmol/L, SD 9.0) was significantly higher when compared to non-malignant inflammatory controls (IC: 11.1 μmol/L, SD 6.1) and healthy controls (HC: 10.3 μmol/L, SD 3.1). Highest area under receiver operating characteristic (AUROC) values were seen for discrimination of TU versus HC (0.89) followed by TU versus IC (0.77). IC and HC could barely be separated indicated by an AUROC value of 0.57. The proteolytic activity towards the RP was conserved in serum specimens that were kept at room temperature for up to 24 hours prior to the analysis. Conclusion The proteolytic cleavage of reporter peptides is a surrogate marker for tumor associated proteolytic activity in serum specimens of cancer patients. A simple, robust and highly reproducible LC/MS method has been developed that allows the quantification of proteolytic fragments in serum specimens. The preanalytical impact of sample handling is minimal as the tumor-associated proteolytic activity towards the reporter peptide is stable for at least up to 24 h. Taken together, the functional protease profiling shows characteristics that are in line with routinely performed diagnostic assays. Further work will focus on the identification of additional reporter peptides for the construction of a multiplex assay to increase diagnostic accuracy of the functional protease profiling. PMID:22682081

Multiplex pyrosequencing assay using AdvISER-MH-PYRO algorithm: a case for rapid and cost-effective genotyping analysis of prostate cancer risk-associated SNPs.

PubMed

Ambroise, Jérôme; Butoescu, Valentina; Robert, Annie; Tombal, Bertrand; Gala, Jean-Luc

2015-06-25

Single Nucleotide Polymorphisms (SNPs) identified in Genome Wide Association Studies (GWAS) have generally moderate association with related complex diseases. Accordingly, Multilocus Genetic Risk Scores (MGRSs) have been computed in previous studies in order to assess the cumulative association of multiple SNPs. When several SNPs have to be genotyped for each patient, using successive uniplex pyrosequencing reactions increases analytical reagent expenses and Turnaround Time (TAT). While a set of several pyrosequencing primers could theoretically be used to analyze multiplex amplicons, this would generate overlapping primer-specific pyro-signals that are visually uninterpretable. In the current study, two multiplex assays were developed consisting of a quadruplex (n=4) and a quintuplex (n=5) polymerase chain reaction (PCR) each followed by multiplex pyrosequencing analysis. The aim was to reliably but rapidly genotype a set of prostate cancer-related SNPs (n=9). The nucleotide dispensation order was selected using SENATOR software. Multiplex pyro-signals were analyzed using the new AdvISER-MH-PYRO software based on a sparse representation of the signal. Using uniplex assays as gold standard, the concordance between multiplex and uniplex assays was assessed on DNA extracted from patient blood samples (n = 10). All genotypes (n=90) generated with the quadruplex and the quintuplex pyroquencing assays were perfectly (100 %) concordant with uniplex pyrosequencing. Using multiplex genotyping approach for analyzing a set of 90 patients allowed reducing TAT by approximately 75 % (i.e., from 2025 to 470 min) while reducing reagent consumption and cost by approximately 70 % (i.e., from ~229 US$ /patient to ~64 US$ /patient). This combination of quadruplex and quintuplex pyrosequencing and PCR assays enabled to reduce the amount of DNA required for multi-SNP analysis, and to lower the global TAT and costs of SNP genotyping while providing results as reliable as uniplex analysis. Using this combined multiplex approach also substantially reduced the production of waste material. These genotyping assays appear therefore to be biologically, economically and ecologically highly relevant, being worth to be integrated in genetic-based predictive strategies for better selecting patients at risk for prostate cancer. In addition, the same approach could now equally be transposed to other clinical/research applications relying on the computation of MGRS based on multi-SNP genotyping.

Evaluation of Multiplexed 16S rRNA Microbial Population Surveys Using Illumina MiSeq Platform (Seventh Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting 2012)

ScienceCinema

Tremblay, Julien

2018-01-22

Julien Tremblay from DOE JGI presents "Evaluation of Multiplexed 16S rRNA Microbial Population Surveys Using Illumina MiSeq Platorm" at the 7th Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting held in June, 2012 in Santa Fe, NM.

Evaluation of Multiplexed 16S rRNA Microbial Population Surveys Using Illumina MiSeq Platform (Seventh Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting 2012)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tremblay, Julien

2012-06-01

Julien Tremblay from DOE JGI presents "Evaluation of Multiplexed 16S rRNA Microbial Population Surveys Using Illumina MiSeq Platorm" at the 7th Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting held in June, 2012 in Santa Fe, NM.

EFFECT OF DIFFERENT REGIONS OF AMPLIFIED 16S RDNA ON A PERFORMANCE OF A MULTIPLEXED, BEAD-BASED METHOD FOR ANALYSIS OF DNA SEQUENCES IN ENVIRONMENTAL SAMPLES.

EPA Science Inventory

Using a bead-based method for multiplexed analysis of community DNA, the dynamics of aquatic microbial communities can be assessed. Capture probes, specific for a genus or species of bacteria, are attached to the surface of uniquely labeled, microscopic polystyrene beads. Primers...

NIST mixed stain study 3: signal intensity balance in commercial short tandem repeat multiplexes.

PubMed

Duewer, David L; Kline, Margaret C; Redman, Janette W; Butler, John M

2004-12-01

Short-tandem repeat (STR) allelic intensities were collected from more than 60 forensic laboratories for a suite of seven samples as part of the National Institute of Standards and Technology-coordinated 2001 Mixed Stain Study 3 (MSS3). These interlaboratory challenge data illuminate the relative importance of intrinsic and user-determined factors affecting the locus-to-locus balance of signal intensities for currently used STR multiplexes. To varying degrees, seven of the eight commercially produced multiplexes used by MSS3 participants displayed very similar patterns of intensity differences among the different loci probed by the multiplexes for all samples, in the hands of multiple analysts, with a variety of supplies and instruments. These systematic differences reflect intrinsic properties of the individual multiplexes, not user-controllable measurement practices. To the extent that quality systems specify minimum and maximum absolute intensities for data acceptability and data interpretation schema require among-locus balance, these intrinsic intensity differences may decrease the utility of multiplex results and surely increase the cost of analysis.

Interferometric space-mode multiplexing based on binary phase plates and refractive phase shifters.

PubMed

Liñares, Jesús; Prieto-Blanco, Xesús; Moreno, Vicente; Montero-Orille, Carlos; Mouriz, Dolores; Nistal, María C; Barral, David

2017-05-15

A Mach-Zehnder interferometer (MZI) that includes in an arm either a reflective image inverter or a Gouy phase shifter (RGPS) can (de)multiplex many types of modes of a few mode fiber without fundamental loss. The use of RGPSs in combination with binary phase plates for multiplexing purposes is studied for the first time, showing that the particular RGPS that shifts π the odd modes only multiplexes accurately low order modes. To overcome such a restriction, we present a new exact refractive image inverter, more compact and flexible than its reflective counterpart. Moreover, we show that these interferometers remove or reduce the crosstalk that the binary phase plates could introduce between the multiplexed modes. Finally, an experimental analysis of a MZI with both an approximated and an exact refractive image inverter is presented for the case of a bimodal multiplexing. Likewise, it is proven experimentally that a RGPS that shifts π/2 demultiplexes two odd modes which can not be achieved by any image inverter.

Multiplex lexical networks reveal patterns in early word acquisition in children

NASA Astrophysics Data System (ADS)

Stella, Massimo; Beckage, Nicole M.; Brede, Markus

2017-04-01

Network models of language have provided a way of linking cognitive processes to language structure. However, current approaches focus only on one linguistic relationship at a time, missing the complex multi-relational nature of language. In this work, we overcome this limitation by modelling the mental lexicon of English-speaking toddlers as a multiplex lexical network, i.e. a multi-layered network where N = 529 words/nodes are connected according to four relationship: (i) free association, (ii) feature sharing, (iii) co-occurrence, and (iv) phonological similarity. We investigate the topology of the resulting multiplex and then proceed to evaluate single layers and the full multiplex structure on their ability to predict empirically observed age of acquisition data of English speaking toddlers. We find that the multiplex topology is an important proxy of the cognitive processes of acquisition, capable of capturing emergent lexicon structure. In fact, we show that the multiplex structure is fundamentally more powerful than individual layers in predicting the ordering with which words are acquired. Furthermore, multiplex analysis allows for a quantification of distinct phases of lexical acquisition in early learners: while initially all the multiplex layers contribute to word learning, after about month 23 free associations take the lead in driving word acquisition.

Fiber-Optic Propagation Effects in Long-Haul HF/VHF/UHF Analog Photonic Links

DTIC Science & Technology

2014-04-17

theoretical analysis of crosstalk in fiber optic wavelength division multiplexed systems is presented for the HF/VHF/UHF (1 MHz to 3 GHz) frequency...Street, Suite 1425 Arlington, VA 22203-1995 EW-271-003 6582 ONR Wavelength division multiplexing Crosstalk 05-03-2013 – 20-08-2014 TABLE OF CONTENTS...in optical fiber that can alter the phase relationship between signals in separate fibers or between signals that are multiplexed onto the same

Protein Multiplexed Immunoassay Analysis with R.

PubMed

Breen, Edmond J

2017-01-01

Plasma samples from 177 control and type 2 diabetes patients collected at three Australian hospitals are screened for 14 analytes using six custom-made multiplex kits across 60 96-well plates. In total 354 samples were collected from the patients, representing one baseline and one end point sample from each patient. R methods and source code for analyzing the analyte fluorescence response obtained from these samples by Luminex Bio-Plex ® xMap multiplexed immunoassay technology are disclosed. Techniques and R procedures for reading Bio-Plex ® result files for statistical analysis and data visualization are also presented. The need for technical replicates and the number of technical replicates are addressed as well as plate layout design strategies. Multinomial regression is used to determine plate to sample covariate balance. Methods for matching clinical covariate information to Bio-Plex ® results and vice versa are given. As well as methods for measuring and inspecting the quality of the fluorescence responses are presented. Both fixed and mixed-effect approaches for immunoassay statistical differential analysis are presented and discussed. A random effect approach to outlier analysis and detection is also shown. The bioinformatics R methodology present here provides a foundation for rigorous and reproducible analysis of the fluorescence response obtained from multiplexed immunoassays.

Longitudinal molecular characterization of endoscopic specimens from colorectal lesions

PubMed Central

Minarikova, Petra; Benesova, Lucie; Halkova, Tereza; Belsanova, Barbora; Suchanek, Stepan; Cyrany, Jiri; Tuckova, Inna; Bures, Jan; Zavoral, Miroslav; Minarik, Marek

2016-01-01

AIM: To compare molecular profiles of proximal colon, distal colon and rectum in large adenomas, early and late carcinomas. To assess feasibility of testing directed at molecular markers from this study in routine clinical practice. METHODS: A prospective 3-year study has resulted in the acquisition of samples from 159 large adenomas and 138 carcinomas along with associated clinical parameters including localization, grade and histological type for adenomas and localization and stage for carcinomas. A complex molecular phenotyping has been performed using multiplex ligation-dependent probe amplification technique for the evaluation of CpG-island methylator phenotype (CIMP), PCR fragment analysis for detection of microsatellite instability and denaturing capillary electrophoresis for sensitive detection of somatic mutations in KRAS, BRAF, TP53 and APC genes. RESULTS: Molecular types according to previously introduced Jass classification have been evaluated for large adenomas and early and late carcinomas. An increase in CIMP+ type, eventually accompanied with KRAS mutations, was notable between large adenomas and early carcinomas. As expected, the longitudinal observations revealed a correlation of the CIMP+/BRAF+ type with proximal location. CONCLUSION: Prospective molecular classification of tissue specimens is feasible in routine endoscopy practice. Increased frequency of some molecular types corresponds to the developmental stages of colorectal tumors. As expected, a clear distinction is notable for tumors located in proximal colon supposedly arising from the serrated (methylation) pathway. PMID:27239120

Clinical implementation of RNA signatures for pharmacogenomic decision-making

PubMed Central

Tang, Weihua; Hu, Zhiyuan; Muallem, Hind; Gulley, Margaret L

2011-01-01

RNA profiling is increasingly used to predict drug response, dose, or toxicity based on analysis of drug pharmacokinetic or pharmacodynamic pathways. Before implementing multiplexed RNA arrays in clinical practice, validation studies are carried out to demonstrate sufficient evidence of analytic and clinical performance, and to establish an assay protocol with quality assurance measures. Pathologists assure quality by selecting input tissue and by interpreting results in the context of the input tissue as well as the technologies that were used and the clinical setting in which the test was ordered. A strength of RNA profiling is the array-based measurement of tens to thousands of RNAs at once, including redundant tests for critical analytes or pathways to promote confidence in test results. Instrument and reagent manufacturers are crucial for supplying reliable components of the test system. Strategies for quality assurance include careful attention to RNA preservation and quality checks at pertinent steps in the assay protocol, beginning with specimen collection and proceeding through the various phases of transport, processing, storage, analysis, interpretation, and reporting. Specimen quality is checked by probing housekeeping transcripts, while spiked and exogenous controls serve as a check on analytic performance of the test system. Software is required to manipulate abundant array data and present it for interpretation by a laboratory physician who reports results in a manner facilitating therapeutic decision-making. Maintenance of the assay requires periodic documentation of personnel competency and laboratory proficiency. These strategies are shepherding genomic arrays into clinical settings to provide added value to patients and to the larger health care system. PMID:23226056

The Seasonality and Ecology of the Anopheles gambiae complex (Dipetra: Culicidae) in Liberia Using Molecular Identification.

PubMed

Fahmy, N T; Villinski, J T; Bolay, F; Stoops, C A; Tageldin, R A; Fakoli, L; Okasha, O; Obenauer, P J; Diclaro, J W

2015-05-01

Members of the Anopheles gambiae sensu lato (Giles) complex define a group of seven morphologically indistinguishable species, including the principal malaria vectors in Sub-Saharan Africa. Members of this complex differ in behavior and ability to transmit malaria; hence, precise identification of member species is critical to monitoring and evaluating malaria threat levels. We collected mosquitoes from five counties in Liberia every other month from May 2011 until May 2012, using various trapping techniques. A. gambiae complex members were identified using molecular techniques based on differences in the ribosomal DNA (rDNA) region between species and the molecular forms (S and M) of A. gambiae sensu stricto (s.s) specimens. In total, 1,696 A. gambiae mosquitoes were collected and identified. DNA was extracted from legs of each specimen with species identification determined by multiplex polymerase chain reaction using specific primers. The molecular forms (M or S) of A. gambiae s.s were determined by restriction fragment length polymorphism. Bivariate and multivariate logistic regression models identified environmental variables associated with genomic differentiation. Our results indicate widespread occurrence of A. gambiae s.s., the principal malaria vector in the complex, although two Anopheles melas Theobald/A. merus Donitz mosquitoes were detected. We found 72.6, 25.5, and 1.9% of A. gambiae s.s specimens were S, M, and hybrid forms, respectively. Statistical analysis indicates that the S form was more likely to be found in rural areas during rainy seasons and indoor catchments. This information will enhance vector control efforts in Liberia. Published by Oxford University Press on behalf of Entomological Society of America 2015. This work is written by US Government employees and is in the public domain in the US.

Prospective evaluation of a high multiplexing real-time polymerase chain reaction array for the rapid identification and characterization of bacteria causative of nosocomial pneumonia from clinical specimens: a proof-of-concept study.

PubMed

Roisin, S; Huang, T-D; de Mendonça, R; Nonhoff, C; Bogaerts, P; Hites, M; Delaere, B; Hamels, S; de Longueville, F; Glupczynski, Y; Denis, O

2018-01-01

The purpose of this study was evaluation of the VAPChip assay based on the "Rapid-Array-PCR-technology" which targets 13 respiratory pathogens and 24 β-lactam resistance genes directly on respiratory clinical specimens. The first step included analysis of 45 respiratory specimens in order to calibrate and determine the threshold for target genes. The second prospective step involved 85 respiratory samples from patients suspected of nosocomial pneumonia collected in two academic hospitals over an 8-month period. Results of the VAPChip assay were compared to routine methods. The first step showed a large proportion of positive signals for H. influenzae and/or S. pneumoniae. For identification, discrepancies were observed in seven samples. Thresholds were adapted and two probes were re-designed to create a new version of the cartridge. In the second phase, sensitivity and specificity of the VAPchip for bacterial identification were 72.9% and 99.1%, respectively. Seventy (82%) pathogens were correctly identified by both methods. Nine pathogens detected by the VAPChip were culture negative and 26 pathogens identified by culture were VAPChip negative. For resistance mechanisms, 11 probes were positive without identification of pathogens with an antimicrobial-susceptibility testing compatible by culture. However, the patient's recent microbiological history was able to explain most of these positive signals. The VAPChip assay simultaneously detects different pathogens and resistance mechanisms directly from clinical samples. This system seems very promising but the extraction process needs to be automated for routine implementation. This kind of rapid point-of-care automated platform permitting a syndromic approach will be the future challenge in the management of infectious diseases.

Quantitative detection of RASSF1A DNA promoter methylation in tumors and serum of patients with serous epithelial ovarian cancer.

PubMed

Bondurant, Amy E; Huang, Zhiqing; Whitaker, Regina S; Simel, Lauren R; Berchuck, Andrew; Murphy, Susan K

2011-12-01

Detection of cell free tumor-specific DNA methylation has been proposed as a potentially useful noninvasive mechanism to detect malignancies, including ovarian cancer, and to monitor response to treatment. However, there are few easily implemented quantitative approaches available for DNA methylation analysis. Our objectives were to develop an absolute quantitative method for detection of DNA methylation using RASSF1A, a known target of promoter methylation in ovarian cancer, and test the ability to detect RASSF1A methylation in tumors and serum specimens of women with ovarian cancer. Bisulfite modified DNAs were subjected to real time PCR using nondiscriminatory PCR primers and a probe with sequence containing a single CpG site, theoretically able to capture the methylation status of that CpG for every allele within a given specimen. Input DNA was normalized to ACTB levels detected simultaneously by assay multiplexing. Methylation levels were established by comparison to results obtained from universally methylated DNA. The assay was able to detect one methylated RASSF1A allele in 100,000 unmethylated alleles. RASSF1A was methylated in 54 of 106 (51%) invasive serous ovarian cancers analyzed and methylation status was concordant in 20/20 matched preoperative serum-tumor pairs. Serial serum specimens taken over the course of treatment for 8 of 9 patients showed fluctuations in RASSF1A methylation concomitant with disease status. This novel assay provides a real-time PCR-based method for absolute quantitation of DNA methylation. Our results support feasibility of monitoring RASSF1A methylation from serum samples taken over the course of treatment from women with ovarian cancer. Copyright © 2011 Elsevier Inc. All rights reserved.

Sensitive and specific detection of EML4-ALK rearrangements in non-small cell lung cancer (NSCLC) specimens by multiplex amplicon RNA massive parallel sequencing.

PubMed

Moskalev, Evgeny A; Frohnauer, Judith; Merkelbach-Bruse, Sabine; Schildhaus, Hans-Ulrich; Dimmler, Arno; Schubert, Thomas; Boltze, Carsten; König, Helmut; Fuchs, Florian; Sirbu, Horia; Rieker, Ralf J; Agaimy, Abbas; Hartmann, Arndt; Haller, Florian

2014-06-01

Recurrent gene fusions of anaplastic lymphoma receptor tyrosine kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) have been recently identified in ∼5% of non-small cell lung cancers (NSCLCs) and are targets for selective tyrosine kinase inhibitors. While fluorescent in situ hybridization (FISH) is the current gold standard for detection of EML4-ALK rearrangements, several limitations exist including high costs, time-consuming evaluation and somewhat equivocal interpretation of results. In contrast, targeted massive parallel sequencing has been introduced as a powerful method for simultaneous and sensitive detection of multiple somatic mutations even in limited biopsies, and is currently evolving as the method of choice for molecular diagnostic work-up of NSCLCs. We developed a novel approach for indirect detection of EML4-ALK rearrangements based on 454 massive parallel sequencing after reverse transcription and subsequent multiplex amplification (multiplex ALK RNA-seq) which takes advantage of unbalanced expression of the 5' and 3' ALK mRNA regions. Two lung cancer cell lines and a selected series of 32 NSCLC samples including 11 cases with EML4-ALK rearrangement were analyzed with this novel approach in comparison to ALK FISH, ALK qRT-PCR and EML4-ALK RT-PCR. The H2228 cell line with known EML4-ALK rearrangement showed 171 and 729 reads for 5' and 3' ALK regions, respectively, demonstrating a clearly unbalanced expression pattern. In contrast, the H1299 cell line with ALK wildtype status displayed no reads for both ALK regions. Considering a threshold of 100 reads for 3' ALK region as indirect indicator of EML4-ALK rearrangement, there was 100% concordance between the novel multiplex ALK RNA-seq approach and ALK FISH among all 32 NSCLC samples. Multiplex ALK RNA-seq is a sensitive and specific method for indirect detection of EML4-ALK rearrangements, and can be easily implemented in panel based molecular diagnostic work-up of NSCLCs by massive parallel sequencing. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Growing multiplex networks with arbitrary number of layers

NASA Astrophysics Data System (ADS)

Momeni, Naghmeh; Fotouhi, Babak

2015-12-01

This paper focuses on the problem of growing multiplex networks. Currently, the results on the joint degree distribution of growing multiplex networks present in the literature pertain to the case of two layers and are confined to the special case of homogeneous growth and are limited to the state state (that is, the limit of infinite size). In the present paper, we first obtain closed-form solutions for the joint degree distribution of heterogeneously growing multiplex networks with arbitrary number of layers in the steady state. Heterogeneous growth means that each incoming node establishes different numbers of links in different layers. We consider both uniform and preferential growth. We then extend the analysis of the uniform growth mechanism to arbitrary times. We obtain a closed-form solution for the time-dependent joint degree distribution of a growing multiplex network with arbitrary initial conditions. Throughout, theoretical findings are corroborated with Monte Carlo simulations. The results shed light on the effects of the initial network on the transient dynamics of growing multiplex networks and takes a step towards characterizing the temporal variations of the connectivity of growing multiplex networks, as well as predicting their future structural properties.

Multiplex polymerase chain reaction on FTA cards vs. flow cytometry for B-lymphocyte clonality.

PubMed

Dictor, Michael; Skogvall, Ingela; Warenholt, Janina; Rambech, Eva

2007-01-01

Two-colour flow cytometry was compared with multiplex PCR with capillary electrophoresis for clonality determination in specific categories of B-cell lymphoma. FTA cards were evaluated for preserving DNA from node imprints and expediting molecular analysis. A single-tube multiplex PCR targeted IGH and lymphoma-specific translocations in DNA extracted from 180 frozen lymphoid tissues and DNA bound to FTA cards from 192 fresh tissues and 137 aspirates. PCR results were compared with flow cytometry in the extracted and aspirated samples. Overall, single-tube multiplex PCR sensitivity was equivalent in the sample groups (intergroup range 79%-91%). False negatives were associated with tumour origin in the follicle centre. Multiplex PCR and flow cytometry were equally sensitive and together detected 98% of B-cell lymphomas. Additional two-tube targeting of IGK suggested an overall molecular sensitivity >90%. False positive (pseudoclonal) single-tube multiplex PCR was associated with necrosis and sparse lymphocytes. Multiplex PCR using template DNA bound to an FTA card effectively detects B-lymphocyte clonality, obviates DNA extraction and refrigeration, and can be used without diminished sensitivity in fine needle aspirates or node imprints as a replacement for or complement to flow cytometry at any point in the diagnostic work-up.

PCA3 Reference Set Application: T2-Erg-Martin Sanda-Emory (2014) — EDRN Public Portal

Cancer.gov

We hypothesize that combining T2:erg (T2:erg) fusion and PCA3 detection in urine collected after digital rectal exam can improve the specificity of identifying clinically significant prostate cancer presence over the standard PSA and DRE. To address this hypothesis we propose to validate the performance of the urinary T2:erg in a multiplex model predicting the diagnosis of clinically significant prostate cancer on subsequent prostate biopsy using post-DRE pre biopsy urine specimens from a cohort of 900 men on the EDRN’s PCA3 trial.

Simultaneous detection of Legionella species and L. anisa, L. bozemanii, L. longbeachae and L. micdadei using conserved primers and multiple probes in a multiplex real-time PCR assay.

PubMed

Cross, Kristen E; Mercante, Jeffrey W; Benitez, Alvaro J; Brown, Ellen W; Diaz, Maureen H; Winchell, Jonas M

2016-07-01

Legionnaires' disease is a severe respiratory disease that is estimated to cause between 8,000 and 18,000 hospitalizations each year, though the exact burden is unknown due to under-utilization of diagnostic testing. Although Legionella pneumophila is the most common species detected in clinical cases (80-90%), other species have also been reported to cause disease. However, little is known about Legionnaires' disease caused by these non-pneumophila species. We designed a multiplex real-time PCR assay for detection of all Legionella spp. and simultaneous specific identification of four clinically-relevant Legionella species, L. anisa, L. bozemanii, L. longbeachae, and L. micdadei, using 5'-hydrolysis probe real-time PCR. The analytical sensitivity for detection of nucleic acid from each target species was ≤50fg per reaction. We demonstrated the utility of this assay in spiked human sputum specimens. This assay could serve as a tool for understanding the scope and impact of non-pneumophila Legionella species in human disease. Published by Elsevier Inc.

Design and laboratory validation of a structural element instrumented with multiplexed interferometric fiber optic sensors

NASA Astrophysics Data System (ADS)

Zonta, Daniele; Pozzi, Matteo; Wu, Huayong; Inaudi, Daniele

2008-03-01

This paper introduces a concept of smart structural elements for the real-time condition monitoring of bridges. These are prefabricated reinforced concrete elements embedding a permanent sensing system and capable of self-diagnosis when in operation. The real-time assessment is automatically controlled by a numerical algorithm founded on Bayesian logic: the method assigns a probability to each possible damage scenario, and estimates the statistical distribution of the damage parameters involved (such as location and extent). To verify the effectiveness of the technology, we produced and tested in the laboratory a reduced-scale smart beam prototype. The specimen is 3.8 m long and has cross-section 0.3 by 0.5m, and has been prestressed using a Dywidag bar, in such a way as to control the preload level. The sensor system includes a multiplexed version of SOFO interferometric sensors mounted on a composite bar, along with a number of traditional metal-foil strain gauges. The method allowed clear recognition of increasing fault states, simulated on the beam by gradually reducing the prestress level.

Biotube

NASA Technical Reports Server (NTRS)

Richards, Stephanie E. (Compiler); Levine, Howard G.; Romero, Vergel

2016-01-01

Biotube was developed for plant gravitropic research investigating the potential for magnetic fields to orient plant roots as they grow in microgravity. Prior to flight, experimental seeds are placed into seed cassettes, that are capable of containing up to 10 seeds, and inserted between two magnets located within one of three Magnetic Field Chamber (MFC). Biotube is stored within an International Space Station (ISS) stowage locker and provides three levels of containment for chemical fixatives. Features include monitoring of temperature, fixative/ preservative delivery to specimens, and real-time video imaging downlink. Biotube's primary subsystems are: (1) The Water Delivery System that automatically activates and controls the delivery of water (to initiate seed germination). (2) The Fixative Storage and Delivery System that stores and delivers chemical fixative or RNA later to each seed cassette. (3) The Digital Imaging System consisting of 4 charge-coupled device (CCD) cameras, a video multiplexer, a lighting multiplexer, and 16 infrared light-emitting diodes (LEDs) that provide illumination while the photos are being captured. (4) The Command and Data Management System that provides overall control of the integrated subsystems, graphical user interface, system status and error message display, image display, and other functions.

Molecular approaches to Taenia asiatica.

PubMed

Jeon, Hyeong-Kyu; Eom, Keeseon S

2013-02-01

Taenia solium, T. saginata, and T. asiatica are taeniid tapeworms that cause taeniasis in humans and cysticercosis in intermediate host animals. Taeniases remain an important public health concerns in the world. Molecular diagnostic methods using PCR assays have been developed for rapid and accurate detection of human infecting taeniid tapeworms, including the use of sequence-specific DNA probes, PCR-RFLP, and multiplex PCR. More recently, DNA diagnosis using PCR based on histopathological specimens such as 10% formalin-fixed paraffin-embedded and stained sections mounted on slides has been applied to cestode infections. The mitochondrial gene sequence is believed to be a very useful molecular marker for not only studying evolutionary relationships among distantly related taxa, but also for investigating the phylo-biogeography of closely related species. The complete sequence of the human Taenia tapeworms mitochondrial genomes were determined, and its organization and structure were compared to other human-tropic Taenia tapeworms for which complete mitochondrial sequence data were available. The multiplex PCR assay with the Ta4978F, Ts5058F, Tso7421F, and Rev7915 primers will be useful for differential diagnosis, molecular characterization, and epidemiological surveys of human Taenia tapeworms.

A novel one-step real-time multiplex PCR assay to detect Streptococcus agalactiae presence and serotypes Ia, Ib, and III.

PubMed

Furfaro, Lucy L; Chang, Barbara J; Payne, Matthew S

2017-09-01

Streptococcus agalactiae is the leading cause of early-onset neonatal sepsis. Culture-based screening methods lack the sensitivity of molecular assays and do not indicate serotype; a potentially important virulence marker. We aimed to develop a multiplex PCR to detect S. agalactiae while simultaneously identifying serotypes Ia, Ib, and III; commonly associated with infant disease. Primers were designed to target S. agalactiae serotype-specific cps genes and the dltS gene. The assay was validated with 512 vaginal specimens from pregnant women. 112 (21.9%) were dltS positive, with 14.3%, 0.9%, and 6.3% of these identified as cps Ia, Ib, and III, respectively. Our assay is a specific and sensitive method to simultaneously detect S. agalactiae and serotypes Ia, Ib, and III in a single step. It is of high significance for clinical diagnostic applications and also provides epidemiological data on serotype, information that may be important for vaccine development and other targeted non-antibiotic therapies. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterization of vaginal Lactobacillus species by rplK -based multiplex qPCR in Russian women.

PubMed

Demkin, Vladimir V; Koshechkin, Stanislav I

2017-10-01

We describe a multiplex qPCR assay for identification and quantitative assessment of a set of vaginal Lactobacillus species, including L. acidophilus, L. crispatus, L. gasseri, L. helveticus, L. iners, and L. jensenii. The assay extends the previously developed qPCR method for Lactobacillus detection and total quantification based on targeting the rplK gene. Both assays use only single pair of primers and a set of probes combined in three reactions, comprising a vaginal Lactobacillus diagnostic assay panel. The utility of the diagnostic panel was evaluated by analyzing of vaginal swab specimens from 145 patients with different status of vaginal health. Most frequently, only one Lactobacillus species was dominant (68,9%), mostly L. crispatus (18,6%) or L. iners (33,1%), but two or three Lactobacillus species were also being simultaneously detected (24,9%). The diagnostic panel will facilitate investigations of the role of Lactobacillus species in the health of the female reproductive system and promote studies of variability of the vaginal microbiota. Copyright © 2017 Elsevier Ltd. All rights reserved.

Fully integrated multiplexed lab-on-a-card assay for enteric pathogens

NASA Astrophysics Data System (ADS)

Weigl, B. H.; Gerdes, J.; Tarr, P.; Yager, P.; Dillman, L.; Peck, R.; Ramachandran, S.; Lemba, M.; Kokoris, M.; Nabavi, M.; Battrell, F.; Hoekstra, D.; Klein, E. J.; Denno, D. M.

2006-01-01

Under this NIH-funded project, we are developing a lab-on-a-card platform to identify enteric bacterial pathogens in patients presenting with acute diarrhea, with special reference to infections that might be encountered in developing countries. Component functions that are integrated on this platform include on-chip immunocapture of live or whole pathogens, multiplexed nucleic acid amplification and on-chip detection, sample processing to support direct use of clinical specimens, and dry reagent storage and handling. All microfluidic functions are contained on the lab card. This new diagnostic test will be able to rapidly identify and differentiate Shigella dysenteriae serotype 1, Shigella toxin-producing Escherichia coli, E. coli 0157, Campylobacter jejuni, and Salmonella and Shigella species. This presentation will report on progress to date on sample and bacteria processing methodologies, identification and validation of capture antibodies and strategy for organism immunocapture, identification and validation of specific polymerase chain reaction (PCR) primer sequences for over 200 clinical isolates of enteric pathogens, and implementation of on-chip nucleic acid extraction for a subset of those pathogens.

Diagnostic Performance of a Multiplex PCR assay for meningitis in an HIV-infected population in Uganda

PubMed Central

Rhein, Joshua; Bahr, Nathan C; Hemmert, Andrew C; Cloud, Joann L; Bellamkonda, Satya; Oswald, Cody; Lo, Eric; Nabeta, Henry; Kiggundu, Reuben; Akampurira, Andrew; Musubire, Abdu; Williams, Darlisha; Meya, David B; Boulware, David R

2015-01-01

Meningitis remains a worldwide problem, and rapid diagnosis is essential to optimize survival. We evaluated the utility of a multiplex PCR test in differentiating possible etiologies of meningitis. Cerebrospinal fluid (CSF) from 69 HIV-infected Ugandan adults with meningitis was collected at diagnosis (n=51) and among persons with cryptococcal meningitis during therapeutic lumbar punctures (n=68). Cryopreserved CSF specimens were analyzed with BioFire FilmArray® Meningitis/Encephalitis panel, which targets 17 pathogens. The panel detected Cryptococcus in the CSF of patients diagnosed with a first-episode of cryptococcal meningitis by fungal culture with 100% sensitivity and specificity, and differentiated between fungal relapse and paradoxical immune reconstitution inflammatory syndrome in recurrent episodes. A negative FilmArray result was predictive of CSF sterility on follow-up lumbar punctures for cryptococcal meningitis. EBV was frequently detected in this immunosuppressed population (n=45). Other pathogens detected included: CMV (n=2), VZV (n=2), HHV-6 (n=1), and Streptococcus pneumoniae (n=1). The FilmArray Meningitis/Encephalitis panel offers a promising platform for rapid meningitis diagnosis. PMID:26711635

Multiplexed and scalable super-resolution imaging of three-dimensional protein localization in size-adjustable tissues.

PubMed

Ku, Taeyun; Swaney, Justin; Park, Jeong-Yoon; Albanese, Alexandre; Murray, Evan; Cho, Jae Hun; Park, Young-Gyun; Mangena, Vamsi; Chen, Jiapei; Chung, Kwanghun

2016-09-01

The biology of multicellular organisms is coordinated across multiple size scales, from the subnanoscale of molecules to the macroscale, tissue-wide interconnectivity of cell populations. Here we introduce a method for super-resolution imaging of the multiscale organization of intact tissues. The method, called magnified analysis of the proteome (MAP), linearly expands entire organs fourfold while preserving their overall architecture and three-dimensional proteome organization. MAP is based on the observation that preventing crosslinking within and between endogenous proteins during hydrogel-tissue hybridization allows for natural expansion upon protein denaturation and dissociation. The expanded tissue preserves its protein content, its fine subcellular details, and its organ-scale intercellular connectivity. We use off-the-shelf antibodies for multiple rounds of immunolabeling and imaging of a tissue's magnified proteome, and our experiments demonstrate a success rate of 82% (100/122 antibodies tested). We show that specimen size can be reversibly modulated to image both inter-regional connections and fine synaptic architectures in the mouse brain.

Enrichment of methylated molecules using enhanced-ice-co-amplification at lower denaturation temperature-PCR (E-ice-COLD-PCR) for the sensitive detection of disease-related hypermethylation.

PubMed

Mauger, Florence; Kernaleguen, Magali; Lallemand, Céline; Kristensen, Vessela N; Deleuze, Jean-François; Tost, Jörg

2018-05-01

The detection of specific DNA methylation patterns bears great promise as biomarker for personalized management of cancer patients. Co-amplification at lower denaturation temperature-PCR (COLD-PCR) assays are sensitive methods, but have previously only been able to analyze loss of DNA methylation. Enhanced (E)-ice-COLD-PCR reactions starting from 2 ng of bisulfite-converted DNA were developed to analyze methylation patterns in two promoters with locked nucleic acid (LNA) probes blocking amplification of unmethylated CpGs. The enrichment of methylated molecules was compared to quantitative (q)PCR and quantified using serial dilutions. E-ice-COLD-PCR allowed the multiplexed enrichment and quantification of methylated DNA. Assays were validated in primary breast cancer specimens and circulating cell-free DNA from cancer patients. E-ice-COLD-PCR could prove a useful tool in the context of DNA methylation analysis for personalized medicine.

Multiplex picodroplet digital PCR to detect KRAS mutations in circulating DNA from the plasma of colorectal cancer patients.

PubMed

Taly, Valerie; Pekin, Deniz; Benhaim, Leonor; Kotsopoulos, Steve K; Le Corre, Delphine; Li, Xinyu; Atochin, Ivan; Link, Darren R; Griffiths, Andrew D; Pallier, Karine; Blons, Hélène; Bouché, Olivier; Landi, Bruno; Hutchison, J Brian; Laurent-Puig, Pierre

2013-12-01

Multiplex digital PCR (dPCR) enables noninvasive and sensitive detection of circulating tumor DNA with performance unachievable by current molecular-detection approaches. Furthermore, picodroplet dPCR facilitates simultaneous screening for multiple mutations from the same sample. We investigated the utility of multiplex dPCR to screen for the 7 most common mutations in codons 12 and 13 of the KRAS (Kirsten rat sarcoma viral oncogene homolog) oncogene from plasma samples of patients with metastatic colorectal cancer. Fifty plasma samples were tested from patients for whom the primary tumor biopsy tissue DNA had been characterized by quantitative PCR. Tumor characterization revealed that 19 patient tumors had KRAS mutations. Multiplex dPCR analysis of the plasma DNA prepared from these samples identified 14 samples that matched the mutation identified in the tumor, 1 sample contained a different KRAS mutation, and 4 samples had no detectable mutation. Among the tumor samples that were wild type for KRAS, 2 KRAS mutations were identified in the corresponding plasma samples. Duplex dPCR (i.e., wild-type and single-mutation assay) was also used to analyze plasma samples from patients with KRAS-mutated tumors and 5 samples expected to contain the BRAF (v-raf murine sarcoma viral oncogene homolog B) V600E mutation. The results for the duplex analysis matched those for the multiplex analysis for KRAS-mutated samples and, owing to its higher sensitivity, enabled detection of 2 additional samples with low levels of KRAS-mutated DNA. All 5 samples with BRAF mutations were detected. This work demonstrates the clinical utility of multiplex dPCR to screen for multiple mutations simultaneously with a sensitivity sufficient to detect mutations in circulating DNA obtained by noninvasive blood collection.

Natural history of polyomaviruses in men: the HPV infection in men (HIM) study.

PubMed

Hampras, Shalaka S; Giuliano, Anna R; Lin, Hui-Yi; Fisher, Kate J; Abrahamsen, Martha E; McKay-Chopin, Sandrine; Gheit, Tarik; Tommasino, Massimo; Rollison, Dana E

2015-05-01

Several new polyomaviruses have been discovered in the last decade, including Merkel cell polyomavirus (MCPyV). Little is known about the natural history of the more recently discovered polyomaviruses. We estimated the incidence, prevalence, and persistence of 9 polyomaviruses (MCPyV, BK polyomavirus, KI polyomavirus, JC polyomavirus, WU polyomavirus, Human polyomavirus 6 [HPyV6], HPyV7, HPyV9, and Trichodysplasia spinulosa-associated polyomavirus) and examined factors associated with MCPyV infection in a prospective cohort of 209 men initially enrolled in the HPV Infection in Men (HIM) study. Participants enrolled at the US site of the HIM study were recruited into a substudy of cutaneous viral infections and followed for a median of 12.6 months. Eyebrow hair and normal skin swab specimens were obtained at each study visit, and the viral DNA load was measured using multiplex polymerase chain reaction. MCPyV infection showed the highest prevalence (65.1% of normal skin swab specimens and 30.6% of eyebrow hair specimens), incidence (81.7 cases per 1000 person-months among normal skin swab specimens, and 24.1 cases per 1000 person-months among eyebrow hair specimens), and persistence (85.8% of normal skin swab specimens and 58.9% of eyebrow hair specimens) among all polyomaviruses examined. Age of >44 years (odds ratio [OR], 2.11; 95% confidence interval [CI], 1.03-4.33) and Hispanic race (OR, 2.64; 95% CI, 1.01-6.88) were associated with an increased prevalence of MCPyV infection in eyebrow hair and normal skin swab specimens, respectively. MCPyV infection is highly prevalent in adults, with age and race being predisposing factors. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Molecular analysis and test of linkage between the FMR-I gene and infantile autism in multiplex families

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hallmayer, J.; Pintado, E.; Lotspeich, L.

Approximately 2%-5% of autistic children show cytogenetic evidence of the fragile X syndrome. This report tests whether infantile autism in multiplex autism families arises from an unusual manifestion of the fragile X syndrome. This could arise either by expansion of the (CGG)n trinucleotide repeat in FMR-1 or from a mutation elsewhere in the gene. We studied 35 families that met stringent criteria for multiplex autism. Amplification of the trinucleotide repeat and analysis of methylation status were performed in 79 autistic children and in 31 of their unaffected siblings by Southern blot analysis. No examples of amplified repeats were seen inmore » the autistic or control children or in their parents or grandparents. We next examined the hypothesis that there was a mutation elsewhere in the FMR-1 gene, by linkage analysis in 32 of these families. We tested four different dominant models and a recessive model. Linkage to FMR-1 could be excluded (lod score between -24 and -62) in all models by using probes DXS548, FRAXAC1, and FRAXAC2 and the CGG repeat itself. Tests for heterogeneity in this sample were negative, and the occurrence of positive lod scores in this data set could be attributed to chance. Analysis of the data by the affected-sib method also did not show evidence for linkage of any marker to autism. These results enable us to reject the hypothesis that multiplex autism arises from expansion of the (CGG)n trinucleotide repeat in FMR-1. Further, because the overall lod scores for all probes in all models tested were highly negative, linkage to FMR-1 can also be ruled out in multiplex autistic families. 35 refs., 2 figs., 5 tabs.« less

Immunization of Epidemics in Multiplex Networks

PubMed Central

Zhao, Dawei; Wang, Lianhai; Li, Shudong; Wang, Zhen; Wang, Lin; Gao, Bo

2014-01-01

Up to now, immunization of disease propagation has attracted great attention in both theoretical and experimental researches. However, vast majority of existing achievements are limited to the simple assumption of single layer networked population, which seems obviously inconsistent with recent development of complex network theory: each node could possess multiple roles in different topology connections. Inspired by this fact, we here propose the immunization strategies on multiplex networks, including multiplex node-based random (targeted) immunization and layer node-based random (targeted) immunization. With the theory of generating function, theoretical analysis is developed to calculate the immunization threshold, which is regarded as the most critical index for the effectiveness of addressed immunization strategies. Interestingly, both types of random immunization strategies show more efficiency in controlling disease spreading on multiplex Erdös-Rényi (ER) random networks; while targeted immunization strategies provide better protection on multiplex scale-free (SF) networks. PMID:25401755

Immunization of epidemics in multiplex networks.

PubMed

Zhao, Dawei; Wang, Lianhai; Li, Shudong; Wang, Zhen; Wang, Lin; Gao, Bo

2014-01-01

Up to now, immunization of disease propagation has attracted great attention in both theoretical and experimental researches. However, vast majority of existing achievements are limited to the simple assumption of single layer networked population, which seems obviously inconsistent with recent development of complex network theory: each node could possess multiple roles in different topology connections. Inspired by this fact, we here propose the immunization strategies on multiplex networks, including multiplex node-based random (targeted) immunization and layer node-based random (targeted) immunization. With the theory of generating function, theoretical analysis is developed to calculate the immunization threshold, which is regarded as the most critical index for the effectiveness of addressed immunization strategies. Interestingly, both types of random immunization strategies show more efficiency in controlling disease spreading on multiplex Erdös-Rényi (ER) random networks; while targeted immunization strategies provide better protection on multiplex scale-free (SF) networks.

Loop-Mediated Isothermal Amplification Label-Based Gold Nanoparticles Lateral Flow Biosensor for Detection of Enterococcus faecalis and Staphylococcus aureus

PubMed Central

Wang, Yi; Li, Hui; Wang, Yan; Zhang, Lu; Xu, Jianguo; Ye, Changyun

2017-01-01

The report describes a simple, rapid and sensitive assay for visual and multiplex detection of Enterococcus faecalis and Staphylococcus aureus based on multiple loop-mediated isothermal amplification (mLAMP) and lateral flow biosensor (LFB). Detection and differentiation of the Ef0027 gene (E. faecalis-specific gene) and nuc gene (S. aureus-specific gene) were determined using fluorescein (FITC)-and digoxin-modified primers in the mLAMP process. In the presence of biotin- and FITC-/digoxin-modified primers, the mLAMP yielded numerous biotin- and FITC-/digoxin-attached duplex products, which were detected by LFB through biotin/streptavidin interaction (biotin on the duplex and streptavidin on the gold nanoparticle) and immunoreactions (FITC/digoxin on the duplex and anti-FITC/digoxin on the LFB test line). The accumulation of gold nanoparticles generated a characteristic red line, enabling visual and multiplex detection of target pathogens without instrumentation. The limit of detection (LoD), analytical specificity and feasibility of LAMP-LFB technique were successfully examined in pure culture and blood samples. The entire procedure, including specimen (blood samples) processing (30 min), isothermal reaction (40 min) and result reporting (within 2 min), could be completed within 75 min. Thus, this assay offers a simple, rapid, sensitive and specific test for multiplex detection of E. faecalis and S. aureus strains. Furthermore, the LAMP-LFB strategy is a universal technique, which can be extended to detect various target sequences by re-designing the specific LAMP primers. PMID:28239371

Establishment of a Molecular Serotyping Scheme and a Multiplexed Luminex-Based Array for Enterobacter aerogenes

PubMed Central

Guo, Xi; Wang, Min; Wang, Lu; Wang, Yao; Chen, Tingting; Wu, Pan; Chen, Min; Liu, Bin; Feng, Lu

2018-01-01

Serotyping based on surface polysaccharide antigens is important for the clinical detection and epidemiological surveillance of pathogens. Polysaccharide gene clusters (PSgcs) are typically responsible for the diversity of bacterial surface polysaccharides. Through whole-genome sequencing and analysis, eight putative PSgc types were identified in 23 Enterobacter aerogenes strains from several geographic areas, allowing us to present the first molecular serotyping system for E. aerogenes. A conventional antigenic scheme was also established and correlated well with the molecular serotyping system that was based on PSgc genetic variation, indicating that PSgc-based molecular typing and immunological serology provide equally valid results. Further, a multiplex Luminex-based array was developed, and a double-blind test was conducted with 97 clinical specimens from Shanghai, China, to validate our array. The results of these analyses indicated that strains containing PSgc4 and PSgc7 comprised the predominant groups. We then examined 86 publicly available E. aerogenes strain genomes and identified an additional seven novel PSgc types, with PSgc10 being the most abundant type. In total, our study identified 15 PSgc types in E. aerogenes, providing the basis for a molecular serotyping scheme. From these results, differing epidemic patterns were identified between strains that were predominant in different regions. Our study highlights the feasibility and reliability of a serotyping system based on PSgc diversity, and for the first time, presents a molecular serotyping system, as well as an antigenic scheme for E. aerogenes, providing the basis for molecular diagnostics and epidemiological surveillance of this important emerging pathogen. PMID:29616012

Establishment of a Molecular Serotyping Scheme and a Multiplexed Luminex-Based Array for Enterobacter aerogenes.

