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Sample records for multiplex pcr-based reverse

  1. Detection of gastroenteritis viruses among pediatric patients in Hiroshima Prefecture, Japan, between 2006 and 2013 using multiplex reverse transcription PCR-based assays involving fluorescent dye-labeled primers.

    PubMed

    Shigemoto, Naoki; Hisatsune, Yuri; Toukubo, Yasushi; Tanizawa, Yukie; Shimazu, Yukie; Takao, Shinichi; Tanaka, Tomoyuki; Noda, Mamoru; Fukuda, Shinji

    2017-05-01

    Multiplex reverse transcription (RT)-polymerase chain reaction (PCR)-based assays involving fluorescent dye-labeled primers were modified to detect 10 types of gastroenteritis viruses by adding two further assays to a previously developed assay. Then, these assays were applied to clinical samples, which were collected between January 2006 and December 2013. All 10 types of viruses were effectively detected in the multiplex RT-PCR-based assays. In addition, various viral parameters, such as the detection rates and age distributions of each viral type, were examined. The frequency and types of mixed infections were also investigated. Among the 186 virus-positive samples, genogroup II noroviruses were found to be the most common type of virus (32.7%), followed by group A rotaviruses (10.6%) and parechoviruses (10.3%). Mixed infections were observed in 37 samples, and many of them were detected in patients who were less than 2 years old. These observations showed that the multiplex RT-PCR-based assays involving fluorescent dye-labeled primers were able to effectively detect the viruses circulating among pediatric acute gastroenteritis patients and contributed to the highly specific and sensitive diagnosis of gastroenteritis. J. Med. Virol. 89:791-800, 2017. © 2016 Wiley Periodicals, Inc.

  2. Genetic characterisation of invasive breast cancer: a comparison of CGH and PCR based multiplex microsatellite analysis.

    PubMed

    Buerger, H; Schmidt, H; Beckmann, A; Zänker, K S; Boecker, W; Brandt, B

    2001-11-01

    Comparative genomic hybridisation (CGH) is a reliable tool to gain an overview of all unbalanced chromosomal alterations within a tumour. Nevertheless, the high numbers of tumour cells required and the comparatively low resolution are drawbacks of this technique. Polymerase chain reaction (PCR) based multiplex microsatellite analysis represents a semi-automated, highly reproducible method, which requires small amounts of tumour cells. This is a comparative study of CGH and microsatellite analysis. Eighty one samples of invasive breast cancer were investigated by two sensitive multiplex PCRs containing three microsatellites each of six markers (D6S261, D11S907, D6S300, D11S927, D8S272, and D11S925), and two additional microsatellite markers located within intron 1 of the epidermal growth factor receptor gene (egfr) and p53 (p53CA). At least one example of loss of heterozygosity was detectable in all breast cancer tissues. However, the overall rate of accordance between the two methods tested was only 61%. An increasing rate of the number of genetic alterations in each case was mirrored by a constantly increasing fractional allelic loss index. PCR based multiplex microsatellite analysis using this panel of eight microsatellite markers not only enables the characterisation of cells that have malignant potential in a high frequency of patients with breast cancer, but can also give an estimate of the degree of genetic progression.

  3. Multiplex PCR based on a universal biotinylated primer to generate templates for pyrosequencing.

    PubMed

    Chen, Zhiyao; Liu, Yunlong; Duan, Wenbang; Ye, Hui; Wu, Haiping; Li, Jinheng; Zhou, Guohua

    2014-06-01

    Pyrosequencing is a powerful tool widely used in genetic analysis, however template preparation prior to pyrosequencing is still costly and time-consuming. To achieve an inexpensive and labor-saving template preparation for pyrosequencing, we have successfully developed a single-tube multiplex PCR including a pre-amplification and a universal amplification. In the process of pre-amplification, a low concentration of target-specific primers tagged with universal ends introduced universal priming regions into amplicons. In the process of universal amplification, a high concentration of universal primers was used for yielding amplicons with various SNPs of interest. As only a universal biotinylated primer and one step of single-stranded DNA preparation were required for typing multiple SNPs located on different sequences, pyrosequencing-based genotyping became time-saving, labor-saving, sample-saving, and cost-saving. By a simple optimization of multiplex PCR condition, only a 4-plex and a 3-plex PCR were required for typing 7 SNPs related to tamoxifen metabolism. Further study showed that pyrosequencing coupled with an improved multiplex PCR protocol allowed around 30% decrease of either typing cost or typing labor. Considering the biotinylated primer and the optimized condition of the multiplex PCR are independent of SNP locus, it is easy to use the same condition and the identical biotinylated primer for typing other SNPs. The preliminary typing results of the 7 SNPs in 11 samples demonstrated that multiplex PCR-based pyrosequencing could be promising in personalized medicine at a low cost.

  4. Genomic Characterization of Flavobacterium psychrophilum Serotypes and Development of a Multiplex PCR-Based Serotyping Scheme.

    PubMed

    Rochat, Tatiana; Fujiwara-Nagata, Erina; Calvez, Ségolène; Dalsgaard, Inger; Madsen, Lone; Calteau, Alexandra; Lunazzi, Aurélie; Nicolas, Pierre; Wiklund, Tom; Bernardet, Jean-François; Duchaud, Eric

    2017-01-01

    Flavobacterium psychrophilum is a devastating bacterial pathogen of salmonids reared in freshwater worldwide. So far, serological diversity between isolates has been described but the underlying molecular factors remain unknown. By combining complete genome sequence analysis and the serotyping method proposed by Lorenzen and Olesen (1997) for a set of 34 strains, we identified key molecular determinants of the serotypes. This knowledge allowed us to develop a robust multiplex PCR-based serotyping scheme, which was applied to 244 bacterial isolates. The results revealed a striking association between PCR-serotype and fish host species and illustrate the use of this approach as a simple and cost-effective method for the determination of F. psychrophilum serogroups. PCR-based serotyping could be a useful tool in a range of applications such as disease surveillance, selection of salmonids for bacterial coldwater disease resistance and future vaccine formulation.

  5. Genetic characterisation of invasive breast cancer: a comparison of CGH and PCR based multiplex microsatellite analysis

    PubMed Central

    Buerger, H; Schmidt, H; Beckmann, A; Zanker, K; Boecker, W; Brandt, B

    2001-01-01

    Aims—Comparative genomic hybridisation (CGH) is a reliable tool to gain an overview of all unbalanced chromosomal alterations within a tumour. Nevertheless, the high numbers of tumour cells required and the comparatively low resolution are drawbacks of this technique. Polymerase chain reaction (PCR) based multiplex microsatellite analysis represents a semi-automated, highly reproducible method, which requires small amounts of tumour cells. This is a comparative study of CGH and microsatellite analysis. Methods—Eighty one samples of invasive breast cancer were investigated by two sensitive multiplex PCRs containing three microsatellites each of six markers (D6S261, D11S907, D6S300, D11S927, D8S272, and D11S925), and two additional microsatellite markers located within intron 1 of the epidermal growth factor recepter gene (egfr) and p53 (p53CA). Results—At least one example of loss of heterozygosity was detectable in all breast cancer tissues. However, the overall rate of accordance between the two methods tested was only 61%. An increasing rate of the number of genetic alterations in each case was mirrored by a constantly increasing fractional allelic loss index. Conclusions—PCR based multiplex microsatellite analysis using this panel of eight microsatellite markers not only enables the characterisation of cells that have malignant potential in a high frequency of patients with breast cancer, but can also give an estimate of the degree of genetic progression. Key Words: breast cancer • comparative genomic hybridisation • microsatellites • epidermal growth factor • p53 PMID:11684716

  6. False positives in multiplex PCR-based next-generation sequencing have unique signatures.

    PubMed

    McCall, Chad M; Mosier, Stacy; Thiess, Michele; Debeljak, Marija; Pallavajjala, Aparna; Beierl, Katie; Deak, Kristen L; Datto, Michael B; Gocke, Christopher D; Lin, Ming-Tseh; Eshleman, James R

    2014-09-01

    Next-generation sequencing shows great promise by allowing rapid mutational analysis of multiple genes in human cancers. Recently, we implemented the multiplex PCR-based Ion AmpliSeq Cancer Hotspot Panel (>200 amplicons in 50 genes) to evaluate EGFR, KRAS, and BRAF in lung and colorectal adenocarcinomas. In 10% of samples, automated analysis identified a novel G873R substitution mutation in EGFR. By examining reads individually, we found this mutation in >5% of reads in 50 of 291 samples and also found similar events in 18 additional amplicons. These apparent mutations are present only in short reads and within 10 bases of either end of the read. We therefore hypothesized that these were from panel primers promiscuously binding to nearly complementary sequences of nontargeted amplicons. Sequences around the mutations matched primer binding sites in the panel in 18 of 19 cases, thus likely corresponding to panel primers. Furthermore, because most primers did not show this effect, we demonstrated that next-generation sequencing may be used to better design multiplex PCR primers through iterative elimination of offending primers to minimize mispriming. Our results indicate the need for careful sequence analysis to avoid false-positive mutations that can arise in multiplex PCR panels. The AmpliSeq Cancer panel is a valuable tool for clinical diagnostics, provided awareness of potential artifacts.