PubMed

Guo, Xi; Wang, Min; Wang, Lu; Wang, Yao; Chen, Tingting; Wu, Pan; Chen, Min; Liu, Bin; Feng, Lu

2018-01-01

Serotyping based on surface polysaccharide antigens is important for the clinical detection and epidemiological surveillance of pathogens. Polysaccharide gene clusters (PSgcs) are typically responsible for the diversity of bacterial surface polysaccharides. Through whole-genome sequencing and analysis, eight putative PSgc types were identified in 23 Enterobacter aerogenes strains from several geographic areas, allowing us to present the first molecular serotyping system for E. aerogenes . A conventional antigenic scheme was also established and correlated well with the molecular serotyping system that was based on PSgc genetic variation, indicating that PSgc-based molecular typing and immunological serology provide equally valid results. Further, a multiplex Luminex-based array was developed, and a double-blind test was conducted with 97 clinical specimens from Shanghai, China, to validate our array. The results of these analyses indicated that strains containing PSgc4 and PSgc7 comprised the predominant groups. We then examined 86 publicly available E. aerogenes strain genomes and identified an additional seven novel PSgc types, with PSgc10 being the most abundant type. In total, our study identified 15 PSgc types in E. aerogenes , providing the basis for a molecular serotyping scheme. From these results, differing epidemic patterns were identified between strains that were predominant in different regions. Our study highlights the feasibility and reliability of a serotyping system based on PSgc diversity, and for the first time, presents a molecular serotyping system, as well as an antigenic scheme for E. aerogenes , providing the basis for molecular diagnostics and epidemiological surveillance of this important emerging pathogen.

Evaluation of Several Biochemical and Molecular Techniques for Identification of Streptococcus pneumoniae and Streptococcus pseudopneumoniae and Their Detection in Respiratory Samples

PubMed Central

Schelfaut, Jacqueline J. G.; Bernards, Alexandra T.; Claas, Eric C. J.

2012-01-01

The identification and detection of mitis group streptococci, which contain Streptococcus pneumoniae, have been hampered by the lack of sensitive and specific assays. In this study, we evaluated several biochemical and molecular assays for the identification of S. pneumoniae and Streptococcus pseudopneumoniae and their distinction from other mitis group streptococci using a collection of 54 isolates obtained by the routine culturing of 53 respiratory specimens from patients with community-acquired pneumonia. The combined results of the biochemical and molecular assays indicated the presence of 23 S. pneumoniae, 2 S. pseudopneumoniae, and 29 other mitis group streptococcal isolates. The tube bile solubility test that is considered gold standard for the identification of S. pneumoniae showed concordant results with optochin susceptibility testing (CO2 atmosphere) and a real-time multiplex PCR assay targeting the Spn9802 fragment and the autolysin gene. Optochin susceptibility testing upon incubation in an O2 atmosphere, bile solubility testing by oxgall disk, matrix-assisted laser desorption ionization–time of flight mass spectrometry, and sequence analysis of the tuf and rpoB genes resulted in several false-positive, false-negative, or inconclusive results. The S. pseudopneumoniae isolates could be identified only by molecular assays, and the multiplex real-time PCR assay was concluded to be most convenient for the identification of S. pneumoniae and S. pseudopneumoniae isolates. Using this method, S. pneumoniae and S. pseudopneumoniae DNA could be detected in the respiratory samples from which they were isolated and in an additional 11 samples from which only other streptococci were isolated. PMID:22278834

Prediction of Response to Preoperative Chemoradiotherapy in Rectal Cancer by Multiplex Kinase Activity Profiling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Folkvord, Sigurd; Flatmark, Kjersti; Department of Cancer and Surgery, Norwegian Radium Hospital, Oslo University Hospital

2010-10-01

Purpose: Tumor response of rectal cancer to preoperative chemoradiotherapy (CRT) varies considerably. In experimental tumor models and clinical radiotherapy, activity of particular subsets of kinase signaling pathways seems to predict radiation response. This study aimed to determine whether tumor kinase activity profiles might predict tumor response to preoperative CRT in locally advanced rectal cancer (LARC). Methods and Materials: Sixty-seven LARC patients were treated with a CRT regimen consisting of radiotherapy, fluorouracil, and, where possible, oxaliplatin. Pretreatment tumor biopsy specimens were analyzed using microarrays with kinase substrates, and the resulting substrate phosphorylation patterns were correlated with tumor response to preoperative treatmentmore » as assessed by histomorphologic tumor regression grade (TRG). A predictive model for TRG scores from phosphosubstrate signatures was obtained by partial-least-squares discriminant analysis. Prediction performance was evaluated by leave-one-out cross-validation and use of an independent test set. Results: In the patient population, 73% and 15% were scored as good responders (TRG 1-2) or intermediate responders (TRG 3), whereas 12% were assessed as poor responders (TRG 4-5). In a subset of 7 poor responders and 12 good responders, treatment outcome was correctly predicted for 95%. Application of the prediction model on the remaining patient samples resulted in correct prediction for 85%. Phosphosubstrate signatures generated by poor-responding tumors indicated high kinase activity, which was inhibited by the kinase inhibitor sunitinib, and several discriminating phosphosubstrates represented proteins derived from signaling pathways implicated in radioresistance. Conclusions: Multiplex kinase activity profiling may identify functional biomarkers predictive of tumor response to preoperative CRT in LARC.« less

Multiplexed and Microparticle-based Analyses: Quantitative Tools for the Large-Scale Analysis of Biological Systems

PubMed Central

Nolan, John P.; Mandy, Francis

2008-01-01

While the term flow cytometry refers to the measurement of cells, the approach of making sensitive multiparameter optical measurements in a flowing sample stream is a very general analytical approach. The past few years have seen an explosion in the application of flow cytometry technology for molecular analysis and measurements using micro-particles as solid supports. While microsphere-based molecular analyses using flow cytometry date back three decades, the need for highly parallel quantitative molecular measurements that has arisen from various genomic and proteomic advances has driven the development in particle encoding technology to enable highly multiplexed assays. Multiplexed particle-based immunoassays are now common place, and new assays to study genes, protein function, and molecular assembly. Numerous efforts are underway to extend the multiplexing capabilities of microparticle-based assays through new approaches to particle encoding and analyte reporting. The impact of these developments will be seen in the basic research and clinical laboratories, as well as in drug development. PMID:16604537

MiniX-STR multiplex system population study in Japan and application to degraded DNA analysis.

PubMed

Asamura, H; Sakai, H; Kobayashi, K; Ota, M; Fukushima, H

2006-05-01

We sought to evaluate a more effective system for analyzing X-chromosomal short tandem repeats (X-STRs) in highly degraded DNA. To generate smaller amplicon lengths, we designed new polymerase chain reaction (PCR) primers for DXS7423, DXS6789, DXS101, GATA31E08, DXS8378, DXS7133, DXS7424, and GATA165B12 at X-linked short tandem repeat (STR) loci, devising two miniX-multiplex PCR systems. Among 333 Japanese individuals, these X-linked loci were detected in amplification products ranging in length from 76 to 169 bp, and statistical analyses of the eight loci indicated a high usefulness for the Japanese forensic practice. Results of tests on highly degraded DNA indicated the miniX-STR multiplex strategies to be an effective system for analyzing degraded DNA. We conclude that analysis by the current miniX-STR multiplex systems offers high effectiveness for personal identification from degraded DNA samples.

Mapping Multiplex Hubs in Human Functional Brain Networks

PubMed Central

De Domenico, Manlio; Sasai, Shuntaro; Arenas, Alex

2016-01-01

Typical brain networks consist of many peripheral regions and a few highly central ones, i.e., hubs, playing key functional roles in cerebral inter-regional interactions. Studies have shown that networks, obtained from the analysis of specific frequency components of brain activity, present peculiar architectures with unique profiles of region centrality. However, the identification of hubs in networks built from different frequency bands simultaneously is still a challenging problem, remaining largely unexplored. Here we identify each frequency component with one layer of a multiplex network and face this challenge by exploiting the recent advances in the analysis of multiplex topologies. First, we show that each frequency band carries unique topological information, fundamental to accurately model brain functional networks. We then demonstrate that hubs in the multiplex network, in general different from those ones obtained after discarding or aggregating the measured signals as usual, provide a more accurate map of brain's most important functional regions, allowing to distinguish between healthy and schizophrenic populations better than conventional network approaches. PMID:27471443

Dynamical Interplay between Awareness and Epidemic Spreading in Multiplex Networks

NASA Astrophysics Data System (ADS)

Granell, Clara; Gómez, Sergio; Arenas, Alex

2013-09-01

We present the analysis of the interrelation between two processes accounting for the spreading of an epidemic, and the information awareness to prevent its infection, on top of multiplex networks. This scenario is representative of an epidemic process spreading on a network of persistent real contacts, and a cyclic information awareness process diffusing in the network of virtual social contacts between the same individuals. The topology corresponds to a multiplex network where two diffusive processes are interacting affecting each other. The analysis using a microscopic Markov chain approach reveals the phase diagram of the incidence of the epidemics and allows us to capture the evolution of the epidemic threshold depending on the topological structure of the multiplex and the interrelation with the awareness process. Interestingly, the critical point for the onset of the epidemics has a critical value (metacritical point) defined by the awareness dynamics and the topology of the virtual network, from which the onset increases and the epidemics incidence decreases.

Dynamical interplay between awareness and epidemic spreading in multiplex networks.

PubMed

Granell, Clara; Gómez, Sergio; Arenas, Alex

2013-09-20

We present the analysis of the interrelation between two processes accounting for the spreading of an epidemic, and the information awareness to prevent its infection, on top of multiplex networks. This scenario is representative of an epidemic process spreading on a network of persistent real contacts, and a cyclic information awareness process diffusing in the network of virtual social contacts between the same individuals. The topology corresponds to a multiplex network where two diffusive processes are interacting affecting each other. The analysis using a microscopic Markov chain approach reveals the phase diagram of the incidence of the epidemics and allows us to capture the evolution of the epidemic threshold depending on the topological structure of the multiplex and the interrelation with the awareness process. Interestingly, the critical point for the onset of the epidemics has a critical value (metacritical point) defined by the awareness dynamics and the topology of the virtual network, from which the onset increases and the epidemics incidence decreases.

RaPToRS Sample Delivery System

NASA Astrophysics Data System (ADS)

Henchen, Robert; Shibata, Kye; Krieger, Michael; Pogozelski, Edward; Padalino, Stephen; Glebov, Vladimir; Sangster, Craig

2010-11-01

At various labs (NIF, LLE, NRL), activated material samples are used to measure reaction properties. The Rapid Pneumatic Transport of Radioactive Samples (RaPToRS) system quickly and safely moves these radioactive samples through a closed PVC tube via airflow. The carrier travels from the reaction chamber to the control and analysis station, pneumatically braking at the outlet. A reversible multiplexer routes samples from various locations near the shot chamber to the analysis station. Also, the multiplexer allows users to remotely load unactivated samples without manually approaching the reaction chamber. All elements of the system (pneumatic drivers, flow control valves, optical position sensors, multiplexers, Geiger counters, and release gates at the analysis station) can be controlled manually or automatically using a custom LabVIEW interface. A prototype is currently operating at NRL in Washington DC. Prospective facilities for Raptors systems include LLE and NIF.

Laboratory Tests of Multiplex Detection of PCR Amplicons Using the Luminex 100 Flow Analyzer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Venkateswaran, K.S.; Nasarabadi, S.; Langlois, R.G.

2000-05-05

Lawrence Livermore National Laboratory (LLNL) demonstrated the power of flow cytometry in detecting the biological agents simulants at JFT III. LLNL pioneered in the development of advanced nucleic acid analyzer (ANM) for portable real time identification. Recent advances in flow cytometry provide a means for multiplexed nucleic acid detection and immunoassay of pathogenic microorganisms. We are presently developing multiplexed immunoassays for the simultaneous detection of different simulants. Our goal is to build an integrated instrument for both nucleic acid analysis and immuno detection. In this study we evaluated the Luminex LX 100 for concurrent identification of more than one PCRmore » amplified product. ANAA has real-time Taqman fluorescent detection capability for rapid identification of field samples. However, its multiplexing ability is limited by the combination of available fluorescent labels. Hence integration of ANAA with flow cytometry can give the rapidity of ANAA amplification and the multiplex capability of flow cytometry. Multiplexed flow cytometric analysis is made possible using a set of fluorescent latex microsphere that are individually identified by their red and infrared fluorescence. A green fluorochrome is used as the assay signal. Methods were developed for the identification of specific nucleic acid sequences from Bacillus globigii (Bg), Bacillus thuringensis (Bt) and Erwinia herbicola (Eh). Detection sensitivity using different reporter fluorochromes was tested with the LX 100, and also different assay formats were evaluated for their suitability for rapid testing. A blind laboratory trial was carried out December 22-27, 1999 to evaluate bead assays for multiplex identification of Bg and Bt PCR products. This report summarizes the assay development, fluorochrome comparisons, and the results of the blind trial conducted at LLNL for the laboratory evaluation of the LX 100 flow analyzer.« less

The new challenges of multiplex networks: Measures and models

NASA Astrophysics Data System (ADS)

Battiston, Federico; Nicosia, Vincenzo; Latora, Vito

2017-02-01

What do societies, the Internet, and the human brain have in common? They are all examples of complex relational systems, whose emerging behaviours are largely determined by the non-trivial networks of interactions among their constituents, namely individuals, computers, or neurons, rather than only by the properties of the units themselves. In the last two decades, network scientists have proposed models of increasing complexity to better understand real-world systems. Only recently we have realised that multiplexity, i.e. the coexistence of several types of interactions among the constituents of a complex system, is responsible for substantial qualitative and quantitative differences in the type and variety of behaviours that a complex system can exhibit. As a consequence, multilayer and multiplex networks have become a hot topic in complexity science. Here we provide an overview of some of the measures proposed so far to characterise the structure of multiplex networks, and a selection of models aiming at reproducing those structural properties and quantifying their statistical significance. Focusing on a subset of relevant topics, this brief review is a quite comprehensive introduction to the most basic tools for the analysis of multiplex networks observed in the real-world. The wide applicability of multiplex networks as a framework to model complex systems in different fields, from biology to social sciences, and the colloquial tone of the paper will make it an interesting read for researchers working on both theoretical and experimental analysis of networked systems.

A Multiplex Snapback Primer System for the Enrichment and Detection of JAK2 V617F and MPL W515L/K Mutations in Philadelphia-Negative Myeloproliferative Neoplasms

PubMed Central

Zhang, Yunqing; Zhang, Xinju; Xu, Xiao; Kang, Zhihua; Li, Shibao; Zhang, Chen; Su, Bing

2014-01-01

A multiplex snapback primer system was developed for the simultaneous detection of JAK2 V617F and MPL W515L/K mutations in Philadelphia chromosome- (Ph-) negative myeloproliferative neoplasms (MPNs). The multiplex system comprises two snapback versus limiting primer sets for JAK2 and MPL mutation enrichment and detection, respectively. Linear-After exponential (LATE) PCR strategy was employed for the primer design to maximize the amplification efficiency of the system. Low ionic strength buffer and rapid PCR protocol allowed for selective amplification of the mutant alleles. Amplification products were analyzed by melting curve analysis for mutation identification. The multiplex system archived 0.1% mutation load sensitivity and <5% coefficient of variation inter-/intra-assay reproducibility. 120 clinical samples were tested by the multiplex snapback primer assay, and verified with amplification refractory system (ARMS), quantitative PCR (qPCR) and Sanger sequencing method. The multiplex system, with a favored versatility, provided the molecular diagnosis of Ph-negative MPNs with a suitable implement and simplified the genetic test process. PMID:24729973

Helicase dependent OnChip-amplification and its use in multiplex pathogen detection.

PubMed

Andresen, Dennie; von Nickisch-Rosenegk, Markus; Bier, Frank F

2009-05-01

The need for fast, specific and sensitive multiparametric detection methods is an ever growing demand in molecular diagnostics. Here we report on a newly developed method, the helicase dependent OnChip amplification (OnChip-HDA). This approach integrates the analysis and detection in one single reaction thus leading to time and cost savings in multiparametric analysis. HDA is an isothermal amplification method that is not depending on thermocycling as known from PCR due to the helicases' ability to unwind DNA double-strands. We have combined the HDA with microarray based detection, making it suitable for multiplex detection. As an example we used the OnChip HDA in single and multiplex amplifications for the detection of the two pathogens N. gonorrhoeae and S. aureus directly on surface bound primers. We have successfully shown the OnChip-HDA and applied it for single- and duplex-detection of the pathogens N. gonorrhoeae and S. aureus. We have developed a new method, the OnChip-HDA for the multiplex detection of pathogens. Its simplicity in reaction setup and potential for miniaturization and multiparametric analysis is advantageous for the integration in miniaturized Lab on Chip systems, e.g. needed in point of care diagnostics.

Developing a Multiplexed Quantitative Cross-Linking Mass Spectrometry Platform for Comparative Structural Analysis of Protein Complexes.

PubMed

Yu, Clinton; Huszagh, Alexander; Viner, Rosa; Novitsky, Eric J; Rychnovsky, Scott D; Huang, Lan

2016-10-18

Cross-linking mass spectrometry (XL-MS) represents a recently popularized hybrid methodology for defining protein-protein interactions (PPIs) and analyzing structures of large protein assemblies. In particular, XL-MS strategies have been demonstrated to be effective in elucidating molecular details of PPIs at the peptide resolution, providing a complementary set of structural data that can be utilized to refine existing complex structures or direct de novo modeling of unknown protein structures. To study structural and interaction dynamics of protein complexes, quantitative cross-linking mass spectrometry (QXL-MS) strategies based on isotope-labeled cross-linkers have been developed. Although successful, these approaches are mostly limited to pairwise comparisons. In order to establish a robust workflow enabling comparative analysis of multiple cross-linked samples simultaneously, we have developed a multiplexed QXL-MS strategy, namely, QMIX (Quantitation of Multiplexed, Isobaric-labeled cross (X)-linked peptides) by integrating MS-cleavable cross-linkers with isobaric labeling reagents. This study has established a new analytical platform for quantitative analysis of cross-linked peptides, which can be directly applied for multiplexed comparisons of the conformational dynamics of protein complexes and PPIs at the proteome scale in future studies.

First ancient DNA sequences from the Late Pleistocene red deer (Cervus elaphus) in the Crimea, Ukraine

NASA Astrophysics Data System (ADS)

Stanković, Ana; Nadachowski, Adam; Doan, Karolina; Stefaniak, Krzysztof; Baca, Mateusz; Socha, Paweł; Wegleński, Piotr; Ridush, Bogdan

2010-05-01

The Late Pleistocene has been a period of significant population and species turnover and extinctions among the large mammal fauna. Massive climatic and environmental changes during Pleistocene significantly influenced the distribution and also genetic diversity of plants and animals. The model of glacial refugia and habitat contraction to southern peninsulas in Europe as areas for the survival of temperate animal species during unfavourable Pleistocene glaciations is at present widely accepted. However, both molecular data and the fossil record indicate the presence of northern and perhaps north-eastern refugia in Europe. In recent years, much new palaeontological data have been obtained in the Crimean Peninsula, Ukraine, following extensive investigations. The red deer (Cervus elaphus) samples for aDNA studies were collected in Emine-Bair-Khosar Cave, situated on the north edge of Lower Plateau of the Chatyrdag Massif (Crimean Mountains). The cave is a vertical shaft, which functioned as a huge mega-trap over a long period of time (probably most of the Pleistocene). The bone assemblages provided about 5000 bones belonging to more than 40 species. The C. elaphus bones were collected from three different stratigraphical levels, radiocarbon dated by accelerator mass spectrometry (AMS) method. The bone fragments of four specimens of red deer were used for the DNA isolation and analysis. The mtDNA (Cytochome b) was successfully isolated from three bone fragments and the cytochrome b sequences were amplified by multiplex PCR. The sequences obtained so far allowed for the reconstruction of only preliminary phylogenetic trees. A fragment of metatarsus from level dated to ca. 48,500±2,000 years BP, yielded a sequence of 513 bp, allowing to locate the specimen on the phylogenetic tree within modern C. elaphus specimens from southern and middle Europe. The second bone fragment, a fragment of mandible, collected from level dated approximately to ca. 33,500±400 years BP, yielded a sequence (696 bp) locating this specimen much closer to the modern C. elaphus specimens from China and Far East. From the third bone fragment (metatarsus), dated between ca. 12,000 years BP and 30,000 years BP, the sequence of only 346 bp has been obtained. It locates this specimen between European and Asiatic haplogroups. The preliminary results of analysis of the DNA from Crimean C. elaphus fossils reveal the great genetic heterogeneity and a complex phylogeographical pattern of the material studied. The obtained results support the opinion that Crimean Peninsula was the most north-eastern refugium in Europe during Late Pleistocene playing a major role in recolonization and dispersal processes of temperate species during and after the Late Pleistocene in this part of the Euro-Asian continent.

East Asian mtDNA haplogroup determination in Koreans: haplogroup-level coding region SNP analysis and subhaplogroup-level control region sequence analysis.

PubMed

Lee, Hwan Young; Yoo, Ji-Eun; Park, Myung Jin; Chung, Ukhee; Kim, Chong-Youl; Shin, Kyoung-Jin

2006-11-01

The present study analyzed 21 coding region SNP markers and one deletion motif for the determination of East Asian mitochondrial DNA (mtDNA) haplogroups by designing three multiplex systems which apply single base extension methods. Using two multiplex systems, all 593 Korean mtDNAs were allocated into 15 haplogroups: M, D, D4, D5, G, M7, M8, M9, M10, M11, R, R9, B, A, and N9. As the D4 haplotypes occurred most frequently in Koreans, the third multiplex system was used to further define D4 subhaplogroups: D4a, D4b, D4e, D4g, D4h, and D4j. This method allowed the complementation of coding region information with control region mutation motifs and the resultant findings also suggest reliable control region mutation motifs for the assignment of East Asian mtDNA haplogroups. These three multiplex systems produce good results in degraded samples as they contain small PCR products (101-154 bp) for single base extension reactions. SNP scoring was performed in 101 old skeletal remains using these three systems to prove their utility in degraded samples. The sequence analysis of mtDNA control region with high incidence of haplogroup-specific mutations and the selective scoring of highly informative coding region SNPs using the three multiplex systems are useful tools for most applications involving East Asian mtDNA haplogroup determination and haplogroup-directed stringent quality control.

Highly Sensitive Multiplex Assay for Detection of Human Immunodeficiency Virus Type 1 and Hepatitis C Virus RNA

PubMed Central

Giachetti, C.; Linnen, J. M.; Kolk, D. P.; Dockter, J.; Gillotte-Taylor, K.; Park, M.; Ho-Sing-Loy, M.; McCormick, M. K.; Mimms, L. T.; McDonough, S. H.

2002-01-01

Various nucleic acid assays have been developed and implemented for diagnostics and therapeutic monitoring of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infections. The high-throughput, semiautomated assays described here were developed to provide a method suitable for screening plasma specimens for the presence of HIV-1 and HCV RNAs. Three assays were developed: a multiplex HIV-1/HCV assay for simultaneous detection of HIV-1 and HCV, and discriminatory assays for specific detection of HIV-1 and HCV. The assay systems utilize three proprietary technologies: (i) target capture-based sample preparation, (ii) transcription-mediated amplification (TMA), and (iii) hybridization protection assay (HPA). An internal control is incorporated into each reaction to control for every step of the assay and identify random false-negative reactions. The assays demonstrated a sensitivity of at least 100 copies/ml for each target, and they detected with similar sensitivity all major variants of HCV and HIV-1, including HIV-1 group O strains. Assay sensitivity for one virus was not affected by the presence of the other. The specificity of these TMA-driven assays was ≥99.5% in both normal donor specimens and plasma containing potentially interfering substances or other blood-borne pathogens. Statistical receiver operating characteristic plots of 1 − specificity versus sensitivity data determined very wide analyte cutoff values for each assay at the point at which the assay specificity and sensitivity were both ≥99.5%. The sensitivity, specificity, and throughput capability predict that these assays will be valuable for large-volume plasma screening, either in a blood bank setting or in other diagnostic applications. PMID:12089255

Multiplex method for initial complex testing of antibodies to blood transmitted diseases agents.

PubMed

Poltavchenko, Alexander G; Nechitaylo, Oleg V; Filatov, Pavel V; Ersh, Anna V; Gureyev, Vadim N

2016-10-01

Initial screening of donors and population at high risk of infection with blood transmitted diseases involves a number of analyses using monospesific diagnostic systems, and therefore is expensive labor- and time-consuming process. The goal of this work is to construct a multiplex test enabling to carry out rapid initial complex testing at a low price. The paper describes a kit making it possible to detect simultaneously antibodies to six agents of the most significant blood transmitted diseases: HIV virus, hepatitis B and C viruses, cytomegalovirus, T. pallidum and T. gondii in blood products. The kit comprises multiplex dot-immunoassay based on plane protein arrays (immune chips) using colloidal gold conjugates and silver development. It provides an opportunity to carry out complex analysis within 70min at room temperature, and there is no need of well-qualified personnel. We compared laboratory findings of the kit with monospecific kits for ELISA produced by two Russian commercial companies. Dot-assay results correlate well with data obtained using commercial kits for ELISA. Furthermore, multiplex analysis is quicker and cheaper in comparison with ELISA and can be carried out in non-laboratory conditions. The kit for multiplex dot-immunoassay of antibodies to blood transmitted agents can significantly simplify initial complex testing. Copyright © 2016 Elsevier B.V. All rights reserved.

Detection of inflammatory cytokines using a fiber optic microsphere immunoassay array

NASA Astrophysics Data System (ADS)

Blicharz, Timothy M.; Walt, David R.

2006-10-01

A multiplexed fiber optic microsphere-based immunoassay array capable of simultaneously measuring five inflammatory cytokines has been developed. Five groups of amine-functionalized 3.1 micron microspheres were internally encoded with five distinct concentrations of a europium dye and converted to cytokine probes by covalently coupling monoclonal capture antibodies specific for human VEGF, IFN-gamma, RANTES, IP-10, and Eotaxin-3 to the microspheres via glutaraldehyde chemistry. The microspheres were pooled and loaded into a 1 mm diameter fiber optic bundle containing ~50,000 individual etched microwells, producing the multiplexed cytokine immunoassay array. Multiple arrays can be created from a single microsphere pool for high throughput sample analysis. Sandwich fluoroimmunoassays were performed by incubating the probe array in a sample, followed by incubation in a mixture of biotin-labeled detection antibodies that are complementary to the five cytokines. Finally, universal detection of each protein was performed using a fluorescence imaging system after briefly immersing the array in a solution of fluorophore-labeled streptavidin. The multiplexed cytokine array has been shown to respond selectively to VEGF, IFNgamma, RANTES, IP-10, and Eotaxin-3, permitting multiplexed quantitative analysis. Ultimately, the multiplexed cytokine array will be utilized to evaluate the potential of using saliva as a noninvasive diagnostic fluid for pulmonary inflammatory diseases such as asthma.

Analysis of the Effects of Phase Noise and Frequency Offset in Orthogonal Frequency Division Multiplexing (OFDM) Systems

DTIC Science & Technology

2004-03-01

Data Communication , http://www.iec.org/, last accessed December 2003. 13. Klaus Witrisal, “Orthogonal Frequency Division Multiplexing (OFDM) for...http://ieeexplore.ieee.org, last accessed 26 February 2003. 12. The International Engineering Consortium, Web Forum Tutorials, OFDM for Mobile

Integrated multiplexed capillary electrophoresis system

DOEpatents

Yeung, Edward S.; Tan, Hongdong

2002-05-14

The present invention provides an integrated multiplexed capillary electrophoresis system for the analysis of sample analytes. The system integrates and automates multiple components, such as chromatographic columns and separation capillaries, and further provides a detector for the detection of analytes eluting from the separation capillaries. The system employs multiplexed freeze/thaw valves to manage fluid flow and sample movement. The system is computer controlled and is capable of processing samples through reaction, purification, denaturation, pre-concentration, injection, separation and detection in parallel fashion. Methods employing the system of the invention are also provided.

Salvage microbiology: opportunities and challenges in the detection of bacterial pathogens following initiation of antimicrobial treatment

PubMed Central

Farrell, John J.; Hujer, Andrea M.; Sampath, Rangarajan; Bonomo, Robert A.

2015-01-01

Broad-range 16S ribosomal RNA gene PCR coupled with Sanger sequencing was originally employed by soil scientists and was subsequently adapted for clinical applications. PCR coupled with electrospray ionization mass spectrometry has also progressed from initial applications in the detection of organisms from environmental samples into the clinical realm and has demonstrated promise in detection of pathogens in clinical specimens obtained from patients with suspected infection but negative cultures. We review studies of multiplex PCR, 16S ribosomal RNA gene PCR and sequencing and PCR coupled with electrospray ionization mass spectrometry for detection of bacteria in specimens that were obtained from patients during or after administration of antibiotic treatment, and examine the role of each for assisting in antimicrobial treatment and stewardship efforts. Following an exploration of the available data in this field we discuss the opportunities that the preliminary investigations reveal, as well as the challenges faced with implementation of these strategies in clinical practice. PMID:25523281

Importance of cholera and other etiologies of acute diarrhea in post-earthquake Port-au-Prince, Haiti.

PubMed

Charles, Macarthur; Delva, Glavdia G; Boutin, Jethro; Severe, Karine; Peck, Mireille; Mabou, Marie Marcelle; Wright, Peter F; Pape, Jean W

2014-03-01

We estimated the proportion of diarrhea attributable to cholera and other pathogens during the rainy and dry seasons in patients seen in two urban health settings: a cholera treatment center (CTC) and oral rehydration points (ORPs). During April 1, 2011-November 30, 2012, stool samples were collected from 1,206 of 10,845 patients who came to the GHESKIO CTC or to the community ORPs with acute diarrhea, cultured for Vibrio cholerae, and tested by multiplex polymerase reaction. Vibrio cholerae was isolated from 409 (41.8%, 95% confidence interval [CI] = 38.7-44.9%) of the 979 specimens from the CTC and in 45 (19.8%, 95% CI = 14.8-25.6%) of the 227 specimens from the ORPs. Frequencies varied from 21.4% (95% CI = 16.6-26.7%) during the dry season to 46.8% (95% CI = 42.9-50.7%) in the rainy season. Shigella, enterotoxigenic Escherichia coli, rotavirus, and Cryptosporidium were frequent causes of diarrhea in children less than five years of age.

Mass cytometry: a highly multiplexed single-cell technology for advancing drug development.

PubMed

Atkuri, Kondala R; Stevens, Jeffrey C; Neubert, Hendrik

2015-02-01

Advanced single-cell analysis technologies (e.g., mass cytometry) that help in multiplexing cellular measurements in limited-volume primary samples are critical in bridging discovery efforts to successful drug approval. Mass cytometry is the state-of-the-art technology in multiparametric single-cell analysis. Mass cytometers (also known as cytometry by time-of-flight or CyTOF) combine the cellular analysis principles of traditional fluorescence-based flow cytometry with the selectivity and quantitative power of inductively coupled plasma-mass spectrometry. Standard flow cytometry is limited in the number of parameters that can be measured owing to the overlap in signal when detecting fluorescently labeled antibodies. Mass cytometry uses antibodies tagged to stable isotopes of rare earth metals, which requires minimal signal compensation between the different metal tags. This unique feature enables researchers to seamlessly multiplex up to 40 independent measurements on single cells. In this overview we first present an overview of mass cytometry and compare it with traditional flow cytometry. We then discuss the emerging and potential applications of CyTOF technology in the pharmaceutical industry, including quantitative and qualitative deep profiling of immune cells and their applications in assessing drug immunogenicity, extensive mapping of signaling networks in single cells, cell surface receptor quantification and multiplexed internalization kinetics, multiplexing sample analysis by barcoding, and establishing cell ontologies on the basis of phenotype and/or function. We end with a discussion of the anticipated impact of this technology on drug development lifecycle with special emphasis on the utility of mass cytometry in deciphering a drug's pharmacokinetics and pharmacodynamics relationship. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Clinical cytomics

NASA Astrophysics Data System (ADS)

Tárnok, Attila; Mittag, Anja; Lenz, Dominik

2006-02-01

The goal of predictive medicine is the detection of changes in patient's state prior to the clinical manifestation of the deterioration of the patients current status. Therefore, both the diagnostic of diseases like cancer, coronary atherosclerosis or congenital heart failure and the prognosis of the effect specific therapeutics on patients outcome are the main fields of predictive medicine. Clinical Cytomcs is based on the analysis of specimens from the patient by Cytomic technologies that are mainly imaging based techniques and their combinations with other assays. Predictive medicine aims at the recognition of the "fate" of each individual patients in order to yield unequivocal indications for decision making (i.e. how does the patient respond to therapy, react to medication etc.). This individualized prediction is based on the Predictive Medicine by Clinical Cytomics concept. These considerations have recently stimulated the idea of the Human Cytome Project. A major focus of the Human Cytome Project is multiplexed cy-tomic analysis of individual cells of the patient, extraction of predictive information and individual prediction that merges into individualized therapy. Although still at the beginning, Clinical Cytomics is a promising new field that may change therapy in the near future for the benefit of the patients.

Multiplex social ecological network analysis reveals how social changes affect community robustness more than resource depletion.

PubMed

Baggio, Jacopo A; BurnSilver, Shauna B; Arenas, Alex; Magdanz, James S; Kofinas, Gary P; De Domenico, Manlio

2016-11-29

Network analysis provides a powerful tool to analyze complex influences of social and ecological structures on community and household dynamics. Most network studies of social-ecological systems use simple, undirected, unweighted networks. We analyze multiplex, directed, and weighted networks of subsistence food flows collected in three small indigenous communities in Arctic Alaska potentially facing substantial economic and ecological changes. Our analysis of plausible future scenarios suggests that changes to social relations and key households have greater effects on community robustness than changes to specific wild food resources.

Precoded spatial multiplexing MIMO system with spatial component interleaver.

PubMed

Gao, Xiang; Wu, Zhanji

In this paper, the performance of precoded bit-interleaved coded modulation (BICM) spatial multiplexing multiple-input multiple-output (MIMO) system with spatial component interleaver is investigated. For the ideal precoded spatial multiplexing MIMO system with spatial component interleaver based on singular value decomposition (SVD) of the MIMO channel, the average pairwise error probability (PEP) of coded bits is derived. Based on the PEP analysis, the optimum spatial Q-component interleaver design criterion is provided to achieve the minimum error probability. For the limited feedback precoded proposed scheme with linear zero forcing (ZF) receiver, in order to minimize a bound on the average probability of a symbol vector error, a novel effective signal-to-noise ratio (SNR)-based precoding matrix selection criterion and a simplified criterion are proposed. Based on the average mutual information (AMI)-maximization criterion, the optimal constellation rotation angles are investigated. Simulation results indicate that the optimized spatial multiplexing MIMO system with spatial component interleaver can achieve significant performance advantages compared to the conventional spatial multiplexing MIMO system.

Data acquisition and analysis in the DOE/NASA Wind Energy Program

NASA Technical Reports Server (NTRS)

Neustadter, H. E.

1980-01-01

Four categories of data systems, each responding to a distinct information need are presented. The categories are: control, technology, engineering and performance. The focus is on the technology data system which consists of the following elements: sensors which measure critical parameters such as wind speed and direction, output power, blade loads and strains, and tower vibrations; remote multiplexing units (RMU) mounted on each wind turbine which frequency modulate, multiplex and transmit sensor outputs; the instrumentation available to record, process and display these signals; and centralized computer analysis of data. The RMU characteristics and multiplexing techniques are presented. Data processing is illustrated by following a typical signal through instruments such as the analog tape recorder, analog to digital converter, data compressor, digital tape recorder, video (CRT) display, and strip chart recorder.

A Liquid Array Platform For the Multiplexed Analysis of Synthetic Molecule-Protein Interactions

PubMed Central

Doran, Todd M.; Kodadek, Thomas

2014-01-01

Synthetic molecule microarrays, consisting of many different compounds spotted onto a planar surface such as modified glass or cellulose, have proven to be useful tools for the multiplexed analysis of small molecule- and peptide-protein interactions. However, these arrays are technically difficult to manufacture and use with high reproducibility and require specialized equipment. Here we report a more convenient alternative comprised of color-encoded beads that display a small molecule protein ligand on the surface. Quantitative, multiplexed assay of protein binding to up to 24 different ligands can be achieved using a common flow cytometer for the readout. This technology should be useful for evaluating hits from library screening efforts, the determination of structure activity relationships and for certain types of serological analyses. PMID:24245981

Dynamic multiplexed analysis method using ion mobility spectrometer

DOEpatents

Belov, Mikhail E [Richland, WA

2010-05-18

A method for multiplexed analysis using ion mobility spectrometer in which the effectiveness and efficiency of the multiplexed method is optimized by automatically adjusting rates of passage of analyte materials through an IMS drift tube during operation of the system. This automatic adjustment is performed by the IMS instrument itself after determining the appropriate levels of adjustment according to the method of the present invention. In one example, the adjustment of the rates of passage for these materials is determined by quantifying the total number of analyte molecules delivered to the ion trap in a preselected period of time, comparing this number to the charge capacity of the ion trap, selecting a gate opening sequence; and implementing the selected gate opening sequence to obtain a preselected rate of analytes within said IMS drift tube.

Homo-FRET Based Biosensors and Their Application to Multiplexed Imaging of Signalling Events in Live Cells

PubMed Central

Warren, Sean C.; Margineanu, Anca; Katan, Matilda; Dunsby, Chris; French, Paul M. W.

2015-01-01

Multiplexed imaging of Förster Resonance Energy Transfer (FRET)-based biosensors potentially presents a powerful approach to monitoring the spatio-temporal correlation of signalling pathways within a single live cell. Here, we discuss the potential of homo-FRET based biosensors to facilitate multiplexed imaging. We demonstrate that the homo-FRET between pleckstrin homology domains of Akt (Akt-PH) labelled with mCherry may be used to monitor 3′-phosphoinositide accumulation in live cells and show how global analysis of time resolved fluorescence anisotropy measurements can be used to quantify this accumulation. We further present multiplexed imaging readouts of calcium concentration, using fluorescence lifetime measurements of TN-L15-a CFP/YFP based hetero-FRET calcium biosensor-with 3′-phosphoinositide accumulation. PMID:26133241

High-throughput tandem mass spectrometry multiplex analysis for newborn urinary screening of creatine synthesis and transport disorders, Triple H syndrome and OTC deficiency.

PubMed

Auray-Blais, Christiane; Maranda, Bruno; Lavoie, Pamela

2014-09-25

Creatine synthesis and transport disorders, Triple H syndrome and ornithine transcarbamylase deficiency are treatable inborn errors of metabolism. Early screening of patients was found to be beneficial. Mass spectrometry analysis of specific urinary biomarkers might lead to early detection and treatment in the neonatal period. We developed a high-throughput mass spectrometry methodology applicable to newborn screening using dried urine on filter paper for these aforementioned diseases. A high-throughput methodology was devised for the simultaneous analysis of creatine, guanidineacetic acid, orotic acid, uracil, creatinine and respective internal standards, using both positive and negative electrospray ionization modes, depending on the compound. The precision and accuracy varied by <15%. Stability during storage at different temperatures was confirmed for three weeks. The limits of detection and quantification for each biomarker varied from 0.3 to 6.3 μmol/l and from 1.0 to 20.9 μmol/l, respectively. Analyses of urine specimens from affected patients revealed abnormal results. Targeted biomarkers in urine were detected in the first weeks of life. This rapid, simple and robust liquid chromatography/tandem mass spectrometry methodology is an efficient tool applicable to urine screening for inherited disorders by biochemical laboratories. Copyright © 2014 Elsevier B.V. All rights reserved.

A portable multi-channel recording system for analysis of acceleration and angular velocity in six dimension.

PubMed

Yamashita, M; Yamashita, A; Ishii, T; Naruo, Y; Nagatomo, M

1998-11-01

A portable recording system was developed for analysis of more than three analog signals collected in field works. Stereo audio recorder, available as consumer products, was made use for a core cornponent of the system. For the two tracks of recording, a multiplexed analog signal is stored on one track, and reference code on the other track. The reference code indicates the start of one cycle for multiplexing and swiching point of each channel. Multiplexed signal is playbacked and decoded with a reference of the code to reconstruct original profiles of the signal. Since commercial stereo recorders have cut DC component off, a fixed reference voltage is inserted in the sequence of multiplexing. Change of voltage at switching from the reference to the data channel is measured from playbacked signal to get the original data with its DC component. Movement of vehicles and human head were analyzed by the system. It was verified to be capable to record and analyze multi-channel signal at a sampling rate more than 10Hz.

Direct measurement of beta-agonists in swine hair extract in multiplexed mode by surface-enhanced Raman spectroscopy and microfluidic paper.

PubMed

Dou, Bin; Luo, Yong; Chen, Xu; Shi, Bo; Du, Yuguang; Gao, Zhigang; Zhao, Weijie; Lin, Bingcheng

2015-02-01

Bare gold nanoparticles selectively enhance the Raman signal of beta-agnonists in swine hair extract at 780 nm, which enables analysis of beta-agonists in swine hair extract without chemical labeling, purification, or separation. The analysis is multiplexable and the LOD of beta-agonists is around ng/mL in the assistance of microfluidic paper. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chemiluminescence microarrays in analytical chemistry: a critical review.

PubMed

Seidel, Michael; Niessner, Reinhard

2014-09-01

Multi-analyte immunoassays on microarrays and on multiplex DNA microarrays have been described for quantitative analysis of small organic molecules (e.g., antibiotics, drugs of abuse, small molecule toxins), proteins (e.g., antibodies or protein toxins), and microorganisms, viruses, and eukaryotic cells. In analytical chemistry, multi-analyte detection by use of analytical microarrays has become an innovative research topic because of the possibility of generating several sets of quantitative data for different analyte classes in a short time. Chemiluminescence (CL) microarrays are powerful tools for rapid multiplex analysis of complex matrices. A wide range of applications for CL microarrays is described in the literature dealing with analytical microarrays. The motivation for this review is to summarize the current state of CL-based analytical microarrays. Combining analysis of different compound classes on CL microarrays reduces analysis time, cost of reagents, and use of laboratory space. Applications are discussed, with examples from food safety, water safety, environmental monitoring, diagnostics, forensics, toxicology, and biosecurity. The potential and limitations of research on multiplex analysis by use of CL microarrays are discussed in this review.

Multiplexing a high-throughput liability assay to leverage efficiencies.

PubMed

Herbst, John; Anthony, Monique; Stewart, Jeremy; Connors, David; Chen, Taosheng; Banks, Martyn; Petrillo, Edward W; Agler, Michele

2009-06-01

In order to identify potential cytochrome P-450 3A4 (drug-metabolizing enzyme) inducers at an early stage of the drug discovery process, a cell-based transactivation high-throughput luciferase reporter assay for the human pregnane X receptor (PXR) in HepG2 cells has been implemented and multiplexed with a viability end point for data interpretation, as part of a Lead Profiling portfolio of assays. As a routine part of Lead Profiling operations, assays are periodically evaluated for utility as well as for potential improvements in technology or process. We used a recent evaluation of our PXR-transactivation assay as a model for the application of Lean Thinking-based process analysis to lab-bench assay optimization and automation. This resulted in the development of a 384-well multiplexed homogeneous assay simultaneously detecting PXR transactivation and HepG2 cell cytotoxicity. In order to multiplex fluorescent and luminescent read-outs, modifications to each assay were necessary, which included optimization of multiple assay parameters such as cell density, plate type, and reagent concentrations. Subsequently, a set of compounds including known cytotoxic compounds and PXR inducers were used to validate the multiplexed assay. Results from the multiplexed assay correlate well with those from the singleplexed assay formats measuring PXR transactivation and viability separately. Implementation of the multiplexed assay for routine compound profiling provides improved data quality, sample conservation, cost savings, and resource efficiencies.

Development of a Multiplexed Liquid Chromatography Multiple-Reaction-Monitoring Mass Spectrometry (LC-MRM/MS) Method for Evaluation of Salivary Proteins as Oral Cancer Biomarkers*

PubMed Central

Chen, Hsiao-Wei; Wu, Chun-Feng; Chu, Lichieh Julie; Chiang, Wei-Fang; Wu, Chih-Ching; Yu, Jau-Song; Tsai, Cheng-Han; Liang, Kung-Hao; Chang, Yu-Sun; Wu, Maureen; Ou Yang, Wei-Ting

2017-01-01

Multiple (selected) reaction monitoring (MRM/SRM) of peptides is a growing technology for target protein quantification because it is more robust, precise, accurate, high-throughput, and multiplex-capable than antibody-based techniques. The technique has been applied clinically to the large-scale quantification of multiple target proteins in different types of fluids. However, previous MRM-based studies have placed less focus on sample-preparation workflow and analytical performance in the precise quantification of proteins in saliva, a noninvasively sampled body fluid. In this study, we evaluated the analytical performance of a simple and robust multiple reaction monitoring (MRM)-based targeted proteomics approach incorporating liquid chromatography with mass spectrometry detection (LC-MRM/MS). This platform was used to quantitatively assess the biomarker potential of a group of 56 salivary proteins that have previously been associated with human cancers. To further enhance the development of this technology for assay of salivary samples, we optimized the workflow for salivary protein digestion and evaluated quantification performance, robustness and technical limitations in analyzing clinical samples. Using a clinically well-characterized cohort of two independent clinical sample sets (total n = 119), we quantitatively characterized these protein biomarker candidates in saliva specimens from controls and oral squamous cell carcinoma (OSCC) patients. The results clearly showed a significant elevation of most targeted proteins in saliva samples from OSCC patients compared with controls. Overall, this platform was capable of assaying the most highly multiplexed panel of salivary protein biomarkers, highlighting the clinical utility of MRM in oral cancer biomarker research. PMID:28235782

Multiplexed detection of serological cancer markers with plasmon-enhanced Raman spectro-immunoassay† †Electronic supplementary information (ESI) available: Fig. S1–S7 and Table S1. See DOI: 10.1039/c5sc01054c Click here for additional data file.

PubMed Central

Kang, Jeon Woong; Sukumar, Saraswati; Dasari, Ramachandra Rao

2015-01-01

Circulating biomarkers have emerged as promising non-invasive, real-time surrogates for cancer diagnosis, prognostication and monitoring of therapeutic response. Emerging data, however, suggest that single markers are inadequate in describing complex pathologic transformations. Architecting assays capable of parallel measurements of multiple biomarkers can help achieve the desired clinical sensitivity and specificity while conserving patient specimen and reducing turn-around time. Here we describe a plasmon-enhanced Raman spectroscopic assay featuring nanostructured biomolecular probes and spectroscopic imaging for multiplexed detection of disseminated breast cancer markers cancer antigen (CA) 15-3, CA 27-29 and cancer embryonic antigen (CEA). In the developed SERS assay, both the assay chip and surface-enhanced Raman spectroscopy (SERS) tags are functionalized with monoclonal antibodies against CA15-3, CA27-29 and CEA, respectively. Sequential addition of biomarkers and functionalized SERS tags onto the functionalized assay chip enable the specific recognition of these biomarkers through the antibody-antigen interactions, leading to a sandwich spectro-immunoassay. In addition to offering extensive multiplexing capability, our method provides higher sensitivity than conventional immunoassays and demonstrates exquisite specificity owing to selective formation of conjugated complexes and fingerprint spectra of the Raman reporter. We envision that clinical translation of this assay may further enable asymptomatic surveillance of cancer survivors and speedy assessment of treatment benefit through a simple blood test. PMID:26405519

Evaluation of a Commercial Multiplex PCR for Rapid Detection of Multi Drug Resistant Gram Negative Infections

PubMed Central

Chavada, Ruchir; Maley, Michael

2015-01-01

Introduction: Community and healthcare associated infections caused by multi-drug resistant gram negative organisms (MDR GN) represent a worldwide threat. Nucleic Acid Detection tests are becoming more common for their detection; however they can be expensive requiring specialised equipment and local expertise. This study was done to evaluate the utility of a commercial multiplex tandem (MT) PCR for detection of MDR GN. Methods: The study was done on stored laboratory MDR GN isolates from sterile and non-sterile specimens (n=126, out of stored 567 organisms). Laboratory validation of the MT PCR was done to evaluate sensitivity, specificity and agreement with the current phenotypic methods used in the laboratory. Amplicon sequencing was also done on selected isolates for assessing performance characteristics. Workflow and cost implications of the MT PCR were evaluated. Results: The sensitivity and specificity of the MT PCR were calculated to be 95% and 96.7% respectively. Agreement with the phenotypic methods was 80%. Major lack of agreement was seen in detection of AmpC beta lactamase in enterobacteriaceae and carbapenemase in non-fermenters. Agreement of the MT PCR with another multiplex PCR was found to be 87%. Amplicon sequencing confirmed the genotype detected by MT PCR in 94.2 % of cases tested. Time to result was faster for the MT PCR but cost per test was higher. Conclusion: This study shows that with carefully chosen targets for detection of resistance genes in MDR GN, rapid and efficient identification is possible. MT PCR was sensitive and specific and likely more accurate than phenotypic methods. PMID:26464612

Diversity of Salmonella isolates from central Florida surface waters.

PubMed

McEgan, Rachel; Chandler, Jeffrey C; Goodridge, Lawrence D; Danyluk, Michelle D

2014-11-01

Identification of Salmonella serotypes is important for understanding the environmental diversity of the genus Salmonella. This study evaluates the diversity of Salmonella isolates recovered from 165 of 202 Central Florida surface water samples and investigates whether the serotype of the environmental Salmonella isolates can be predicted by a previously published multiplex PCR assay (S. Kim, J. G. Frye, J. Hu, P. J. Fedorka-Cray, R. Gautom, and D. S. Boyle, J. Clin. Microbiol. 44:3608-3615, 2006, http://dx.doi.org/10.1128/JCM.00701-06). Multiplex PCR was performed on 562 Salmonella isolates (as many as 36 isolates per water sample) to predict serotypes. Kauffmann-White serogrouping was used to confirm multiplex PCR pattern groupings before isolates were serotyped, analyzed by pulsed-field gel electrophoresis, and assayed for antimicrobial susceptibility. In 41.2% of the Salmonella-positive water samples, all Salmonella isolates had identical multiplex PCR patterns; in the remaining 58.8%, two or more multiplex PCR patterns were identified. Within each sample, isolates with matching multiplex PCR patterns had matching serogroups. The multiplex patterns of 495 isolates (88.1%) did not match any previously reported pattern. The remaining 68 isolates matched reported patterns but did not match the serotypes for those patterns. The use of the multiplex PCR allowed the number of isolates requiring further analysis to be reduced to 223. Thirty-three Salmonella enterica serotypes were identified; the most frequent included serotypes Muenchen, Rubislaw, Anatum, Gaminara, and IV_50:z4,z23:-. A majority (141/223) of Salmonella isolates clustered into one genotypic group. Salmonella isolates in Central Florida surface waters are serotypically, genotypically, and phenotypically (in terms of antimicrobial susceptibility) diverse. While isolates could be grouped as different or potentially the same using multiplex PCR, the multiplex PCR pattern did not predict the Salmonella serotype. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Diversity of Salmonella Isolates from Central Florida Surface Waters

PubMed Central

McEgan, Rachel; Chandler, Jeffrey C.; Goodridge, Lawrence D.

2014-01-01

Identification of Salmonella serotypes is important for understanding the environmental diversity of the genus Salmonella. This study evaluates the diversity of Salmonella isolates recovered from 165 of 202 Central Florida surface water samples and investigates whether the serotype of the environmental Salmonella isolates can be predicted by a previously published multiplex PCR assay (S. Kim, J. G. Frye, J. Hu, P. J. Fedorka-Cray, R. Gautom, and D. S. Boyle, J. Clin. Microbiol. 44:3608–3615, 2006, http://dx.doi.org/10.1128/JCM.00701-06). Multiplex PCR was performed on 562 Salmonella isolates (as many as 36 isolates per water sample) to predict serotypes. Kauffmann-White serogrouping was used to confirm multiplex PCR pattern groupings before isolates were serotyped, analyzed by pulsed-field gel electrophoresis, and assayed for antimicrobial susceptibility. In 41.2% of the Salmonella-positive water samples, all Salmonella isolates had identical multiplex PCR patterns; in the remaining 58.8%, two or more multiplex PCR patterns were identified. Within each sample, isolates with matching multiplex PCR patterns had matching serogroups. The multiplex patterns of 495 isolates (88.1%) did not match any previously reported pattern. The remaining 68 isolates matched reported patterns but did not match the serotypes for those patterns. The use of the multiplex PCR allowed the number of isolates requiring further analysis to be reduced to 223. Thirty-three Salmonella enterica serotypes were identified; the most frequent included serotypes Muenchen, Rubislaw, Anatum, Gaminara, and IV_50:z4,z23:−. A majority (141/223) of Salmonella isolates clustered into one genotypic group. Salmonella isolates in Central Florida surface waters are serotypically, genotypically, and phenotypically (in terms of antimicrobial susceptibility) diverse. While isolates could be grouped as different or potentially the same using multiplex PCR, the multiplex PCR pattern did not predict the Salmonella serotype. PMID:25172861

Cooperative spreading processes in multiplex networks.

PubMed

Wei, Xiang; Chen, Shihua; Wu, Xiaoqun; Ning, Di; Lu, Jun-An

2016-06-01

This study is concerned with the dynamic behaviors of epidemic spreading in multiplex networks. A model composed of two interacting complex networks is proposed to describe cooperative spreading processes, wherein the virus spreading in one layer can penetrate into the other to promote the spreading process. The global epidemic threshold of the model is smaller than the epidemic thresholds of the corresponding isolated networks. Thus, global epidemic onset arises in the interacting networks even though an epidemic onset does not arise in each isolated network. Simulations verify the analysis results and indicate that cooperative spreading processes in multiplex networks enhance the final infection fraction.

Rapid and inexpensive analysis of genetic variability in Arapaima gigas by PCR multiplex panel of eight microsatellites.

PubMed

Hamoy, I G; Santos, E J M; Santos, S E B

2008-01-22

The aim of the present study was the development of a multiplex genotyping panel of eight microsatellite markers of Arapaima gigas, previously described. Specific primer pairs were developed, each one of them marked with either FAM-6, HEX or NED. The amplification conditions using the new primers were standardized for a single reaction. The results obtained demonstrate high heterozygosity (average of 0.69) in a Lower Amazon population. The multiplex system described can thus be considered a fast, efficient and inexpensive method for the investigation of genetic variability in Arapaima populations.

Multiplexed Imaging of Protein Phosphorylation on Membranes Based on Ti(IV) Functionalized Nanopolymers.

PubMed

Iliuk, Anton; Li, Li; Melesse, Michael; Hall, Mark C; Tao, W Andy

2016-05-17

Accurate protein phosphorylation analysis reveals dynamic cellular signaling events not evident from protein expression levels. The most dominant biochemical assay, western blotting, suffers from the inadequate availability and poor quality of phospho-specific antibodies for phosphorylated proteins. Furthermore, multiplexed assays based on antibodies are limited by steric interference between the antibodies. Here we introduce a multifunctionalized nanopolymer for the universal detection of phosphoproteins that, in combination with regular antibodies, allows multiplexed imaging and accurate determination of protein phosphorylation on membranes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of three automated nucleic acid extraction systems for identification of respiratory viruses in clinical specimens by multiplex real-time PCR.

PubMed

Kim, Yoonjung; Han, Mi-Soon; Kim, Juwon; Kwon, Aerin; Lee, Kyung-A

2014-01-01

A total of 84 nasopharyngeal swab specimens were collected from 84 patients. Viral nucleic acid was extracted by three automated extraction systems: QIAcube (Qiagen, Germany), EZ1 Advanced XL (Qiagen), and MICROLAB Nimbus IVD (Hamilton, USA). Fourteen RNA viruses and two DNA viruses were detected using the Anyplex II RV16 Detection kit (Seegene, Republic of Korea). The EZ1 Advanced XL system demonstrated the best analytical sensitivity for all the three viral strains. The nucleic acids extracted by EZ1 Advanced XL showed higher positive rates for virus detection than the others. Meanwhile, the MICROLAB Nimbus IVD system was comprised of fully automated steps from nucleic extraction to PCR setup function that could reduce human errors. For the nucleic acids recovered from nasopharyngeal swab specimens, the QIAcube system showed the fewest false negative results and the best concordance rate, and it may be more suitable for detecting various viruses including RNA and DNA virus strains. Each system showed different sensitivity and specificity for detection of certain viral pathogens and demonstrated different characteristics such as turnaround time and sample capacity. Therefore, these factors should be considered when new nucleic acid extraction systems are introduced to the laboratory.

A Real-Time PCR Assay to Identify and Discriminate Among Wild-Type and Vaccine Strains of Varicella-Zoster Virus and Herpes Simplex Virus in Clinical Specimens, and Comparison With the Clinical Diagnoses

PubMed Central

Harbecke, Ruth; Oxman, Michael N.; Arnold, Beth A.; Ip, Charlotte; Johnson, Gary R.; Levin, Myron J.; Gelb, Lawrence D.; Schmader, Kenneth E.; Straus, Stephen E.; Wang, Hui; Wright, Peter F.; Pachucki, Constance T.; Gershon, Anne A.; Arbeit, Robert D.; Davis, Larry E.; Simberkoff, Michael S.; Weinberg, Adriana; Williams, Heather M.; Cheney, Carol; Petrukhin, Luba; Abraham, Katalin G.; Shaw, Alan; Manoff, Susan; Antonello, Joseph M.; Green, Tina; Wang, Yue; Tan, Charles; Keller, Paul M.

2014-01-01

A real-time PCR assay was developed to identify varicella-zoster virus (VZV) and herpes simplex virus (HSV) DNA in clinical specimens from subjects with suspected herpes zoster (HZ; shingles). Three sets of primers and probes were used in separate PCR reactions to detect and discriminate among wild-type VZV (VZV-WT), Oka vaccine strain VZV (VZV-Oka), and HSV DNA, and the reaction for each virus DNA was multiplexed with primers and probe specific for the human β-globin gene to assess specimen adequacy. Discrimination of all VZV-WT strains, including Japanese isolates and the Oka parent strain, from VZV-Oka was based upon a single nucleotide polymorphism at position 106262 in ORF 62, resulting in preferential amplification by the homologous primer pair. The assay was highly sensitive and specific for the target virus DNA, and no cross-reactions were detected with any other infectious agent. With the PCR assay as the gold standard, the sensitivity of virus culture was 53% for VZV and 77% for HSV. There was 92% agreement between the clinical diagnosis of HZ by the Clinical Evaluation Committee and the PCR assay results. PMID:19475609

A Pan-HIV Strategy for Complete Genome Sequencing

PubMed Central

Yamaguchi, Julie; Alessandri-Gradt, Elodie; Tell, Robert W.; Brennan, Catherine A.

2015-01-01

Molecular surveillance is essential to monitor HIV diversity and track emerging strains. We have developed a universal library preparation method (HIV-SMART [i.e., switching mechanism at 5′ end of RNA transcript]) for next-generation sequencing that harnesses the specificity of HIV-directed priming to enable full genome characterization of all HIV-1 groups (M, N, O, and P) and HIV-2. Broad application of the HIV-SMART approach was demonstrated using a panel of diverse cell-cultured virus isolates. HIV-1 non-subtype B-infected clinical specimens from Cameroon were then used to optimize the protocol to sequence directly from plasma. When multiplexing 8 or more libraries per MiSeq run, full genome coverage at a median ∼2,000× depth was routinely obtained for either sample type. The method reproducibly generated the same consensus sequence, consistently identified viral sequence heterogeneity present in specimens, and at viral loads of ≤4.5 log copies/ml yielded sufficient coverage to permit strain classification. HIV-SMART provides an unparalleled opportunity to identify diverse HIV strains in patient specimens and to determine phylogenetic classification based on the entire viral genome. Easily adapted to sequence any RNA virus, this technology illustrates the utility of next-generation sequencing (NGS) for viral characterization and surveillance. PMID:26699702

Methods for Multiplex Template Sampling in Digital PCR Assays

PubMed Central

Petriv, Oleh I.; Heyries, Kevin A.; VanInsberghe, Michael; Walker, David; Hansen, Carl L.

2014-01-01

The efficient use of digital PCR (dPCR) for precision copy number analysis requires high concentrations of target molecules that may be difficult or impossible to obtain from clinical samples. To solve this problem we present a strategy, called Multiplex Template Sampling (MTS), that effectively increases template concentrations by detecting multiple regions of fragmented target molecules. Three alternative assay approaches are presented for implementing MTS analysis of chromosome 21, providing a 10-fold concentration enhancement while preserving assay precision. PMID:24854517

Methods for multiplex template sampling in digital PCR assays.

PubMed

Petriv, Oleh I; Heyries, Kevin A; VanInsberghe, Michael; Walker, David; Hansen, Carl L

2014-01-01

The efficient use of digital PCR (dPCR) for precision copy number analysis requires high concentrations of target molecules that may be difficult or impossible to obtain from clinical samples. To solve this problem we present a strategy, called Multiplex Template Sampling (MTS), that effectively increases template concentrations by detecting multiple regions of fragmented target molecules. Three alternative assay approaches are presented for implementing MTS analysis of chromosome 21, providing a 10-fold concentration enhancement while preserving assay precision.

Detection of high-risk mucosal human papillomavirus DNA in human specimens by a novel and sensitive multiplex PCR method combined with DNA microarray.

PubMed

Gheit, Tarik; Tommasino, Massimo

2011-01-01

Epidemiological and functional studies have clearly demonstrated that certain types of human papillomavirus (HPV) from the genus alpha of the HPV phylogenetic tree, referred to as high-risk (HR) types, are the etiological cause of cervical cancer. Several methods for HPV detection and typing have been developed, and their importance in clinical and epidemiological studies has been well demonstrated. However, comparative studies have shown that several assays have different sensitivities for the detection of specific HPV types, particularly in the case of multiple infections. In this chapter, we describe a novel one-shot method for the detection and typing of 19 mucosal HR HPV types (types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, and 82). The assay combines the advantages of the multiplex PCR methods, i.e., high sensitivity and the possibility to perform multiple amplifications in a single reaction, with an array primer extension (APEX) assay. The latter method offers the benefits of Sanger dideoxy sequencing with the high-throughput potential of the microarray. Initial studies have revealed that the assay is very sensitive in detecting multiple HPV infections.

Simple Identification of Human Taenia Species by Multiplex Loop-Mediated Isothermal Amplification in Combination with Dot Enzyme-Linked Immunosorbent Assay

PubMed Central

Nkouawa, Agathe; Sako, Yasuhito; Okamoto, Munehiro; Ito, Akira

2016-01-01

For differential detection of Taenia solium, Taenia saginata, and Taenia asiatica, loop-mediated isothermal amplification (LAMP) assay targeting the cytochrome c oxidase subunit 1 gene has been recently developed and shown to be sensitive, specific, and effective. However, to achieve differential identification, one specimen requires three reaction mixtures containing a primer set of each Taenia species separately, which is complex and time consuming and increases the risk of cross-contamination. In this study, we developed a simple differential identification of human Taenia species using multiplex LAMP (mLAMP) in combination with dot enzyme-linked immunosorbent assay (dot-ELISA). Forward inner primers of T. solium, T. saginata, and T. asiatica labeled with fluorescein isothiocyanate (FITC), digoxigenin (DIG), and tetramethylrhodamine (TAMRA), respectively, and biotin-labeled backward inner primers were used in mLAMP. The mLAMP assay succeeded in specific amplification of each respective target gene in a single tube. Furthermore, the mLAMP product from each species was easily distinguished by dot-ELISA with an antibody specific for FITC, DIG, or TAMRA. The mLAMP assay in combination with dot-ELISA will make identification of human Taenia species simpler, easier, and more practical. PMID:27044566

Molecular Approaches to Taenia asiatica

PubMed Central

Jeon, Hyeong-Kyu

2013-01-01

Taenia solium, T. saginata, and T. asiatica are taeniid tapeworms that cause taeniasis in humans and cysticercosis in intermediate host animals. Taeniases remain an important public health concerns in the world. Molecular diagnostic methods using PCR assays have been developed for rapid and accurate detection of human infecting taeniid tapeworms, including the use of sequence-specific DNA probes, PCR-RFLP, and multiplex PCR. More recently, DNA diagnosis using PCR based on histopathological specimens such as 10% formalin-fixed paraffin-embedded and stained sections mounted on slides has been applied to cestode infections. The mitochondrial gene sequence is believed to be a very useful molecular marker for not only studying evolutionary relationships among distantly related taxa, but also for investigating the phylo-biogeography of closely related species. The complete sequence of the human Taenia tapeworms mitochondrial genomes were determined, and its organization and structure were compared to other human-tropic Taenia tapeworms for which complete mitochondrial sequence data were available. The multiplex PCR assay with the Ta4978F, Ts5058F, Tso7421F, and Rev7915 primers will be useful for differential diagnosis, molecular characterization, and epidemiological surveys of human Taenia tapeworms. PMID:23467738

A Multiplex PCR Assay for Differentiating Coconut Rhinoceros Beetle (Coleoptera: Scarabaeidae) From Oriental Flower Beetle (Coleoptera: Scarabaeidae) in Early Life Stages and Excrement.

PubMed

Watanabe, S; Melzer, M J

2017-04-01

The coconut rhinoceros beetle, Oryctes rhinoceros (L.), is a major pest of coconut and other palm trees. An incipient coconut rhinoceros beetle population was recently discovered on the island of Oahu, Hawaii and is currently the target of a large, mutiagency eradication program. Confounding this program is the widespread presence of another scarab beetle on Oahu, the oriental flower beetle, Protaetia orientalis (Gory and Percheron 1833). Eggs, early life stages, and fecal excrement of coconut rhinoceros beetle and oriental flower beetle are morphologically indistinguishable, thereby creating uncertainty when such specimens are discovered in the field. Here, we report the development of a multiplex PCR assay targeting cytochrome oxidase I of coconut rhinoceros beetle and oriental flower beetle that can rapidly detect and distinguish between these insects. This assay also features an internal positive control to ensure DNA of sufficient quantity and quality is used in the assay, increasing its reliability and reducing the chances of false negative results. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Molecular Identification of Adult and Juvenile Linyphiid and Theridiid Spiders in Alpine Glacier Foreland Communities

PubMed Central

Raso, Lorna; Sint, Daniela; Rief, Alexander; Kaufmann, Rüdiger; Traugott, Michael

2014-01-01

In glacier forelands spiders constitute a large proportion of the invertebrate community. Therefore, it is important to be able to determine the species that can be found in these areas. Linyphiid and theridiid spider identification is currently not possible in juvenile specimens using traditional morphological based methods, however, a large proportion of the population in these areas are usually juveniles. Molecular methods permit identification of species at different life stages, making juvenile identification possible. In this study we tested a molecular tool to identify the 10 most common species of Linyphiidae and Theridiidae found in three glacier foreland communities of the Austrian Alps. Two multiplex PCR systems were developed and over 90% of the 753 field-collected spiders were identified successfully. The species targeted were found to be common in all three valleys during the summer of 2010. A comparison between the molecular and morphological data showed that although there was a slight difference in the results, the overall outcome was the same independently of the identification method used. We believe the quick and reliable identification of the spiders via the multiplex PCR assays developed here will aid the study of these families in Alpine habitats. PMID:25050841

A nested multiplex polymerase chain reaction assay for the differential identification of three zooanthroponotic chlamydial strains in porcine swab samples.

PubMed

Li, Yingguo; Wang, Yu; Nie, Fuping; Xiao, Jinwen; Wang, Guoming; Yuan, Ling; Li, Zhengguo

2011-07-01

Porcine chlamydial infection is an enzootic infectious disease caused by multiple members of the family Chlamydiaceae (e.g. Chlamydophila abortus, Chlamydia suis, and Chlamydophila pneumoniae). Rapid and accurate differentiation of these pathogens is critical in the control and prevention of disease. The aim of the current study was to develop a nested multiplex polymerase chain reaction (nmPCR) assay to simultaneously detect the 3 chlamydial pathogens in clinical samples. In the first round of the nmPCR, 1 pair of family-specific primers were used to amplify the 1,100 base pair (bp) fragment of chlamydial ompA gene. In the second round of the nmPCR, 4 inner primers were designed for Ch. abortus, C. suis, and Ch. pneumoniae. Each pathogen produced a specific amplicon with a size of 340 bp, 526 bp, and 267 bp respectively. The assay was sensitive and specific for detecting target pathogens in both cell cultures and clinical specimens. The results, incorporated with the improved rapid DNA extraction protocol, suggest that the nmPCR could be a promising assay for differential identification of different chlamydial strains in pigs.

Multiplex PCR for diagnosis of Theileria uilenbergi, Theileria luwenshuni, and Theileria ovis in small ruminants.

PubMed

Zhang, Xiao; Liu, Zhijie; Yang, Jifei; Chen, Ze; Guan, Guiquan; Ren, Qiaoyun; Liu, Aihong; Luo, Jianxun; Yin, Hong; Li, Youquan

2014-02-01

Infections with Theileria sp. may cause significant economic losses to the sheep industry. Species identification based on microscopic examination is difficult, and more suitable methods are required for the rapid detection and identification of Theileria sp, in clinical specimens. In this study, a multiplex polymerase chain reaction (mPCR) assay was developed to simultaneously identify three individual Theileria species in small ruminants. Three pairs of specific, sensitive primers were designed on the basis of the 5.8S ribosomal RNA gene (Theileria luwenshuni and Theileria ovis) and the 18S ribosomal RNA gene (Theileria uilenbergi) to generate target products of 303, 884, and 530 bp, respectively. Standard DNA for each of the three species was extracted from blood recovered from infected sheep, and a preliminary study was conducted on 56 sheep to verify the reliability of the system. Optimal PCR conditions, including primer concentration, annealing time, and the number of amplification cycles, were established. The assay sensitivity under these conditions was 10(-3) % parasitemia, and its specificity was 100 %. The results of the study suggest that mPCR represents a simple, efficient test method as a practical alternative for the rapid detection and identification of Theileria species in small ruminants.

Antibiotic resistant Escherichia coli in southeastern Australian pig herds and implications for surveillance.

PubMed

van Breda, L K; Dhungyel, O P; Ward, M P

2018-02-01

To investigate public health implications of antibiotics to control post-weaning scours, we surveyed 22 commercial pig herds in southeastern Australia. Fifty faecal samples per herd were collected from pre- and post-weaned piglets. Presumptive Escherichia coli isolates were confirmed by MALDI-TOF MS. Isolates (n = 325) were screened for susceptibility to 19 veterinary antibiotics using MIC broth microdilution. All 325 E. coli isolates underwent further testing against 27 antibiotics used in human medicine and were screened for ETEC adhesin and enterotoxin genes (F4 (K88), F5 (K99), F6 (987P), F18, F41, STa, STb, Stx2e and LT) by multiplex PCR. Isolates identified as phenotypically resistant to third-generation cephalosporin (3GC) and aminoglycoside antibiotics were screened by multiplex PCR/reverse line blot to detect common β-lactam and aminoglycosides resistance genes, confirmed by sequencing. Twenty (6.1%) of the E. coli isolates were resistant to 3GC antibiotics and 24 (7.4%) to the aminoglycoside antibiotic gentamicin. Genetic analysis revealed six different extended spectrum β-lactamase (ESBL) genes (blaCTX-M-1, -14, -15, -27, blaSHV-12 and blaCMY-2-like genes), four of which have not been previously reported in Australian pigs. Critically, the prevalence of 3GC resistance was higher in non-pathogenic (non-ETEC) isolates and those from clinically normal (non-diarrhoeal) samples. This highlights the importance of non-ETECE. coli as reservoirs of antimicrobial resistance genes in piglet pens. Antimicrobial resistance surveillance in pig production focused on diagnostic specimens from clinically-affected animals might be potentially misleading. We recommend that surveillance for emerging antimicrobial resistance such as to 3GC antibiotics should include clinically healthy pigs. © 2017 Blackwell Verlag GmbH.

Comparison between Urine and Cervical Samples for HPV DNA Detection and Typing in Young Women in Colombia.

PubMed

Cómbita, Alba Lucía; Gheit, Tarik; González, Paula; Puerto, Devi; Murillo, Raúl Hernando; Montoya, Luisa; Vorsters, Alex; Van Keer, Severien; Van Damme, Pierre; Tommasino, Massimo; Hernández-Suárez, Gustavo; Sánchez, Laura; Herrero, Rolando; Wiesner, Carolina

2016-09-01

Urine sampling for HPV DNA detection has been proposed as an effective method for monitoring the impact of HPV vaccination programs; however, conflicting results have been reported. The goal of this study was to evaluate the performance of optimized urine HPV DNA testing in women aged 19 to 25 years. Optimization process included the use of first void urine, immediate mixing of urine with DNA preservative, and the concentration of all HPV DNA, including cell-free DNA fragments. Urine and cervical samples were collected from 535 young women attending cervical screening at health centers from two Colombian cities. HPV DNA detection and genotyping was performed using an HPV type-specific multiplex genotyping assay, which combines multiplex polymerase chain reaction with bead-based Luminex technology. Concordance between HPV DNA detection in urine and cervical samples was determined using kappa statistics and McNemar tests. The accuracy of HPV DNA testing in urine samples was evaluated measuring sensitivity and specificity using as reference the results obtained from cervical samples. Statistical analysis was performed using STATA11.2 software. The findings revealed an overall HPV prevalence of 60.00% in cervical samples and 64.72% in urine samples, HPV-16 being the most frequent HPV type detected in both specimens. Moreover, our results indicate that detection of HPV DNA in first void urine provides similar results to those obtained with cervical samples and can be used to monitor HPV vaccination trials and programs as evidenced by the substantial concordance found for the detection of the four vaccine types. Cancer Prev Res; 9(9); 766-71. ©2016 AACR. ©2016 American Association for Cancer Research.

Specific detection of common pathogens of acute bacterial meningitis using an internally controlled tetraplex-PCR assay.

PubMed

Farahani, Hamidreza; Ghaznavi-Rad, Ehsanollah; Mondanizadeh, Mahdieh; MirabSamiee, Siamak; Khansarinejad, Behzad

2016-08-01

Accurate and timely diagnosis of acute bacterial meningitis is critical for antimicrobial treatment of patients. Although PCR-based methods have been widely used for the diagnosis of acute meningitis caused by bacterial pathogens, the main disadvantage of these methods is their high cost. This disadvantage has hampered the widespread use of molecular assays in many developing countries. The application of multiplex assays and "in-house" protocols are two main approaches that can reduce the overall cost of a molecular test. In the present study, an internally controlled tetraplex-PCR was developed and validated for the specific detection of Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae in cerebrospinal fluid (CSF) samples. The analysis of a panel of other human pathogens showed no cross-reactivity in the assay. The analytical sensitivity of the in-house assay was 792.3 copies/ml, when all three bacteria were presentin the specimens. This value was calculated as 444.5, 283.7, 127.8 copies/ml when only S. pneumoniae, N. meningitidis and H. influenzae, respectively, were present. To demonstrate the diagnostic performance of the assay, a total of 150 archival CSF samples were tested and compared with a commercial multiplex real-time PCR kit. A diagnostic sensitivity of 92.8% and a specificity of 95.1% were determined for the present tetraplex-PCR assay. The results indicate that the established method is sensitive, specific and cost-effective, and can be used particularly in situations where the high cost of commercial kits prevents the use of molecular methods for the diagnosis of bacterial meningitis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Photon nonlinear mixing in subcarrier multiplexed quantum key distribution systems.

PubMed

Capmany, José

2009-04-13

We provide, for the first time to our knowledge, an analysis of the influence of nonlinear photon mixing on the end to end quantum bit error rate (QBER) performance of subcarrier multiplexed quantum key distribution systems. The results show that negligible impact is to be expected for modulation indexes in the range of 2%.

On-chip wavelength multiplexed detection of cancer DNA biomarkers in blood

PubMed Central

Cai, H.; Stott, M. A.; Ozcelik, D.; Parks, J. W.; Hawkins, A. R.; Schmidt, H.

2016-01-01

We have developed an optofluidic analysis system that processes biomolecular samples starting from whole blood and then analyzes and identifies multiple targets on a silicon-based molecular detection platform. We demonstrate blood filtration, sample extraction, target enrichment, and fluorescent labeling using programmable microfluidic circuits. We detect and identify multiple targets using a spectral multiplexing technique based on wavelength-dependent multi-spot excitation on an antiresonant reflecting optical waveguide chip. Specifically, we extract two types of melanoma biomarkers, mutated cell-free nucleic acids —BRAFV600E and NRAS, from whole blood. We detect and identify these two targets simultaneously using the spectral multiplexing approach with up to a 96% success rate. These results point the way toward a full front-to-back chip-based optofluidic compact system for high-performance analysis of complex biological samples. PMID:28058082

Development of a One-Step Multiplex PCR Assay for Differential Detection of Major Mycobacterium Species

PubMed Central

Chae, Hansong; Han, Seung Jung; Kim, Su-Young; Ki, Chang-Seok; Huh, Hee Jae; Yong, Dongeun

2017-01-01

ABSTRACT The prevalence of tuberculosis continues to be high, and nontuberculous mycobacterial (NTM) infection has also emerged worldwide. Moreover, differential and accurate identification of mycobacteria to the species or subspecies level is an unmet clinical need. Here, we developed a one-step multiplex PCR assay using whole-genome analysis and bioinformatics to identify novel molecular targets. The aims of this assay were to (i) discriminate between the Mycobacterium tuberculosis complex (MTBC) and NTM using rv0577 or RD750, (ii) differentiate M. tuberculosis (M. tuberculosis) from MTBC using RD9, (iii) selectively identify the widespread M. tuberculosis Beijing genotype by targeting mtbk_20680, and (iv) simultaneously detect five clinically important NTM (M. avium, M. intracellulare, M. abscessus, M. massiliense, and M. kansasii) by targeting IS1311, DT1, mass_3210, and mkan_rs12360. An initial evaluation of the multiplex PCR assay using reference strains demonstrated 100% specificity for the targeted Mycobacterium species. Analytical sensitivity ranged from 1 to 10 pg for extracted DNA and was 103 and 104 CFU for pure cultures and nonhomogenized artificial sputum cultures, respectively, of the targeted species. The accuracy of the multiplex PCR assay was further evaluated using 55 reference strains and 94 mycobacterial clinical isolates. Spoligotyping, multilocus sequence analysis, and a commercial real-time PCR assay were employed as standard assays to evaluate the multiplex PCR assay with clinical M. tuberculosis and NTM isolates. The PCR assay displayed 100% identification agreement with the standard assays. Our multiplex PCR assay is a simple, convenient, and reliable technique for differential identification of MTBC, M. tuberculosis, M. tuberculosis Beijing genotype, and major NTM species. PMID:28659320

Development of a One-Step Multiplex PCR Assay for Differential Detection of Major Mycobacterium Species.

PubMed

Chae, Hansong; Han, Seung Jung; Kim, Su-Young; Ki, Chang-Seok; Huh, Hee Jae; Yong, Dongeun; Koh, Won-Jung; Shin, Sung Jae

2017-09-01

The prevalence of tuberculosis continues to be high, and nontuberculous mycobacterial (NTM) infection has also emerged worldwide. Moreover, differential and accurate identification of mycobacteria to the species or subspecies level is an unmet clinical need. Here, we developed a one-step multiplex PCR assay using whole-genome analysis and bioinformatics to identify novel molecular targets. The aims of this assay were to (i) discriminate between the Mycobacterium tuberculosis complex (MTBC) and NTM using rv0577 or RD750, (ii) differentiate M. tuberculosis ( M. tuberculosis ) from MTBC using RD9, (iii) selectively identify the widespread M. tuberculosis Beijing genotype by targeting mtbk_20680 , and (iv) simultaneously detect five clinically important NTM ( M. avium , M. intracellulare , M. abscessus , M. massiliense , and M. kansasii ) by targeting IS 1311 , DT1, mass_3210 , and mkan_rs12360 An initial evaluation of the multiplex PCR assay using reference strains demonstrated 100% specificity for the targeted Mycobacterium species. Analytical sensitivity ranged from 1 to 10 pg for extracted DNA and was 10 3 and 10 4 CFU for pure cultures and nonhomogenized artificial sputum cultures, respectively, of the targeted species. The accuracy of the multiplex PCR assay was further evaluated using 55 reference strains and 94 mycobacterial clinical isolates. Spoligotyping, multilocus sequence analysis, and a commercial real-time PCR assay were employed as standard assays to evaluate the multiplex PCR assay with clinical M. tuberculosis and NTM isolates. The PCR assay displayed 100% identification agreement with the standard assays. Our multiplex PCR assay is a simple, convenient, and reliable technique for differential identification of MTBC, M. tuberculosis , M. tuberculosis Beijing genotype, and major NTM species. Copyright © 2017 American Society for Microbiology.

Enhanced capillary electrophoretic screening of Alzheimer based on direct apolipoprotein E genotyping and one-step multiplex PCR.

PubMed

Woo, Nain; Kim, Su-Kang; Sun, Yucheng; Kang, Seong Ho

2018-01-01

Human apolipoprotein E (ApoE) is associated with high cholesterol levels, coronary artery disease, and especially Alzheimer's disease. In this study, we developed an ApoE genotyping and one-step multiplex polymerase chain reaction (PCR) based-capillary electrophoresis (CE) method for the enhanced diagnosis of Alzheimer's. The primer mixture of ApoE genes enabled the performance of direct one-step multiplex PCR from whole blood without DNA purification. The combination of direct ApoE genotyping and one-step multiplex PCR minimized the risk of DNA loss or contamination due to the process of DNA purification. All amplified PCR products with different DNA lengths (112-, 253-, 308-, 444-, and 514-bp DNA) of the ApoE genes were analyzed within 2min by an extended voltage programming (VP)-based CE under the optimal conditions. The extended VP-based CE method was at least 120-180 times faster than conventional slab gel electrophoresis methods In particular, all amplified DNA fragments were detected in less than 10 PCR cycles using a laser-induced fluorescence detector. The detection limits of the ApoE genes were 6.4-62.0pM, which were approximately 100-100,000 times more sensitive than previous Alzheimer's diagnosis methods In addition, the combined one-step multiplex PCR and extended VP-based CE method was also successfully applied to the analysis of ApoE genotypes in Alzheimer's patients and normal samples and confirmed the distribution probability of allele frequencies. This combination of direct one-step multiplex PCR and an extended VP-based CE method should increase the diagnostic reliability of Alzheimer's with high sensitivity and short analysis time even with direct use of whole blood. Copyright © 2017 Elsevier B.V. All rights reserved.

Prevalence of Listeria monocytogenes, Yersinia enterocolitica, Staphylococcus aureus, and Salmonella enterica Typhimurium in meat and meat products using multiplex polymerase chain reaction

PubMed Central

Latha, C.; Anu, C. J.; Ajaykumar, V. J.; Sunil, B.

2017-01-01

Aim: The objective of the study was to investigate the occurrence of Listeria monocytogenes, Yersinia enterocolitica, Staphylococcus aureus, and Salmonella enterica Typhimurium in meat and meat products using the multiplex polymerase chain reaction (PCR) method. Materials and Methods: The assay combined an enrichment step in tryptic soy broth with yeast extract formulated for the simultaneous growth of target pathogens, DNA isolation and multiplex PCR. A total of 1134 samples including beef (n=349), chicken (n=325), pork (n=310), chevon (n=50), and meat products (n=100) were collected from different parts of Kerala, India. All the samples were subjected to multiplex PCR analysis and culture-based detection for the four pathogens in parallel. Results: Overall occurrence of L. monocytogenes was 0.08 % by cultural method. However, no L. monocytogenes was obtained by multiplex PCR method. Yersinia enterocolitica was obtained from beef and pork samples. A high prevalence of S. aureus (46.7%) was found in all types of meat samples tested. None of the samples was positive for S. Typhimurium. Conclusion: Multiplex PCR assay used in this study can detect more than one pathogen simultaneously by amplifying more than one target gene in a single reaction, which can save time and labor cost. PMID:28919685

Massively parallel sequencing of 17 commonly used forensic autosomal STRs and amelogenin with small amplicons.

PubMed

Kim, Eun Hye; Lee, Hwan Young; Yang, In Seok; Jung, Sang-Eun; Yang, Woo Ick; Shin, Kyoung-Jin

2016-05-01

The next-generation sequencing (NGS) method has been utilized to analyze short tandem repeat (STR) markers, which are routinely used for human identification purposes in the forensic field. Some researchers have demonstrated the successful application of the NGS system to STR typing, suggesting that NGS technology may be an alternative or additional method to overcome limitations of capillary electrophoresis (CE)-based STR profiling. However, there has been no available multiplex PCR system that is optimized for NGS analysis of forensic STR markers. Thus, we constructed a multiplex PCR system for the NGS analysis of 18 markers (13CODIS STRs, D2S1338, D19S433, Penta D, Penta E and amelogenin) by designing amplicons in the size range of 77-210 base pairs. Then, PCR products were generated from two single-sources, mixed samples and artificially degraded DNA samples using a multiplex PCR system, and were prepared for sequencing on the MiSeq system through construction of a subsequent barcoded library. By performing NGS and analyzing the data, we confirmed that the resultant STR genotypes were consistent with those of CE-based typing. Moreover, sequence variations were detected in targeted STR regions. Through the use of small-sized amplicons, the developed multiplex PCR system enables researchers to obtain successful STR profiles even from artificially degraded DNA as well as STR loci which are analyzed with large-sized amplicons in the CE-based commercial kits. In addition, successful profiles can be obtained from mixtures up to a 1:19 ratio. Consequently, the developed multiplex PCR system, which produces small size amplicons, can be successfully applied to STR NGS analysis of forensic casework samples such as mixtures and degraded DNA samples. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Wavelet-based compression of M-FISH images.

PubMed

Hua, Jianping; Xiong, Zixiang; Wu, Qiang; Castleman, Kenneth R

2005-05-01

Multiplex fluorescence in situ hybridization (M-FISH) is a recently developed technology that enables multi-color chromosome karyotyping for molecular cytogenetic analysis. Each M-FISH image set consists of a number of aligned images of the same chromosome specimen captured at different optical wavelength. This paper presents embedded M-FISH image coding (EMIC), where the foreground objects/chromosomes and the background objects/images are coded separately. We first apply critically sampled integer wavelet transforms to both the foreground and the background. We then use object-based bit-plane coding to compress each object and generate separate embedded bitstreams that allow continuous lossy-to-lossless compression of the foreground and the background. For efficient arithmetic coding of bit planes, we propose a method of designing an optimal context model that specifically exploits the statistical characteristics of M-FISH images in the wavelet domain. Our experiments show that EMIC achieves nearly twice as much compression as Lempel-Ziv-Welch coding. EMIC also performs much better than JPEG-LS and JPEG-2000 for lossless coding. The lossy performance of EMIC is significantly better than that of coding each M-FISH image with JPEG-2000.

Multiplex PCR analysis of fumonisin biosynthetic genes in fumonisin-nonproducing Aspergillus niger and A. awamori strains

USDA-ARS?s Scientific Manuscript database

In order to determine the genetic basis for loss of fumonisin B¬2 (FB2) biosynthesis in FB2 non-producing A. niger strains, we developed multiplex PCR primer sets to amplify fragments of eight fumonisin biosynthetic pathway (fum) genes. Fragments of all eight fum genes were amplified in FB2-produci...

Social contagions on correlated multiplex networks

NASA Astrophysics Data System (ADS)

Wang, Wei; Cai, Meng; Zheng, Muhua

2018-06-01

The existence of interlayer degree correlations has been disclosed by abundant multiplex network analysis. However, how they impose on the dynamics of social contagions are remain largely unknown. In this paper, we propose a non-Markovian social contagion model in multiplex networks with inter-layer degree correlations to delineate the behavior spreading, and develop an edge-based compartmental (EBC) theory to describe the model. We find that multiplex networks promote the final behavior adoption size. Remarkably, it can be observed that the growth pattern of the final behavior adoption size, versus the behavioral information transmission probability, changes from discontinuous to continuous once decreasing the behavior adoption threshold in one layer. We finally unravel that the inter-layer degree correlations play a role on the final behavior adoption size but have no effects on the growth pattern, which is coincidence with our prediction by using the suggested theory.

Security analysis of orthogonal-frequency-division-multiplexing-based continuous-variable quantum key distribution with imperfect modulation

NASA Astrophysics Data System (ADS)

Zhang, Hang; Mao, Yu; Huang, Duan; Li, Jiawei; Zhang, Ling; Guo, Ying

2018-05-01

We introduce a reliable scheme for continuous-variable quantum key distribution (CV-QKD) by using orthogonal frequency division multiplexing (OFDM). As a spectrally efficient multiplexing technique, OFDM allows a large number of closely spaced orthogonal subcarrier signals used to carry data on several parallel data streams or channels. We place emphasis on modulator impairments which would inevitably arise in the OFDM system and analyze how these impairments affect the OFDM-based CV-QKD system. Moreover, we also evaluate the security in the asymptotic limit and the Pirandola-Laurenza-Ottaviani-Banchi upper bound. Results indicate that although the emergence of imperfect modulation would bring about a slight decrease in the secret key bit rate of each subcarrier, the multiplexing technique combined with CV-QKD results in a desirable improvement on the total secret key bit rate which can raise the numerical value about an order of magnitude.

Development of multiplex microsatellite PCR panels for the seagrass Thalassia hemprichii (Hydrocharitaceae).

PubMed

van Dijk, Kor-Jent; Mellors, Jane; Waycott, Michelle

2014-11-01

New microsatellites were developed for the seagrass Thalassia hemprichii (Hydrocharitaceae), a long-lived seagrass species that is found throughout the shallow waters of tropical and subtropical Indo-West Pacific. Three multiplex PCR panels were designed utilizing new and previously developed markers, resulting in a toolkit for generating a 16-locus genotype. • Through the use of microsatellite enrichment and next-generation sequencing, 16 new, validated, polymorphic microsatellite markers were isolated. Diversity was between two and four alleles per locus totaling 36 alleles. These markers, plus previously developed microsatellite markers for T. hemprichii and T. testudinum, were tested for suitability in multiplex PCR panels. • The generation of an easily replicated suite of multiplex panels of codominant molecular markers will allow for high-resolution and detailed genetic structure analysis and clonality assessment with minimal genotyping costs. We suggest the establishment of a T. hemprichii primer convention for the unification of future data sets.

ddPCRclust - An R package and Shiny app for automated analysis of multiplexed ddPCR data.

PubMed

Brink, Benedikt G; Meskas, Justin; Brinkman, Ryan R

2018-03-09

Droplet digital PCR (ddPCR) is an emerging technology for quantifying DNA. By partitioning the target DNA into ∼20000 droplets, each serving as its own PCR reaction compartment, a very high sensitivity of DNA quantification can be achieved. However, manual analysis of the data is time consuming and algorithms for automated analysis of non-orthogonal, multiplexed ddPCR data are unavailable, presenting a major bottleneck for the advancement of ddPCR transitioning from low-throughput to high- throughput. ddPCRclust is an R package for automated analysis of data from Bio-Rad's droplet digital PCR systems (QX100 and QX200). It can automatically analyse and visualise multiplexed ddPCR experiments with up to four targets per reaction. Results are on par with manual analysis, but only take minutes to compute instead of hours. The accompanying Shiny app ddPCRvis provides easy access to the functionalities of ddPCRclust through a web-browser based GUI. R package: https://github.com/bgbrink/ddPCRclust; Interface: https://github.com/bgbrink/ddPCRvis/; Web: https://bibiserv.cebitec.uni-bielefeld.de/ddPCRvis/. bbrink@cebitec.uni-bielefeld.de.

Network structure of multivariate time series.

PubMed

Lacasa, Lucas; Nicosia, Vincenzo; Latora, Vito

2015-10-21

Our understanding of a variety of phenomena in physics, biology and economics crucially depends on the analysis of multivariate time series. While a wide range tools and techniques for time series analysis already exist, the increasing availability of massive data structures calls for new approaches for multidimensional signal processing. We present here a non-parametric method to analyse multivariate time series, based on the mapping of a multidimensional time series into a multilayer network, which allows to extract information on a high dimensional dynamical system through the analysis of the structure of the associated multiplex network. The method is simple to implement, general, scalable, does not require ad hoc phase space partitioning, and is thus suitable for the analysis of large, heterogeneous and non-stationary time series. We show that simple structural descriptors of the associated multiplex networks allow to extract and quantify nontrivial properties of coupled chaotic maps, including the transition between different dynamical phases and the onset of various types of synchronization. As a concrete example we then study financial time series, showing that a multiplex network analysis can efficiently discriminate crises from periods of financial stability, where standard methods based on time-series symbolization often fail.

An integrated passive micromixer-magnetic separation-capillary electrophoresis microdevice for rapid and multiplex pathogen detection at the single-cell level.

PubMed

Jung, Jae Hwan; Kim, Gha-Young; Seo, Tae Seok

2011-10-21

Here we report an integrated microdevice consisting of an efficient passive mixer, a magnetic separation chamber, and a capillary electrophoretic microchannel in which DNA barcode assay, target pathogen separation, and barcode DNA capillary electrophoretic analysis were performed sequentially within 30 min for multiplex pathogen detection at the single-cell level. The intestine-shaped serpentine 3D micromixer provides a high mixing rate to generate magnetic particle-pathogenic bacteria-DNA barcode labelled AuNP complexes quantitatively. After magnetic separation and purification of those complexes, the barcode DNA strands were released and analyzed by the microfluidic capillary electrophoresis within 5 min. The size of the barcode DNA strand was controlled depending on the target bacteria (Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella typhimurium), and the different elution time of the barcode DNA peak in the electropherogram allows us to recognize the target pathogen with ease in the monoplex as well as in the multiplex analysis. In addition, the quantity of the DNA barcode strand (∼10(4)) per AuNP is enough to be observed in the laser-induced confocal fluorescence detector, thereby making single-cell analysis possible. This novel integrated microdevice enables us to perform rapid, sensitive, and multiplex pathogen detection with sample-in-answer-out capability to be applied for biosafety testing, environmental screening, and clinical trials.

[Application of multiplex rt-PCR assay for screening rare or cryptic chromosome translocations in de novo patients with acute myeloid leukemia].

PubMed

Chen, Hai-Min; Yuan, Hai-Yang; Fan, Xing; He, Hai-Yan; Chen, Bing; Shi, Jing-Yi; Zhu, Yong-Mei

2010-10-01

This study was aimed to investigate the clinical feasibility of using multiplex PT-PCR assay for screening rare/cryptic chromosome translocations in patients with de novo acute myeloid leukemia. For 126 patients with de novo AML-M4/M5 without common chromosome translocations including t(15;17), t(8;21) and t(16;16), 3 parallel multiplex RT-PCR assays were set up to detect 6 mll-related gene rearrangements (mll/af10, mll/af17, mll/ell, mll/af9, mll/af6 and mll/enl) with low detection rate and 4 rare fusion genes (dek/can, tls/erg, aml1/mds (evi1) and npm/mlf1). The results showed that 11 patients with positive result from 126 patients were detected which involved in 5 molecular abnormalities. Among them, 10 cases were AML-M5 (16.67%), 1 cases AML-M4 (1.51%). The marker chromosomes were observed in 2 cases out of 11 cases through conventional karyotyping analysis, the karyotyping analysis in 1 case was not performed because this case had 1 mitotic figure only, no any cytogenetic aberrations were found in other 8 cases through R-band karyotyping analysis. It is concluded that multiplex RT-PCR designed in this study can quickly, effectively and accurately identify the rare/cryptic chromosome translocations and can be used in clinical detection.

Photocleavable DNA Barcoding Antibodies for Multiplexed Protein Analysis in Single Cells.

PubMed

Ullal, Adeeti V; Weissleder, Ralph

2015-01-01

We describe a DNA-barcoded antibody sensing technique for single cell protein analysis in which the barcodes are photocleaved and digitally detected without amplification steps (Ullal et al., Sci Transl Med 6:219, 2014). After photocleaving the unique ~70 mer DNA barcodes we use a fluorescent hybridization technology for detection, similar to what is commonly done for nucleic acid readouts. This protocol offers a simple method for multiplexed protein detection using 100+ antibodies and can be performed on clinical samples as well as single cells.

The SNPforID Assay as a Supplementary Method in Kinship and Trace Analysis

PubMed Central

Schwark, Thorsten; Meyer, Patrick; Harder, Melanie; Modrow, Jan-Hendrick; von Wurmb-Schwark, Nicole

2012-01-01

Objective Short tandem repeat (STR) analysis using commercial multiplex PCR kits is the method of choice for kinship testing and trace analysis. However, under certain circumstances (deficiency testing, mutations, minute DNA amounts), STRs alone may not suffice. Methods We present a 50-plex single nucleotide polymorphism (SNP) assay based on the SNPs chosen by the SNPforID consortium as an additional method for paternity and for trace analysis. The new assay was applied to selected routine paternity and trace cases from our laboratory. Results and Conclusions Our investigation shows that the new SNP multiplex assay is a valuable method to supplement STR analysis, and is a powerful means to solve complicated genetic analyses. PMID:22851934

Multiplex molecular testing for management of infectious gastroenteritis in a hospital setting: a comparative diagnostic and clinical utility study.

PubMed

Halligan, E; Edgeworth, J; Bisnauthsing, K; Bible, J; Cliff, P; Aarons, E; Klein, J; Patel, A; Goldenberg, S

2014-08-01

Laboratory diagnosis and clinical management of inpatients with diarrhoea is complex and time consuming. Tests are often requested sequentially and undertaken in different laboratories. This causes prolonged unnecessary presumptive isolation of patients, because most cases are non-infectious. A molecular multiplex test (Luminex(®) Gastrointestinal Pathogen Panel (GPP)) was compared with conventional testing over 8 months to determine diagnostic accuracy, turnaround times, laboratory costs, use of isolation facilities and user acceptability. A total of 262 (12%) patients had a pathogen detected by conventional methods compared with 483 (22.1%) by GPP. Most additional cases were detected in patients developing symptoms in the first 4 days of admission. Additional cases were detected because of presumed improved diagnostic sensitivity but also because clinicians had not requested the correct pathogen. Turnaround time (41.8 h) was faster than bacterial culture (66.5 h) and parasite investigation (66.5 h) but slower than conventional testing for Clostridium difficile (17.3 h) and viruses (27 h). The test could allow simplified requesting by clinicians and a consolidated laboratory workflow, reducing the overall number of specimens received by the laboratory. A total of 154 isolation days were saved at an estimated cost of £30 800. Consumables and labour were estimated at £150 641 compared with £63 431 for conventional testing. Multiplex molecular testing using a panel of targets allowed enhanced detection and a consolidated laboratory workflow. This is likely to be of greater benefit to cases that present within the first 4 days of hospital admission. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

Development of a Multiplexed Liquid Chromatography Multiple-Reaction-Monitoring Mass Spectrometry (LC-MRM/MS) Method for Evaluation of Salivary Proteins as Oral Cancer Biomarkers.

PubMed

Chen, Yi-Ting; Chen, Hsiao-Wei; Wu, Chun-Feng; Chu, Lichieh Julie; Chiang, Wei-Fang; Wu, Chih-Ching; Yu, Jau-Song; Tsai, Cheng-Han; Liang, Kung-Hao; Chang, Yu-Sun; Wu, Maureen; Ou Yang, Wei-Ting

2017-05-01

Multiple (selected) reaction monitoring (MRM/SRM) of peptides is a growing technology for target protein quantification because it is more robust, precise, accurate, high-throughput, and multiplex-capable than antibody-based techniques. The technique has been applied clinically to the large-scale quantification of multiple target proteins in different types of fluids. However, previous MRM-based studies have placed less focus on sample-preparation workflow and analytical performance in the precise quantification of proteins in saliva, a noninvasively sampled body fluid. In this study, we evaluated the analytical performance of a simple and robust multiple reaction monitoring (MRM)-based targeted proteomics approach incorporating liquid chromatography with mass spectrometry detection (LC-MRM/MS). This platform was used to quantitatively assess the biomarker potential of a group of 56 salivary proteins that have previously been associated with human cancers. To further enhance the development of this technology for assay of salivary samples, we optimized the workflow for salivary protein digestion and evaluated quantification performance, robustness and technical limitations in analyzing clinical samples. Using a clinically well-characterized cohort of two independent clinical sample sets (total n = 119), we quantitatively characterized these protein biomarker candidates in saliva specimens from controls and oral squamous cell carcinoma (OSCC) patients. The results clearly showed a significant elevation of most targeted proteins in saliva samples from OSCC patients compared with controls. Overall, this platform was capable of assaying the most highly multiplexed panel of salivary protein biomarkers, highlighting the clinical utility of MRM in oral cancer biomarker research. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Brief Report: A mass spectrometry assay to simultaneously analyze ROS1 and RET fusion gene expression in non-small cell lung cancer

PubMed Central

Wijesinghe, Priyanga; Bepler, Gerold

2014-01-01

Introduction ROS1 and RET gene fusions were recently discovered in non-small cell lung cancer (NSCLC) as potential therapeutic targets with small molecule kinase inhibitors. The conventional screening methods of these fusions are time consuming and require samples of high quality and quantity. Here, we describe a novel and efficient method by coupling the power of multiplexing PCR and the sensitivity of mass spectrometry. Methods The multiplex mass spectrometry platform simultaneously tests samples for the expression of nine ROS1 and six RET fusion genes. The assay incorporates detection of wild-type exon junctions immediately upstream and downstream of the fusion junction to exclude false negative results. To flag false positives, the system also comprises two independent assays for each fusion gene junction. Results The characteristic mass spectrometric peaks of the gene fusions were obtained using engineered plasmid constructs. Specific assays targeting the wild-type gene exon junctions were validated using cDNA from lung tissue of healthy individuals. The system was further validated using cDNA derived from NSCLC cell lines that express endogenous fusion genes. The expressed ROS1-SLC34A2 and CCDC6-RET gene fusions from the NSCLC cell lines HCC78 and LC-2/ad, respectively, were accurately detected by the novel assay. The assay is extremely sensitive, capable of detecting an event in test specimens containing 0.5% positive tumors. Conclusion The novel multiplexed assay is robustly capable of detecting 15 different clinically relevant RET and ROS1 fusion variants. The benefits of this detection method include exceptionally low sample input, high cost efficiency, flexibility, and rapid turnover. PMID:25384172

Development of single-step multiplex real-time RT-PCR assays for rapid diagnosis of enterovirus 71, coxsackievirus A6, and A16 in patients with hand, foot, and mouth disease.

PubMed

Puenpa, Jiratchaya; Suwannakarn, Kamol; Chansaenroj, Jira; Vongpunsawad, Sompong; Poovorawan, Yong

2017-10-01

Real-time reverse-transcription polymerase chain reaction (rRT-PCR) to detect enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16) has facilitated the rapid and accurate identification of the two most common etiological agents underlying hand, foot, and mouth disease (HFMD). However, the worldwide emergence of CV-A6 infection in HFMD necessitates development of an improved multiplex rRT-PCR method. To rapidly determine the etiology of HFMD, two rRT-PCR assays using TaqMan probes were developed to differentiate among three selected common enteroviruses (EV-A71, CV-A16 and CV-A6) and to enable broad detection of enteroviruses (pan-enterovirus assay). No cross-reactions were observed with other RNA viruses examined. The detection limits of both assays were 10 copies per microliter for EV-A71, CV-A6 and CV-A16, and pan-enterovirus. The methods showed high accuracy (EV-A71, 90.6%; CV-A6, 92.0%; CV-A16, 100%), sensitivity (EV-A71, 96.5%; CV-A6, 95.8%; CV-A16, 99.0%), and specificity (EV-A71, 100%; CV-A6, 99.9%; CV-A16, 99.9%) in testing clinical specimens (n=1049) during 2014-2016, superior to those of conventional RT-PCR. Overall, the multiplex rRT-PCR assays enabled highly sensitive detection and rapid simultaneous typing of EV-A71, CV-A6 and CV-A16, and enteroviruses, rendering them feasible and attractive methods for large-scale surveillance of enteroviruses associated with HFMD outbreaks. Copyright © 2017 Elsevier B.V. All rights reserved.

MT-PCR panel detection of canine parvovirus (CPV-2): Vaccine and wild-type CPV-2 can be difficult to differentiate in canine diagnostic fecal samples.

PubMed

Meggiolaro, Maira N; Ly, Anna; Rysnik-Steck, Benjamin; Silva, Carolina; Zhang, Joshua; Higgins, Damien P; Muscatello, Gary; Norris, Jacqueline M; Krockenberger, Mark; Šlapeta, Jan

2017-06-01

Canine parvovirus (CPV-2) remains an important cause of devastating enteritis in young dogs. It can be successfully prevented with live attenuated CPV-2 vaccines when given at the appropriate age and in the absence of maternal antibody interference. Rapid diagnosis of parvoviral enteritis in young dogs is essential to ensuring suitable barrier nursing protocols within veterinary hospitals. The current diagnostic trend is to use multiplexed PCR panels to detect an array of pathogens commonly responsible for diarrhea in dogs. The multiplexed PCR assays do not distinguish wild from vaccine CPV-2. They are highly sensitive and detect even a low level of virus shedding, such as those caused by the CPV-2 vaccine. The aim of this study was to identify the CPV-2 subtypes detected in diagnostic specimens and rule out occult shedding of CPV-2 vaccine strains. For a total of 21 samples that tested positive for CPV-2 in a small animal fecal pathogens diagnostic multiplexed tandem PCR (MT-PCR) panel during 2014-2016 we partially characterized the VP2 gene of CPV-2. Vaccine CPV-2 strain, wild type CPV-2a subtypes and vaccine-like CPV-2b subtypes were detected. High copy number was indicative of wild-type CPV-2a presence, but presence of vaccine-like CPV-2b had a variable copy number in fecal samples. A yardstick approach to a copy number or C t -value to discriminate vaccine strain from a wild type virus of CPV-2 can be, in some cases, potentially misleading. Therefore, discriminating vaccine strain from a wild type subtype of CPV-2 remains ambitious. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evaluation of the Roche LightMix Gastro parasites multiplex PCR assay detecting Giardia duodenalis, Entamoeba histolytica, cryptosporidia, Dientamoeba fragilis, and Blastocystis hominis.

PubMed

Friesen, J; Fuhrmann, J; Kietzmann, H; Tannich, E; Müller, M; Ignatius, R

2018-03-23

Multiplex PCR assays offer highly sensitive and specific tools for the detection of enteric pathogens. This prospective study aimed at comparing the novel Roche LightMix Modular Assay Gastro Parasites (LMAGP) detecting Giardia duodenalis, Entamoeba histolytica, Cryptosporidium spp., Blastocystishominis, and Dientamoebafragilis with routine laboratory procedures. Stool specimens (n = 1062 from 1009 patients) were consecutively examined by LMAGP, R-Biopharm Ridascreen enzyme immunoassays (EIAs) detecting G. duodenalis or E. histolytica/dispar, and microscopy of wet mounts. Discrepant results were analysed by in-house PCR. D. fragilis or B. hominis were detected by LMAGP in 131 (14.4%) and 179 (19.9%; 16 samples positive by microscopy; p < 0.0001) of 909 samples, respectively. Of 918 samples analysed for Cryptosporidium spp., six were positive by LMAGP (three could be confirmed by Kinyoun staining and one by in-house PCR). G. duodenalis was detected by LMAGP, EIA, or microscopy in 20, 16, or 9 of 1039 stool samples, respectively; all four samples missed by EIA were confirmed by in-house PCR. In total, 938 stool samples were analysed for E. histolytica/dispar. Nine of ten EIA-positive samples were negative by LMAGP but positive by in-house PCR for E. dispar. One E. histolytica infection (positive by both LMAGP and in-house PCR) was missed by EIA and microscopy. Parasites only detected by microscopy included Enterobius vermicularis eggs (n = 3) and apathogenic amoebae (n = 27). The data call for routine use of multiplex PCR assays for the detection of enteric protozoan parasites in laboratory diagnostics. Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Hi-Plex for Simple, Accurate, and Cost-Effective Amplicon-based Targeted DNA Sequencing.

PubMed

Pope, Bernard J; Hammet, Fleur; Nguyen-Dumont, Tu; Park, Daniel J

2018-01-01

Hi-Plex is a suite of methods to enable simple, accurate, and cost-effective highly multiplex PCR-based targeted sequencing (Nguyen-Dumont et al., Biotechniques 58:33-36, 2015). At its core is the principle of using gene-specific primers (GSPs) to "seed" (or target) the reaction and universal primers to "drive" the majority of the reaction. In this manner, effects on amplification efficiencies across the target amplicons can, to a large extent, be restricted to early seeding cycles. Product sizes are defined within a relatively narrow range to enable high-specificity size selection, replication uniformity across target sites (including in the context of fragmented input DNA such as that derived from fixed tumor specimens (Nguyen-Dumont et al., Biotechniques 55:69-74, 2013; Nguyen-Dumont et al., Anal Biochem 470:48-51, 2015), and application of high-specificity genetic variant calling algorithms (Pope et al., Source Code Biol Med 9:3, 2014; Park et al., BMC Bioinformatics 17:165, 2016). Hi-Plex offers a streamlined workflow that is suitable for testing large numbers of specimens without the need for automation.

Investigation of Echinococcus multilocularis in Environmental Definitive Host Feces in the Asian and the European Parts of Turkey

PubMed Central

Gürler, Ali Tümay; Gori, Francesca; Bölükbas¸, Cenk Soner; Umur, Şinasi; Açıcı, Mustafa; Deplazes, Peter

2018-01-01

A study was carried out to investigate the presence of Echinococcus multilocularis in red foxes (Vulpes vulpes) in two regions of Turkey—central Anatolia (in Asia Minor) and Thrace (in the European part of Turkey). A total of 405 putative fox feces were collected from central Anatolia (186 specimens in 59 locations) and from Thrace (219 specimens in 114 locations). All samples were examined by the flotation and sieving method for taeniid eggs, and positive and putative samples were further analyzed by multiplex PCR. In seven samples from three locations in central Anatolia (5.1%) and in one (0.9%) from Thrace, E. multilocularis DNA was amplified, and this result was confirmed with another PCR specific for E. multilocularis. In addition, Echinococcus granulosus s.l. was found in two (0.5%) of the samples. Although alveolar echinococcosis (AE) is known as a serious zoonosis in Turkey, this is the first field study detecting E. multilocularis in collected fecal samples documenting the environmental contamination with eggs of this zoonotic parasite. PMID:29662885

Investigation of Echinococcus multilocularis in Environmental Definitive Host Feces in the Asian and the European Parts of Turkey.

PubMed

Gürler, Ali Tümay; Gori, Francesca; Bölükbas, Cenk Soner; Umur, Şinasi; Açıcı, Mustafa; Deplazes, Peter

2018-01-01

A study was carried out to investigate the presence of Echinococcus multilocularis in red foxes ( Vulpes vulpes ) in two regions of Turkey-central Anatolia (in Asia Minor) and Thrace (in the European part of Turkey). A total of 405 putative fox feces were collected from central Anatolia (186 specimens in 59 locations) and from Thrace (219 specimens in 114 locations). All samples were examined by the flotation and sieving method for taeniid eggs, and positive and putative samples were further analyzed by multiplex PCR. In seven samples from three locations in central Anatolia (5.1%) and in one (0.9%) from Thrace, E . multilocularis DNA was amplified, and this result was confirmed with another PCR specific for E. multilocularis . In addition, Echinococcus granulosus s.l. was found in two (0.5%) of the samples. Although alveolar echinococcosis (AE) is known as a serious zoonosis in Turkey, this is the first field study detecting E. multilocularis in collected fecal samples documenting the environmental contamination with eggs of this zoonotic parasite.

Etiology of community acquired pneumonia among children in India: prospective, cohort study

PubMed Central

Mathew, Joseph L.; Singhi, Sunit; Ray, Pallab; Hagel, Eva; Saghafian–Hedengren, Shanie; Bansal, Arun; Ygberg, Sofia; Sodhi, Kushaljit Singh; Kumar, B V Ravi; Nilsson, Anna

2015-01-01

Background Childhood community acquired pneumonia (CAP) is a significant problem in developing countries, and confirmation of microbial etiology is important for individual, as well as public health. However, there is paucity of data from a large cohort, examining multiple biological specimens for diverse pathogens (bacteria and viruses). The Community Acquired Pneumonia Etiology Study (CAPES) was designed to address this knowledge gap. Methods We enrolled children with CAP (based on WHO IMCI criteria of tachypnea with cough or breathing difficulty) over 24 consecutive months, and recorded presenting symptoms, risk factors, clinical signs, and chest radiography. We performed blood and nasopharyngeal aspirate (NPA) bacterial cultures, and serology (Mycoplasma pneumoniae, Chlamydophila pneumoniae). We also performed multiplex PCR for 25 bacterial/viral species in a subgroup representing 20% of the cohort. Children requiring endotracheal intubation underwent culture and PCR of bronchoalveolar lavage (BAL) specimens. Findings We enrolled 2345 children. NPA and blood cultures yielded bacteria in only 322 (13.7%) and 49 (2.1%) children respectively. In NPA, Streptococcus pneumoniae (79.1%) predominated, followed by Haemophilus influenzae (9.6%) and Staphylococcus aureus (6.8%). In blood, S. aureus (30.6%) dominated, followed by S. pneumoniae (20.4%) and Klebsiella pneumoniae (12.2%). M. pneumoniae and C. pneumoniae serology were positive in 4.3% and 1.1% respectively. Multiplex PCR in 428 NPA specimens identified organisms in 422 (98.6%); of these 352 (82.2%) had multiple organisms and only 70 (16.4%) had a single organism viz. S. pneumoniae: 35 (50%), Cytomegalovirus (CMV): 13 (18.6%), Respiratory Syncytial Virus (RSV): 9 (12.9%), other viruses: 6 (8.7%), S. aureus: 5 (7.1%), and H. influenzae: 2 (2.9%). BAL PCR (n = 30) identified single pathogens in 10 (S. pneumoniae–3, CMV–3, S. aureus–2, H. influenzae–2) and multiple pathogens in 18 children. There were 108 (4.6%) deaths. The pattern of pathogens identified did not correlate with pneumonia severity or mortality. Conclusions The majority of children with CAP have multiple pathogens (bacteria and viruses). S. pneumoniae and S. aureus predominate in NPA and blood respectively. CMV and RSV were the dominant respiratory viruses in NPA and BAL. The presence of multiple pathogens, especially organisms associated with nasopharyngeal carriage, precludes confirmation of a causal relationship in most cases. PMID:26528392

Multiplexed fluorescence detector system for capillary electrophoresis

DOEpatents

Yeung, E.S.; Taylor, J.A.

1996-03-12

A fluorescence detection system for capillary electrophoresis is provided wherein the detection system can simultaneously excite fluorescence and substantially simultaneously monitor separations in multiple capillaries. This multiplexing approach involves laser irradiation of a sample in a plurality of capillaries through optical fibers that are coupled individually with the capillaries. The array is imaged orthogonally through a microscope onto a charge-coupled device camera for signal analysis. 14 figs.

Multiplexed fluorescence detector system for capillary electrophoresis

DOEpatents

Yeung, E.S.; Taylor, J.A.

1994-06-28

A fluorescence detection system for capillary electrophoresis is provided wherein the detection system can simultaneously excite fluorescence and substantially simultaneously monitor separations in multiple capillaries. This multiplexing approach involves laser irradiation of a sample in a plurality of capillaries through optical fibers that are coupled individually with the capillaries. The array is imaged orthogonally through a microscope onto a charge-coupled device camera for signal analysis. 14 figures.

Multiplexed fluorescence detector system for capillary electrophoresis

DOEpatents

Yeung, Edward S.; Taylor, John A.

1996-03-12

A fluorescence detection system for capillary electrophoresis is provided wherein the detection system can simultaneously excite fluorescence and substantially simultaneously monitor separations in multiple capillaries. This multiplexing approach involves laser irradiation of a sample in a plurality of capillaries through optical fibers that are coupled individually with the capillaries. The array is imaged orthogonally through a microscope onto a charge-coupled device camera for signal analysis.

Multiplexed fluorescence detector system for capillary electrophoresis

DOEpatents

Yeung, Edward S.; Taylor, John A.

1994-06-28

A fluorescence detection system for capillary electrophoresis is provided wherein the detection system can simultaneously excite fluorescence and substantially simultaneously monitor separations in multiple capillaries. This multiplexing approach involves laser irradiation of a sample in a plurality of capillaries through optical fibers that are coupled individually with the capillaries. The array is imaged orthogonally through a microscope onto a charge-coupled device camera for signal analysis.

Evaluation of an Internally Controlled Multiplex Tth Endonuclease Cleavage Loop-Mediated Isothermal Amplification (TEC-LAMP) Assay for the Detection of Bacterial Meningitis Pathogens

PubMed Central

Clancy, Eoin; Cormican, Martin; Boo, Teck Wee; Cunney, Robert

2018-01-01

Bacterial meningitis infection is a leading global health concern for which rapid and accurate diagnosis is essential to reduce associated morbidity and mortality. Loop-mediated isothermal amplification (LAMP) offers an effective low-cost diagnostic approach; however, multiplex LAMP is difficult to achieve, limiting its application. We have developed novel real-time multiplex LAMP technology, TEC-LAMP, using Tth endonuclease IV and a unique LAMP primer/probe. This study evaluates the analytical specificity, limit of detection (LOD) and clinical application of an internally controlled multiplex TEC-LAMP assay for detection of leading bacterial meningitis pathogens: Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae. Analytical specificities were established by testing 168 bacterial strains, and LODs were determined using Probit analysis. The TEC-LAMP assay was 100% specific, with LODs for S. pneumoniae, N. meningitidis and H. influenzae of 39.5, 17.3 and 25.9 genome copies per reaction, respectively. Clinical performance was evaluated by testing 65 archived PCR-positive samples. Compared to singleplex real-time PCR, the multiplex TEC-LAMP assay demonstrated diagnostic sensitivity and specificity of 92.3% and 100%, respectively. This is the first report of a single-tube internally controlled multiplex LAMP assay for bacterial meningitis pathogen detection, and the first report of Tth endonuclease IV incorporation into nucleic acid amplification diagnostic technology. PMID:29425124

Evaluation of an Internally Controlled Multiplex Tth Endonuclease Cleavage Loop-Mediated Isothermal Amplification (TEC-LAMP) Assay for the Detection of Bacterial Meningitis Pathogens.

PubMed

Higgins, Owen; Clancy, Eoin; Cormican, Martin; Boo, Teck Wee; Cunney, Robert; Smith, Terry J

2018-02-09

Bacterial meningitis infection is a leading global health concern for which rapid and accurate diagnosis is essential to reduce associated morbidity and mortality. Loop-mediated isothermal amplification (LAMP) offers an effective low-cost diagnostic approach; however, multiplex LAMP is difficult to achieve, limiting its application. We have developed novel real-time multiplex LAMP technology, TEC-LAMP, using Tth endonuclease IV and a unique LAMP primer/probe. This study evaluates the analytical specificity, limit of detection (LOD) and clinical application of an internally controlled multiplex TEC-LAMP assay for detection of leading bacterial meningitis pathogens: Streptococcus pneumoniae , Neisseria meningitidis and Haemophilus influenzae . Analytical specificities were established by testing 168 bacterial strains, and LODs were determined using Probit analysis. The TEC-LAMP assay was 100% specific, with LODs for S. pneumoniae , N. meningitidis and H. influenzae of 39.5, 17.3 and 25.9 genome copies per reaction, respectively. Clinical performance was evaluated by testing 65 archived PCR-positive samples. Compared to singleplex real-time PCR, the multiplex TEC-LAMP assay demonstrated diagnostic sensitivity and specificity of 92.3% and 100%, respectively. This is the first report of a single-tube internally controlled multiplex LAMP assay for bacterial meningitis pathogen detection, and the first report of Tth endonuclease IV incorporation into nucleic acid amplification diagnostic technology.

Single Fluorescence Channel-based Multiplex Detection of Avian Influenza Virus by Quantitative PCR with Intercalating Dye

PubMed Central

Ahberg, Christian D.; Manz, Andreas; Neuzil, Pavel

2015-01-01

Since its invention in 1985 the polymerase chain reaction (PCR) has become a well-established method for amplification and detection of segments of double-stranded DNA. Incorporation of fluorogenic probe or DNA intercalating dyes (such as SYBR Green) into the PCR mixture allowed real-time reaction monitoring and extraction of quantitative information (qPCR). Probes with different excitation spectra enable multiplex qPCR of several DNA segments using multi-channel optical detection systems. Here we show multiplex qPCR using an economical EvaGreen-based system with single optical channel detection. Previously reported non quantitative multiplex real-time PCR techniques based on intercalating dyes were conducted once the PCR is completed by performing melting curve analysis (MCA). The technique presented in this paper is both qualitative and quantitative as it provides information about the presence of multiple DNA strands as well as the number of starting copies in the tested sample. Besides important internal control, multiplex qPCR also allows detecting concentrations of more than one DNA strand within the same sample. Detection of the avian influenza virus H7N9 by PCR is a well established method. Multiplex qPCR greatly enhances its specificity as it is capable of distinguishing both haemagglutinin (HA) and neuraminidase (NA) genes as well as their ratio. PMID:26088868

Detection of α-thalassemia-1 Southeast Asian and Thai Type Deletions and β-thalassemia 3.5-kb Deletion by Single-tube Multiplex Real-time PCR with SYBR Green1 and High-resolution Melting Analysis

PubMed Central

Wiengkum, Thanatcha; Srithep, Sarinee; Chainoi, Isarapong; Singboottra, Panthong; Wongwiwatthananukit, Sanchai

2011-01-01

Background Prevention and control of thalassemia requires simple, rapid, and accurate screening tests for carrier couples who are at risk of conceiving fetuses with severe thalassemia. Methods Single-tube multiplex real-time PCR with SYBR Green1 and high-resolution melting (HRM) analysis were used for the identification of α-thalassemia-1 Southeast Asian (SEA) and Thai type deletions and β-thalassemia 3.5-kb gene deletion. The results were compared with those obtained using conventional gap-PCR. DNA samples were derived from 28 normal individuals, 11 individuals with α-thalassemia-1 SEA type deletion, 2 with α-thalassemia-1 Thai type deletion, and 2 with heterozygous β-thalassemia 3.5-kb gene deletion. Results HRM analysis indicated that the amplified fragments from α-thalassemia-1 SEA type deletion, α-thalassemia-1 Thai type deletion, β-thalassemia 3.5-kb gene deletion, and the wild-type β-globin gene had specific peak heights at mean melting temperature (Tm) values of 86.89℃, 85.66℃, 77.24℃, and 74.92℃, respectively. The results obtained using single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis showed 100% consistency with those obtained using conventional gap-PCR. Conclusions Single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis is a potential alternative for routine clinical screening of the common types of α- and β-thalassemia large gene deletions, since it is simple, cost-effective, and highly accurate. PMID:21779184

Interlaboratory Evaluation of a Multiplexed High Information Content In Vitro Genotoxicity Assay

PubMed Central

Bryce, Steven M.; Bernacki, Derek T.; Bemis, Jeffrey C.; Spellman, Richard A.; Engel, Maria E.; Schuler, Maik; Lorge, Elisabeth; Heikkinen, Pekka T.; Hemmann, Ulrike; Thybaud, Véronique; Wilde, Sabrina; Queisser, Nina; Sutter, Andreas; Zeller, Andreas; Guérard, Melanie; Kirkland, David; Dertinger, Stephen D.

2017-01-01

We previously described a multiplexed in vitro genotoxicity assay based on flow cytometric analysis of detergent-liberated nuclei that are simultaneously stained with propidium iodide and labeled with fluorescent antibodies against p53, γH2AX, and phospho-histone H3. Inclusion of a known number of microspheres provides absolute nuclei counts. The work described herein was undertaken to evaluate the interlaboratory transferability of this assay, commercially known as MultiFlow™ DNA Damage Kit— p53, γH2AX, Phospho-histone H3. For these experiments seven laboratories studied reference chemicals from a group of 84 representing clastogens, aneugens, and non-genotoxicants. TK6 cells were exposed to chemicals in 96-well plates over a range of concentrations for 24 hrs. At 4 and 24 hrs cell aliquots were added to the MultiFlow reagent mix and following a brief incubation period flow cytometric analysis occurred, in most cases directly from a 96-well plate via a robotic walk-away data acquisition system. Multiplexed response data were evaluated using two analysis approaches, one based on global evaluation factors (i.e., cutoff values derived from all inter-laboratory data), and a second based on multinomial logistic regression that considers multiple biomarkers simultaneously. Both data analysis strategies were devised to categorize chemicals as predominately exhibiting a clastogenic, aneugenic, or non-genotoxic mode of action (MoA). Based on the aggregate 231 experiments that were performed, assay sensitivity, specificity, and concordance in relation to a priori MoA grouping were ≥ 92%. These results are encouraging as they suggest that two distinct data analysis strategies can rapidly and reliably predict new chemicals’ predominant genotoxic MoA based on data from an efficient and transferable multiplexed in vitro assay. PMID:28370322

Monitoring for Human Papillomavirus Vaccine Impact Among Gay, Bisexual, and Other Men Who Have Sex With Men—United States, 2012–2014

PubMed Central

Meites, Elissa; Gorbach, Pamina M.; Gratzer, Beau; Panicker, Gitika; Steinau, Martin; Collins, Tom; Parrish, Adam; Randel, Cody; McGrath, Mark; Carrasco, Steven; Moore, Janell; Zaidi, Akbar; Braxton, Jim; Kerndt, Peter R.; Unger, Elizabeth R.; Crosby, Richard A.; Markowitz, Lauri E.

2016-01-01

Background Gay, bisexual, and other men who have sex with men (MSM) are at high risk for human papillomavirus (HPV) infection; vaccination is recommended for US males, including MSM through age 26 years. We assessed evidence of HPV among vaccine-eligible MSM and transgender women to monitor vaccine impact. Methods During 2012–2014, MSM aged 18–26 years at select clinics completed a computer-assisted self-interview regarding sexual behavior, human immunodeficiency virus (HIV) status, and vaccinations. Self-collected anal swab and oral rinse specimens were tested for HPV DNA (37 types) by L1 consensus polymerase chain reaction; serum was tested for HPV antibodies (4 types) by a multiplexed virus-like particle–based immunoglobulin G direct enzyme-linked immunosorbent assay. Results Among 922 vaccine-eligible participants, the mean age was 23 years, and the mean number of lifetime sex partners was 37. Among 834 without HIV infection, any anal HPV was detected in 69.4% and any oral HPV in 8.4%, yet only 8.5% had evidence of exposure to all quadrivalent vaccine types. In multivariate analysis, HPV prevalence varied significantly (P < .05) by HIV status, sexual orientation, and lifetime number of sex partners, but not by race/ethnicity. Discussions Most young MSM lacked evidence of current or past infection with all vaccine-type HPV types, suggesting that they could benefit from vaccination. The impact of vaccination among MSM may be assessed by monitoring HPV prevalence, including in self-collected specimens. PMID:27296847

Versatile label free biochip for the detection of circulating tumor cells from peripheral blood in cancer patients.

PubMed

Tan, Swee Jin; Lakshmi, Rumkumar Lalitha; Chen, Pengfei; Lim, Wan-Teck; Yobas, Levent; Lim, Chwee Teck

2010-12-15

The isolation of circulating tumor cells (CTCs) using microfluidics is attractive as the flow conditions can be accurately manipulated to achieve an efficient separation. CTCs are rare events within the peripheral blood of metastatic cancer patients which makes them hard to detect. The presence of CTCs is likely to indicate the severity of the disease and increasing evidences show its use for prognostic and treatment monitoring purposes. We demonstrated an effective separation using a microfluidic device to utilize the unique differences in size and deformability of cancer cells to blood cells. Using physical structures placed in the path of blood specimens in a microchannel, CTCs which are generally larger and stiffer are retained while most blood constituents are removed. The placements of the structures are optimized by computational analysis to enhance the isolation efficiency. With blood specimens from metastatic lung cancer patients, we confirmed the successful detection of CTCs. The operations for processing blood are straightforward and permit multiplexing of the microdevices to concurrently work with different samples. The microfluidic device is optically transparent which makes it simple to be integrated to existing laboratory microscopes and immunofluorescence staining can be done in situ to distinguish cancer cells from hematopoietic cells. This also minimizes the use of expensive staining reagents, given the small size of the microdevice. Identification of CTCs will aid in the detection of malignancy and disease stage as well as understanding the phenotypic and genotypic expressions of cancer cells. Copyright © 2010 Elsevier B.V. All rights reserved.

Clinical Validation and Implementation of a Targeted Next-Generation Sequencing Assay to Detect Somatic Variants in Non-Small Cell Lung, Melanoma, and Gastrointestinal Malignancies

PubMed Central

Fisher, Kevin E.; Zhang, Linsheng; Wang, Jason; Smith, Geoffrey H.; Newman, Scott; Schneider, Thomas M.; Pillai, Rathi N.; Kudchadkar, Ragini R.; Owonikoko, Taofeek K.; Ramalingam, Suresh S.; Lawson, David H.; Delman, Keith A.; El-Rayes, Bassel F.; Wilson, Malania M.; Sullivan, H. Clifford; Morrison, Annie S.; Balci, Serdar; Adsay, N. Volkan; Gal, Anthony A.; Sica, Gabriel L.; Saxe, Debra F.; Mann, Karen P.; Hill, Charles E.; Khuri, Fadlo R.; Rossi, Michael R.

2017-01-01

We tested and clinically validated a targeted next-generation sequencing (NGS) mutation panel using 80 formalin-fixed, paraffin-embedded (FFPE) tumor samples. Forty non-small cell lung carcinoma (NSCLC), 30 melanoma, and 30 gastrointestinal (12 colonic, 10 gastric, and 8 pancreatic adenocarcinoma) FFPE samples were selected from laboratory archives. After appropriate specimen and nucleic acid quality control, 80 NGS libraries were prepared using the Illumina TruSight tumor (TST) kit and sequenced on the Illumina MiSeq. Sequence alignment, variant calling, and sequencing quality control were performed using vendor software and laboratory-developed analysis workflows. TST generated ≥500× coverage for 98.4% of the 13,952 targeted bases. Reproducible and accurate variant calling was achieved at ≥5% variant allele frequency with 8 to 12 multiplexed samples per MiSeq flow cell. TST detected 112 variants overall, and confirmed all known single-nucleotide variants (n = 27), deletions (n = 5), insertions (n = 3), and multinucleotide variants (n = 3). TST detected at least one variant in 85.0% (68/80), and two or more variants in 36.2% (29/80), of samples. TP53 was the most frequently mutated gene in NSCLC (13 variants; 13/32 samples), gastrointestinal malignancies (15 variants; 13/25 samples), and overall (30 variants; 28/80 samples). BRAF mutations were most common in melanoma (nine variants; 9/23 samples). Clinically relevant NGS data can be obtained from routine clinical FFPE solid tumor specimens using TST, benchtop instruments, and vendor-supplied bioinformatics pipelines. PMID:26801070

Morphologic and genetic identification of Taenia tapeworms in Tanzania and DNA genotyping of Taenia solium.

PubMed

Eom, Keeseon S; Chai, Jong-Yil; Yong, Tai-Soon; Min, Duk-Young; Rim, Han-Jong; Kihamia, Charles; Jeon, Hyeong-Kyu

2011-12-01

Species identification of Taenia tapeworms was performed using morphologic observations and multiplex PCR and DNA sequencing of the mitochondrial cox1 gene. In 2008 and 2009, a total of 1,057 fecal samples were collected from residents of Kongwa district of Dodoma region, Tanzania, and examined microscopically for helminth eggs and proglottids. Of these, 4 Taenia egg positive cases were identified, and the eggs were subjected to DNA analysis. Several proglottids of Taenia solium were recovered from 1 of the 4 cases. This established that the species were T. solium (n = 1) and T. saginata (n = 3). One further T. solium specimen was found among 128 fecal samples collected from Mbulu district in Arusha, and this had an intact strobila with the scolex. Phylegenetic analysis of the mtDNA cox1 gene sequences of these 5 isolates showed that T. saginata was basal to the T. solium clade. The mitochondrial cox1 gene sequences of 3 of these Tanzanian isolates showed 99% similarity to T. saginata, and the other 2 isolates showed 100% similarity to T. solium. The present study has shown that Taenia tapeworms are endemic in Kongwa district of Tanzania, as well as in a previously identified Mbulu district. Both T. solium isolates were found to have an "African/Latin American" genotype (cox1).

Cell type specific gene expression analysis of prostate needle biopsies resolves tumor tissue heterogeneity

PubMed Central

Krönig, Malte; Walter, Max; Drendel, Vanessa; Werner, Martin; Jilg, Cordula A.; Richter, Andreas S.; Backofen, Rolf; McGarry, David; Follo, Marie; Schultze-Seemann, Wolfgang; Schüle, Roland

2015-01-01

A lack of cell surface markers for the specific identification, isolation and subsequent analysis of living prostate tumor cells hampers progress in the field. Specific characterization of tumor cells and their microenvironment in a multi-parameter molecular assay could significantly improve prognostic accuracy for the heterogeneous prostate tumor tissue. Novel functionalized gold-nano particles allow fluorescence-based detection of absolute mRNA expression levels in living cells by fluorescent activated flow cytometry (FACS). We use of this technique to separate prostate tumor and benign cells in human prostate needle biopsies based on the expression levels of the tumor marker alpha-methylacyl-CoA racemase (AMACR). We combined RNA and protein detection of living cells by FACS to gate for epithelial cell adhesion molecule (EPCAM) positive tumor and benign cells, EPCAM/CD45 double negative mesenchymal cells and CD45 positive infiltrating lymphocytes. EPCAM positive epithelial cells were further sub-gated into AMACR high and low expressing cells. Two hundred cells from each population and several biopsies from the same patient were analyzed using a multiplexed gene expression profile to generate a cell type resolved profile of the specimen. This technique provides the basis for the clinical evaluation of cell type resolved gene expression profiles as pre-therapeutic prognostic markers for prostate cancer. PMID:25514598

Optimized protocol for quantitative multiple reaction monitoring-based proteomic analysis of formalin-fixed, paraffin embedded tissues

PubMed Central

Kennedy, Jacob J.; Whiteaker, Jeffrey R.; Schoenherr, Regine M.; Yan, Ping; Allison, Kimberly; Shipley, Melissa; Lerch, Melissa; Hoofnagle, Andrew N.; Baird, Geoffrey Stuart; Paulovich, Amanda G.

2016-01-01

Despite a clinical, economic, and regulatory imperative to develop companion diagnostics, precious few new biomarkers have been successfully translated into clinical use, due in part to inadequate protein assay technologies to support large-scale testing of hundreds of candidate biomarkers in formalin-fixed paraffin embedded (FFPE) tissues. While the feasibility of using targeted, multiple reaction monitoring-mass spectrometry (MRM-MS) for quantitative analyses of FFPE tissues has been demonstrated, protocols have not been systematically optimized for robust quantification across a large number of analytes, nor has the performance of peptide immuno-MRM been evaluated. To address this gap, we used a test battery approach coupled to MRM-MS with the addition of stable isotope labeled standard peptides (targeting 512 analytes) to quantitatively evaluate the performance of three extraction protocols in combination with three trypsin digestion protocols (i.e. 9 processes). A process based on RapiGest buffer extraction and urea-based digestion was identified to enable similar quantitation results from FFPE and frozen tissues. Using the optimized protocols for MRM-based analysis of FFPE tissues, median precision was 11.4% (across 249 analytes). There was excellent correlation between measurements made on matched FFPE and frozen tissues, both for direct MRM analysis (R2 = 0.94) and immuno-MRM (R2 = 0.89). The optimized process enables highly reproducible, multiplex, standardizable, quantitative MRM in archival tissue specimens. PMID:27462933

Robust tumor morphometry in multispectral fluorescence microscopy

NASA Astrophysics Data System (ADS)

Tabesh, Ali; Vengrenyuk, Yevgen; Teverovskiy, Mikhail; Khan, Faisal M.; Sapir, Marina; Powell, Douglas; Mesa-Tejada, Ricardo; Donovan, Michael J.; Fernandez, Gerardo

2009-02-01

Morphological and architectural characteristics of primary tissue compartments, such as epithelial nuclei (EN) and cytoplasm, provide important cues for cancer diagnosis, prognosis, and therapeutic response prediction. We propose two feature sets for the robust quantification of these characteristics in multiplex immunofluorescence (IF) microscopy images of prostate biopsy specimens. To enable feature extraction, EN and cytoplasm regions were first segmented from the IF images. Then, feature sets consisting of the characteristics of the minimum spanning tree (MST) connecting the EN and the fractal dimension (FD) of gland boundaries were obtained from the segmented compartments. We demonstrated the utility of the proposed features in prostate cancer recurrence prediction on a multi-institution cohort of 1027 patients. Univariate analysis revealed that both FD and one of the MST features were highly effective for predicting cancer recurrence (p <= 0.0001). In multivariate analysis, an MST feature was selected for a model incorporating clinical and image features. The model achieved a concordance index (CI) of 0.73 on the validation set, which was significantly higher than the CI of 0.69 for the standard multivariate model based solely on clinical features currently used in clinical practice (p < 0.0001). The contributions of this work are twofold. First, it is the first demonstration of the utility of the proposed features in morphometric analysis of IF images. Second, this is the largest scale study of the efficacy and robustness of the proposed features in prostate cancer prognosis.

Technical Considerations for Reduced Representation Bisulfite Sequencing with Multiplexed Libraries

PubMed Central

Chatterjee, Aniruddha; Rodger, Euan J.; Stockwell, Peter A.; Weeks, Robert J.; Morison, Ian M.

2012-01-01

Reduced representation bisulfite sequencing (RRBS), which couples bisulfite conversion and next generation sequencing, is an innovative method that specifically enriches genomic regions with a high density of potential methylation sites and enables investigation of DNA methylation at single-nucleotide resolution. Recent advances in the Illumina DNA sample preparation protocol and sequencing technology have vastly improved sequencing throughput capacity. Although the new Illumina technology is now widely used, the unique challenges associated with multiplexed RRBS libraries on this platform have not been previously described. We have made modifications to the RRBS library preparation protocol to sequence multiplexed libraries on a single flow cell lane of the Illumina HiSeq 2000. Furthermore, our analysis incorporates a bioinformatics pipeline specifically designed to process bisulfite-converted sequencing reads and evaluate the output and quality of the sequencing data generated from the multiplexed libraries. We obtained an average of 42 million paired-end reads per sample for each flow-cell lane, with a high unique mapping efficiency to the reference human genome. Here we provide a roadmap of modifications, strategies, and trouble shooting approaches we implemented to optimize sequencing of multiplexed libraries on an a RRBS background. PMID:23193365

Image Decoding of Photonic Crystal Beads Array in the Microfluidic Chip for Multiplex Assays

PubMed Central

Yuan, Junjie; Zhao, Xiangwei; Wang, Xiaoxia; Gu, Zhongze

2014-01-01

Along with the miniaturization and intellectualization of biomedical instruments, the increasing demand of health monitoring at anywhere and anytime elevates the need for the development of point of care testing (POCT). Photonic crystal beads (PCBs) as one kind of good encoded microcarriers can be integrated with microfluidic chips in order to realize cost-effective and high sensitive multiplex bioassays. However, there are difficulties in analyzing them towards automated analysis due to the characters of the PCBs and the unique detection manner. In this paper, we propose a strategy to take advantage of automated image processing for the color decoding of the PCBs array in the microfluidic chip for multiplex assays. By processing and alignment of two modal images of epi-fluorescence and epi-white light, every intact bead in the image is accurately extracted and decoded by PC colors, which stand for the target species. This method, which shows high robustness and accuracy under various configurations, eliminates the high hardware requirement of spectroscopy analysis and user-interaction software, and provides adequate supports for the general automated analysis of POCT based on PCBs array. PMID:25341876

Use of multiplex polymerase chain reaction-based assay to conduct epidemiological studies on bovine hemoparasites in Mexico.

PubMed

Figueroa, J V; Alvarez, J A; Ramos, J A; Vega, C A; Buening, G M

1993-01-01

A study was conducted to test the applicability of a Polymerase Chain Reaction (PCR)-based approach for the simultaneous detection of the bovine hemoparasites Babesia bigemina, B. bovis and Anaplasma marginale. Bovine blood samples from cattle ranches of a previously determined enzootic zone in the Yucatan Peninsula of Mexico, were collected from peripheral blood and processed for PCR analysis. Blood samples were subjected to DNA amplification by placing an aliquot in a reaction tube containing oligonucleotide primers specific for DNA of each hemoparasite species. The PCR products were detected by Dot-Blot nucleic acid hybridization utilizing nonradioactive, species-specific, digoxigenin PCR-labeled DNA probes. Four hundred twenty one field samples analyzed by the multiplex PCR-DNA probe assay showed 66.7%, 60.1% and 59.6% prevalence rates for B. bigemina, B. bovis and A. marginale, respectively. The multiplex PCR analysis showed that animals with single, double or triple infection could be detected with the parasite specific DNA probes. The procedure is proposed as a valuable tool for the epidemiological analysis in regions where the hemoparasite species are concurrently infecting cattle.

Using the epigenetic field defect to detect prostate cancer in biopsy negative patients.

PubMed

Truong, Matthew; Yang, Bing; Livermore, Andrew; Wagner, Jennifer; Weeratunga, Puspha; Huang, Wei; Dhir, Rajiv; Nelson, Joel; Lin, Daniel W; Jarrard, David F

2013-06-01

We determined whether a novel combination of field defect DNA methylation markers could predict the presence of prostate cancer using histologically normal transrectal ultrasound guided biopsy cores. Methylation was assessed using quantitative Pyrosequencing® in a training set consisting of 65 nontumor and tumor associated prostate tissues from University of Wisconsin. A multiplex model was generated using multivariate logistic regression and externally validated in blinded fashion in a set of 47 nontumor and tumor associated biopsy specimens from University of Washington. We observed robust methylation differences in all genes at all CpGs assayed (p <0.0001). Regression models incorporating individual genes (EVX1, CAV1 and FGF1) and a gene combination (EVX1 and FGF1) discriminated nontumor from tumor associated tissues in the original training set (AUC 0.796-0.898, p <0.001). On external validation uniplex models incorporating EVX1, CAV1 or FGF1 discriminated tumor from nontumor associated biopsy negative specimens (AUC 0.702, 0.696 and 0.658, respectively, p <0.05). A multiplex model (EVX1 and FGF1) identified patients with prostate cancer (AUC 0.774, p = 0.001) and had a negative predictive value of 0.909. Comparison between 2 separate cores in patients in this validation set revealed similar methylation defects, indicating detection of a widespread field defect. A widespread epigenetic field defect can be used to detect prostate cancer in patients with histologically negative biopsies. To our knowledge this assay is unique, in that it detects alterations in nontumor cells. With further validation this marker combination (EVX1 and FGF1) has the potential to decrease the need for repeat prostate biopsies, a procedure associated with cost and complications. Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Species-Specific Serological Detection for Schistosomiasis by Serine Protease Inhibitor (SERPIN) in Multiplex Assay.

PubMed

Tanigawa, Chihiro; Fujii, Yoshito; Miura, Masashi; Nzou, Samson Muuo; Mwangi, Anne Wanjiru; Nagi, Sachiyo; Hamano, Shinjiro; Njenga, Sammy M; Mbanefo, Evaristus Chibunna; Hirayama, Kenji; Mwau, Matilu; Kaneko, Satoshi

2015-01-01

Both Schistosoma mansoni and Schistosoma haematobium cause schistosomiasis in sub-Saharan Africa. We assessed the diagnostic value of selected Schistosoma antigens for the development of a multiplex serological immunoassay for sero-epidemiological surveillance. Diagnostic ability of recombinant antigens from S. mansoni and S. haematobium was assessed by Luminex multiplex immunoassay using plasma from school children in two areas of Kenya, endemic for different species of schistosomiasis. S. mansoni serine protease inhibitor (SERPIN) and Sm-RP26 showed significantly higher reactivity to patient plasma as compared to the control group. Sm-Filamin, Sm-GAPDH, Sm-GST, Sm-LAP1, Sm-LAP2, Sm-Sm31, Sm-Sm32 and Sm-Tropomyosin did not show difference in reactivity between S. mansoni infected and uninfected pupils. Sm-RP26 was cross-reactive to plasma from S. haematobium patients, whereas Sm-SERPIN was species-specific. Sh-SEPRIN was partially cross-reactive to S. mansoni infected patients. ROC analysis for Sm-RP26, Sm-SERPIN and Sh-SERPIN showed AUC values of 0.833, 0.888 and 0.947, respectively. Using Spearman's rank correlation coefficient analysis, we also found significant positive correlation between the number of excreted eggs and median fluorescence intensity (MFI) from the multiplex immunoassays for Sm-SERPIN (ρ = 0.430, p-value = 0.003) and Sh-SERPIN (ρ = 0.433, p-value = 0.006). Sm-SERPIN is a promising species-specific diagnostic antigen. Sh-SEPRIN was partially cross-reactive to S. mansoni infected patients. SERPINs showed correlation with the number of excreted eggs. These indicate prospects for inclusion of SERPINs in the multiplex serological immunoassay system.

A study of multiplex data bus techniques for the space shuttle

NASA Technical Reports Server (NTRS)

Kearney, R. J.; Kalange, M. A.

1972-01-01

A comprehensive technology base for the design of a multiplexed data bus subsystem is provided. Extensive analyses, both analytical and empirical, were performed. Subjects covered are classified under the following headings: requirements identification and analysis; transmission media studies; signal design and detection studies; synchronization, timing, and control studies; user-subsystem interface studies; operational reliability analyses; design of candidate data bus configurations; and evaluation of candidate data bus designs.

Optimization of ultrahigh-speed multiplex PCR for forensic analysis.

PubMed

Gibson-Daw, Georgiana; Crenshaw, Karin; McCord, Bruce

2018-01-01

In this paper, we demonstrate the design and optimization of an ultrafast PCR amplification technique, used with a seven-locus multiplex that is compatible with conventional capillary electrophoresis systems as well as newer microfluidic chip devices. The procedure involves the use of a high-speed polymerase and a rapid cycling protocol to permit multiplex PCR amplification of forensic short tandem repeat loci in 6.5 min. We describe the selection and optimization of master mix reagents such as enzyme, buffer, MgCl 2 , and dNTPs, as well as primer ratios, total volume, and cycle conditions, in order to get the best profile in the shortest time possible. Sensitivity and reproducibility studies are also described. The amplification process utilizes a small high-speed thermocycler and compact laptop, making it portable and potentially useful for rapid, inexpensive on-site genotyping. The seven loci of the multiplex were taken from conventional STR genotyping kits and selected for their size and lack of overlap. Analysis was performed using conventional capillary electrophoresis and microfluidics with fluorescent detection. Overall, this technique provides a more rapid method for rapid sample screening of suspects and victims. Graphical abstract Rapid amplification of forensic DNA using high speed thermal cycling followed by capillary or microfluidic electrophoresis.

Qualis-SIS: automated standard curve generation and quality assessment for multiplexed targeted quantitative proteomic experiments with labeled standards.

PubMed

Mohammed, Yassene; Percy, Andrew J; Chambers, Andrew G; Borchers, Christoph H

2015-02-06

Multiplexed targeted quantitative proteomics typically utilizes multiple reaction monitoring and allows the optimized quantification of a large number of proteins. One challenge, however, is the large amount of data that needs to be reviewed, analyzed, and interpreted. Different vendors provide software for their instruments, which determine the recorded responses of the heavy and endogenous peptides and perform the response-curve integration. Bringing multiplexed data together and generating standard curves is often an off-line step accomplished, for example, with spreadsheet software. This can be laborious, as it requires determining the concentration levels that meet the required accuracy and precision criteria in an iterative process. We present here a computer program, Qualis-SIS, that generates standard curves from multiplexed MRM experiments and determines analyte concentrations in biological samples. Multiple level-removal algorithms and acceptance criteria for concentration levels are implemented. When used to apply the standard curve to new samples, the software flags each measurement according to its quality. From the user's perspective, the data processing is instantaneous due to the reactivity paradigm used, and the user can download the results of the stepwise calculations for further processing, if necessary. This allows for more consistent data analysis and can dramatically accelerate the downstream data analysis.

Using Next Generation Sequencing for Multiplexed Trait-Linked Markers in Wheat

PubMed Central

Bernardo, Amy; Wang, Shan; St. Amand, Paul; Bai, Guihua

2015-01-01

With the advent of next generation sequencing (NGS) technologies, single nucleotide polymorphisms (SNPs) have become the major type of marker for genotyping in many crops. However, the availability of SNP markers for important traits of bread wheat ( Triticum aestivum L.) that can be effectively used in marker-assisted selection (MAS) is still limited and SNP assays for MAS are usually uniplex. A shift from uniplex to multiplex assays will allow the simultaneous analysis of multiple markers and increase MAS efficiency. We designed 33 locus-specific markers from SNP or indel-based marker sequences that linked to 20 different quantitative trait loci (QTL) or genes of agronomic importance in wheat and analyzed the amplicon sequences using an Ion Torrent Proton Sequencer and a custom allele detection pipeline to determine the genotypes of 24 selected germplasm accessions. Among the 33 markers, 27 were successfully multiplexed and 23 had 100% SNP call rates. Results from analysis of "kompetitive allele-specific PCR" (KASP) and sequence tagged site (STS) markers developed from the same loci fully verified the genotype calls of 23 markers. The NGS-based multiplexed assay developed in this study is suitable for rapid and high-throughput screening of SNPs and some indel-based markers in wheat. PMID:26625271

Frequency-Modulated Continuous Flow Analysis Electrospray Ionization Mass Spectrometry (FM-CFA-ESI-MS) for Sample Multiplexing.

PubMed

Filla, Robert T; Schrell, Adrian M; Coulton, John B; Edwards, James L; Roper, Michael G

2018-02-20

A method for multiplexed sample analysis by mass spectrometry without the need for chemical tagging is presented. In this new method, each sample is pulsed at unique frequencies, mixed, and delivered to the mass spectrometer while maintaining a constant total flow rate. Reconstructed ion currents are then a time-dependent signal consisting of the sum of the ion currents from the various samples. Spectral deconvolution of each reconstructed ion current reveals the identity of each sample, encoded by its unique frequency, and its concentration encoded by the peak height in the frequency domain. This technique is different from other approaches that have been described, which have used modulation techniques to increase the signal-to-noise ratio of a single sample. As proof of concept of this new method, two samples containing up to 9 analytes were multiplexed. The linear dynamic range of the calibration curve was increased with extended acquisition times of the experiment and longer oscillation periods of the samples. Because of the combination of the samples, salt had little effect on the ability of this method to achieve relative quantitation. Continued development of this method is expected to allow for increased numbers of samples that can be multiplexed.

Simple Identification of Human Taenia Species by Multiplex Loop-Mediated Isothermal Amplification in Combination with Dot Enzyme-Linked Immunosorbent Assay.

PubMed

Nkouawa, Agathe; Sako, Yasuhito; Okamoto, Munehiro; Ito, Akira

2016-06-01

For differential detection of Taenia solium, Taenia saginata, and Taenia asiatica, loop-mediated isothermal amplification (LAMP) assay targeting the cytochrome c oxidase subunit 1 gene has been recently developed and shown to be sensitive, specific, and effective. However, to achieve differential identification, one specimen requires three reaction mixtures containing a primer set of each Taenia species separately, which is complex and time consuming and increases the risk of cross-contamination. In this study, we developed a simple differential identification of human Taenia species using multiplex LAMP (mLAMP) in combination with dot enzyme-linked immunosorbent assay (dot-ELISA). Forward inner primers of T. solium, T. saginata, and T. asiatica labeled with fluorescein isothiocyanate (FITC), digoxigenin (DIG), and tetramethylrhodamine (TAMRA), respectively, and biotin-labeled backward inner primers were used in mLAMP. The mLAMP assay succeeded in specific amplification of each respective target gene in a single tube. Furthermore, the mLAMP product from each species was easily distinguished by dot-ELISA with an antibody specific for FITC, DIG, or TAMRA. The mLAMP assay in combination with dot-ELISA will make identification of human Taenia species simpler, easier, and more practical. © The American Society of Tropical Medicine and Hygiene.

Multiplex PCR assay discriminates rabbit, rat and squirrel meat in food chain.

PubMed

Ahamad, Mohammad Nasir Uddin; Ali, Md Eaqub; Hossain, M A Motalib; Asing, Asing; Sultana, Sharmin; Jahurul, M H A

2017-12-01

Rabbit meat is receiving increasing attention because it contains a high level of proteins with relatively little fat. On the other hand, squirrel meat is served in upper-class meals in certain countries, so is sold at higher prices. The other side of the coin is rat meat, which has family ties with rabbit and squirrel but poses substantial threats to public health because it is a potential carrier of several zoonotic organisms. Recently, rat meat was mislabelled and sold as lamb after chemical modification. Thus, the chances of rabbit and squirrel meat substitution by rat meat cannot be ruled out. For the first time, a multiplex PCR assay was developed in Malaysia for the discriminatory identification of rat, rabbit and squirrel in the food chain. Rabbit (123 bp), rat (108 bp) and squirrel (243 bp) targets were amplified from ATP6 and cytb genes, along with a eukaryotic internal control (141bp). The products were sequenced and cross-tested against 22 species. A total of 81 reference samples and 72 meatball specimens were screened to validate the assay. Analyte stability was evaluated through boiling, autoclaving and micro-oven cooking. The tested lower limits of detection were 0.01 ng DNA for pure meat and 0.1% for meatballs.

Frozen tissue preparation for high-resolution multiplex histological analyses of human brain specimens.

PubMed

Shao, Fangjie; Jiang, Wenhong; Gao, Qingqing; Li, Baizhou; Sun, Chongran; Wang, Qiyuan; Chen, Qin; Sun, Bing; Shen, Hong; Zhu, Keqing; Zhang, Jianmin; Liu, Chong

2017-10-01

The availability of a comprehensive tissue library is essential for elucidating the function and pathology of human brains. Considering the irreplaceable status of the formalin-fixation-paraffin-embedding (FFPE) preparation in routine pathology and the advantage of ultra-low temperature to preserve nucleic acids and proteins for multi-omics studies, these methods have become major modalities for the construction of brain tissue libraries. Nevertheless, the use of FFPE and snap-frozen samples is limited in high-resolution histological analyses because the preparation destroys tissue integrity and/or many important cellular markers. To overcome these limitations, we detailed a protocol to prepare and analyze frozen human brain samples that is particularly suitable for high-resolution multiplex immunohistological studies. As an alternative, we offered an optimized procedure to rescue snap-frozen tissues for the same purpose. Importantly, we provided a guideline to construct libraries of frozen tissue with minimal effort, cost and space. Taking advantage of this new tissue preparation modality to nicely preserve the cellular information that was otherwise damaged using conventional methods and to effectively remove tissue autofluorescence, we described the high-resolution landscape of the cellular composition in both lower-grade gliomas and glioblastoma multiforme samples. Our work showcases the great value of fixed frozen tissue in understanding the cellular mechanisms of CNS functions and abnormalities.

Highly Multiplexed, Single Cell Transcriptomic Analysis of T-Cells by Microfluidic PCR.

PubMed

Dominguez, Maria; Roederer, Mario; Chattopadhyay, Pratip K

2017-01-01

Recently, technologies have been developed to measure expression of 96 (or more) mRNA transcripts at once from a single cell. Here we describe methods and important considerations for use of Fluidigm's BioMark platform for multiplexed single cell gene expression. We describe how to qualify primer/probes, select genes to examine in 96-parameter panels, perform the reverse transcription/cDNA synthesis step, and operate the instrument. In addition, we describe data analysis considerations. This technology has enormous value for characterizing the heterogeneity of T-cells, thereby providing a useful tool for immune monitoring.

Development of multiplex microsatellite PCR panels for the seagrass Thalassia hemprichii (Hydrocharitaceae)1

PubMed Central

van Dijk, Kor-jent; Mellors, Jane; Waycott, Michelle

2014-01-01

• Premise of the study: New microsatellites were developed for the seagrass Thalassia hemprichii (Hydrocharitaceae), a long-lived seagrass species that is found throughout the shallow waters of tropical and subtropical Indo-West Pacific. Three multiplex PCR panels were designed utilizing new and previously developed markers, resulting in a toolkit for generating a 16-locus genotype. • Methods and Results: Through the use of microsatellite enrichment and next-generation sequencing, 16 new, validated, polymorphic microsatellite markers were isolated. Diversity was between two and four alleles per locus totaling 36 alleles. These markers, plus previously developed microsatellite markers for T. hemprichii and T. testudinum, were tested for suitability in multiplex PCR panels. • Conclusions: The generation of an easily replicated suite of multiplex panels of codominant molecular markers will allow for high-resolution and detailed genetic structure analysis and clonality assessment with minimal genotyping costs. We suggest the establishment of a T. hemprichii primer convention for the unification of future data sets. PMID:25383269

Photocleavage-based affinity purification of biomarkers from serum: Application to multiplex allergy testing.

PubMed

Wan, Zhi; Ostendorff, Heather P; Liu, Ziying; Schneider, Lynda C; Rothschild, Kenneth J; Lim, Mark J

2018-01-01

Multiplex serological immunoassays, such as implemented on microarray or microsphere-based platforms, provide greater information content and higher throughput, while lowering the cost and blood volume required. These features are particularly attractive in pediatric food allergy testing to facilitate high throughput multi-allergen analysis from finger- or heel-stick collected blood. However, the miniaturization and microfluidics necessary for creating multiplex assays make them highly susceptible to the "matrix effect" caused by interference from non-target agents in serum and other biofluids. Such interference can result in lower sensitivity, specificity, reproducibility and quantitative accuracy. These problems have in large part prevented wide-spread implementation of multiplex immunoassays in clinical laboratories. We report the development of a novel method to eliminate the matrix effect by utilizing photocleavable capture antibodies to purify and concentrate blood-based biomarkers (a process termed PC-PURE) prior to detection in a multiplex immunoassay. To evaluate this approach, it was applied to blood-based allergy testing. Patient total IgE was purified and enriched using PC-PURE followed by multiplex microsphere-based detection of allergen-specific IgEs (termed the AllerBead assay). AllerBead was formatted to detect the eight most common pediatric food allergens: milk, soy, wheat, egg, peanuts, tree nuts, fin fish and shellfish, which account for >90% of all pediatric food allergies. 205 serum samples obtained from Boston Children's Hospital were evaluated. When PC-PURE was employed with AllerBead, excellent agreement was obtained with the standard, non-multiplex, ImmunoCAP® assay (average sensitivity above published negative predictive cutoffs = 96% and average Pearson r = 0.90; average specificity = 97%). In contrast, poor ImmunoCAP®-correlation was observed when PC-PURE was not utilized (average sensitivity above published negative predictive cutoffs = 59% and average Pearson r = 0.61; average specificity = 97%). This approach should be adaptable to improve a wide range of multiplex immunoassays such as in cancer, infectious disease and autoimmune disease.

Photocleavage-based affinity purification of biomarkers from serum: Application to multiplex allergy testing

PubMed Central

Wan, Zhi; Ostendorff, Heather P.; Liu, Ziying; Schneider, Lynda C.; Rothschild, Kenneth J.

2018-01-01

Multiplex serological immunoassays, such as implemented on microarray or microsphere-based platforms, provide greater information content and higher throughput, while lowering the cost and blood volume required. These features are particularly attractive in pediatric food allergy testing to facilitate high throughput multi-allergen analysis from finger- or heel-stick collected blood. However, the miniaturization and microfluidics necessary for creating multiplex assays make them highly susceptible to the “matrix effect” caused by interference from non-target agents in serum and other biofluids. Such interference can result in lower sensitivity, specificity, reproducibility and quantitative accuracy. These problems have in large part prevented wide-spread implementation of multiplex immunoassays in clinical laboratories. We report the development of a novel method to eliminate the matrix effect by utilizing photocleavable capture antibodies to purify and concentrate blood-based biomarkers (a process termed PC-PURE) prior to detection in a multiplex immunoassay. To evaluate this approach, it was applied to blood-based allergy testing. Patient total IgE was purified and enriched using PC-PURE followed by multiplex microsphere-based detection of allergen-specific IgEs (termed the AllerBead assay). AllerBead was formatted to detect the eight most common pediatric food allergens: milk, soy, wheat, egg, peanuts, tree nuts, fin fish and shellfish, which account for >90% of all pediatric food allergies. 205 serum samples obtained from Boston Children’s Hospital were evaluated. When PC-PURE was employed with AllerBead, excellent agreement was obtained with the standard, non-multiplex, ImmunoCAP® assay (average sensitivity above published negative predictive cutoffs = 96% and average Pearson r = 0.90; average specificity = 97%). In contrast, poor ImmunoCAP®-correlation was observed when PC-PURE was not utilized (average sensitivity above published negative predictive cutoffs = 59% and average Pearson r = 0.61; average specificity = 97%). This approach should be adaptable to improve a wide range of multiplex immunoassays such as in cancer, infectious disease and autoimmune disease. PMID:29389948

The Microwave SQUID Multiplexer

NASA Astrophysics Data System (ADS)

Mates, John Arthur Benson

2011-12-01

This thesis describes a multiplexer of Superconducting Quantum Interference Devices (SQUIDs) with low-noise, ultra-low power dissipation, and great scalability. The multiplexer circuit measures the magnetic flux in a large number of unshunted rf SQUIDs by coupling each SQUID to a superconducting microwave resonator tuned to a unique resonance frequency and driving the resonators from a common feedline. A superposition of microwave tones measures each SQUID simultaneously using only two coaxial cables between the cryogenic device and room temperature. This multiplexer will enable the instrumentation of arrays with hundreds of thousands of low-temperature detectors for new applications in cosmology, materials analysis, and nuclear non-proliferation. The driving application of the Microwave SQUID Multiplexer is the readout of large arrays of superconducting transition-edge sensors, by some figures of merit the most sensitive detectors of electromagnetic signals over a span of more than nine orders of magnitude in energy, from 40 GHz microwaves to 200 keV gamma rays. Modern transition-edge sensors have noise-equivalent power as low as 10-20 W / Hz1/2 and energy resolution as good as 2 eV at 6 keV. These per-pixel sensitivities approach theoretical limits set by the underlying signals, motivating a rapid increase in pixel count to access new science. Compelling applications, like the non-destructive assay of nuclear material for treaty verification or the search for primordial gravity waves from inflation use arrays of these detectors to increase collection area or tile a focal plane. We developed three generations of SQUID multiplexers, optimizing the first for flux noise 0.17 muPhi0 / Hz1/2, the second for input current noise 19 pA / Hz1/2, and the last for practical multiplexing of large arrays of cosmic microwave background polarimeters based on transition-edge sensors. Using the last design we demonstrated multiplexed readout of prototype polarimeters with the performance required for the future development of a large-scale astronomical instrument.

Multiplex Networks of Cortical and Hippocampal Neurons Revealed at Different Timescales

PubMed Central

Timme, Nicholas; Ito, Shinya; Myroshnychenko, Maxym; Yeh, Fang-Chin; Hiolski, Emma; Hottowy, Pawel; Beggs, John M.

2014-01-01

Recent studies have emphasized the importance of multiplex networks – interdependent networks with shared nodes and different types of connections – in systems primarily outside of neuroscience. Though the multiplex properties of networks are frequently not considered, most networks are actually multiplex networks and the multiplex specific features of networks can greatly affect network behavior (e.g. fault tolerance). Thus, the study of networks of neurons could potentially be greatly enhanced using a multiplex perspective. Given the wide range of temporally dependent rhythms and phenomena present in neural systems, we chose to examine multiplex networks of individual neurons with time scale dependent connections. To study these networks, we used transfer entropy – an information theoretic quantity that can be used to measure linear and nonlinear interactions – to systematically measure the connectivity between individual neurons at different time scales in cortical and hippocampal slice cultures. We recorded the spiking activity of almost 12,000 neurons across 60 tissue samples using a 512-electrode array with 60 micrometer inter-electrode spacing and 50 microsecond temporal resolution. To the best of our knowledge, this preparation and recording method represents a superior combination of number of recorded neurons and temporal and spatial recording resolutions to any currently available in vivo system. We found that highly connected neurons (“hubs”) were localized to certain time scales, which, we hypothesize, increases the fault tolerance of the network. Conversely, a large proportion of non-hub neurons were not localized to certain time scales. In addition, we found that long and short time scale connectivity was uncorrelated. Finally, we found that long time scale networks were significantly less modular and more disassortative than short time scale networks in both tissue types. As far as we are aware, this analysis represents the first systematic study of temporally dependent multiplex networks among individual neurons. PMID:25536059

Multiplexity and multireciprocity in directed multiplexes.

PubMed

Gemmetto, Valerio; Squartini, Tiziano; Picciolo, Francesco; Ruzzenenti, Franco; Garlaschelli, Diego

2016-10-01

Real-world multilayer networks feature nontrivial dependencies among links of different layers. Here we argue that if links are directed, then dependencies are twofold. Besides the ordinary tendency of links of different layers to align as the result of "multiplexity," there is also a tendency to antialign as a result of what we call "multireciprocity," i.e., the fact that links in one layer can be reciprocated by opposite links in a different layer. Multireciprocity generalizes the scalar definition of single-layer reciprocity to that of a square matrix involving all pairs of layers. We introduce multiplexity and multireciprocity matrices for both binary and weighted multiplexes and validate their statistical significance against maximum-entropy null models that filter out the effects of node heterogeneity. We then perform a detailed empirical analysis of the world trade multiplex (WTM), representing the import-export relationships between world countries in different commodities. We show that the WTM exhibits strong multiplexity and multireciprocity, an effect which is, however, largely encoded into the degree or strength sequences of individual layers. The residual effects are still significant and allow us to classify pairs of commodities according to their tendency to be traded together in the same direction and/or in opposite ones. We also find that the multireciprocity of the WTM is significantly lower than the usual reciprocity measured on the aggregate network. Moreover, layers with low (high) internal reciprocity are embedded within sets of layers with comparably low (high) mutual multireciprocity. This suggests that, in the WTM, reciprocity is inherent to groups of related commodities rather than to individual commodities. We discuss the implications for international trade research focusing on product taxonomies, the product space, and fitness and complexity metrics.

Molecular allergy diagnostics using multiplex assays: methodological and practical considerations for use in research and clinical routine: Part 21 of the Series Molecular Allergology.

PubMed

Jakob, Thilo; Forstenlechner, Peter; Matricardi, Paolo; Kleine-Tebbe, Jörg

The availability of single allergens and their use in microarray technology enables the simultaneous determination of specific IgE (sIgE) to a multitude of different allergens (> 100) in a multiplex procedure requiring only minute amounts of serum. This allows extensive individual sensitization profiles to be determined from a single analysis. Combined with a patient's medical history, these profiles simplify identification of cross-reactivity; permit a more accurate estimation of the risk of severe reactions; and enable the indication for specific immunotherapy to be more precisely established, particularly in cases of polysensitization. Strictly speaking, a multiplex assay is not a single test, but instead more than 100 simultaneous tests. This places considerable demands on the production, quality assurance, and interpretation of data. The following chapter describes the multiplex test systems currently available and discusses their characteristics. Performance data are presented and the sIgE values obtained from multiplex and singleplex assays are compared. Finally, the advantages and limitations of molecular allergy diagnostics using multiplex assays in clinical routine are discussed, and innovative possibilities for clinical research are described. The multiplex diagnostic tests available for clinical routine have now become well established. The interpretation of test results is demanding, particularly since all individual results need to be checked for their plausibility and clinical relevance on the basis of previous history (patient history, clinical symptoms, challenge test results). There is still room for improvement in certain areas, for example with respect to the overall test sensitivity of the method, as well as the availability and quality of particular allergens. The current test systems are just the beginning of a continuous development that will influence and most likely change clinical allergology in the coming years.

Molecular Analysis-Based Genetic Characterization of a Cohort of Patients with Duchenne and Becker Muscular Dystrophy in Eastern China.

PubMed

Zhao, Hui-Hui; Sun, Xue-Ping; Shi, Ming-Chao; Yi, Yong-Xiang; Cheng, Hong; Wang, Xing-Xia; Xu, Qing-Cheng; Ma, Hong-Ming; Wu, Hao-Quan; Jin, Qing-Wen; Niu, Qi

2018-04-05

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are common X-linked recessive neuromuscular disorders caused by mutations in dystrophin gene. Multiplex polymerase chain reaction (multiplex PCR) and multiplex ligation-dependent probe amplification (MLPA) are the most common methods for detecting dystrophin gene mutations. This study aimed to contrast the two methods and discern the genetic characterization of patients with DMD/BMD in Eastern China. We collected 121 probands, 64 mothers of probands, and 15 fetuses in our study. The dystrophin gene was detected by multiplex PCR primarily in 28 probands, and MLPA was used in multiplex PCR-negative cases subsequently. The dystrophin gene of the remaining 93 probands and 62 female potential carriers was tested by MLPA directly. In fetuses, multiplex PCR and MLPA were performed on 4 fetuses and 10 fetuses, respectively. In addition, sequencing was also performed in 4 probands with negative MLPA. We found that 61.98% of the subjects had genetic mutations including deletions (50.41%) and duplications (11.57%). There were 43.75% of mothers as carriers of the mutation. In 15 fetuses, 2 out of 7 male fetuses were found to be unhealthy and 2 out of 8 female fetuses were found to be carriers. Exons 3-26 and 45-52 have the maximum frequency in mutation regions. In the frequency of exons individually, exon 47 and exon 50 were the most common in deleted regions and exons 5, 6, and 7 were found most frequently in duplicated regions. MLPA has better productivity and sensitivity than multiplex PCR. Prenatal diagnosis should be applied in DMD high-risk fetuses to reduce the disease incidence. Furthermore, it is the responsibility of physicians to inform female carriers the importance of prenatal diagnosis.

Multiplex gas chromatography for use in space craft

NASA Technical Reports Server (NTRS)

Valentin, J. R.

1985-01-01

Gas chromatography is a powerful technique for the analysis of gaseous mixtures. Some limitations in this technique still exist which can be alleviated with multiplex gas chromatography (MGC). In MGC, rapid multiple sample injections are made into the column without having to wait for one determination to be finished before taking a new sample. The resulting data must then be reduced using computational methods such as cross correlation. In order to efficiently perform multiplexgas chromatography, experiments in the laboratory and on board future space craft, skills, equipment, and computer software were developed. Three new techniques for modulating, i.e., changing, sample concentrations were demonstrated by using desorption, decomposition, and catalytic modulators. In all of them, the need for a separate gas stream as the carrier was avoided by placing the modulator at the head of the column to directly modulate a sample stream. Finally, the analysis of an environmental sample by multiplex chromatography was accomplished by employing silver oxide to catalytically modulate methane in ambient air.

Developmental validation of a Cannabis sativa STR multiplex system for forensic analysis.

PubMed

Howard, Christopher; Gilmore, Simon; Robertson, James; Peakall, Rod

2008-09-01

A developmental validation study based on recommendations of the Scientific Working Group on DNA Analysis Methods (SWGDAM) was conducted on a multiplex system of 10 Cannabis sativa short tandem repeat loci. Amplification of the loci in four multiplex reactions was tested across DNA from dried root, stem, and leaf sources, and DNA from fresh, frozen, and dried leaf tissue with a template DNA range of 10.0-0.01 ng. The loci were amplified and scored consistently for all DNA sources when DNA template was in the range of 10.0-1.0 ng. Some allelic dropout and PCR failure occurred in reactions with lower template DNA amounts. Overall, amplification was best using 10.0 ng of template DNA from dried leaf tissue indicating that this is the optimal source material. Cross species amplification was observed in Humulus lupulus for three loci but there was no allelic overlap. This is the first study following SWGDAM validation guidelines to validate short tandem repeat markers for forensic use in plants.

Recent developments in Förster resonance energy transfer (FRET) diagnostics using quantum dots.

PubMed

Geißler, Daniel; Hildebrandt, Niko

2016-07-01

The exceptional photophysical properties and the nanometric dimensions of colloidal semiconductor quantum dots (QD) have strongly attracted the bioanalytical community over the last approximately 20 y. In particular, the integration of QDs in the analysis of biological components and interactions, and the related diagnostics using Förster resonance energy transfer (FRET), have allowed researchers to significantly improve and diversify fluorescence-based biosensing. In this TRENDS article, we review some recent developments in QD-FRET biosensing that have implemented this technology in electronic consumer products, multiplexed analysis, and detection without light excitation for diagnostic applications. In selected examples of smartphone-based imaging, single- and multistep FRET, steady-state and time-resolved spectroscopy, and bio/chemiluminescence detection of QDs used as both FRET donors and acceptors, we highlight the advantages of QD-based FRET biosensing for multiplexed and sensitive diagnostics. Graphical Abstract Quantum dots (QDs) can be applied as donors and/or acceptors for Förster resonance energy transfer- (FRET-) based biosensing for multiplexed and sensitive diagnostics in various assay formats.

'Mitominis': multiplex PCR analysis of reduced size amplicons for compound sequence analysis of the entire mtDNA control region in highly degraded samples.

PubMed

Eichmann, Cordula; Parson, Walther

2008-09-01

The traditional protocol for forensic mitochondrial DNA (mtDNA) analyses involves the amplification and sequencing of the two hypervariable segments HVS-I and HVS-II of the mtDNA control region. The primers usually span fragment sizes of 300-400 bp each region, which may result in weak or failed amplification in highly degraded samples. Here we introduce an improved and more stable approach using shortened amplicons in the fragment range between 144 and 237 bp. Ten such amplicons were required to produce overlapping fragments that cover the entire human mtDNA control region. These were co-amplified in two multiplex polymerase chain reactions and sequenced with the individual amplification primers. The primers were carefully selected to minimize binding on homoplasic and haplogroup-specific sites that would otherwise result in loss of amplification due to mis-priming. The multiplexes have successfully been applied to ancient and forensic samples such as bones and teeth that showed a high degree of degradation.

Multiplex cDNA quantification method that facilitates the standardization of gene expression data

PubMed Central

Gotoh, Osamu; Murakami, Yasufumi; Suyama, Akira

2011-01-01

Microarray-based gene expression measurement is one of the major methods for transcriptome analysis. However, current microarray data are substantially affected by microarray platforms and RNA references because of the microarray method can provide merely the relative amounts of gene expression levels. Therefore, valid comparisons of the microarray data require standardized platforms, internal and/or external controls and complicated normalizations. These requirements impose limitations on the extensive comparison of gene expression data. Here, we report an effective approach to removing the unfavorable limitations by measuring the absolute amounts of gene expression levels on common DNA microarrays. We have developed a multiplex cDNA quantification method called GEP-DEAN (Gene expression profiling by DCN-encoding-based analysis). The method was validated by using chemically synthesized DNA strands of known quantities and cDNA samples prepared from mouse liver, demonstrating that the absolute amounts of cDNA strands were successfully measured with a sensitivity of 18 zmol in a highly multiplexed manner in 7 h. PMID:21415008

A novel multiplex PCR assay for simultaneous detection of nine clinically significant bacterial pathogens associated with bovine mastitis.

PubMed

Ashraf, Aqeela; Imran, Muhammad; Yaqub, Tahir; Tayyab, Muhammad; Shehzad, Wasim; Thomson, Peter C

2017-06-01

For rapid and simultaneous detection of nine bovine mastitic pathogens, a sensitive and specific multiplex PCR assay was developed. The assay was standardized using reference strains and validated on mastitic milk cultures which were identified to species level based on 16S rRNA sequencing. Multiplex PCR assay also efficiently detected the target bacterial strains directly from milk. The detection limit of the assay was up to 50 pg for DNA isolated from pure cultures and 10 4  CFU/ml for spiked milk samples. As estimated by latent class analysis, the assay was sensitive up to 88% and specific up to 98% for targeted mastitic pathogens, compared with the bacterial culture method and the 16S rRNA sequence analysis. This novel molecular assay could be useful for monitoring and maintaining the bovine udder health, ensuring the bacteriological safety of milk, and conducting epidemiological studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

Single quantum dot analysis enables multiplexed point mutation detection by gap ligase chain reaction.

PubMed

Song, Yunke; Zhang, Yi; Wang, Tza-Huei

2013-04-08

Gene point mutations present important biomarkers for genetic diseases. However, existing point mutation detection methods suffer from low sensitivity, specificity, and a tedious assay processes. In this report, an assay technology is proposed which combines the outstanding specificity of gap ligase chain reaction (Gap-LCR), the high sensitivity of single-molecule coincidence detection, and the superior optical properties of quantum dots (QDs) for multiplexed detection of point mutations in genomic DNA. Mutant-specific ligation products are generated by Gap-LCR and subsequently captured by QDs to form DNA-QD nanocomplexes that are detected by single-molecule spectroscopy (SMS) through multi-color fluorescence burst coincidence analysis, allowing for multiplexed mutation detection in a separation-free format. The proposed assay is capable of detecting zeptomoles of KRAS codon 12 mutation variants with near 100% specificity. Its high sensitivity allows direct detection of KRAS mutation in crude genomic DNA without PCR pre-amplification. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators

PubMed Central

Bodenmiller, Bernd; Zunder, Eli R.; Finck, Rachel; Chen, Tiffany J.; Savig, Erica S.; Bruggner, Robert V.; Simonds, Erin F.; Bendall, Sean C.; Sachs, Karen; Krutzik, Peter O.; Nolan, Garry P.

2013-01-01

The ability to comprehensively explore the impact of bio-active molecules on human samples at the single-cell level can provide great insight for biomedical research. Mass cytometry enables quantitative single-cell analysis with deep dimensionality, but currently lacks high-throughput capability. Here we report a method termed mass-tag cellular barcoding (MCB) that increases mass cytometry throughput by sample multiplexing. 96-well format MCB was used to characterize human peripheral blood mononuclear cell (PBMC) signaling dynamics, cell-to-cell communication, the signaling variability between 8 donors, and to define the impact of 27 inhibitors on this system. For each compound, 14 phosphorylation sites were measured in 14 PBMC types, resulting in 18,816 quantified phosphorylation levels from each multiplexed sample. This high-dimensional systems-level inquiry allowed analysis across cell-type and signaling space, reclassified inhibitors, and revealed off-target effects. MCB enables high-content, high-throughput screening, with potential applications for drug discovery, pre-clinical testing, and mechanistic investigation of human disease. PMID:22902532

Characterization and multiplexing of EST-SSR primers in Cynodon (Poaceae) species1.

PubMed

Jewell, Margaret C; Frere, Celine H; Prentis, Peter J; Lambrides, Christopher J; Godwin, Ian D

2010-10-01

Cynodon species are multiple-use grasses that display varying levels of adaptation to biotic and abiotic stress. Previously identified EST-SSR primers were characterized and multiplexed to assess the level of genetic diversity present within a collection of almost 1200 Cynodon accessions from across Australia. • Two multiplex reactions were developed comprising a total of 16 EST-SSR markers. All SSR markers amplified across different Cynodon species and different levels of ploidy. The number of alleles ranged from one to eight per locus and the total number of alleles for the germplasm collection was 79. • The 16 markers show sufficient variation for the characterization of Cynodon core collections and analysis of population genetic diversity in Cynodon grasses.

Scout-MRM: Multiplexed Targeted Mass Spectrometry-Based Assay without Retention Time Scheduling Exemplified by Dickeya dadantii Proteomic Analysis during Plant Infection.

PubMed

Rougemont, Blandine; Bontemps Gallo, Sébastien; Ayciriex, Sophie; Carrière, Romain; Hondermarck, Hubert; Lacroix, Jean Marie; Le Blanc, J C Yves; Lemoine, Jérôme

2017-02-07

Targeted mass spectrometry of a surrogate peptide panel is a powerful method to study the dynamics of protein networks, but chromatographic time scheduling remains a major limitation for dissemination and implementation of robust and large multiplexed assays. We unveil a Multiple Reaction Monitoring method (Scout-MRM) where the use of spiked scout peptides triggers complex transition lists, regardless of the retention time of targeted surrogate peptides. The interest of Scout-MRM method regarding the retention time independency, multiplexing capability, reproducibility, and putative interest in facilitating method transfer was illustrated by a 782-peptide-plex relative assay targeting 445 proteins of the phytopathogen Dickeya dadantii during plant infection.

Influence of enrichment broths on multiplex PCR detection of total coliform bacteria, Escherichia coli and Clostridium perfringens, in spiked water samples.

PubMed

Worakhunpiset, S; Tharnpoophasiam, P

2009-07-01

Although multiplex PCR amplification condition for simultaneous detection of total coliform bacteria, Escherichia coli and Clostridium perfringens in water sample has been developed, results with high sensitivity are obtained when amplifying purified DNA, but the sensitivity is low when applied to spiked water samples. An enrichment broth culture prior PCR analysis increases sensitivity of the test but the specific nature of enrichment broth can affect the PCR results. Three enrichment broths, lactose broth, reinforced clostridial medium and fluid thioglycollate broth, were compared for their influence on sensitivity and on time required with multiplex PCR assay. Fluid thioglycollate broth was the most effective with shortest enrichment time and lowest detection limit.

Simultaneous frequency stabilization and high-power dense wavelength division multiplexing (HP-DWDM) using an external cavity based on volume Bragg gratings (VBGs)

NASA Astrophysics Data System (ADS)

Hengesbach, Stefan; Klein, Sarah; Holly, Carlo; Witte, Ulrich; Traub, Martin; Hoffmann, Dieter

2016-03-01

Multiplexing technologies enable the development of high-brightness diode lasers for direct industrial applications. We present a High-Power Dense Wavelength Division Multiplexer (HP-DWDM) with an average channel spacing of 1.7 (1.5) nm and a subsequent external cavity mirror to provide feedback for frequency stabilization and multiplexing in one step. The "self-optimizing" multiplexing unit consists of four reflective Volume Bragg Gratings (VBGs) with 99% diffraction efficiency and seven dielectric mirrors to overlay the radiation of five input channels with an adjustable channel spacing of 1-2 nm. In detail, we focus on the analysis of the overall optical efficiency, the change of the beam parameter product and the spectral width. The performance is demonstrated using five 90 μm multimode 9xx single emitters with M2<=17. Because of the feedback the lateral (multimodal) spatial and angular intensity distribution changes strongly and the beam parameter product decreases by a factor of 1.2 to 1.9. Thereby the angular intensity distribution is more affected than the width of the beam waist. The spectral width per emitter decreases to 3-200 pm (FWHM) depending on the injection current and the reflectance of the feedback mirror (0.75%, 1.5%, 4%, 6% or 8%). The overall optical multiplexing efficiency ranges between 77% and 86%. With some modifications (e.g. enhanced AR-coatings) we expect 90-95%.

Multiplex Ultrasensitive Genotyping of Patients with Non-Small Cell Lung Cancer for Epidermal Growth Factor Receptor (EGFR) Mutations by Means of Picodroplet Digital PCR.

PubMed

Watanabe, Masaru; Kawaguchi, Tomoya; Isa, Shun-Ichi; Ando, Masahiko; Tamiya, Akihiro; Kubo, Akihito; Saka, Hideo; Takeo, Sadanori; Adachi, Hirofumi; Tagawa, Tsutomu; Kawashima, Osamu; Yamashita, Motohiro; Kataoka, Kazuhiko; Ichinose, Yukito; Takeuchi, Yukiyasu; Watanabe, Katsuya; Matsumura, Akihide; Koh, Yasuhiro

2017-07-01

Epidermal growth factor receptor (EGFR) mutations have been used as the strongest predictor of effectiveness of treatment with EGFR tyrosine kinase inhibitors (TKIs). Three most common EGFR mutations (L858R, exon 19 deletion, and T790M) are known to be major selection markers for EGFR-TKIs therapy. Here, we developed a multiplex picodroplet digital PCR (ddPCR) assay to detect 3 common EGFR mutations in 1 reaction. Serial-dilution experiments with genomic DNA harboring EGFR mutations revealed linear performance, with analytical sensitivity ~0.01% for each mutation. All 33 EGFR-activating mutations detected in formalin-fixed paraffin-embedded (FFPE) tissue samples by the conventional method were also detected by this multiplex assay. Owing to the higher sensitivity, an additional mutation (T790M; including an ultra-low-level mutation, <0.1%) was detected in the same reaction. Regression analysis of the duplex assay and multiplex assay showed a correlation coefficient (R 2 ) of 0.9986 for L858R, 0.9844 for an exon 19 deletion, and 0.9959 for T790M. Using ddPCR, we designed a multiplex ultrasensitive genotyping platform for 3 common EGFR mutations. Results of this proof-of-principle study on clinical samples indicate clinical utility of multiplex ddPCR for screening for multiple EGFR mutations concurrently with an ultra-rare pretreatment mutation (T790M). Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Multiplexed SNP genotyping using the Qbead™ system: a quantum dot-encoded microsphere-based assay

PubMed Central

Xu, Hongxia; Sha, Michael Y.; Wong, Edith Y.; Uphoff, Janet; Xu, Yanzhang; Treadway, Joseph A.; Truong, Anh; O’Brien, Eamonn; Asquith, Steven; Stubbins, Michael; Spurr, Nigel K.; Lai, Eric H.; Mahoney, Walt

2003-01-01

We have developed a new method using the Qbead™ system for high-throughput genotyping of single nucleotide polymorphisms (SNPs). The Qbead system employs fluorescent Qdot™ semiconductor nanocrystals, also known as quantum dots, to encode microspheres that subsequently can be used as a platform for multiplexed assays. By combining mixtures of quantum dots with distinct emission wavelengths and intensities, unique spectral ‘barcodes’ are created that enable the high levels of multiplexing required for complex genetic analyses. Here, we applied the Qbead system to SNP genotyping by encoding microspheres conjugated to allele-specific oligonucleotides. After hybridization of oligonucleotides to amplicons produced by multiplexed PCR of genomic DNA, individual microspheres are analyzed by flow cytometry and each SNP is distinguished by its unique spectral barcode. Using 10 model SNPs, we validated the Qbead system as an accurate and reliable technique for multiplexed SNP genotyping. By modifying the types of probes conjugated to microspheres, the Qbead system can easily be adapted to other assay chemistries for SNP genotyping as well as to other applications such as analysis of gene expression and protein–protein interactions. With its capability for high-throughput automation, the Qbead system has the potential to be a robust and cost-effective platform for a number of applications. PMID:12682378

PrimerSuite: A High-Throughput Web-Based Primer Design Program for Multiplex Bisulfite PCR.

PubMed

Lu, Jennifer; Johnston, Andrew; Berichon, Philippe; Ru, Ke-Lin; Korbie, Darren; Trau, Matt

2017-01-24

The analysis of DNA methylation at CpG dinucleotides has become a major research focus due to its regulatory role in numerous biological processes, but the requisite need for assays which amplify bisulfite-converted DNA represents a major bottleneck due to the unique design constraints imposed on bisulfite-PCR primers. Moreover, a review of the literature indicated no available software solutions which accommodated both high-throughput primer design, support for multiplex amplification assays, and primer-dimer prediction. In response, the tri-modular software package PrimerSuite was developed to support bisulfite multiplex PCR applications. This software was constructed to (i) design bisulfite primers against multiple regions simultaneously (PrimerSuite), (ii) screen for primer-primer dimerizing artefacts (PrimerDimer), and (iii) support multiplex PCR assays (PrimerPlex). Moreover, a major focus in the development of this software package was the emphasis on extensive empirical validation, and over 1300 unique primer pairs have been successfully designed and screened, with over 94% of them producing amplicons of the expected size, and an average mapping efficiency of 93% when screened using bisulfite multiplex resequencing. The potential use of the software in other bisulfite-based applications such as methylation-specific PCR is under consideration for future updates. This resource is freely available for use at PrimerSuite website (www.primer-suite.com).

Sensitive and quantitative measurement of gene expression directly from a small amount of whole blood.

PubMed

Zheng, Zhi; Luo, Yuling; McMaster, Gary K

2006-07-01

Accurate and precise quantification of mRNA in whole blood is made difficult by gene expression changes during blood processing, and by variations and biases introduced by sample preparations. We sought to develop a quantitative whole-blood mRNA assay that eliminates blood purification, RNA isolation, reverse transcription, and target amplification while providing high-quality data in an easy assay format. We performed single- and multiplex gene expression analysis with multiple hybridization probes to capture mRNA directly from blood lysate and used branched DNA to amplify the signal. The 96-well plate singleplex assay uses chemiluminescence detection, and the multiplex assay combines Luminex-encoded beads with fluorescent detection. The single- and multiplex assays could quantitatively measure as few as 6000 and 24,000 mRNA target molecules (0.01 and 0.04 amoles), respectively, in up to 25 microL of whole blood. Both formats had CVs < 10% and dynamic ranges of 3-4 logs. Assay sensitivities allowed quantitative measurement of gene expression in the minority of cells in whole blood. The signals from whole-blood lysate correlated well with signals from purified RNA of the same sample, and absolute mRNA quantification results from the assay were similar to those obtained by quantitative reverse transcription-PCR. Both single- and multiplex assay formats were compatible with common anticoagulants and PAXgene-treated samples; however, PAXgene preparations induced expression of known antiapoptotic genes in whole blood. Both the singleplex and the multiplex branched DNA assays can quantitatively measure mRNA expression directly from small volumes of whole blood. The assay offers an alternative to current technologies that depend on RNA isolation and is amenable to high-throughput gene expression analysis of whole blood.

A multiplexable, microfluidic platform for the rapid quantitation of a biomarker panel for early ovarian cancer detection at the point-of-care

PubMed Central

Shadfan, Basil H.; Simmons, Archana R.; Simmons, Glennon W.; Ho, Andy; Wong, Jorge; Lu, Karen H.; Bast, Robert C.; McDevitt, John T.

2015-01-01

Point-of-care (POC) diagnostic platforms have the potential to enable low-cost, large-scale screening. As no single biomarker is shed by all ovarian cancers, multiplexed biomarker panels promise improved sensitivity and specificity to address the unmet need for early detection of ovarian cancer. We have configured the programmable bio-nano-chip (p-BNC) - a multiplexable, microfluidic, modular platform - to quantify a novel multimarker panel comprising CA125, HE4, MMP-7 and CA72-4. The p-BNC is a bead-based immunoanalyzer system with a credit-card-sized footprint that integrates automated sample metering, bubble and debris removal, reagent storage and waste disposal, permitting POC analysis. Multiplexed p-BNC immunoassays demonstrated high specificity, low cross-reactivity, low limits of detection suitable for early detection, and a short analysis time of 43 minutes. Day-to-day variability, a critical factor for longitudinally monitoring biomarkers, ranged between 5.4–10.5%, well below the biological variation for all four markers. Biomarker concentrations for 31 late-stage sera correlated well (R2 = 0.71 to 0.93 for various biomarkers) with values obtained on the Luminex® platform. In a 31 patient cohort encompassing early- and late-stage ovarian cancers along with benign and healthy controls, the multiplexed p-BNC panel was able to distinguish cases from controls with 68.7% sensitivity at 80% specificity. Utility for longitudinal biomarker monitoring was demonstrated with pre-diagnostic sera from 2 cases and 4 controls. Taken together, the p-BNC shows strong promise as a diagnostic tool for large-scale screening that takes advantage of faster results and lower costs while leveraging possible improvement in sensitivity and specificity from biomarker panels. PMID:25388014

Forensic validation of the PowerPlex® ESI 16 STR Multiplex and comparison of performance with AmpFlSTR® SGM Plus®.

PubMed

Tucker, Valerie C; Kirkham, Amanda J; Hopwood, Andrew J

2012-05-01

We describe the forensic validation of Promega's PowerPlex® European Standard Investigator 16 (ESI 16) multiplex kit and compare results generated with the AmpFlSTR® SGM Plus® (SGM+) multiplex. ESI 16 combines the loci contained within the SGM+ multiplex with five additional loci: D2S441, D10S1248, D22S1045, D1S1656, and D12S391. A relative reduction in amplicon size of the SGM+ loci facilitates an increased robustness and amplification success of these amplicons with degraded DNA samples. Tests performed herein supplement ESI 16 data published previously with sensitivity, profile quality, mock casework, inhibitor and mixture study data collected in our laboratories in alignment with our internal technical and quality guidelines and those issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM), the DNA Advisory Board (DAB) and the DNA working group (DNAWG) of the European Network of Forensic Science Institutes (ENFSI). Full profiles were routinely generated from a fully heterozygous single source DNA template using 62.5 pg for ESI 16 and 500 pg for SGM+. This increase in sensitivity has a consequent effect on mixture analyses and the detection of minor mixture components. The improved PCR chemistry confers enhanced tolerance to high levels of laboratory prepared inhibitors compared with SGM+ results. In summary, our results demonstrate that the ESI 16 multiplex kit is more robust and sensitive compared with SGM+ and will be a suitable replacement system for the analysis of forensic DNA samples providing compliance with the European standard set of STR loci.

A novel multiplex bead-based platform highlights the diversity of extracellular vesicles

PubMed Central

Koliha, Nina; Wiencek, Yvonne; Heider, Ute; Jüngst, Christian; Kladt, Nikolay; Krauthäuser, Susanne; Johnston, Ian C. D.; Bosio, Andreas; Schauss, Astrid; Wild, Stefan

2016-01-01

The surface protein composition of extracellular vesicles (EVs) is related to the originating cell and may play a role in vesicle function. Knowledge of the protein content of individual EVs is still limited because of the technical challenges to analyse small vesicles. Here, we introduce a novel multiplex bead-based platform to investigate up to 39 different surface markers in one sample. The combination of capture antibody beads with fluorescently labelled detection antibodies allows the analysis of EVs that carry surface markers recognized by both antibodies. This new method enables an easy screening of surface markers on populations of EVs. By combining different capture and detection antibodies, additional information on relative expression levels and potential vesicle subpopulations is gained. We also established a protocol to visualize individual EVs by stimulated emission depletion (STED) microscopy. Thereby, markers on single EVs can be detected by fluorophore-conjugated antibodies. We used the multiplex platform and STED microscopy to show for the first time that NK cell–derived EVs and platelet-derived EVs are devoid of CD9 or CD81, respectively, and that EVs isolated from activated B cells comprise different EV subpopulations. We speculate that, according to our STED data, tetraspanins might not be homogenously distributed but may mostly appear as clusters on EV subpopulations. Finally, we demonstrate that EV mixtures can be separated by magnetic beads and analysed subsequently with the multiplex platform. Both the multiplex bead-based platform and STED microscopy revealed subpopulations of EVs that have been indistinguishable by most analysis tools used so far. We expect that an in-depth view on EV heterogeneity will contribute to our understanding of different EVs and functions. PMID:26901056

Simultaneous detection of the three ilarviruses affecting stone fruit trees by nonisotopic molecular hybridization and multiplex reverse-transcription polymerase chain reaction.

PubMed

Saade, M; Aparicio, F; Sánchez-Navarro, J A; Herranz, M C; Myrta, A; Di Terlizzi, B; Pallás, V

2000-12-01

ABSTRACT The three most economically damaging ilarviruses affecting stone fruit trees on a worldwide scale are the related Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), and Apple mosaic virus (ApMV). Nonisotopic molecular hybridization and multiplex reverse-transcription polymerase chain reaction (RT-PCR) methodologies were developed that could detect all these viruses simultaneously. The latter technique was advantageous because it was discriminatory. For RT-PCR, a degenerate antisense primer was designed which was used in conjunction with three virus-specific sense primers. The amplification efficiencies for the detection of the three viruses in the multiplex RT-PCR reaction were identical to those obtained in the single RT-PCR reactions for individual viruses. This cocktail of primers was able to amplify sequences from all of the PNRSV, ApMV, and PDV isolates tested in five Prunus spp. hosts (almond, apricot, cherry, peach, and plum) occurring naturally in single or multiple infections. For ApMV isolates, differences in the electrophoretic mobilities of the PCR products were observed. The nucleotide sequence of the amplified products of two representative ApMV isolates was determined, and comparative analysis revealed the existence of a 28-nucleotide deletion in the sequence of isolates showing the faster electrophoretic mobility. To our knowledge, this is the first report on the simultaneous detection of three plant viruses by multiplex RT-PCR in woody hosts. This multiplex RT-PCR could be a useful time and cost saving method for indexing these three ilarviruses, which damage stone fruit tree yields, and for the analysis of mother plants in certification programs.

Antimicrobial susceptibility patterns of Ureaplasma species and Mycoplasma hominis in pregnant women.

PubMed

Redelinghuys, Mathys J; Ehlers, Marthie M; Dreyer, Andries W; Lombaard, Hennie A; Kock, Marleen M

2014-03-28

Genital mycoplasmas colonise up to 80% of sexually mature women and may invade the amniotic cavity during pregnancy and cause complications. Tetracyclines and fluoroquinolones are contraindicated in pregnancy and erythromycin is often used to treat patients. However, increasing resistance to common antimicrobial agents is widely reported. The purpose of this study was to investigate antimicrobial susceptibility patterns of genital mycoplasmas in pregnant women. Self-collected vaginal swabs were obtained from 96 pregnant women attending an antenatal clinic in Gauteng, South Africa. Specimens were screened with the Mycofast Revolution assay for the presence of Ureaplasma species and Mycoplasma hominis. The antimicrobial susceptibility to levofloxacin, moxifloxacin, erythromycin, clindamycin and tetracycline were determined at various breakpoints. A multiplex polymerase chain reaction assay was used to speciate Ureaplasma positive specimens as either U. parvum or U. urealyticum. Seventy-six percent (73/96) of specimens contained Ureaplasma spp., while 39.7% (29/73) of Ureaplasma positive specimens were also positive for M. hominis. Susceptibilities of Ureaplasma spp. to levofloxacin and moxifloxacin were 59% (26/44) and 98% (43/44) respectively. Mixed isolates (Ureaplasma species and M. hominis) were highly resistant to erythromycin and tetracycline (both 97% resistance). Resistance of Ureaplasma spp. to erythromycin was 80% (35/44) and tetracycline resistance was detected in 73% (32/44) of Ureaplasma spp. Speciation indicated that U. parvum was the predominant Ureaplasma spp. conferring antimicrobial resistance. Treatment options for genital mycoplasma infections are becoming limited. More elaborative studies are needed to elucidate the diverse antimicrobial susceptibility patterns found in this study when compared to similar studies. To prevent complications in pregnant women, the foetus and the neonate, routine screening for the presence of genital mycoplasmas is recommended. In addition, it is recommended that antimicrobial susceptibility patterns are determined.

Antimicrobial susceptibility patterns of Ureaplasma species and Mycoplasma hominis in pregnant women

PubMed Central

2014-01-01

Background Genital mycoplasmas colonise up to 80% of sexually mature women and may invade the amniotic cavity during pregnancy and cause complications. Tetracyclines and fluoroquinolones are contraindicated in pregnancy and erythromycin is often used to treat patients. However, increasing resistance to common antimicrobial agents is widely reported. The purpose of this study was to investigate antimicrobial susceptibility patterns of genital mycoplasmas in pregnant women. Methods Self-collected vaginal swabs were obtained from 96 pregnant women attending an antenatal clinic in Gauteng, South Africa. Specimens were screened with the Mycofast Revolution assay for the presence of Ureaplasma species and Mycoplasma hominis. The antimicrobial susceptibility to levofloxacin, moxifloxacin, erythromycin, clindamycin and tetracycline were determined at various breakpoints. A multiplex polymerase chain reaction assay was used to speciate Ureaplasma positive specimens as either U. parvum or U. urealyticum. Results Seventy-six percent (73/96) of specimens contained Ureaplasma spp., while 39.7% (29/73) of Ureaplasma positive specimens were also positive for M. hominis. Susceptibilities of Ureaplasma spp. to levofloxacin and moxifloxacin were 59% (26/44) and 98% (43/44) respectively. Mixed isolates (Ureaplasma species and M. hominis) were highly resistant to erythromycin and tetracycline (both 97% resistance). Resistance of Ureaplasma spp. to erythromycin was 80% (35/44) and tetracycline resistance was detected in 73% (32/44) of Ureaplasma spp. Speciation indicated that U. parvum was the predominant Ureaplasma spp. conferring antimicrobial resistance. Conclusions Treatment options for genital mycoplasma infections are becoming limited. More elaborative studies are needed to elucidate the diverse antimicrobial susceptibility patterns found in this study when compared to similar studies. To prevent complications in pregnant women, the foetus and the neonate, routine screening for the presence of genital mycoplasmas is recommended. In addition, it is recommended that antimicrobial susceptibility patterns are determined. PMID:24679107

Comparison of Automated Microarray Detection with Real-Time PCR Assays for Detection of Respiratory Viruses in Specimens Obtained from Children▿

PubMed Central

Raymond, Frédéric; Carbonneau, Julie; Boucher, Nancy; Robitaille, Lynda; Boisvert, Sébastien; Wu, Whei-Kuo; De Serres, Gaston; Boivin, Guy; Corbeil, Jacques

2009-01-01

Respiratory virus infections are a major health concern and represent the primary cause of testing consultation and hospitalization for young children. We developed and compared two assays that allow the detection of up to 23 different respiratory viruses that frequently infect children. The first method consisted of single TaqMan quantitative real-time PCR assays in a 96-well-plate format. The second consisted of a multiplex PCR followed by primer extension and microarray hybridization in an integrated molecular diagnostic device, the Infiniti analyzer. Both of our assays can detect adenoviruses of groups A, B, C, and E; coronaviruses HKU1, 229E, NL63, and OC43; enteroviruses A, B, C, and D; rhinoviruses of genotypes A and B; influenza viruses A and B; human metapneumoviruses (HMPV) A and B, human respiratory syncytial viruses (HRSV) A and B; and parainfluenza viruses of types 1, 2, and 3. These tests were used to identify viruses in 221 nasopharyngeal aspirates obtained from children hospitalized for respiratory tract infections. Respiratory viruses were detected with at least one of the two methods in 81.4% of the 221 specimens: 10.0% were positive for HRSV A, 38.0% for HRSV B, 13.1% for influenzavirus A, 8.6% for any coronaviruses, 13.1% for rhinoviruses or enteroviruses, 7.2% for adenoviruses, 4.1% for HMPV, and 1.5% for parainfluenzaviruses. Multiple viral infections were found in 13.1% of the specimens. The two methods yielded concordant results for 94.1% of specimens. These tests allowed a thorough etiological assessment of respiratory viruses infecting children in hospital settings and would assist public health interventions. PMID:19158263

Comparison of automated microarray detection with real-time PCR assays for detection of respiratory viruses in specimens obtained from children.

PubMed

Raymond, Frédéric; Carbonneau, Julie; Boucher, Nancy; Robitaille, Lynda; Boisvert, Sébastien; Wu, Whei-Kuo; De Serres, Gaston; Boivin, Guy; Corbeil, Jacques

2009-03-01

Respiratory virus infections are a major health concern and represent the primary cause of testing consultation and hospitalization for young children. We developed and compared two assays that allow the detection of up to 23 different respiratory viruses that frequently infect children. The first method consisted of single TaqMan quantitative real-time PCR assays in a 96-well-plate format. The second consisted of a multiplex PCR followed by primer extension and microarray hybridization in an integrated molecular diagnostic device, the Infiniti analyzer. Both of our assays can detect adenoviruses of groups A, B, C, and E; coronaviruses HKU1, 229E, NL63, and OC43; enteroviruses A, B, C, and D; rhinoviruses of genotypes A and B; influenza viruses A and B; human metapneumoviruses (HMPV) A and B, human respiratory syncytial viruses (HRSV) A and B; and parainfluenza viruses of types 1, 2, and 3. These tests were used to identify viruses in 221 nasopharyngeal aspirates obtained from children hospitalized for respiratory tract infections. Respiratory viruses were detected with at least one of the two methods in 81.4% of the 221 specimens: 10.0% were positive for HRSV A, 38.0% for HRSV B, 13.1% for influenzavirus A, 8.6% for any coronaviruses, 13.1% for rhinoviruses or enteroviruses, 7.2% for adenoviruses, 4.1% for HMPV, and 1.5% for parainfluenzaviruses. Multiple viral infections were found in 13.1% of the specimens. The two methods yielded concordant results for 94.1% of specimens. These tests allowed a thorough etiological assessment of respiratory viruses infecting children in hospital settings and would assist public health interventions.

Molecular serotyping, virulence gene profiling and pathogenicity of Streptococcus agalactiae isolated from tilapia farms in Thailand by multiplex PCR.

PubMed

Kannika, K; Pisuttharachai, D; Srisapoome, P; Wongtavatchai, J; Kondo, H; Hirono, I; Unajak, S; Areechon, N

2017-06-01

This study aimed to biotype Streptococcus agalactiae isolated from tilapia farms in Thailand based on molecular biotyping methods and to determine the correlation between the serotype and virulence of bacteria. In addition to a biotyping (serotyping) technique based on multiplex PCR of cps genes, in this study, we developed multiplex PCR typing of Group B streptococcus (GBS) virulence genes to examine three clusters of virulence genes and their correlation with the pathogenicity of S. agalactiae. The epidemiology of S. agalactiae in Thailand was analysed to provide bacterial genetic information towards a future rational vaccine strategy for tilapia culture systems. Streptococcus agalactiae were isolated from diseased tilapia from different areas of Thailand. A total of 124 S. agalactiae isolates were identified by phenotypic analysis and confirmed by 16S rRNA PCR. Bacterial genotyping was conducted based on (i) molecular serotyping of the capsular polysaccharide (cps) gene cluster and (ii) virulence gene profiling using multiplex PCR analysis of 14 virulence genes (lmb, scpB, pavA, cspA, spb1, cyl, bca, rib, fbsA, fbsB, cfb, hylB, bac and pbp1A/ponA). Only serotypes Ia and III were found in this study; serotype Ia lacks the lmb, scpB and spb1 genes, whereas serotype III lacks only the bac gene. Virulence tests in juvenile Nile tilapia demonstrated a correlation between the pathogenicity of the bacteria and their virulence gene profile, with serotype III showing higher virulence than serotype Ia. Epidemiological analysis showed an almost equal distribution in all regions of Thailand, except serotype III was found predominantly in the southern areas. Only two serotypes of S. agalactiae were isolated from diseased tilapia in Thailand. Serotype Ia showed fewer virulence genes and lower virulence than serotype III. Both serotypes showed a similar distribution throughout Thailand. We identified two major serotypes of S. agalactiae isolates associated with the outbreak in tilapia culture in Thailand. We developed multiplex PCR assays for 14 virulence genes, which may be used to predict the pathogenicity of the isolates and track future infections. Multiplex PCR typing of the GBS virulence genes was developed and might be further used to predict the pathogenicity of S. agalactiae. © 2017 The Society for Applied Microbiology.

Propionibacterium acnes biofilm is present in intervertebral discs of patients undergoing microdiscectomy.

PubMed

Capoor, Manu N; Ruzicka, Filip; Schmitz, Jonathan E; James, Garth A; Machackova, Tana; Jancalek, Radim; Smrcka, Martin; Lipina, Radim; Ahmed, Fahad S; Alamin, Todd F; Anand, Neel; Baird, John C; Bhatia, Nitin; Demir-Deviren, Sibel; Eastlack, Robert K; Fisher, Steve; Garfin, Steven R; Gogia, Jaspaul S; Gokaslan, Ziya L; Kuo, Calvin C; Lee, Yu-Po; Mavrommatis, Konstantinos; Michu, Elleni; Noskova, Hana; Raz, Assaf; Sana, Jiri; Shamie, A Nick; Stewart, Philip S; Stonemetz, Jerry L; Wang, Jeffrey C; Witham, Timothy F; Coscia, Michael F; Birkenmaier, Christof; Fischetti, Vincent A; Slaby, Ondrej

2017-01-01

In previous studies, Propionibacterium acnes was cultured from intervertebral disc tissue of ~25% of patients undergoing microdiscectomy, suggesting a possible link between chronic bacterial infection and disc degeneration. However, given the prominence of P. acnes as a skin commensal, such analyses often struggled to exclude the alternate possibility that these organisms represent perioperative microbiologic contamination. This investigation seeks to validate P. acnes prevalence in resected disc cultures, while providing microscopic evidence of P. acnes biofilm in the intervertebral discs. Specimens from 368 patients undergoing microdiscectomy for disc herniation were divided into several fragments, one being homogenized, subjected to quantitative anaerobic culture, and assessed for bacterial growth, and a second fragment frozen for additional analyses. Colonies were identified by MALDI-TOF mass spectrometry and P. acnes phylotyping was conducted by multiplex PCR. For a sub-set of specimens, bacteria localization within the disc was assessed by microscopy using confocal laser scanning and FISH. Bacteria were cultured from 162 discs (44%), including 119 cases (32.3%) with P. acnes. In 89 cases, P. acnes was cultured exclusively; in 30 cases, it was isolated in combination with other bacteria (primarily coagulase-negative Staphylococcus spp.) Among positive specimens, the median P. acnes bacterial burden was 350 CFU/g (12 - ~20,000 CFU/g). Thirty-eight P. acnes isolates were subjected to molecular sub-typing, identifying 4 of 6 defined phylogroups: IA1, IB, IC, and II. Eight culture-positive specimens were evaluated by fluorescence microscopy and revealed P. acnes in situ. Notably, these bacteria demonstrated a biofilm distribution within the disc matrix. P. acnes bacteria were more prevalent in males than females (39% vs. 23%, p = 0.0013). This study confirms that P. acnes is prevalent in herniated disc tissue. Moreover, it provides the first visual evidence of P. acnes biofilms within such specimens, consistent with infection rather than microbiologic contamination.

Propionibacterium acnes biofilm is present in intervertebral discs of patients undergoing microdiscectomy

PubMed Central

Ruzicka, Filip; Schmitz, Jonathan E.; James, Garth A.; Machackova, Tana; Jancalek, Radim; Smrcka, Martin; Lipina, Radim; Ahmed, Fahad S.; Alamin, Todd F.; Anand, Neel; Baird, John C.; Bhatia, Nitin; Demir-Deviren, Sibel; Eastlack, Robert K.; Fisher, Steve; Garfin, Steven R.; Gogia, Jaspaul S.; Gokaslan, Ziya L.; Kuo, Calvin C.; Lee, Yu-Po; Mavrommatis, Konstantinos; Michu, Elleni; Noskova, Hana; Raz, Assaf; Sana, Jiri; Shamie, A. Nick; Stewart, Philip S.; Stonemetz, Jerry L.; Wang, Jeffrey C.; Witham, Timothy F.; Coscia, Michael F.; Birkenmaier, Christof; Fischetti, Vincent A.; Slaby, Ondrej

2017-01-01

Background In previous studies, Propionibacterium acnes was cultured from intervertebral disc tissue of ~25% of patients undergoing microdiscectomy, suggesting a possible link between chronic bacterial infection and disc degeneration. However, given the prominence of P. acnes as a skin commensal, such analyses often struggled to exclude the alternate possibility that these organisms represent perioperative microbiologic contamination. This investigation seeks to validate P. acnes prevalence in resected disc cultures, while providing microscopic evidence of P. acnes biofilm in the intervertebral discs. Methods Specimens from 368 patients undergoing microdiscectomy for disc herniation were divided into several fragments, one being homogenized, subjected to quantitative anaerobic culture, and assessed for bacterial growth, and a second fragment frozen for additional analyses. Colonies were identified by MALDI-TOF mass spectrometry and P. acnes phylotyping was conducted by multiplex PCR. For a sub-set of specimens, bacteria localization within the disc was assessed by microscopy using confocal laser scanning and FISH. Results Bacteria were cultured from 162 discs (44%), including 119 cases (32.3%) with P. acnes. In 89 cases, P. acnes was cultured exclusively; in 30 cases, it was isolated in combination with other bacteria (primarily coagulase-negative Staphylococcus spp.) Among positive specimens, the median P. acnes bacterial burden was 350 CFU/g (12 - ~20,000 CFU/g). Thirty-eight P. acnes isolates were subjected to molecular sub-typing, identifying 4 of 6 defined phylogroups: IA1, IB, IC, and II. Eight culture-positive specimens were evaluated by fluorescence microscopy and revealed P. acnes in situ. Notably, these bacteria demonstrated a biofilm distribution within the disc matrix. P. acnes bacteria were more prevalent in males than females (39% vs. 23%, p = 0.0013). Conclusions This study confirms that P. acnes is prevalent in herniated disc tissue. Moreover, it provides the first visual evidence of P. acnes biofilms within such specimens, consistent with infection rather than microbiologic contamination. PMID:28369127

Fully integrated wearable sensor arrays for multiplexed in situ perspiration analysis

DOE PAGES

Gao, Wei; Emaminejad, Sam; Nyein, Hnin Yin Yin; ...

2016-01-27

We report that wearable sensor technologies are essential to the realization of personalized medicine through continuously monitoring an individual’s state of health. Sampling human sweat, which is rich in physiological information13, could enable non-invasive monitoring. Previously reported sweat-based and other noninvasive biosensors either can only monitor a single analyte at a time or lack on-site signal processing circuitry and sensor calibration mechanisms for accurate analysis of the physiological state14–18. Given the complexity of sweat secretion, simultaneous and multiplexed screening of target biomarkers is critical and requires full system integration to ensure the accuracy of measurements. Here we present a mechanicallymore » flexible and fully integrated (that is, no external analysis is needed) sensor array for multiplexed in situ perspiration analysis, which simultaneously and selectively measures sweat metabolites (such as glucose and lactate) and electrolytes (such as sodium and potassium ions), as well as the skin temperature (to calibrate the response of the sensors). Lastly, our work bridges the technological gap between signal transduction, conditioning (amplification and filtering), processing and wireless transmission in wearable biosensors by merging plasticbased sensors that interface with the skin with silicon integrated circuits consolidated on a flexible circuit board for complex signal processing.« less

Fully integrated wearable sensor arrays for multiplexed in situ perspiration analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao, Wei; Emaminejad, Sam; Nyein, Hnin Yin Yin

We report that wearable sensor technologies are essential to the realization of personalized medicine through continuously monitoring an individual’s state of health. Sampling human sweat, which is rich in physiological information13, could enable non-invasive monitoring. Previously reported sweat-based and other noninvasive biosensors either can only monitor a single analyte at a time or lack on-site signal processing circuitry and sensor calibration mechanisms for accurate analysis of the physiological state14–18. Given the complexity of sweat secretion, simultaneous and multiplexed screening of target biomarkers is critical and requires full system integration to ensure the accuracy of measurements. Here we present a mechanicallymore » flexible and fully integrated (that is, no external analysis is needed) sensor array for multiplexed in situ perspiration analysis, which simultaneously and selectively measures sweat metabolites (such as glucose and lactate) and electrolytes (such as sodium and potassium ions), as well as the skin temperature (to calibrate the response of the sensors). Lastly, our work bridges the technological gap between signal transduction, conditioning (amplification and filtering), processing and wireless transmission in wearable biosensors by merging plasticbased sensors that interface with the skin with silicon integrated circuits consolidated on a flexible circuit board for complex signal processing.« less

Molecular prey identification in Central European piscivores.

PubMed

Thalinger, Bettina; Oehm, Johannes; Mayr, Hannes; Obwexer, Armin; Zeisler, Christiane; Traugott, Michael

2016-01-01

Diet analysis is an important aspect when investigating the ecology of fish-eating animals and essential for assessing their functional role in food webs across aquatic and terrestrial ecosystems. The identification of fish remains in dietary samples, however, can be time-consuming and unsatisfying using conventional morphological analysis of prey remains. Here, we present a two-step multiplex PCR system, comprised of six assays, allowing for rapid, sensitive and specific detection of fish DNA in dietary samples. This approach encompasses 78 fish and lamprey species native to Central European freshwaters and enables the identification of 31 species, six genera, two families, two orders and two fish family clusters. All targeted taxa were successfully amplified from 25 template molecules, and each assay was specific when tested against a wide range of invertebrates and vertebrates inhabiting aquatic environments. The applicability of the multiplex PCR system was evaluated in a feeding trial, wherein it outperformed morphological prey analysis regarding species-specific prey identification in faeces of Eurasian otters. Additionally, a wide spectrum of fish species was detected in field-collected faecal samples and regurgitated pellets of Common Kingfishers and Great Cormorants, demonstrating the broad applicability of the approach. In conclusion, this multiplex PCR system provides an efficient, easy to use and cost-effective tool for assessing the trophic ecology of piscivores in Central Europe. Furthermore, the multiplex PCRs and the primers described therein will be applicable wherever DNA of the targeted fish species needs to be detected at high sensitivity and specificity. © 2015 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

Rapid detection of Wuchereria bancrofti and Brugia malayi in mosquito vectors (Diptera: Culicidae) using a real-time fluorescence resonance energy transfer multiplex PCR and melting curve analysis.

PubMed

Intapan, Pewpan M; Thanchomnang, Tongjit; Lulitanond, Viraphong; Maleewong, Wanchai

2009-01-01

We developed a single-step real-time fluorescence resonance energy transfer (FRET) multiplex polymerase chain reaction (PCR) merged with melting curve analysis for the detection of Wuchereria bancrofti and Brugia malayi DNA in blood-fed mosquitoes. Real-time FRET multiplex PCR is based on fluorescence melting curve analysis of a hybrid of amplicons generated from two families of repeated DNA elements: the 188 bp SspI repeated sequence, specific to W. bancrofti, and the 153-bp HhaI repeated sequence, specific to the genus Brugia and two pairs of specific fluorophore-labeled probes. Both W. bancrofti and B. malayi can be differentially detected in infected vectors by this process through their different fluorescence channel and melting temperatures. The assay could distinguish both human filarial DNAs in infected vectors from the DNAs of Dirofilaria immitis- and Plasmodium falciparum-infected human red blood cells and noninfected mosquitoes and human leukocytes. The technique showed 100% sensitivity and specificity and offers a rapid and reliable procedure for differentially identifying lymphatic filariasis. The introduced real-time FRET multiplex PCR can reduce labor time and reagent costs and is not prone to carry over contamination. The test can be used to screen mosquito vectors in endemic areas and therefore should be a useful diagnostic tool for the evaluation of infection rate of the mosquito populations and for xenomonitoring in the community after eradication programs such as the Global Program to Eliminate Lymphatic Filariasis.

Phage-protease-peptide: a novel trifecta enabling multiplex detection of viable bacterial pathogens.

PubMed

Alcaine, S D; Tilton, L; Serrano, M A C; Wang, M; Vachet, R W; Nugen, S R

2015-10-01

Bacteriophages represent rapid, readily targeted, and easily produced molecular probes for the detection of bacterial pathogens. Molecular biology techniques have allowed researchers to make significant advances in the bioengineering of bacteriophage to further improve speed and sensitivity of detection. Despite their host specificity, bacteriophages have not been meaningfully leveraged in multiplex detection of bacterial pathogens. We propose a proof-of-principal phage-based scheme to enable multiplex detection. Our scheme involves bioengineering bacteriophage to carry a gene for a specific protease, which is expressed during infection of the target cell. Upon lysis, the protease is released to cleave a reporter peptide, and the signal detected. Here we demonstrate the successful (i) modification of T7 bacteriophage to carry tobacco etch virus (TEV) protease; (ii) expression of TEV protease by Escherichia coli following infection by our modified T7, an average of 2000 units of protease per phage are produced during infection; and (iii) proof-of-principle detection of E. coli in 3 h after a primary enrichment via TEV protease activity using a fluorescent peptide and using a designed target peptide for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI-TOF MS) analysis. This proof-of-principle can be translated to other phage-protease-peptide combinations to enable multiplex bacterial detection and readily adopted on multiple platforms, like MALDI-TOF MS or fluorescent readers, commonly found in labs.

Applications of the DOE/NASA wind turbine engineering information system

NASA Technical Reports Server (NTRS)

Neustadter, H. E.; Spera, D. A.

1981-01-01

A statistical analysis of data obtained from the Technology and Engineering Information Systems was made. The systems analyzed consist of the following elements: (1) sensors which measure critical parameters (e.g., wind speed and direction, output power, blade loads and component vibrations); (2) remote multiplexing units (RMUs) on each wind turbine which frequency-modulate, multiplex and transmit sensor outputs; (3) on-site instrumentation to record, process and display the sensor output; and (4) statistical analysis of data. Two examples of the capabilities of these systems are presented. The first illustrates the standardized format for application of statistical analysis to each directly measured parameter. The second shows the use of a model to estimate the variability of the rotor thrust loading, which is a derived parameter.

Few-mode fiber, splice and SDM component characterization by spatially-diverse optical vector network analysis.

PubMed

Rommel, Simon; Mendinueta, José Manuel Delgado; Klaus, Werner; Sakaguchi, Jun; Olmos, Juan José Vegas; Awaji, Yoshinari; Monroy, Idelfonso Tafur; Wada, Naoya

2017-09-18

This paper discusses spatially diverse optical vector network analysis for space division multiplexing (SDM) component and system characterization, which is becoming essential as SDM is widely considered to increase the capacity of optical communication systems. Characterization of a 108-channel photonic lantern spatial multiplexer, coupled to a 36-core 3-mode fiber, is experimentally demonstrated, extracting the full impulse response and complex transfer function matrices as well as insertion loss (IL) and mode-dependent loss (MDL) data. Moreover, the mode-mixing behavior of fiber splices in the few-mode multi-core fiber and their impact on system IL and MDL are analyzed, finding splices to cause significant mode-mixing and to be non-negligible in system capacity analysis.

Study of cellular oncometabolism via multidimensional protein identification technology.

PubMed

Aukim-Hastie, Claire; Garbis, Spiros D

2014-01-01

Cellular proteomics is becoming a widespread clinical application, matching the definition of bench-to-bedside translation. Among various fields of investigation, this approach can be applied to the study of the metabolic alterations that accompany oncogenesis and tumor progression, which are globally referred to as oncometabolism. Here, we describe a multidimensional protein identification technology (MuDPIT)-based strategy that can be employed to study the cellular proteome of malignant cells and tissues. This method has previously been shown to be compatible with the reproducible, in-depth analysis of up to a thousand proteins in clinical samples. The possibility to employ this technique to study clinical specimens demonstrates its robustness. MuDPIT is advantageous as compared to other approaches because it is direct, highly sensitive, and reproducible, it provides high resolution with ultra-high mass accuracy, it allows for relative quantifications, and it is compatible with multiplexing (thus limiting costs).This method enables the direct assessment of the proteomic profile of neoplastic cells and tissues and could be employed in the near future as a high-throughput, rapid, quantitative, and cost-effective screening platform for clinical samples. © 2014 Elsevier Inc. All rights reserved.

Genetic Fingerprinting Using Microsatellite Markers in a Multiplex PCR Reaction: A Compilation of Methodological Approaches from Primer Design to Detection Systems.

PubMed

Krüger, Jacqueline; Schleinitz, Dorit

2017-01-01

Microsatellites are polymorphic DNA loci comprising repeated sequence motifs of two to five base pairs which are dispersed throughout the genome. Genotyping of microsatellites is a widely accepted tool for diagnostic and research purposes such as forensic investigations and parentage testing, but also in clinics (e.g. monitoring of bone marrow transplantation), as well as for the agriculture and food industries. The co-amplification of several short tandem repeat (STR) systems in a multiplex reaction with simultaneous detection helps to obtain more information from a DNA sample where its availability may be limited. Here, we introduce and describe this commonly used genotyping technique, providing an overview on available resources on STRs, multiplex design, and analysis.

Bridging online and offline social networks: Multiplex analysis

NASA Astrophysics Data System (ADS)

Filiposka, Sonja; Gajduk, Andrej; Dimitrova, Tamara; Kocarev, Ljupco

2017-04-01

We show that three basic actor characteristics, namely normalized reciprocity, three cycles, and triplets, can be expressed using an unified framework that is based on computing the similarity index between two sets associated with the actor: the set of her/his friends and the set of those considering her/him as a friend. These metrics are extended to multiplex networks and then computed for two friendship networks generated by collecting data from two groups of undergraduate students. We found that in offline communication strong and weak ties are (almost) equally presented, while in online communication weak ties are dominant. Moreover, weak ties are much less reciprocal than strong ties. However, across different layers of the multiplex network reciprocities are preserved, while triads (measured with normalized three cycles and triplets) are not significant.

Multiplex Microsphere Immunoassays for the Detection of IgM and IgG to Arboviral Diseases

PubMed Central

Basile, Alison J.; Horiuchi, Kalanthe; Panella, Amanda J.; Laven, Janeen; Kosoy, Olga; Lanciotti, Robert S.; Venkateswaran, Neeraja; Biggerstaff, Brad J.

2013-01-01

Serodiagnosis of arthropod-borne viruses (arboviruses) at the Division of Vector-Borne Diseases, CDC, employs a combination of individual enzyme-linked immunosorbent assays and microsphere immunoassays (MIAs) to test for IgM and IgG, followed by confirmatory plaque-reduction neutralization tests. Based upon the geographic origin of a sample, it may be tested concurrently for multiple arboviruses, which can be a cumbersome task. The advent of multiplexing represents an opportunity to streamline these types of assays; however, because serologic cross-reactivity of the arboviral antigens often confounds results, it is of interest to employ data analysis methods that address this issue. Here, we constructed 13-virus multiplexed IgM and IgG MIAs that included internal and external controls, based upon the Luminex platform. Results from samples tested using these methods were analyzed using 8 different statistical schemes to identify the best way to classify the data. Geographic batteries were also devised to serve as a more practical diagnostic format, and further samples were tested using the abbreviated multiplexes. Comparative error rates for the classification schemes identified a specific boosting method based on logistic regression “Logitboost” as the classification method of choice. When the data from all samples tested were combined into one set, error rates from the multiplex IgM and IgG MIAs were <5% for all geographic batteries. This work represents both the most comprehensive, validated multiplexing method for arboviruses to date, and also the most systematic attempt to determine the most useful classification method for use with these types of serologic tests. PMID:24086608

Preliminary Assessment of Microwave Readout Multiplexing Factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Croce, Mark Philip; Koehler, Katrina Elizabeth; Rabin, Michael W.

2017-01-23

Ultra-high resolution microcalorimeter gamma spectroscopy is a new non-destructive assay technology for measurement of plutonium isotopic composition, with the potential to reduce total measurement uncertainty to a level competitive with destructive analysis methods [1-4]. Achieving this level of performance in practical applications requires not only the energy resolution now routinely achieved with transition-edge sensor microcalorimeter arrays (an order of magnitude better than for germanium detectors) but also high throughput. Microcalorimeter gamma spectrometers have not yet achieved detection efficiency and count rate capability that is comparable to germanium detectors, largely because of limits from existing readout technology. Microcalorimeter detectors must bemore » operated at low temperature to achieve their exceptional energy resolution. Although the typical 100 mK operating temperatures can be achieved with reliable, cryogen-free systems, the cryogenic complexity and heat load from individual readout channels for large sensor arrays is prohibitive. Multiplexing is required for practical systems. The most mature multiplexing technology at present is time-division multiplexing (TDM) [3, 5-6]. In TDM, the sensor outputs are switched by applying bias current to one SQUID amplifier at a time. Transition-edge sensor (TES) microcalorimeter arrays as large as 256 pixels have been developed for X-ray and gamma-ray spectroscopy using TDM technology. Due to bandwidth limits and noise scaling, TDM is limited to a maximum multiplexing factor of approximately 32-40 sensors on one readout line [8]. Increasing the size of microcalorimeter arrays above the kilopixel scale, required to match the throughput of germanium detectors, requires the development of a new readout technology with a much higher multiplexing factor.« less

An epigenetically derived monoclonal origin for recurrent respiratory papillomatosis.

PubMed

Stephen, Josena Kunjoonju; Vaught, Lori E; Chen, Kang Mei; Shah, Veena; Schweitzer, Vanessa G; Gardner, Glendon; Benninger, Michael S; Worsham, Maria J

2007-07-01

To investigate the contribution of promoter methylation-mediated epigenetic events in recurrent respiratory papillomatosis tumorigenesis. Archival tissue DNA, extracted from microdissected papilloma lesions, was interrogated for methylation status by means of the novel, multigene methylation-specific multiplex ligation-dependent probe amplification assay. Fifteen subjects with recurrent respiratory papillomatosis, 3 females and 12 males, all with adult onset of illness (age range, 23-73 years) except for 1 female patient with juvenile onset (1 year old). Promoter hypermethylation was recorded in 14 of 15 cases, and 19 of 22 unique methylation-prone cancer genes in the multigene panel had altered DNA methylation in at least 1 laryngeal papilloma biopsy specimen. Identical abnormally methylated genes were found in 5 of 15 recurrent cases, of which the CDKN2B gene was hypermethylated in all 5 cases. Dissimilar epigenetic events were noted in the remaining cases. A clonal origin was derived for 5 of 15 recurrent respiratory papillomatosis biopsy specimens based on identical epigenetic events. The high frequency of epigenetic events, characterized by consistent promoter hypermethylation of multiple tumor suppressor genes, points to the use of gene silencing mechanisms in the pathogenesis of recurrent respiratory papillomatosis.

Monitoring for Human Papillomavirus Vaccine Impact Among Gay, Bisexual, and Other Men Who Have Sex With Men-United States, 2012-2014.

PubMed

Meites, Elissa; Gorbach, Pamina M; Gratzer, Beau; Panicker, Gitika; Steinau, Martin; Collins, Tom; Parrish, Adam; Randel, Cody; McGrath, Mark; Carrasco, Steven; Moore, Janell; Zaidi, Akbar; Braxton, Jim; Kerndt, Peter R; Unger, Elizabeth R; Crosby, Richard A; Markowitz, Lauri E

2016-09-01

Gay, bisexual, and other men who have sex with men (MSM) are at high risk for human papillomavirus (HPV) infection; vaccination is recommended for US males, including MSM through age 26 years. We assessed evidence of HPV among vaccine-eligible MSM and transgender women to monitor vaccine impact. During 2012-2014, MSM aged 18-26 years at select clinics completed a computer-assisted self-interview regarding sexual behavior, human immunodeficiency virus (HIV) status, and vaccinations. Self-collected anal swab and oral rinse specimens were tested for HPV DNA (37 types) by L1 consensus polymerase chain reaction; serum was tested for HPV antibodies (4 types) by a multiplexed virus-like particle-based immunoglobulin G direct enzyme-linked immunosorbent assay. Among 922 vaccine-eligible participants, the mean age was 23 years, and the mean number of lifetime sex partners was 37. Among 834 without HIV infection, any anal HPV was detected in 69.4% and any oral HPV in 8.4%, yet only 8.5% had evidence of exposure to all quadrivalent vaccine types. In multivariate analysis, HPV prevalence varied significantly (P < .05) by HIV status, sexual orientation, and lifetime number of sex partners, but not by race/ethnicity. Most young MSM lacked evidence of current or past infection with all vaccine-type HPV types, suggesting that they could benefit from vaccination. The impact of vaccination among MSM may be assessed by monitoring HPV prevalence, including in self-collected specimens. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Aqueous two-phase system patterning of detection antibody solutions for cross-reaction-free multiplex ELISA

NASA Astrophysics Data System (ADS)

Frampton, John P.; White, Joshua B.; Simon, Arlyne B.; Tsuei, Michael; Paczesny, Sophie; Takayama, Shuichi

2014-05-01

Accurate disease diagnosis, patient stratification and biomarker validation require the analysis of multiple biomarkers. This paper describes cross-reactivity-free multiplexing of enzyme-linked immunosorbent assays (ELISAs) using aqueous two-phase systems (ATPSs) to confine detection antibodies at specific locations in fully aqueous environments. Antibody cross-reactions are eliminated because the detection antibody solutions are co-localized only to corresponding surface-immobilized capture antibody spots. This multiplexing technique is validated using plasma samples from allogeneic bone marrow recipients. Patients with acute graft versus host disease (GVHD), a common and serious condition associated with allogeneic bone marrow transplantation, display higher mean concentrations for four multiplexed biomarkers (HGF, elafin, ST2 and TNFR1) relative to healthy donors and transplant patients without GVHD. The antibody co-localization capability of this technology is particularly useful when using inherently cross-reactive reagents such as polyclonal antibodies, although monoclonal antibody cross-reactivity can also be reduced. Because ATPS-ELISA adapts readily available antibody reagents, plate materials and detection instruments, it should be easily transferable into other research and clinical settings.

A valveless rotary microfluidic device for multiplex point mutation identification based on ligation-rolling circle amplification.

PubMed

Heo, Hyun Young; Chung, Soyi; Kim, Yong Tae; Kim, Do Hyun; Seo, Tae Seok

2016-04-15

Genetic variations such as single nucleotide polymorphism (SNP) and point mutations are important biomarkers to monitor disease prognosis and diagnosis. In this study, we developed a novel rotary microfluidic device which can perform multiplex SNP typing on the mutation sites of TP53 genes. The microdevice consists of three glass layers: a channel wafer, a Ti/Pt electrode-patterned resistance temperature detector (RTD) wafer, and a rotary plate in which twelve reaction chambers were fabricated. A series of sample injection, ligation-rolling circle amplification (L-RCA) reaction, and fluorescence detection of the resultant amplicons could be executed by rotating the top rotary plate, identifying five mutation points related with cancer prognosis. The use of the rotary plate eliminates the necessity of microvalves and micropumps to control the microfluidic flow in the channel, simplifying the chip design and chip operation for multiplex SNP detection. The proposed microdevice provides an advanced genetic analysis platform in terms of multiplexity, simplicity, and portability in the fields of biomedical diagnostics. Copyright © 2015 Elsevier B.V. All rights reserved.

Orbital angular momentum in four channel spatial domain multiplexing system for multi-terabit per second communication architectures

NASA Astrophysics Data System (ADS)

Murshid, Syed H.; Muralikrishnan, Hari P.; Kozaitis, Samuel P.

2012-06-01

Bandwidth increase has always been an important area of research in communications. A novel multiplexing technique known as Spatial Domain Multiplexing (SDM) has been developed at the Optronics Laboratory of Florida Institute of Technology to increase the bandwidth to T-bits/s range. In this technique, space inside the fiber is used effectively to transmit up to four channels of same wavelength at the same time. Experimental and theoretical analysis shows that these channels follow independent helical paths inside the fiber without interfering with each other. Multiple pigtail laser sources of exactly the same wavelength are used to launch light into a single carrier fiber in a fashion that resulting channels follow independent helical trajectories. These helically propagating light beams form optical vortices inside the fiber and carry their own Orbital Angular Momentum (OAM). The outputs of these beams appear as concentric donut shaped rings when projected on a screen. This endeavor presents the experimental outputs and simulated results for a four channel spatially multiplexed system effectively increasing the system bandwidth by a factor of four.

Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections

PubMed Central

Kishen, Ria E. B.; Kluth, David C.; Bellamy, Christopher O. C.

2016-01-01

The recent availability of novel dyes and alternative light sources to facilitate complex tissue immunofluorescence studies such as multiplex labelling has not been matched by reports critically evaluating the considerations and relative benefits of these new tools, particularly in combination. Product information is often limited to wavelengths used for older fluorophores (FITC, TRITC & corresponding Alexa dyes family). Consequently, novel agents such as Quantum dots are not widely appreciated or used, despite highly favourable properties including extremely bright emission, stability and potentially reduced tissue autofluorescence at the excitation wavelength. Using spectral analysis, we report here a detailed critical appraisal and comparative evaluation of different light sources and fluorophores in multiplex immunofluorescence of clinical biopsy sections. The comparison includes mercury light, metal halide and 3 different LED-based systems, using 7 Qdots (525, 565, 585, 605, 625, 705), Cy3 and Cy5. We discuss the considerations relevant to achieving the best combination of light source and fluorophore for accurate multiplex fluorescence quantitation. We highlight practical limitations and confounders to quantitation with filter-based approaches. PMID:27632367

Simultaneous mutation and copy number variation (CNV) detection by multiplex PCR-based GS-FLX sequencing.

PubMed

Goossens, Dirk; Moens, Lotte N; Nelis, Eva; Lenaerts, An-Sofie; Glassee, Wim; Kalbe, Andreas; Frey, Bruno; Kopal, Guido; De Jonghe, Peter; De Rijk, Peter; Del-Favero, Jurgen

2009-03-01

We evaluated multiplex PCR amplification as a front-end for high-throughput sequencing, to widen the applicability of massive parallel sequencers for the detailed analysis of complex genomes. Using multiplex PCR reactions, we sequenced the complete coding regions of seven genes implicated in peripheral neuropathies in 40 individuals on a GS-FLX genome sequencer (Roche). The resulting dataset showed highly specific and uniform amplification. Comparison of the GS-FLX sequencing data with the dataset generated by Sanger sequencing confirmed the detection of all variants present and proved the sensitivity of the method for mutation detection. In addition, we showed that we could exploit the multiplexed PCR amplicons to determine individual copy number variation (CNV), increasing the spectrum of detected variations to both genetic and genomic variants. We conclude that our straightforward procedure substantially expands the applicability of the massive parallel sequencers for sequencing projects of a moderate number of amplicons (50-500) with typical applications in resequencing exons in positional or functional candidate regions and molecular genetic diagnostics. 2008 Wiley-Liss, Inc.

Aqueous two-phase system patterning of detection antibody solutions for cross-reaction-free multiplex ELISA

PubMed Central

Frampton, John P.; White, Joshua B.; Simon, Arlyne B.; Tsuei, Michael; Paczesny, Sophie; Takayama, Shuichi

2014-01-01

Accurate disease diagnosis, patient stratification and biomarker validation require the analysis of multiple biomarkers. This paper describes cross-reactivity-free multiplexing of enzyme-linked immunosorbent assays (ELISAs) using aqueous two-phase systems (ATPSs) to confine detection antibodies at specific locations in fully aqueous environments. Antibody cross-reactions are eliminated because the detection antibody solutions are co-localized only to corresponding surface-immobilized capture antibody spots. This multiplexing technique is validated using plasma samples from allogeneic bone marrow recipients. Patients with acute graft versus host disease (GVHD), a common and serious condition associated with allogeneic bone marrow transplantation, display higher mean concentrations for four multiplexed biomarkers (HGF, elafin, ST2 and TNFR1) relative to healthy donors and transplant patients without GVHD. The antibody co-localization capability of this technology is particularly useful when using inherently cross-reactive reagents such as polyclonal antibodies, although monoclonal antibody cross-reactivity can also be reduced. Because ATPS-ELISA adapts readily available antibody reagents, plate materials and detection instruments, it should be easily transferable into other research and clinical settings. PMID:24786974

The -(α)(5.2) Deletion Detected in a Uruguayan Family: First Case Report in the Americas.

PubMed

Soler, Ana María; Schelotto, Magdalena; de Oliveira Mota, Natalia; Dorta Ferreira, Roberta; Sonati, Maria de Fatima; da Luz, Julio Abayubá

2016-08-01

In Uruguay, α-thalassemia (α-thal) mutations were introduced predominantly by Mediterranean European immigrant populations and by slave trade of African populations. A patient with anemia with hypochromia and microcytosis, refractory to iron treatment and with normal hemoglobin (Hb) electrophoresis was analyzed for α-thal mutations by multiplex gap-polymerase chain reaction (gap-PCR), automated sequencing and multiplex ligation-dependent probe amplification (MLPA) analyses. Agarose gel electrophoresis of the multiplex gap-PCR showed a band of unexpected size (approximately 700 bp) in the samples from the proband and mother. Automated sequencing of the amplified fragment showed the presence of the -(α)(5.2) deletion (NG_000006.1: g.32867_38062del5196) [an α-thal-1 deletion of 5196 nucleotides (nts)]. The MLPA analysis of the proband's sample also showed the presence of the -(α)(5.2) deletion in heterozygous state. We report here the presence of the -(α)(5.2) deletion, for the first time in the Americas, in a Uruguayan family with Italian ancestry, detected with a previously described multiplex gap-PCR.

Multiplex Immunoassay Profiling of Hormones Involved in Metabolic Regulation.

PubMed

Stephen, Laurie; Guest, Paul C

2018-01-01

Multiplex immunoassays are used for rapid profiling of biomarker proteins and small molecules in biological fluids. The advantages over single immunoassays include lower sample consumption, cost, and labor. This chapter details a protocol to develop a 5-plex assay for glucagon-like peptide 1, growth hormone, insulin, leptin, and thyroid-stimulating hormone on the Luminex ® platform. The results of the analysis of insulin in normal control subjects are given due to the important role of this hormone in nutritional programming diseases.

Multiplex sequence analysis demonstrates the competitive growth advantage of the A-to-G mutants of clarithromycin-resistant Helicobacter pylori.

PubMed

Wang, G; Rahman, M S; Humayun, M Z; Taylor, D E

1999-03-01

Clarithromycin resistance in Helicobacter pylori is due to point mutation within the 23S rRNA. We examined the growth rates of different types of site-directed mutants and demonstrated quantitatively the competitive growth advantage of A-to-G mutants over other types of mutants by a multiplex sequencing assay. The results provide a rational explanation of why A-to-G mutants are predominantly observed among clarithromycin-resistant clinical isolates.

Multiplex Sequence Analysis Demonstrates the Competitive Growth Advantage of the A-to-G Mutants of Clarithromycin-Resistant Helicobacter pylori

PubMed Central

Wang, Ge; Rahman, M. Sayeedur; Humayun, M. Zafri; Taylor, Diane E.

1999-01-01

Clarithromycin resistance in Helicobacter pylori is due to point mutation within the 23S rRNA. We examined the growth rates of different types of site-directed mutants and demonstrated quantitatively the competitive growth advantage of A-to-G mutants over other types of mutants by a multiplex sequencing assay. The results provide a rational explanation of why A-to-G mutants are predominantly observed among clarithromycin-resistant clinical isolates. PMID:10049289

Embedded fiber optic sensors for monitoring processing, quality and structural health of resin transfer molded components

NASA Astrophysics Data System (ADS)

Keulen, C.; Rocha, B.; Yildiz, M.; Suleman, A.

2011-07-01

Due to their small size and flexibility fiber optics can be embedded into composite materials with little negative effect on strength and reliability of the host material. Fiber optic sensors such as Fiber Bragg Gratings (FBG) or Etched Fiber Sensors (EFS) can be used to detect a number of relevant parameters such as flow, degree of cure, quality and structural health throughout the life of a composite component. With a detection algorithm these embedded sensors can be used to detect damage in real time while the component remains in service. This paper presents the research being conducted on the use of fiber optic sensors for process and Structural Health Monitoring (SHM) of Resin Transfer Molded (RTM) composite structures. Fiber optic sensors are used at all life stages of an RTM composite panel. A laboratory scale RTM apparatus was developed with the capability of visually monitoring the resin filling process. A technique for embedding fiber optic sensors with this apparatus has also been developed. Both FBGs and EFSs have been embedded in composite panels using the apparatus. EFSs to monitor the fabrication process, specifically resin flow have been embedded and shown to be capable of detecting the presence of resin at various locations as it is injected into the mold. Simultaneously these sensors were multiplexed on the same fiber with FBGs, which have the ability to measure strain. Since multiple sensors can be multiplexed on a single fiber the number of ingress/egress locations required per sensor can be significantly reduced. To characterize the FBGs for strain detection tensile test specimens with embedded FBG sensors have been produced. These specimens have been instrumented with a resistive strain gauge for benchmarking. Both specimens and embedded sensors were characterized through tensile testing. Furthermore FBGs have been embedded into composite panels in a manner that is conducive to detection of Lamb waves generated with a centrally located PZT. To sense Lamb waves a high speed, high precision sensing technique is required to acquire data from embedded FBGs due to the high velocities and small strain amplitudes of these guided waves. A technique based on a filter consisting of a tunable FBG was developed. Since this filter is not dependant on moving parts, tests executed with this filter concluded with the detection of Lamb waves, removing the influence of temperature and operational strains. A damage detection algorithm was developed to detect and localize cracks and delaminations.

The complete mitochondrial genome of an 11,450-year-old aurochsen (Bos primigenius) from Central Italy.

PubMed

Lari, Martina; Rizzi, Ermanno; Mona, Stefano; Corti, Giorgio; Catalano, Giulio; Chen, Kefei; Vernesi, Cristiano; Larson, Greger; Boscato, Paolo; De Bellis, Gianluca; Cooper, Alan; Caramelli, David; Bertorelle, Giorgio

2011-01-31

Bos primigenius, the aurochs, is the wild ancestor of modern cattle breeds and was formerly widespread across Eurasia and northern Africa. After a progressive decline, the species became extinct in 1627. The origin of modern taurine breeds in Europe is debated. Archaeological and early genetic evidence point to a single Near Eastern origin and a subsequent spread during the diffusion of herding and farming. More recent genetic data are instead compatible with local domestication events or at least some level of local introgression from the aurochs. Here we present the analysis of the complete mitochondrial genome of a pre-Neolithic Italian aurochs. In this study, we applied a combined strategy employing both multiplex PCR amplifications and 454 pyrosequencing technology to sequence the complete mitochondrial genome of an 11,450-year-old aurochs specimen from Central Italy. Phylogenetic analysis of the aurochs mtDNA genome supports the conclusions from previous studies of short mtDNA fragments--namely that Italian aurochsen were genetically very similar to modern cattle breeds, but highly divergent from the North-Central European aurochsen. Complete mitochondrial genome sequences are now available for several modern cattle and two pre-Neolithic mtDNA genomes from very different geographic areas. These data suggest that previously identified sub-groups within the widespread modern cattle mitochondrial T clade are polyphyletic, and they support the hypothesis that modern European breeds have multiple geographic origins.

Morphologic and Genetic Identification of Taenia Tapeworms in Tanzania and DNA Genotyping of Taenia solium

PubMed Central

Eom, Keeseon S.; Chai, Jong-Yil; Yong, Tai-Soon; Min, Duk-Young; Rim, Han-Jong; Kihamia, Charles

2011-01-01

Species identification of Taenia tapeworms was performed using morphologic observations and multiplex PCR and DNA sequencing of the mitochondrial cox1 gene. In 2008 and 2009, a total of 1,057 fecal samples were collected from residents of Kongwa district of Dodoma region, Tanzania, and examined microscopically for helminth eggs and proglottids. Of these, 4 Taenia egg positive cases were identified, and the eggs were subjected to DNA analysis. Several proglottids of Taenia solium were recovered from 1 of the 4 cases. This established that the species were T. solium (n=1) and T. saginata (n=3). One further T. solium specimen was found among 128 fecal samples collected from Mbulu district in Arusha, and this had an intact strobila with the scolex. Phylegenetic analysis of the mtDNA cox1 gene sequences of these 5 isolates showed that T. saginata was basal to the T. solium clade. The mitochondrial cox1 gene sequences of 3 of these Tanzanian isolates showed 99% similarity to T. saginata, and the other 2 isolates showed 100% similarity to T. solium. The present study has shown that Taenia tapeworms are endemic in Kongwa district of Tanzania, as well as in a previously identified Mbulu district. Both T. solium isolates were found to have an "African/Latin American" genotype (cox1). PMID:22355207

Whole genome sequencing identifies influenza A H3N2 transmission and offers superior resolution to classical typing methods.

PubMed

Meinel, Dominik M; Heinzinger, Susanne; Eberle, Ute; Ackermann, Nikolaus; Schönberger, Katharina; Sing, Andreas

2018-02-01

Influenza with its annual epidemic waves is a major cause of morbidity and mortality worldwide. However, only little whole genome data are available regarding the molecular epidemiology promoting our understanding of viral spread in human populations. We implemented a RT-PCR strategy starting from patient material to generate influenza A whole genome sequences for molecular epidemiological surveillance. Samples were obtained within the Bavarian Influenza Sentinel. The complete influenza virus genome was amplified by a one-tube multiplex RT-PCR and sequenced on an Illumina MiSeq. We report whole genomic sequences for 50 influenza A H3N2 viruses, which was the predominating virus in the season 2014/15, directly from patient specimens. The dataset included random samples from Bavaria (Germany) throughout the influenza season and samples from three suspected transmission clusters. We identified the outbreak samples based on sequence identity. Whole genome sequencing (WGS) was superior in resolution compared to analysis of single segments or partial segment analysis. Additionally, we detected manifestation of substantial amounts of viral quasispecies in several patients, carrying mutations varying from the dominant virus in each patient. Our rapid whole genome sequencing approach for influenza A virus shows that WGS can effectively be used to detect and understand outbreaks in large communities. Additionally, the genomic data provide in-depth details about the circulating virus within one season.

Selective impairment of hippocampus and posterior hub areas in Alzheimer's disease: an MEG-based multiplex network study.

PubMed

Yu, Meichen; Engels, Marjolein M A; Hillebrand, Arjan; van Straaten, Elisabeth C W; Gouw, Alida A; Teunissen, Charlotte; van der Flier, Wiesje M; Scheltens, Philip; Stam, Cornelis J

2017-05-01

Although frequency-specific network analyses have shown that functional brain networks are altered in patients with Alzheimer's disease, the relationships between these frequency-specific network alterations remain largely unknown. Multiplex network analysis is a novel network approach to study complex systems consisting of subsystems with different types of connectivity patterns. In this study, we used magnetoencephalography to integrate five frequency-band specific brain networks in a multiplex framework. Previous structural and functional brain network studies have consistently shown that hub brain areas are selectively disrupted in Alzheimer's disease. Accordingly, we hypothesized that hub regions in the multiplex brain networks are selectively targeted in patients with Alzheimer's disease in comparison to healthy control subjects. Eyes-closed resting-state magnetoencephalography recordings from 27 patients with Alzheimer's disease (60.6 ± 5.4 years, 12 females) and 26 controls (61.8 ± 5.5 years, 14 females) were projected onto atlas-based regions of interest using beamforming. Subsequently, source-space time series for both 78 cortical and 12 subcortical regions were reconstructed in five frequency bands (delta, theta, alpha 1, alpha 2 and beta band). Multiplex brain networks were constructed by integrating frequency-specific magnetoencephalography networks. Functional connections between all pairs of regions of interests were quantified using a phase-based coupling metric, the phase lag index. Several multiplex hub and heterogeneity metrics were computed to capture both overall importance of each brain area and heterogeneity of the connectivity patterns across frequency-specific layers. Different nodal centrality metrics showed consistently that several hub regions, particularly left hippocampus, posterior parts of the default mode network and occipital regions, were vulnerable in patients with Alzheimer's disease compared to control subjects. Of note, these detected vulnerable hubs in Alzheimer's disease were absent in each individual frequency-specific network, thus showing the value of integrating the networks. The connectivity patterns of these vulnerable hub regions in the patients were heterogeneously distributed across layers. Perturbed cognitive function and abnormal cerebrospinal fluid amyloid-β42 levels correlated positively with the vulnerability of the hub regions in patients with Alzheimer's disease. Our analysis therefore demonstrates that the magnetoencephalography-based multiplex brain networks contain important information that cannot be revealed by frequency-specific brain networks. Furthermore, this indicates that functional networks obtained in different frequency bands do not act as independent entities. Overall, our multiplex network study provides an effective framework to integrate the frequency-specific networks with different frequency patterns and reveal neuropathological mechanism of hub disruption in Alzheimer's disease. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Reconstructing the world trade multiplex: The role of intensive and extensive biases

NASA Astrophysics Data System (ADS)

Mastrandrea, Rossana; Squartini, Tiziano; Fagiolo, Giorgio; Garlaschelli, Diego

2014-12-01

In economic and financial networks, the strength of each node has always an important economic meaning, such as the size of supply and demand, import and export, or financial exposure. Constructing null models of networks matching the observed strengths of all nodes is crucial in order to either detect interesting deviations of an empirical network from economically meaningful benchmarks or reconstruct the most likely structure of an economic network when the latter is unknown. However, several studies have proved that real economic networks and multiplexes topologically differ from configurations inferred only from node strengths. Here we provide a detailed analysis of the world trade multiplex by comparing it to an enhanced null model that simultaneously reproduces the strength and the degree of each node. We study several temporal snapshots and almost 100 layers (commodity classes) of the multiplex and find that the observed properties are systematically well reproduced by our model. Our formalism allows us to introduce the (static) concept of extensive and intensive bias, defined as a measurable tendency of the network to prefer either the formation of extra links or the reinforcement of link weights, with respect to a reference case where only strengths are enforced. Our findings complement the existing economic literature on (dynamic) intensive and extensive trade margins. More generally, they show that real-world multiplexes can be strongly shaped by layer-specific local constraints.

A multiplex calibrated real-time PCR assay for quantitation of DNA of EBV-1 and 2.

PubMed

Gatto, Francesca; Cassina, Giulia; Broccolo, Francesco; Morreale, Giuseppe; Lanino, Edoardo; Di Marco, Eddi; Vardas, Efthiya; Bernasconi, Daniela; Buttò, Stefano; Principi, Nicola; Esposito, Susanna; Scarlatti, Gabriella; Lusso, Paolo; Malnati, Mauro S

2011-12-01

Accurate and highly sensitive tests for the diagnosis of active Epstein-Barr virus (EBV) infection are essential for the clinical management of individuals infected with EBV. A calibrated quantitative real-time PCR assay for the measurement of EBV DNA of both EBV-1 and 2 subtypes was developed, combining the detection of the EBV DNA and a synthetic DNA calibrator in a multiplex PCR format. The assay displays a wide dynamic range and a high degree of accuracy even in the presence of 1μg of human genomic DNA. This assay measures with the same efficiency EBV DNA from strains prevalent in different geographic areas. The clinical sensitivity and specificity of the system were evaluated by testing 181 peripheral blood mononuclear cell (PBMCs) and plasma specimens obtained from 21 patients subjected to bone marrow transplantation, 70 HIV-seropositive subjects and 23 healthy controls. Patients affected by EBV-associated post-transplant lymphoprolipherative disorders had the highest frequency of EBV detection and the highest viral load. Persons infected with HIV had higher levels of EBV DNA load in PBMCs and a higher frequency of EBV plasma viremia compared to healthy controls. In conclusion, this new assay provides a reliable high-throughput method for the quantitation of EBV DNA in clinical samples. Copyright © 2011 Elsevier B.V. All rights reserved.

Automated methods for multiplexed pathogen detection.

PubMed

Straub, Timothy M; Dockendorff, Brian P; Quiñonez-Díaz, Maria D; Valdez, Catherine O; Shutthanandan, Janani I; Tarasevich, Barbara J; Grate, Jay W; Bruckner-Lea, Cynthia J

2005-09-01

Detection of pathogenic microorganisms in environmental samples is a difficult process. Concentration of the organisms of interest also co-concentrates inhibitors of many end-point detection methods, notably, nucleic acid methods. In addition, sensitive, highly multiplexed pathogen detection continues to be problematic. The primary function of the BEADS (Biodetection Enabling Analyte Delivery System) platform is the automated concentration and purification of target analytes from interfering substances, often present in these samples, via a renewable surface column. In one version of BEADS, automated immunomagnetic separation (IMS) is used to separate cells from their samples. Captured cells are transferred to a flow-through thermal cycler where PCR, using labeled primers, is performed. PCR products are then detected by hybridization to a DNA suspension array. In another version of BEADS, cell lysis is performed, and community RNA is purified and directly labeled. Multiplexed detection is accomplished by direct hybridization of the RNA to a planar microarray. The integrated IMS/PCR version of BEADS can successfully purify and amplify 10 E. coli O157:H7 cells from river water samples. Multiplexed PCR assays for the simultaneous detection of E. coli O157:H7, Salmonella, and Shigella on bead suspension arrays was demonstrated for the detection of as few as 100 cells for each organism. Results for the RNA version of BEADS are also showing promising results. Automation yields highly purified RNA, suitable for multiplexed detection on microarrays, with microarray detection specificity equivalent to PCR. Both versions of the BEADS platform show great promise for automated pathogen detection from environmental samples. Highly multiplexed pathogen detection using PCR continues to be problematic, but may be required for trace detection in large volume samples. The RNA approach solves the issues of highly multiplexed PCR and provides "live vs. dead" capabilities. However, sensitivity of the method will need to be improved for RNA analysis to replace PCR.

Evaluation of a multiplex immunoassay for bovine respiratory syncytial virus and bovine coronavirus antibodies in bulk tank milk against two indirect ELISAs using latent class analysis.

PubMed

Toftaker, Ingrid; Toft, Nils; Stokstad, Maria; Sølverød, Liv; Harkiss, Gordon; Watt, Neil; O' Brien, Amanda; Nødtvedt, Ane

2018-06-01

Bovine respiratory syncytial virus (BRSV) and bovine coronavirus (BCV) are responsible for respiratory disease and diarrhea in cattle worldwide. The Norwegian control program against these infections is based on herd-level diagnosis using a new multiplex immunoassay. The objective of this study was to estimate sensitivity and specificity across different cut-off values for the MVD-Enferplex BCV/BRSV multiplex, by comparing them to a commercially available ELISA, the SVANOVIR ® BCV-Ab and SVANOVIR ® BRSV-Ab, respectively. We analyzed bulk tank milk samples from 360 herds in a low- and 360 herds in a high-prevalence area. As none of the tests were considered perfect, estimation of test characteristics was performed using Bayesian latent class models. At the manufacturers' recommended cut-off values, the median sensitivity for the BRSV multiplex and the BRSV ELISA was 94.4 [89.8-98.7 95% Posterior Credibility Interval (PCI)] and 99.8 [98.7-100 95% PCI], respectively. The median specificity for the BRSV multiplex was 90.6 [85.5-94.4 95% PCI], but only 57.4 [50.5-64.4 95% PCI] for the BRSV ELISA. However, increasing the cut-off of the BRSV ELISA increased specificity without compromising sensitivity. For the BCV multiplex we found that by using only one of the three antigens included in the test, the specificity increased, without concurrent loss in sensitivity. At the recommended cut-off this resulted in a sensitivity of 99.9 [99.3-100 95% PCI] and specificity of 93.7 [88.8-97.8 95% PCI] for the multiplex and a sensitivity of 99.5 [98.1-100 95% PCI] and a specificity of 99.6 [97.6-100 95% PCI] for the BCV ELISA. Copyright © 2018 Elsevier B.V. All rights reserved.

Automated Methods for Multiplexed Pathogen Detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Straub, Tim M.; Dockendorff, Brian P.; Quinonez-Diaz, Maria D.

2005-09-01

Detection of pathogenic microorganisms in environmental samples is a difficult process. Concentration of the organisms of interest also co-concentrates inhibitors of many end-point detection methods, notably, nucleic acid methods. In addition, sensitive, highly multiplexed pathogen detection continues to be problematic. The primary function of the BEADS (Biodetection Enabling Analyte Delivery System) platform is the automated concentration and purification of target analytes from interfering substances, often present in these samples, via a renewable surface column. In one version of BEADS, automated immunomagnetic separation (IMS) is used to separate cells from their samples. Captured cells are transferred to a flow-through thermal cyclermore » where PCR, using labeled primers, is performed. PCR products are then detected by hybridization to a DNA suspension array. In another version of BEADS, cell lysis is performed, and community RNA is purified and directly labeled. Multiplexed detection is accomplished by direct hybridization of the RNA to a planar microarray. The integrated IMS/PCR version of BEADS can successfully purify and amplify 10 E. coli O157:H7 cells from river water samples. Multiplexed PCR assays for the simultaneous detection of E. coli O157:H7, Salmonella, and Shigella on bead suspension arrays was demonstrated for the detection of as few as 100 cells for each organism. Results for the RNA version of BEADS are also showing promising results. Automation yields highly purified RNA, suitable for multiplexed detection on microarrays, with microarray detection specificity equivalent to PCR. Both versions of the BEADS platform show great promise for automated pathogen detection from environmental samples. Highly multiplexed pathogen detection using PCR continues to be problematic, but may be required for trace detection in large volume samples. The RNA approach solves the issues of highly multiplexed PCR and provides ''live vs. dead'' capabilities. However, sensitivity of the method will need to be improved for RNA analysis to replace PCR.« less

Dual function microscope for quantitative DIC and birefringence imaging

NASA Astrophysics Data System (ADS)

Li, Chengshuai; Zhu, Yizheng

2016-03-01

A spectral multiplexing interferometry (SXI) method is presented for integrated birefringence and phase gradient measurement on label-free biological specimens. With SXI, the retardation and orientation of sample birefringence are simultaneously encoded onto two separate spectral carrier waves, generated by a crystal retarder oriented at a specific angle. Thus sufficient information for birefringence determination can be obtained from a single interference spectrum, eliminating the need for multiple acquisitions with mechanical rotation or electrical modulation. In addition, with the insertion of a Nomarski prism, the setup can then acquire quantitative differential interference contrast images. Red blood cells infected by malaria parasites are imaged for birefringence retardation as well as phase gradient. The results demonstrate that the SXI approach can achieve both quantitative phase imaging and birefringence imaging with a single, high-sensitivity system.

Syndromic Panel-Based Testing in Clinical Microbiology.

PubMed

Ramanan, Poornima; Bryson, Alexandra L; Binnicker, Matthew J; Pritt, Bobbi S; Patel, Robin

2018-01-01

The recent development of commercial panel-based molecular diagnostics for the rapid detection of pathogens in positive blood culture bottles, respiratory specimens, stool, and cerebrospinal fluid has resulted in a paradigm shift in clinical microbiology and clinical practice. This review focuses on U.S. Food and Drug Administration (FDA)-approved/cleared multiplex molecular panels with more than five targets designed to assist in the diagnosis of bloodstream, respiratory tract, gastrointestinal, or central nervous system infections. While these panel-based assays have the clear advantages of a rapid turnaround time and the detection of a large number of microorganisms and promise to improve health care, they present certain challenges, including cost and the definition of ideal test utilization strategies (i.e., optimal ordering) and test interpretation. Copyright © 2017 American Society for Microbiology.

Polymerase chain reaction identification of three members of the Anopheles sundaicus (Diptera: Culicidae) complex, malaria vectors in Southeast Asia.

PubMed

Dusfour, Isabelle; Blondeau, Johanna; Harbach, Ralph E; Vythilingham, Indra; Baimai, Visut; Trung, Ho D; Sochanta, Tho; Bangs, Michael J; Manguin, Sylvie

2007-09-01

Anopheles sundaicus s.l., a major malaria vector taxon, occurs primarily along coastal areas and on islands in Southeast Asia. Our previous studies using cytochrome oxidase I, cytochrome-b, and internal transcribed spacer 2 markers discriminated three allopatric species: An. sundaicus s.s. in northern Borneo, An. epiroticus in Southeast Asia, and An. sundaicus E on Sumatra and Java, Indonesia. Morphological comparisons of three developmental stages did not reveal unique diagnostic characters that could reliably distinguish the three species. Therefore, we developed a multiplex polymerase chain reaction (PCR) assay based on two mitochondrial DNA markers to unambiguously identify them. This PCR was tested on 374 specimens from 24 different geographical populations, expanding our knowledge of the distribution of these species.

EDRN Pre-Validation of Metabolic Biomarkers of Prostate Cancer: Follow up EDRN Challenge — EDRN Public Portal

Cancer.gov

The goal of this EDRN set-asides proposal is to carry out pre-validation studies on sarcosine as a metabolomic biomarker of prostate cancer in urine. Not only does sarcosine have potential as a marker for the early detection of prostate cancer in post-DRE urine specimens-- but since its highest levels are in metastatic disease it might have utility in predicting aggressiveness of clinically localized disease. We will also use these funds to determine if we can add additional metabolites to sarcosine in order to develop a multiplex metabolomic biomarker panel in prostate cancer. In addition to sarcosine, we have 10-12 additional candidate metabolomic biomarkers that could be developed (as seen from our preliminary global metabolite studies).

Multiplexed Analysis of Serum Breast and Ovarian Cancer Markers by Means of Suspension Bead-quantum Dot Microarrays

NASA Astrophysics Data System (ADS)

Brazhnik, Kristina; Sokolova, Zinaida; Baryshnikova, Maria; Bilan, Regina; Nabiev, Igor; Sukhanova, Alyona

Multiplexed analysis of cancer markers is crucial for early tumor diagnosis and screening. We have designed lab-on-a-bead microarray for quantitative detection of three breast cancer markers in human serum. Quantum dots were used as bead-bound fluorescent tags for identifying each marker by means of flow cytometry. Antigen-specific beads reliably detected CA 15-3, CEA, and CA 125 in serum samples, providing clear discrimination between the samples with respect to the antigen levels. The novel microarray is advantageous over the routine single-analyte ones due to the simultaneous detection of various markers. Therefore the developed microarray is a promising tool for serum tumor marker profiling.

Centrality in earthquake multiplex networks

NASA Astrophysics Data System (ADS)

Lotfi, Nastaran; Darooneh, Amir Hossein; Rodrigues, Francisco A.

2018-06-01

Seismic time series has been mapped as a complex network, where a geographical region is divided into square cells that represent the nodes and connections are defined according to the sequence of earthquakes. In this paper, we map a seismic time series to a temporal network, described by a multiplex network, and characterize the evolution of the network structure in terms of the eigenvector centrality measure. We generalize previous works that considered the single layer representation of earthquake networks. Our results suggest that the multiplex representation captures better earthquake activity than methods based on single layer networks. We also verify that the regions with highest seismological activities in Iran and California can be identified from the network centrality analysis. The temporal modeling of seismic data provided here may open new possibilities for a better comprehension of the physics of earthquakes.

Fundamentals of multiplexing with digital PCR.

PubMed

Whale, Alexandra S; Huggett, Jim F; Tzonev, Svilen

2016-12-01

Over the past decade numerous publications have demonstrated how digital PCR (dPCR) enables precise and sensitive quantification of nucleic acids in a wide range of applications in both healthcare and environmental analysis. This has occurred in parallel with the advances in partitioning fluidics that enable a reaction to be subdivided into an increasing number of partitions. As the majority of dPCR systems are based on detection in two discrete optical channels, most research to date has focused on quantification of one or two targets within a single reaction. Here we describe 'higher order multiplexing' that is the unique ability of dPCR to precisely measure more than two targets in the same reaction. Using examples, we describe the different types of duplex and multiplex reactions that can be achieved. We also describe essential experimental considerations to ensure accurate quantification of multiple targets.

Observability of Boolean multiplex control networks

NASA Astrophysics Data System (ADS)

Wu, Yuhu; Xu, Jingxue; Sun, Xi-Ming; Wang, Wei

2017-04-01

Boolean multiplex (multilevel) networks (BMNs) are currently receiving considerable attention as theoretical arguments for modeling of biological systems and system level analysis. Studying control-related problems in BMNs may not only provide new views into the intrinsic control in complex biological systems, but also enable us to develop a method for manipulating biological systems using exogenous inputs. In this article, the observability of the Boolean multiplex control networks (BMCNs) are studied. First, the dynamical model and structure of BMCNs with control inputs and outputs are constructed. By using of Semi-Tensor Product (STP) approach, the logical dynamics of BMCNs is converted into an equivalent algebraic representation. Then, the observability of the BMCNs with two different kinds of control inputs is investigated by giving necessary and sufficient conditions. Finally, examples are given to illustrate the efficiency of the obtained theoretical results.

An in vivo multiplexed small molecule screening platform

PubMed Central

Yang, Dian; Ogasawara, Daisuke; Dix, Melissa M.; Rogers, Zoë N.; Chuang, Chen-Hua; McFarland, Christopher D.; Chiou, Shin-Heng; Brown, J. Mark; Cravatt, Benjamin F.; Bogyo, Matthew; Winslow, Monte M.

2016-01-01

Phenotype-based small molecule screening is a powerful method to identify regulators of cellular function. However, such screens are generally performed in vitro using conditions that do not necessarily model complex physiological conditions or disease states. Here, we use molecular cell barcoding to enable direct in vivo phenotypic screening of libraries of small molecules. The multiplexed nature of this approach allows rapid in vivo analysis of hundreds to thousands of compounds. Using this platform, we screened >700 covalent inhibitors directed towards hydrolases for their effect on pancreatic cancer metastatic seeding. We identified multiple hits and confirmed the relevant target of one compound as the lipase ABHD6. Pharmacological and genetic studies confirmed the role of this enzyme as a regulator of metastatic fitness. Our results highlight the applicability of this multiplexed screening platform for investigating complex processes in vivo. PMID:27617390

A Spread-Spectrum SQUID Multiplexer

NASA Astrophysics Data System (ADS)

Irwin, K. D.; Chaudhuri, S.; Cho, H.-M.; Dawson, C.; Kuenstner, S.; Li, D.; Titus, C. J.; Young, B. A.

2018-06-01

The transition-edge sensor (TES) is a mature, high-resolution x-ray spectrometer technology that provides a much higher efficiency than dispersive spectrometers such as gratings and crystal spectrometers. As larger arrays are developed, time-division multiplexing schemes operating at MHz frequencies are being replaced by microwave SQUID multiplexers using frequency-division multiplexing at GHz frequencies. However, the multiplexing factor achievable with microwave SQUIDs is limited by the high slew rate on the leading edge of x-ray pulses. In this paper, we propose a new multiplexing scheme for high-slew-rate TES x-ray calorimeters: the spread-spectrum SQUID multiplexer, which has the potential to enable higher multiplexing factors, especially in applications with lower photon-arrival rates.

Multiplexed image storage by electromagnetically induced transparency in a solid

NASA Astrophysics Data System (ADS)

Heinze, G.; Rentzsch, N.; Halfmann, T.

2012-11-01

We report on frequency- and angle-multiplexed image storage by electromagnetically induced transparency (EIT) in a Pr3+:Y2SiO5 crystal. Frequency multiplexing by EIT relies on simultaneous storage of light pulses in atomic coherences, driven in different frequency ensembles of the inhomogeneously broadened solid medium. Angular multiplexing by EIT relies on phase matching of the driving laser beams, which permits simultaneous storage of light pulses propagating under different angles into the crystal. We apply the multiplexing techniques to increase the storage capacity of the EIT-driven optical memory, in particular to implement multiplexed storage of larger two-dimensional amounts of data (images). We demonstrate selective storage and readout of images by frequency-multiplexed EIT and angular-multiplexed EIT, as well as the potential to combine both multiplexing approaches towards further enhanced storage capacities.

Development of melting temperature-based SYBR Green I polymerase chain reaction methods for multiplex genetically modified organism detection.

PubMed

Hernández, Marta; Rodríguez-Lázaro, David; Esteve, Teresa; Prat, Salomé; Pla, Maria

2003-12-15

Commercialization of several genetically modified crops has been approved worldwide to date. Uniplex polymerase chain reaction (PCR)-based methods to identify these different insertion events have been developed, but their use in the analysis of all commercially available genetically modified organisms (GMOs) is becoming progressively insufficient. These methods require a large number of assays to detect all possible GMOs present in the sample and thereby the development of multiplex PCR systems using combined probes and primers targeted to sequences specific to various GMOs is needed for detection of this increasing number of GMOs. Here we report on the development of a multiplex real-time PCR suitable for multiple GMO identification, based on the intercalating dye SYBR Green I and the analysis of the melting curves of the amplified products. Using this method, different amplification products specific for Maximizer 176, Bt11, MON810, and GA21 maize and for GTS 40-3-2 soybean were obtained and identified by their specific Tm. We have combined amplification of these products in a number of multiplex reactions and show the suitability of the methods for identification of GMOs with a sensitivity of 0.1% in duplex reactions. The described methods offer an economic and simple alternative to real-time PCR systems based on sequence-specific probes (i.e., TaqMan chemistry). These methods can be used as selection tests and further optimized for uniplex GMO quantification.

Core Needle Lung Biopsy Specimens: Adequacy for EGFR and KRAS Mutational Analysis

PubMed Central

Zakowski, Maureen F.; Pao, William; Thornton, Raymond H.; Ladanyi, Marc; Kris, Mark G.; Rusch, Valerie W.; Rizvi, Naiyer A.

2013-01-01

OBJECTIVE The purpose of this study was to prospectively compare the adequacy of core needle biopsy specimens with the adequacy of specimens from resected tissue, the histologic reference standard, for mutational analysis of malignant tumors of the lung. SUBJECTS AND METHODS The first 18 patients enrolled in a phase 2 study of gefitinib for lung cancer in July 2004 through August 2005 underwent CT- or fluoroscopy-guided lung biopsy before the start of gefitinib therapy. Three weeks after gefitinib therapy, the patients underwent lung tumor resection. The results of EGFR and KRAS mutational analysis of the core needle biopsy specimens were compared with those of EGFR and KRAS mutational analysis of the surgical specimens. RESULTS Two specimens were unsatisfactory for mutational analysis. The results of mutational assay results of the other 16 specimens were the same as those of analysis of the surgical specimens obtained an average of 31 days after biopsy. CONCLUSION Biopsy with small (18- to 20-gauge) core needles can yield sufficient and reliable samples for mutational analysis. This technique is likely to become an important tool with the increasing use of pharmacotherapy based on the genetics of specific tumors in individual patients. PMID:20028932

Rapid and accurate identification of Mycobacterium tuberculosis complex and common non-tuberculous mycobacteria by multiplex real-time PCR targeting different housekeeping genes.

PubMed

Nasr Esfahani, Bahram; Rezaei Yazdi, Hadi; Moghim, Sharareh; Ghasemian Safaei, Hajieh; Zarkesh Esfahani, Hamid

2012-11-01

Rapid and accurate identification of mycobacteria isolates from primary culture is important due to timely and appropriate antibiotic therapy. Conventional methods for identification of Mycobacterium species based on biochemical tests needs several weeks and may remain inconclusive. In this study, a novel multiplex real-time PCR was developed for rapid identification of Mycobacterium genus, Mycobacterium tuberculosis complex (MTC) and the most common non-tuberculosis mycobacteria species including M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and the M. gordonae in three reaction tubes but under same PCR condition. Genetic targets for primer designing included the 16S rDNA gene, the dnaJ gene, the gyrB gene and internal transcribed spacer (ITS). Multiplex real-time PCR was setup with reference Mycobacterium strains and was subsequently tested with 66 clinical isolates. Results of multiplex real-time PCR were analyzed with melting curves and melting temperature (T (m)) of Mycobacterium genus, MTC, and each of non-tuberculosis Mycobacterium species were determined. Multiplex real-time PCR results were compared with amplification and sequencing of 16S-23S rDNA ITS for identification of Mycobacterium species. Sensitivity and specificity of designed primers were each 100 % for MTC, M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and M. gordonae. Sensitivity and specificity of designed primer for genus Mycobacterium was 96 and 100 %, respectively. According to the obtained results, we conclude that this multiplex real-time PCR with melting curve analysis and these novel primers can be used for rapid and accurate identification of genus Mycobacterium, MTC, and the most common non-tuberculosis Mycobacterium species.

Development of the first standardised panel of two new microsatellite multiplex PCRs for gilthead seabream (Sparus aurata L.).

PubMed

Lee-Montero, I; Navarro, A; Borrell, Y; García-Celdrán, M; Martín, N; Negrín-Báez, D; Blanco, G; Armero, E; Berbel, C; Zamorano, M J; Sánchez, J J; Estévez, A; Ramis, G; Manchado, M; Afonso, J M

2013-08-01

The high number of multiplex PCRs developed for gilthead seabream (Sparus aurata L.) from many different microsatellite markers does not allow comparison among populations. This highlights the need for developing a reproducible panel of markers, which can be used with safety and reliability by all users. In this study, the first standardised panel of two new microsatellite multiplex PCRs was developed for this species. Primers of 138 specific microsatellites from the genetic linkage map were redesigned and evaluated according to their genetic variability, allele size range and genotyping reliability. A protocol to identify and classify genotyping errors or potential errors was proposed to assess the reliability of each marker. Two new multiplex PCRs from the best assessed markers were designed with 11 markers in each, named SMsa1 and SMsa2 (SuperMultiplex Sparus aurata). Three broodstocks (59, 47 and 98 breeders) from different Spanish companies, and a sample of 80 offspring from each one, were analysed to validate the usefulness of these multiplexes in the parental assignation. It was possible to assign each offspring to a single parent pair (100% success) using the exclusion method with SMsa1 and/or SMsa2. In each genotyped a reference sample (Ref-sa) was used, and its DNA is available on request similar to the kits of bin set to genotype by genemapper (v.3.7) software (kit-SMsa1 and kit-SMsa2). This will be a robust and effective tool for pedigree analysis or characterisation of populations and will be proposed as an international panel for this species. © 2013 The Authors, Animal Genetics © 2013 Stichting International Foundation for Animal Genetics.

Multiplex fluorescent PCR for noninvasive prenatal detection of fetal-derived paternally inherited diseases using circulatory fetal DNA in maternal plasma.

PubMed

Tang, Dong-ling; Li, Yan; Zhou, Xin; Li, Xia; Zheng, Fang

2009-05-01

To develop a fluorescent polymerase chain reaction (PCR) assay for the detection of circulating fetal DNA in maternal plasma and use the established multiplex in noninvasive prenatal genetic diagnosis and its further applications in forensic casework. The DNA template was extracted from 47 pregnant women and the whole blood samples from the stated biological fathers were used to detect genotype. Using multiplex fluorescent PCR at 16 different polymorphic short tandem repeat (STR) loci, maternal DNA extracted from plasma samples at early pregnancy, medium pregnancy and late pregnancy were used to detect genotype. Their husbands' DNA was also used for fetal genotype ascertainment. Multiplex fluorescent PCR with 16 polymorphic short tandem repeats revealed the presence of fetal DNA in all cases. Every pregnant women/husband pair was informative in at least 3 of 16 loci. The chances of detecting paternally inherited fetal alleles ranged from 66.67 to 94.12%. They are 66.67% in early pregnancy, 85.71% in medium pregnancy and 94.12% in late pregnancy. The accuracy of Multiplex PCR assay to detect fetal DNA was 100%. Circulating fetal DNA analysis can be used as a possible alternative tool in routine laboratory prenatal diagnosis in the near future; this highly polymorphic STR multiplex has greatly improved the chances of detecting paternally inherited fetal alleles compared with other fetal DNA detection systems that use fetus-derived Y sequences to detect only male fetal DNA in maternal plasma. Our proposed technique can be applied to both female and male fetuses, which provides a sensitive, accurate and efficient method for noninvasive prenatal genetic diagnosis and forensic casework.

Reconfigurable data path processor

NASA Technical Reports Server (NTRS)

Donohoe, Gregory (Inventor)

2005-01-01

A reconfigurable data path processor comprises a plurality of independent processing elements. Each of the processing elements advantageously comprising an identical architecture. Each processing element comprises a plurality of data processing means for generating a potential output. Each processor is also capable of through-putting an input as a potential output with little or no processing. Each processing element comprises a conditional multiplexer having a first conditional multiplexer input, a second conditional multiplexer input and a conditional multiplexer output. A first potential output value is transmitted to the first conditional multiplexer input, and a second potential output value is transmitted to the second conditional multiplexer output. The conditional multiplexer couples either the first conditional multiplexer input or the second conditional multiplexer input to the conditional multiplexer output, according to an output control command. The output control command is generated by processing a set of arithmetic status-bits through a logical mask. The conditional multiplexer output is coupled to a first processing element output. A first set of arithmetic bits are generated according to the processing of the first processable value. A second set of arithmetic bits may be generated from a second processing operation. The selection of the arithmetic status-bits is performed by an arithmetic-status bit multiplexer selects the desired set of arithmetic status bits from among the first and second set of arithmetic status bits. The conditional multiplexer evaluates the select arithmetic status bits according to logical mask defining an algorithm for evaluating the arithmetic status bits.

Multiplexer and time duration measuring circuit

DOEpatents

Gray, Jr., James

1980-01-01

A multiplexer device is provided for multiplexing data in the form of randomly developed, variable width pulses from a plurality of pulse sources to a master storage. The device includes a first multiplexer unit which includes a plurality of input circuits each coupled to one of the pulse sources, with all input circuits being disabled when one input circuit receives an input pulse so that only one input pulse is multiplexed by the multiplexer unit at any one time.

Retrospective Species Identification of Microsporidian Spores in Diarrheic Fecal Samples from Human Immunodeficiency Virus/AIDS Patients by Multiplexed Fluorescence In Situ Hybridization▿

PubMed Central

Graczyk, Thaddeus K.; Johansson, Michael A.; Tamang, Leena; Visvesvara, Govinda S.; Moura, Laci S.; DaSilva, Alexandre J.; Girouard, Autumn S.; Matos, Olga

2007-01-01

In order to assess the applicability of multiplexed fluorescence in situ hybridization (FISH) assay for the clinical setting, we conducted retrospective analysis of 110 formalin-stored diarrheic stool samples from human immunodeficiency virus (HIV)/AIDS patients with intestinal microsporidiosis collected between 1992 and 2003. The multiplexed FISH assay identified microsporidian spores in 94 of 110 (85.5%) samples: 49 (52.1%) were positive for Enterocytozoon bieneusi, 43 (45.8%) were positive for Encephalitozoon intestinalis, 2 (2.1%) were positive for Encephalitozoon hellem, and 9 samples (9.6%) contained both E. bieneusi and E. intestinalis spores. Quantitative spore counts per ml of stool yielded concentration values from 3.5 × 103 to 4.4 × 105 for E. bieneusi (mean, 8.8 × 104/ml), 2.3 × 102 to 7.8 × 104 (mean, 1.5 × 104/ml) for E. intestinalis, and 1.8 × 102 to 3.6 × 102 for E. hellem (mean, 2.7 × 102/ml). Identification of microsporidian spores by multiplex FISH assay was more sensitive than both Chromotrope-2R and CalcoFluor White M2R stains; 85.5% versus 72.7 and 70.9%, respectively. The study demonstrated that microsporidian coinfection in HIV/AIDS patients with intestinal microsporidiosis is not uncommon and that formalin-stored fecal samples older than 10 years may not be suitable for retrospective analysis by techniques targeting rRNA. Multiplexed FISH assay is a reliable, quantitative fluorescence microscopy method for the simultaneous identification of E. bieneusi, E. intestinalis, and E. hellem, as well as Encephalitozoon cuniculi, spores in fecal samples and is a useful tool for assessing spore shedding intensity in intestinal microsporidiosis. The method can be used for epidemiological investigations and applied in clinical settings. PMID:17287331

Results of a remote multiplexer/digitizer unit accuracy and environmental study

NASA Technical Reports Server (NTRS)

Wilner, D. O.

1977-01-01

A remote multiplexer/digitizer unit (RMDU), a part of the airborne integrated flight test data system, was subjected to an accuracy study. The study was designed to show the effects of temperature, altitude, and vibration on the RMDU. The RMDU was subjected to tests at temperatures from -54 C (-65 F) to 71 C (160 F), and the resulting data are presented here, along with a complete analysis of the effects. The methods and means used for obtaining correctable data and correcting the data are also discussed.

Free-space wavelength-multiplexed optical scanner.

PubMed

Yaqoob, Z; Rizvi, A A; Riza, N A

2001-12-10

A wavelength-multiplexed optical scanning scheme is proposed for deflecting a free-space optical beam by selection of the wavelength of the light incident on a wavelength-dispersive optical element. With fast tunable lasers or optical filters, this scanner features microsecond domain scan setting speeds and large- diameter apertures of several centimeters or more for subdegree angular scans. Analysis performed indicates an optimum scan range for a given diffraction order and grating period. Limitations include beam-spreading effects based on the varying scanner aperture sizes and the instantaneous information bandwidth of the data-carrying laser beam.

Comprehensive protein profiling by multiplexed capillary zone electrophoresis using cross-linked polyacrylamide coated capillaries.

PubMed

Liu, Shaorong; Gao, Lin; Pu, Qiaosheng; Lu, Joann J; Wang, Xingjia

2006-02-01

We have recently developed a new process to create cross-linked polyacrylamide (CPA) coatings on capillary walls to suppress protein-wall interactions. Here, we demonstrate CPA-coated capillaries for high-efficiency (>2 x 10(6) plates per meter) protein separations by capillary zone electrophoresis (CZE). Because CPA virtually eliminates electroosmotic flow, positive and negative proteins cannot be analyzed in a single run. A "one-sample-two-separation" approach is developed to achieve a comprehensive protein analysis. High throughput is achieved through a multiplexed CZE system.

A macrochip interconnection network enabled by silicon nanophotonic devices.

PubMed

Zheng, Xuezhe; Cunningham, John E; Koka, Pranay; Schwetman, Herb; Lexau, Jon; Ho, Ron; Shubin, Ivan; Krishnamoorthy, Ashok V; Yao, Jin; Mekis, Attila; Pinguet, Thierry

2010-03-01

We present an advanced wavelength-division multiplexing point-to-point network enabled by silicon nanophotonic devices. This network offers strictly non-blocking all-to-all connectivity while maximizing bisection bandwidth, making it ideal for multi-core and multi-processor interconnections. We introduce one of the key components, the nanophotonic grating coupler, and discuss, for the first time, how this device can be useful for practical implementations of the wavelength-division multiplexing network using optical proximity communications. Finite difference time-domain simulation of the nanophotonic grating coupler device indicates that it can be made compact (20 microm x 50 microm), low loss (3.8 dB), and broadband (100 nm). These couplers require subwavelength material modulation at the nanoscale to achieve the desired functionality. We show that optical proximity communication provides unmatched optical I/O bandwidth density to electrical chips, which enables the application of wavelength-division multiplexing point-to-point network in macrochip with unprecedented bandwidth-density. The envisioned physical implementation is discussed. The benefits of such an interconnect network include a 5-6x improvement in latency when compared to a purely electronic implementation. Performance analysis shows that the wavelength-division multiplexing point-to-point network offers better overall performance over other optical network architectures.

Serotype and mating type characterization of Cryptococcus neoformans by multiplex PCR.

PubMed

Carvalho, Vívian Gonçalves; Terceti, Mateus Souza; Dias, Amanda Latercia Tranches; Paula, Claudete Rodrigues; Lyon, Juliana Pereira; de Siqueira, Antônio Martins; Franco, Marília Caixeta

2007-01-01

Cryptococcus neoformans is an encapsulated yeast, etiological agent of cryptococcosis. The species is commonly associated with pigeon droppings and plant materials. The aim of the present work was to verify the presence of the yeast in pigeon droppings, and to identify the isolates obtained in serotypes and mating types (MAT). Ten samples of pigeon droppings were collected in the rural area of the city of Alfenas, Brazil. Samples were inoculated in agar Niger medium for fungal isolation and 22 isolates with characteristics of C. neoformans were obtained. The serotypes and MAT were determined by multiplex PCR using specific primers. Serotypes were also determined by using the Kit Crypto Check. Among the 22 samples evaluated, eight were identified as C. neoformans by classic identification tests. These samples were characterized as serotype A by the Kit Crypto check and as serotype A MAT alpha by the multiplex PCR. The present study reinforces the evidence that pigeon droppings are a reservoir for C. neoformans and confirms the prevalence of C. neoformans var. grubii (A alpha) among environmental isolates. It also demonstrates that multiplex PCR is an acceptable alternative for serotype analysis because it reduces the costs for each reaction and analyses serotype and MAT simultaneously.

Dynamic stress analysis of smooth and notched fiber composite flexural specimens

NASA Technical Reports Server (NTRS)

Murthy, P. L. N.; Chamis, C. C.

1984-01-01

A detailed analysis of the dynamic stress field in smooth and notched fiber composite (Charpy-type) specimens is reported in this paper. The analysis is performed with the aid of the direct transient response analysis solution sequence of MSC/NASTRAN. Three unidirectional composites were chosen for the study. They are S-Glass/Epoxy, Kevlar/Epoxy and T-300/Epoxy composite systems. The specimens are subjected to an impact load which is modeled as a triangular impulse with a maximum of 2000 lb and a duration of 1 ms. The results are compared with those of static analysis of the specimens subjected to a peak load of 2000 lb. For the geometry and type of materials studied, the static analysis results gave close conservative estimates for the dynamic stresses. Another interesting inference from the study is that the impact induced effects are felt by S-Glass/Epoxy specimens sooner than Kevlar/Epoxy or T-300/Epoxy specimens.

Biosensors in Health Care: The Milestones Achieved in Their Development towards Lab-on-Chip-Analysis

PubMed Central

Patel, Suprava; Nanda, Rachita; Sahoo, Sibasish; Mohapatra, Eli

2016-01-01

Immense potentiality of biosensors in medical diagnostics has driven scientists in evolution of biosensor technologies and innovating newer tools in time. The cornerstone of the popularity of biosensors in sensing wide range of biomolecules in medical diagnostics is due to their simplicity in operation, higher sensitivity, ability to perform multiplex analysis, and capability to be integrated with different function by the same chip. There remains a huge challenge to meet the demands of performance and yield to its simplicity and affordability. Ultimate goal stands for providing point-of-care testing facility to the remote areas worldwide, particularly the developing countries. It entails continuous development in technology towards multiplexing ability, fabrication, and miniaturization of biosensor devices so that they can provide lab-on-chip-analysis systems to the community. PMID:27042353

Frequency-domain-independent vector analysis for mode-division multiplexed transmission

NASA Astrophysics Data System (ADS)

Liu, Yunhe; Hu, Guijun; Li, Jiao

2018-04-01

In this paper, we propose a demultiplexing method based on frequency-domain independent vector analysis (FD-IVA) algorithm for mode-division multiplexing (MDM) system. FD-IVA extends frequency-domain independent component analysis (FD-ICA) from unitary variable to multivariate variables, and provides an efficient method to eliminate the permutation ambiguity. In order to verify the performance of FD-IVA algorithm, a 6 ×6 MDM system is simulated. The simulation results show that the FD-IVA algorithm has basically the same bit-error-rate(BER) performance with the FD-ICA algorithm and frequency-domain least mean squares (FD-LMS) algorithm. Meanwhile, the convergence speed of FD-IVA algorithm is the same as that of FD-ICA. However, compared with the FD-ICA and the FD-LMS, the FD-IVA has an obviously lower computational complexity.

CO2 Supermarket Refrigeration Systems for Southeast Asia and the USA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sharma, Vishaldeep; Fricke, Brian A; Bansal, Pradeep

This paper presents a comparative analysis of the annual energy consumption of these refrigeration systems in eighty eight cities from all climate zones in Southeast Asia. Also, the performance of the CO2 refrigeration systems is compared to the baseline R404A multiplex direct expansion (DX) system. Finally, the overall performance of the CO2 refrigeration systems in various climatic conditions in Southeast Asia is compared to that in the United States. For the refrigeration systems investigated, it was found that the Transcritical Booster System with Bypass Compressor (TBS-BC) performs better or equivalent to the R404A multiplex DX system in the northern regionsmore » of Southeast Asia (China and Japan). In the southern regions of Southeast Asia (India, Bangladesh, Burma), the R404A multiplex DX system and the Combined Secondary Cascade (CSC) system performs better than the TBS-BC.« less

High frequency of ribosomal protein gene deletions in Italian Diamond-Blackfan anemia patients detected by multiplex ligation-dependent probe amplification assay

PubMed Central

Quarello, Paola; Garelli, Emanuela; Brusco, Alfredo; Carando, Adriana; Mancini, Cecilia; Pappi, Patrizia; Vinti, Luciana; Svahn, Johanna; Dianzani, Irma; Ramenghi, Ugo

2012-01-01

Diamond-Blackfan anemia is an autosomal dominant disease due to mutations in nine ribosomal protein encoding genes. Because most mutations are loss of function and detected by direct sequencing of coding exons, we reasoned that part of the approximately 50% mutation negative patients may have carried a copy number variant of ribosomal protein genes. As a proof of concept, we designed a multiplex ligation-dependent probe amplification assay targeted to screen the six genes that are most frequently mutated in Diamond-Blackfan anemia patients: RPS17, RPS19, RPS26, RPL5, RPL11, and RPL35A. Using this assay we showed that deletions represent approximately 20% of all mutations. The combination of sequencing and multiplex ligation-dependent probe amplification analysis of these six genes allows the genetic characterization of approximately 65% of patients, showing that Diamond-Blackfan anemia is indisputably a ribosomopathy. PMID:22689679

Development and evaluation of a Hadamard transform imaging spectrometer and a Hadamard transform thermal imager

NASA Technical Reports Server (NTRS)

Harwit, M.; Swift, R.; Wattson, R.; Decker, J.; Paganetti, R.

1976-01-01

A spectrometric imager and a thermal imager, which achieve multiplexing by the use of binary optical encoding masks, were developed. The masks are based on orthogonal, pseudorandom digital codes derived from Hadamard matrices. Spatial and/or spectral data is obtained in the form of a Hadamard transform of the spatial and/or spectral scene; computer algorithms are then used to decode the data and reconstruct images of the original scene. The hardware, algorithms and processing/display facility are described. A number of spatial and spatial/spectral images are presented. The achievement of a signal-to-noise improvement due to the signal multiplexing was also demonstrated. An analysis of the results indicates both the situations for which the multiplex advantage may be gained, and the limitations of the technique. A number of potential applications of the spectrometric imager are discussed.

High-Definition Optical Velocimetry: A New Diagnostic Paradigm for Nuclear Security

DOE Office of Scientific and Technical Information (OSTI.GOV)

Daykin, E; Diaz, A; Gallegos, C

This slide-show describes work done to address the challenge of high-definition optical velocimetry with hundred(s) of high-fidelity velocity vs. time measurements. After a review of the historical context and a general technical description of how optical velocimetry, particularly photonic Doppler velocimetry, works, the innovation of multiplexed photonic Doppler velocimetry (MPDV) is described as implemented with commercially available telecom products and dense wavelength division multiplexing (DWDM). High amplification of small signals allows for laser-safe operations. The authors have evaluated and leveraged telecom components– optical amplifiers, wavelength multiplexers, and seed lasers–to provide an economical, compact and rugged approach to system architecture. Fouriermore » transform data analysis is seen to be robust and capable of discriminating simultaneous data traces recorded onto a single digitizer channel. The authors successfully fielded demonstration MPDV system on shock driven experiments.« less

Multiplex Particle Focusing via Hydrodynamic Force in Viscoelastic Fluids

NASA Astrophysics Data System (ADS)

Lee, Doo Jin; Brenner, Howard; Youn, Jae Ryoun; Song, Young Seok

2013-11-01

We introduce a multiplex particle focusing phenomenon that arises from the hydrodynamic interaction between the viscoelastic force and the Dean drag force in a microfluidic device. In a confined microchannel, the first normal stress difference of viscoelastic fluids results in a lateral migration of suspended particles. Such a viscoelastic force was harnessed to focus different sized particles in the middle of a microchannel, and spiral channel geometry was also considered in order to take advantage of the counteracting force, Dean drag force that induces particle migration in the outward direction. For theoretical understanding, we performed a numerical analysis of viscoelastic fluids in the spiral microfluidic channel. From these results, a concept of the `Dean-coupled Elasto-inertial Focusing band (DEF)' was proposed. This study provides in-depth physical insight into the multiplex focusing of particles that can open a new venue for microfluidic particle dynamics for a concrete high throughput platform at microscale.

Pigment phenotype and biogeographical ancestry from ancient skeletal remains: inferences from multiplexed autosomal SNP analysis.

PubMed

Bouakaze, Caroline; Keyser, Christine; Crubézy, Eric; Montagnon, Daniel; Ludes, Bertrand

2009-07-01

In the present study, a multiplexed genotyping assay for ten single nucleotide polymorphisms (SNPs) located within six pigmentation candidate genes was developed on modern biological samples and applied to DNA retrieved from 25 archeological human remains from southern central Siberia dating from the Bronze and Iron Ages. SNP genotyping was successful for the majority of ancient samples and revealed that most probably had typical European pigment features, i.e., blue or green eye color, light hair color and skin type, and were likely of European individual ancestry. To our knowledge, this study reports for the first time the multiplexed typing of autosomal SNPs on aged and degraded DNA. By providing valuable information on pigment traits of an individual and allowing individual biogeographical ancestry estimation, autosomal SNP typing can improve ancient DNA studies and aid human identification in some forensic casework situations when used to complement conventional molecular markers.

Rapid and simultaneous detection of Salmonella spp., Escherichia coli O157, and Listeria monocytogenes by magnetic capture hybridization and multiplex real-time PCR.

PubMed

Carloni, Elisa; Rotundo, Luca; Brandi, Giorgio; Amagliani, Giulia

2018-05-25

The application of rapid, specific, and sensitive methods for pathogen detection and quantification is very advantageous in diagnosis of human pathogens in several applications, including food analysis. The aim of this study was the evaluation of a method for the multiplexed detection and quantification of three significant foodborne pathogenic species (Escherichia coli O157, Salmonella spp., and Listeria monocytogenes). The assay combines specific DNA extraction by multiplex magnetic capture hybridization (mMCH) with multiplex real-time PCR. The amplification assay showed linearity in the range 10 6 -10 genomic units (GU)/PCR for each co-amplified species. The sensitivity corresponded to 1 GU/PCR for E. coli O157 and L. monocytogenes, and 10 GU/PCR for Salmonella spp. The immobilization process and the hybrid capture of the MCH showed good efficiency and reproducibility for all targets, allowing the combination in equal amounts of the different nanoparticle types in mMCH. MCH and mMCH efficiencies were similar. The detection limit of the method was 10 CFU in samples with individual pathogens and 10 2  CFU in samples with combination of the three pathogens in unequal amounts (amount's differences of 2 or 3 log). In conclusion, this multiplex molecular platform can be applied to determine the presence of target species in food samples after culture enrichment. In this way, this method could be a time-saving and sensitive tool to be used in routine diagnosis.

Piglet colibacillosis diagnosis based on multiplex polymerase chain reaction and immunohistochemistry of paraffin-embedded tissues

PubMed Central

de Andrade, Caroline P.; Machado, Verônica S. L.; Bianchi, Matheus V.; Rolim, Veronica M.; Cruz, Raquel A. S.; Driemeier, David

2018-01-01

Enterotoxigenic Escherichia coli (ETEC) causes diarrhea in pigs, referred to as colibacillosis. The aim of this study was to optimize multiplex polymerase chain reaction (PCR) and immunohistochemistry (IHC) analyses of paraffin-embedded material to detect pathogenic E. coli strains causing colibacillosis in pigs. Multiplex PCR was optimized for fimbriae (F18, F4, F6, F5, and F41) and toxins (types A and B heat-stable toxins [STaP and STb], heat-labile toxin [LT], and type 2 Shiga toxin [STx2e]), and IHC was optimized for an anti-E. coli polyclonal antibody. Samples (132) from pigs received between 2006 and 2014 with clinical and histopathological diagnoses of colibacillosis were analyzed. E. coli was detected by IHC in 78.7%, and at least one virulence factor gene was detected in 71.2%. Pathogenic strains of ETEC with at least one fimbria and one toxin were detected in 40% of the samples in multiplex PCR. The most frequent virulence types were F18-STaP (7.5%), F18-STaP-STb (5.7%), and F4-STaP (3.8%). A statistically significant association was noted between virulence factors F4, F18, STaP, and STb and positive immunostaining results. Colibacillosis diagnosis through multiplex PCR and IHC of paraffin-embedded tissues is a practical approach, as samples can be fixed and stored for long periods before analysis. PMID:28693311

Detection and Typing of Human Papilloma Viruses by Nested Multiplex Polymerase Chain Reaction Assay in Cervical Cancer

PubMed Central

Jalal Kiani, Seyed; Shatizadeh Malekshahi, Somayeh; Yousefi Ghalejoogh, Zohreh; Ghavvami, Nastaran; Shafiei Jandaghi, Nazanin Zahra; Shahsiah, Reza; Jahanzad, Isa; Yavarian, Jila

2015-01-01

Background: Cervical cancer is the leading cause of death from cancer in under-developed countries. Human papilloma virus (HPV) 16 and 18 are the most prevalent types associated with carcinogenesis in the cervix. Conventional Polymerase Chain Reaction (PCR), type-specific and consensus primer-based PCR followed by sequencing, Restriction Fragment Length Polymorphism (RFLP) or hybridization by specific probes are common methods for HPV detection and typing. In addition, some researchers have developed a multiplex PCR for simultaneous detection and typing of different HPVs. Objectives: The aim of the present study was to investigate the prevalence of HPV infection and its types in cervical Squamous Cell Carcinoma (SCC) using the Nested Multiplex PCR (NMPCR) assay. Patients and Methods: Sixty-six samples with histologically confirmed SCC were evaluated. Total DNA was isolated by phenol–chloroform extraction and ethanol precipitation. Nested multiplex PCR was performed with first-round PCR by GP-E6/E7 consensus primers for amplification of the genomic DNA of all known mucosal HPV genotypes and second-round PCR by type-specific multiplex PCR primer cocktails. Results: Human papilloma virus infection was detected in 78.8% of samples, with the highest prevalence of HPV 16 (60.6%) while concurrent infections with two types was detected in 10.6%. Conclusions: The NMPCR assay is more convenient and easy for analysis of results, which is important for fast diagnosis and patient management, in a type-specific manner. PMID:26865940

Inter-laboratory evaluation of the EUROFORGEN Global ancestry-informative SNP panel by massively parallel sequencing using the Ion PGM™.

PubMed

Eduardoff, M; Gross, T E; Santos, C; de la Puente, M; Ballard, D; Strobl, C; Børsting, C; Morling, N; Fusco, L; Hussing, C; Egyed, B; Souto, L; Uacyisrael, J; Syndercombe Court, D; Carracedo, Á; Lareu, M V; Schneider, P M; Parson, W; Phillips, C; Parson, W; Phillips, C

2016-07-01

The EUROFORGEN Global ancestry-informative SNP (AIM-SNPs) panel is a forensic multiplex of 128 markers designed to differentiate an individual's ancestry from amongst the five continental population groups of Africa, Europe, East Asia, Native America, and Oceania. A custom multiplex of AmpliSeq™ PCR primers was designed for the Global AIM-SNPs to perform massively parallel sequencing using the Ion PGM™ system. This study assessed individual SNP genotyping precision using the Ion PGM™, the forensic sensitivity of the multiplex using dilution series, degraded DNA plus simple mixtures, and the ancestry differentiation power of the final panel design, which required substitution of three original ancestry-informative SNPs with alternatives. Fourteen populations that had not been previously analyzed were genotyped using the custom multiplex and these studies allowed assessment of genotyping performance by comparison of data across five laboratories. Results indicate a low level of genotyping error can still occur from sequence misalignment caused by homopolymeric tracts close to the target SNP, despite careful scrutiny of candidate SNPs at the design stage. Such sequence misalignment required the exclusion of component SNP rs2080161 from the Global AIM-SNPs panel. However, the overall genotyping precision and sensitivity of this custom multiplex indicates the Ion PGM™ assay for the Global AIM-SNPs is highly suitable for forensic ancestry analysis with massively parallel sequencing. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Synovial fluid multiplex PCR is superior to culture for detection of low-virulent pathogens causing periprosthetic joint infection.

PubMed

Morgenstern, Christian; Cabric, Sabrina; Perka, Carsten; Trampuz, Andrej; Renz, Nora

2018-02-01

Analysis of joint aspirate is the standard preoperative investigation for diagnosis of periprosthetic joint infection (PJI). We compared the diagnostic performance of culture and multiplex polymerase chain reaction (PCR) of synovial fluid for diagnosis of PJI. Patients in whom aspiration of the prosthetic hip or knee joint was performed before revision arthroplasty were prospectively included. The performance of synovial fluid culture and multiplex PCR was compared by McNemar's chi-squared test. A total of 142 patients were included, 82 with knee and 60 with hip prosthesis. PJI was diagnosed in 77 patients (54%) and aseptic failure in 65 patients (46%). The sensitivity of synovial fluid culture and PCR was 52% and 60%, respectively, showing concordant results in 116 patients (82%). In patients with PJI, PCR missed 6 high-virulent pathogens (S. aureus, streptococci, E. faecalis, E. coli) which grew in synovial fluid culture, whereas synovial fluid culture missed 12 pathogens detected by multiplex PCR, predominantly low-virulent pathogens (Cutibacterium acnes and coagulase-negative staphylococci). In patients with aseptic failure, PCR detected 6 low-virulent organisms (predominantly C. acnes). While the overall performance of synovial fluid PCR was comparable to culture, PCR was superior for detection of low-virulent bacteria such as Cutibacterium spp. and coagulase-negative staphylococci. In addition, synovial fluid culture required several days for growth, whereas multiplex PCR provided results within 5hours in an automated manner. Copyright © 2017 Elsevier Inc. All rights reserved.

Multiplex ligation-dependent probe amplification analysis on capillary electrophoresis instruments for a rapid gene copy number study.

PubMed

Jankowski, Stéphane; Currie-Fraser, Erica; Xu, Licen; Coffa, Jordy

2008-09-01

Annotated DNA samples that had been previously analyzed were tested using multiplex ligation-dependent probe amplification (MLPA) assays containing probes targeting BRCA1, BRCA2, and MMR (MLH1/MSH2 genes) and the 9p21 chromosomal region. MLPA polymerase chain reaction products were separated on a capillary electrophoresis platform, and the data were analyzed using GeneMapper v4.0 software (Applied Biosystems, Foster City, CA). After signal normalization, loci regions that had undergone deletions or duplications were identified using the GeneMapper Report Manager and verified using the DyeScale functionality. The results highlight an easy-to-use, optimal sample preparation and analysis workflow that can be used for both small- and large-scale studies.

Self-calibrating multiplexer circuit

DOEpatents

Wahl, Chris P.

1997-01-01

A time domain multiplexer system with automatic determination of acceptable multiplexer output limits, error determination, or correction is comprised of a time domain multiplexer, a computer, a constant current source capable of at least three distinct current levels, and two series resistances employed for calibration and testing. A two point linear calibration curve defining acceptable multiplexer voltage limits may be defined by the computer by determining the voltage output of the multiplexer to very accurately known input signals developed from predetermined current levels across the series resistances. Drift in the multiplexer may be detected by the computer when the output voltage limits, expected during normal operation, are exceeded, or the relationship defined by the calibration curve is invalidated.

Multiplex PCR Tests for Detection of Pathogens Associated with Gastroenteritis

PubMed Central

Zhang, Hongwei; Morrison, Scott; Tang, Yi-Wei

2016-01-01

Synopsis A wide range of enteric pathogens can cause infectious gastroenteritis. Conventional diagnostic algorithms including culture, biochemical identification, immunoassay and microscopic examination are time consuming and often lack sensitivity and specificity. Advances in molecular technology have as allowed its use as clinical diagnostic tools. Multiplex PCR based testing has made its way to gastroenterology diagnostic arena in recent years. In this article we present a review of recent laboratory developed multiplex PCR tests and current commercial multiplex gastrointestinal pathogen tests. We will focus on two FDA cleared commercial syndromic multiplex tests: Luminex xTAG GPP and Biofire FimArray GI test. These multiplex tests can detect and identify multiple enteric pathogens in one test and provide results within hours. Multiplex PCR tests have shown superior sensitivity to conventional methods for detection of most pathogens. The high negative predictive value of these multiplex tests has led to the suggestion that they be used as screening tools especially in outbreaks. Although the clinical utility and benefit of multiplex PCR test are to be further investigated, implementing these multiplex PCR tests in gastroenterology diagnostic algorithm has the potential to improve diagnosis of infectious gastroenteritis. PMID:26004652

Equivalence of time-multiplexed and frequency-multiplexed signals in digital communications.

NASA Technical Reports Server (NTRS)

Timor, U.

1972-01-01

In comparing different techniques for multiplexing N binary data signals into a single channel, time-division multiplexing (TDM) is known to have a theoretic efficiency of 100 percent (neglecting sync power) and thus seems to outperform frequency-division multiplexing systems (FDM). By considering more general FDM systems, we will show that both TDM and FDM are equivalent and have an efficiency of 100 percent. The difference between the systems is in the multiplexing and demultiplexing subsystems, but not in the performance or in the generated waveforms.

Multiple-Locus Variable-Number Tandem-Repeats Analysis of Escherichia coli O157 using PCR multiplexing and multi-colored capillary electrophoresis.

PubMed

Lindstedt, Bjørn-Arne; Vardund, Traute; Kapperud, Georg

2004-08-01

The Multiple-Locus Variable-Number Tandem-Repeats Analysis (MLVA) method is currently being used as the primary typing tool for Shiga-toxin-producing Escherichia coli (STEC) O157 isolates in our laboratory. The initial assay was performed using a single fluorescent dye and the different patterns were assigned using a gel image. Here, we present a significantly improved assay using multiple dye colors and enhanced PCR multiplexing to increase speed, and ease the interpretation of the results. The different MLVA patterns are now based on allele sizes entered as character values, thus removing the uncertainties introduced when analyzing band patterns from the gel image. We additionally propose an easy numbering scheme for the identification of separate isolates that will facilitate exchange of typing data. Seventy-two human and animal strains of Shiga-toxin-producing E. coli O157 were used for the development of the improved MLVA assay. The method is based on capillary separation of multiplexed PCR products of VNTR loci in the E. coli O157 genome labeled with multiple fluorescent dyes. The different alleles at each locus were then assigned to allele numbers, which were used for strain comparison.

[Applying healthcare failure mode and effect analysis to improve the surgical specimen transportation process and rejection rate].

PubMed

Hu, Pao-Hsueh; Hu, Hsiao-Chen; Huang, Hui-Ju; Chao, Hui-Lin; Lei, Ei-Fang

2014-04-01

Because surgical pathology specimens are crucial to the diagnosis and treatment of disease, it is critical that they be collected and transported safely and securely. Due to recent near-miss events in our department, we used the healthcare failure model and effect analysis to identify 14 potential perils in the specimen collection and transportation process. Improvement and prevention strategies were developed accordingly to improve quality of care. Using health care failure mode and effect analysis (HFMEA) may improve the surgical specimen transportation process and reduce the rate of surgical specimen rejection. Rectify standard operating procedures for surgical pathology specimen collection and transportation. Create educational videos and posters. Rectify methods of specimen verification. Organize and create an online and instantaneous management system for specimen tracking and specimen rejection. Implementation of the new surgical specimen transportation process effectively eliminated the 14 identified potential perils. In addition, the specimen rejection fell from 0.86% to 0.03%. This project was applied to improve the specimen transportation process, enhance interdisciplinary cooperation, and improve the patient-centered healthcare system. The creation and implementation of an online information system significantly facilitates specimen tracking, hospital cost reductions, and patient safety improvements. The success in our department is currently being replicated across all departments in our hospital that transport specimens. Our experience and strategy may be applied to inter-hospital specimen transportation in the future.

Multiplex APLP System for High-Resolution Haplogrouping of Extremely Degraded East-Asian Mitochondrial DNAs

PubMed Central

Kakuda, Tsuneo; Shojo, Hideki; Tanaka, Mayumi; Nambiar, Phrabhakaran; Minaguchi, Kiyoshi; Umetsu, Kazuo; Adachi, Noboru

2016-01-01

Mitochondrial DNA (mtDNA) serves as a powerful tool for exploring matrilineal phylogeographic ancestry, as well as for analyzing highly degraded samples, because of its polymorphic nature and high copy numbers per cell. The recent advent of complete mitochondrial genome sequencing has led to improved techniques for phylogenetic analyses based on mtDNA, and many multiplex genotyping methods have been developed for the hierarchical analysis of phylogenetically important mutations. However, few high-resolution multiplex genotyping systems for analyzing East-Asian mtDNA can be applied to extremely degraded samples. Here, we present a multiplex system for analyzing mitochondrial single nucleotide polymorphisms (mtSNPs), which relies on a novel amplified product-length polymorphisms (APLP) method that uses inosine-flapped primers and is specifically designed for the detailed haplogrouping of extremely degraded East-Asian mtDNAs. We used fourteen 6-plex polymerase chain reactions (PCRs) and subsequent electrophoresis to examine 81 haplogroup-defining SNPs and 3 insertion/deletion sites, and we were able to securely assign the studied mtDNAs to relevant haplogroups. Our system requires only 1×10−13 g (100 fg) of crude DNA to obtain a full profile. Owing to its small amplicon size (<110 bp), this new APLP system was successfully applied to extremely degraded samples for which direct sequencing of hypervariable segments using mini-primer sets was unsuccessful, and proved to be more robust than conventional APLP analysis. Thus, our new APLP system is effective for retrieving reliable data from extremely degraded East-Asian mtDNAs. PMID:27355212

Charging YOYO-1 on Capillary Wall for Online DNA Intercalation and Integrating This Approach with Multiplex PCR and Bare Narrow Capillary–Hydrodynamic Chromatography for Online DNA Analysis

PubMed Central

2016-01-01

Multiplex polymerase chain reaction (PCR) has been widely utilized for high-throughput pathogen identification. Often, a dye is used to intercalate the amplified DNA fragments, and identifications of the pathogens are carried out by DNA melting curve analysis or gel electrophoresis. Integrating DNA amplification and identification is a logic path toward maximizing the benefit of multiplex PCR. Although PCR and gel electrophoresis have been integrated, replenishing the gels after each run is tedious and time-consuming. In this technical note, we develop an approach to address this issue. We perform multiplex PCR inside a capillary, transfer the amplified fragments to a bare narrow capillary, and measure their lengths online using bare narrow capillary–hydrodynamic chromatography (BaNC-HDC), a new technique recently developed in our laboratory for free-solution DNA separation. To intercalate the DNA with YOYO-1 (a fluorescent dye) for BaNC-HDC, we flush the capillary column with a YOYO-1 solution; positively charged YOYO-1 is adsorbed (or charged) onto the negatively charged capillary wall. As DNA molecules are driven down the column for separation, they react with the YOYO-1 stored on the capillary wall and are online-intercalated with the dye. With a single YOYO-1 charging, the column can be used for more than 40 runs, although the fluorescence signal intensities of the DNA peaks decrease gradually. Although the dye-DNA intercalation occurs during the separation, it does not affect the retention times, separation efficiencies, or resolutions. PMID:25555111

Multiplex APLP System for High-Resolution Haplogrouping of Extremely Degraded East-Asian Mitochondrial DNAs.

PubMed

Kakuda, Tsuneo; Shojo, Hideki; Tanaka, Mayumi; Nambiar, Phrabhakaran; Minaguchi, Kiyoshi; Umetsu, Kazuo; Adachi, Noboru

2016-01-01

Mitochondrial DNA (mtDNA) serves as a powerful tool for exploring matrilineal phylogeographic ancestry, as well as for analyzing highly degraded samples, because of its polymorphic nature and high copy numbers per cell. The recent advent of complete mitochondrial genome sequencing has led to improved techniques for phylogenetic analyses based on mtDNA, and many multiplex genotyping methods have been developed for the hierarchical analysis of phylogenetically important mutations. However, few high-resolution multiplex genotyping systems for analyzing East-Asian mtDNA can be applied to extremely degraded samples. Here, we present a multiplex system for analyzing mitochondrial single nucleotide polymorphisms (mtSNPs), which relies on a novel amplified product-length polymorphisms (APLP) method that uses inosine-flapped primers and is specifically designed for the detailed haplogrouping of extremely degraded East-Asian mtDNAs. We used fourteen 6-plex polymerase chain reactions (PCRs) and subsequent electrophoresis to examine 81 haplogroup-defining SNPs and 3 insertion/deletion sites, and we were able to securely assign the studied mtDNAs to relevant haplogroups. Our system requires only 1×10-13 g (100 fg) of crude DNA to obtain a full profile. Owing to its small amplicon size (<110 bp), this new APLP system was successfully applied to extremely degraded samples for which direct sequencing of hypervariable segments using mini-primer sets was unsuccessful, and proved to be more robust than conventional APLP analysis. Thus, our new APLP system is effective for retrieving reliable data from extremely degraded East-Asian mtDNAs.

TES Detector Noise Limited Readout Using SQUID Multiplexers

NASA Technical Reports Server (NTRS)

Staguhn, J. G.; Benford, D. J.; Chervenak, J. A.; Khan, S. A.; Moseley, S. H.; Shafer, R. A.; Deiker, S.; Grossman, E. N.; Hilton, G. C.; Irwin, K. D.

2004-01-01

The availability of superconducting Transition Edge Sensors (TES) with large numbers of individual detector pixels requires multiplexers for efficient readout. The use of multiplexers reduces the number of wires needed between the cryogenic electronics and the room temperature electronics and cuts the number of required cryogenic amplifiers. We are using an 8 channel SQUID multiplexer to read out one-dimensional TES arrays which are used for submillimeter astronomical observations. We present results from test measurements which show that the low noise level of the SQUID multiplexers allows accurate measurements of the TES Johnson noise, and that in operation, the readout noise is dominated by the detector noise. Multiplexers for large number of channels require a large bandwidth for the multiplexed readout signal. We discuss the resulting implications for the noise performance of these multiplexers which will be used for the readout of two dimensional TES arrays in next generation instruments.

Extracting information from multiplex networks

NASA Astrophysics Data System (ADS)

Iacovacci, Jacopo; Bianconi, Ginestra

2016-06-01

Multiplex networks are generalized network structures that are able to describe networks in which the same set of nodes are connected by links that have different connotations. Multiplex networks are ubiquitous since they describe social, financial, engineering, and biological networks as well. Extending our ability to analyze complex networks to multiplex network structures increases greatly the level of information that is possible to extract from big data. For these reasons, characterizing the centrality of nodes in multiplex networks and finding new ways to solve challenging inference problems defined on multiplex networks are fundamental questions of network science. In this paper, we discuss the relevance of the Multiplex PageRank algorithm for measuring the centrality of nodes in multilayer networks and we characterize the utility of the recently introduced indicator function Θ ˜ S for describing their mesoscale organization and community structure. As working examples for studying these measures, we consider three multiplex network datasets coming for social science.

Three-mode all-optical (de)multiplexing on a SOI chip

NASA Astrophysics Data System (ADS)

Le, Yan-Si; Wang, Zhi; Li, Zhi-Yong; Li, Ying; Li, Qiang; Cui, Can; Wu, Chong-Qing

2018-01-01

An on-chip three-mode division multiplexing circuit using a simple ADC-based TE0 & TE1 & TE2 (de)multiplexer is demonstrated to improve the link capacity of on-chip optical interconnects. The proposed (de)multiplexer does not contain any tapered waveguide which is different from the previous mode (de)multiplexer based on ADCs. Here, we choose multimode waveguide width first and then confirm corresponding width of the other two waveguides. Thus the bus waveguide without any tapers can not only reduce complexity of (de)multiplexer but also reduce difficulty of the fabrication. Our simulation results show that the hybrid multiplexer has relatively low loss and low crosstalk about -40 dB, -26.99 dB and -28.72 dB for each mode around 1550 nm with a width-variation w =± 25 nm. These properties make the proposed mode-(de)multiplexer suitable for application in high-capacity data transmission.

High-performance single cell genetic analysis using microfluidic emulsion generator arrays.

PubMed

Zeng, Yong; Novak, Richard; Shuga, Joe; Smith, Martyn T; Mathies, Richard A

2010-04-15

High-throughput genetic and phenotypic analysis at the single cell level is critical to advance our understanding of the molecular mechanisms underlying cellular function and dysfunction. Here we describe a high-performance single cell genetic analysis (SCGA) technique that combines high-throughput microfluidic emulsion generation with single cell multiplex polymerase chain reaction (PCR). Microfabricated emulsion generator array (MEGA) devices containing 4, 32, and 96 channels are developed to confer a flexible capability of generating up to 3.4 x 10(6) nanoliter-volume droplets per hour. Hybrid glass-polydimethylsiloxane diaphragm micropumps integrated into the MEGA chips afford uniform droplet formation, controlled generation frequency, and effective transportation and encapsulation of primer functionalized microbeads and cells. A multiplex single cell PCR method is developed to detect and quantify both wild type and mutant/pathogenic cells. In this method, microbeads functionalized with multiple forward primers targeting specific genes from different cell types are used for solid-phase PCR in droplets. Following PCR, the droplets are lysed and the beads are pooled and rapidly analyzed by multicolor flow cytometry. Using Escherichia coli bacterial cells as a model, we show that this technique enables digital detection of pathogenic E. coli O157 cells in a high background of normal K12 cells, with a detection limit on the order of 1/10(5). This result demonstrates that multiplex SCGA is a promising tool for high-throughput quantitative digital analysis of genetic variation in complex populations.

High-Performance Single Cell Genetic Analysis Using Microfluidic Emulsion Generator Arrays

PubMed Central

Zeng, Yong; Novak, Richard; Shuga, Joe; Smith, Martyn T.; Mathies, Richard A.

2010-01-01

High-throughput genetic and phenotypic analysis at the single cell level is critical to advance our understanding of the molecular mechanisms underlying cellular function and dysfunction. Here we describe a high-performance single cell genetic analysis (SCGA) technique that combines high-throughput microfluidic emulsion generation with single cell multiplex PCR. Microfabricated emulsion generator array (MEGA) devices containing 4, 32 and 96 channels are developed to confer a flexible capability of generating up to 3.4 × 106 nanoliter-volume droplets per hour. Hybrid glass-polydimethylsiloxane diaphragm micropumps integrated into the MEGA chips afford uniform droplet formation, controlled generation frequency, and effective transportation and encapsulation of primer functionalized microbeads and cells. A multiplex single cell PCR method is developed to detect and quantify both wild type and mutant/pathogenic cells. In this method, microbeads functionalized with multiple forward primers targeting specific genes from different cell types are used for solid-phase PCR in droplets. Following PCR, the droplets are lysed, the beads are pooled and rapidly analyzed by multi-color flow cytometry. Using E. coli bacterial cells as a model, we show that this technique enables digital detection of pathogenic E. coli O157 cells in a high background of normal K12 cells, with a detection limit on the order of 1:105. This result demonstrates that multiplex SCGA is a promising tool for high-throughput quantitative digital analysis of genetic variation in complex populations. PMID:20192178

Point-Counterpoint: A Nucleic Acid Amplification Test for Streptococcus pyogenes Should Replace Antigen Detection and Culture for Detection of Bacterial Pharyngitis.

PubMed

Pritt, Bobbi S; Patel, Robin; Kirn, Thomas J; Thomson, Richard B

2016-10-01

Nucleic acid amplification tests (NAATs) have frequently been the standard diagnostic approach when specific infectious agents are sought in a clinic specimen. They can be applied for specific agents such as S. pyogenes, or commercial multiplex NAATs for detection of a variety of pathogens in gastrointestinal, bloodstream, and respiratory infections may be used. NAATs are both rapid and sensitive. For many years, S. pyogenes testing algorithms used a rapid and specific group A streptococcal antigen test to screen throat specimens, followed, in some clinical settings, by a throat culture for S. pyogenes to increase the sensitivity of its detection. Now S. pyogenes NAATs are being used with increasing frequency. Given their accuracy, rapidity, and ease of use, should they replace antigen detection and culture for the detection of bacterial pharyngitis? Bobbi Pritt and Robin Patel of the Mayo Clinic, where S. pyogenes NAATs have been used for well over a decade with great success, will explain the advantages of this approach, while Richard (Tom) Thomson and Tom Kirn of the NorthShore University HealthSystem will discuss their concerns about this approach to diagnosing bacterial pharyngitis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

46 CFR 4.06-50 - Specimen analysis and follow-up procedures.

Code of Federal Regulations, 2011 CFR

2011-10-01

... to develop all relevant information and to produce a complete analysis report. (b) Reports shall be... 46 Shipping 1 2011-10-01 2011-10-01 false Specimen analysis and follow-up procedures. 4.06-50... Involving Vessels in Commercial Service § 4.06-50 Specimen analysis and follow-up procedures. (a) Each...

46 CFR 4.06-50 - Specimen analysis and follow-up procedures.

Code of Federal Regulations, 2012 CFR

2012-10-01

... to develop all relevant information and to produce a complete analysis report. (b) Reports shall be... 46 Shipping 1 2012-10-01 2012-10-01 false Specimen analysis and follow-up procedures. 4.06-50... Involving Vessels in Commercial Service § 4.06-50 Specimen analysis and follow-up procedures. (a) Each...

Quantitative, multiplexed workflow for deep analysis of human blood plasma and biomarker discovery by mass spectrometry.

PubMed

Keshishian, Hasmik; Burgess, Michael W; Specht, Harrison; Wallace, Luke; Clauser, Karl R; Gillette, Michael A; Carr, Steven A

2017-08-01

Proteomic characterization of blood plasma is of central importance to clinical proteomics and particularly to biomarker discovery studies. The vast dynamic range and high complexity of the plasma proteome have, however, proven to be serious challenges and have often led to unacceptable tradeoffs between depth of coverage and sample throughput. We present an optimized sample-processing pipeline for analysis of the human plasma proteome that provides greatly increased depth of detection, improved quantitative precision and much higher sample analysis throughput as compared with prior methods. The process includes abundant protein depletion, isobaric labeling at the peptide level for multiplexed relative quantification and ultra-high-performance liquid chromatography coupled to accurate-mass, high-resolution tandem mass spectrometry analysis of peptides fractionated off-line by basic pH reversed-phase (bRP) chromatography. The overall reproducibility of the process, including immunoaffinity depletion, is high, with a process replicate coefficient of variation (CV) of <12%. Using isobaric tags for relative and absolute quantitation (iTRAQ) 4-plex, >4,500 proteins are detected and quantified per patient sample on average, with two or more peptides per protein and starting from as little as 200 μl of plasma. The approach can be multiplexed up to 10-plex using tandem mass tags (TMT) reagents, further increasing throughput, albeit with some decrease in the number of proteins quantified. In addition, we provide a rapid protocol for analysis of nonfractionated depleted plasma samples analyzed in 10-plex. This provides ∼600 quantified proteins for each of the ten samples in ∼5 h of instrument time.