  7. Phylogeographic analysis of hemorrhagic fever with renal syndrome patients using multiplex PCR-based next generation sequencing

    PubMed Central

    Kim, Won-Keun; Kim, Jeong-Ah; Song, Dong Hyun; Lee, Daesang; Kim, Yong Chul; Lee, Sook-Young; Lee, Seung-Ho; No, Jin Sun; Kim, Ji Hye; Kho, Jeong Hoon; Gu, Se Hun; Jeong, Seong Tae; Wiley, Michael; Kim, Heung-Chul; Klein, Terry A.; Palacios, Gustavo; Song, Jin-Won

    2016-01-01

    Emerging and re-emerging infectious diseases caused by RNA viruses pose a critical public health threat. Next generation sequencing (NGS) is a powerful technology to define genomic sequences of the viruses. Of particular interest is the use of whole genome sequencing (WGS) to perform phylogeographic analysis, that allows the detection and tracking of the emergence of viral infections. Hantaviruses, Bunyaviridae, cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) in humans. We propose to use WGS for the phylogeographic analysis of human hantavirus infections. A novel multiplex PCR-based NGS was developed to gather whole genome sequences of Hantaan virus (HTNV) from HFRS patients and rodent hosts in endemic areas. The obtained genomes were described for the spatial and temporal links between cases and their sources. Phylogenetic analyses demonstrated geographic clustering of HTNV strains from clinical specimens with the HTNV strains circulating in rodents, suggesting the most likely site and time of infection. Recombination analysis demonstrated a genome organization compatible with recombination of the HTNV S segment. The multiplex PCR-based NGS is useful and robust to acquire viral genomic sequences and may provide important ways to define the phylogeographical association and molecular evolution of hantaviruses. PMID:27221218

  8. Multiplex PCR-based identification of Streptococcus canis, Streptococcus zooepidemicus and Streptococcus dysgalactiae subspecies from dogs.

    PubMed

    Moriconi, M; Acke, E; Petrelli, D; Preziuso, S

    2017-02-01

    Streptococcus canis (S. canis), Streptococcus equi subspecies zooepidemicus (S. zooepidemicus) and Streptococcus dysgalactiae subspecies (S. dysgalactiae subspecies) are β-haemolytic Gram positive bacteria infecting animals and humans. S. canis and S. zooepidemicus are considered as two of the major zoonotic species of Streptococcus, while more research is needed on S. dysgalactiae subspecies bacteria. In this work, a multiplex-PCR protocol was tested on strains and clinical samples to detect S. canis, S. dysgalactiae subspecies and S. equi subspecies bacteria in dogs. All strains were correctly identified as S. canis, S. equi subspecies or S. dysgalactiae subspecies by the multiplex-PCR. The main Streptococcus species isolated from symptomatic dogs were confirmed S. canis. The multiplex-PCR protocol described is a rapid, accurate and efficient method for identifying S. canis, S. equi subspecies and S. dysgalactiae subspecies in dogs and could be used for diagnostic purposes and for epidemiological studies.

  9. A Colony Multiplex Quantitative PCR-Based 3S3DBC Method and Variations of It for Screening DNA Libraries

    PubMed Central

    An, Yang; Toyoda, Atsushi; Zhao, Chen; Fujiyama, Asao; Agata, Kiyokazu

    2015-01-01

    A DNA library is a collection of DNA fragments cloned into vectors and stored individually in host cells, and is a valuable resource for molecular cloning, gene physical mapping, and genome sequencing projects. To take the best advantage of a DNA library, a good screening method is needed. After describing pooling strategies and issues that should be considered in DNA library screening, here we report an efficient colony multiplex quantitative PCR-based 3-step, 3-dimension, and binary-code (3S3DBC) method we used to screen genes from a planarian genomic DNA fosmid library. This method requires only 3 rounds of PCR reactions and only around 6 hours to distinguish one or more desired clones from a large DNA library. According to the particular situations in different research labs, this method can be further modified and simplified to suit their requirements. PMID:25646755

  10. Development of a PCR-Based Reverse Genetics System for an Attenuated Duck Tembusu Virus Strain

    PubMed Central

    Wu, Xiaogang; Shi, Ying; Yan, Dawei; Li, Xuesong; Yan, Pixi; Gao, Xuyuan; Zhang, Yuee; Yu, Lei; Ren, Chaochao; Li, Guoxin; Yan, Liping; Teng, Qiaoyang; Li, Zejun

    2016-01-01

    The infectious disease caused by the duck Tembusu virus (DTMUV) has resulted in massive economic losses to the Chinese duck industry in China since 2010. Research on the molecular basis of DTMUV pathogenicity has been hampered by the lack of a reliable reverse genetics system for this virus. Here we developed a PCR-based reverse genetics system with high fidelity for the attenuated DTMUV strain FX2010-180P. The rescued virus was characterized by using both indirect immunofluorescence assays (IFA) and whole genome sequencing. The rescued virus (rFX2010-180P) grew to similar titers as compared with the wild-type virus in DF-1 cells, and had similar replication and immunogenicity properties in ducks. To determine whether exogenous proteins could be expressed from DTMUV, both an internal ribosomal entry site (IRES) and the enhanced green fluorescent protein (eGFP) gene were introduced between the NS5 gene and the 3' non-coding sequence of FX2010-180P. A recombinant DTMUV expressing eGFP was rescued, but eGFP expression was unstable after 4 passages in DF-1 cells due to a deletion of 1,294 nucleotides. The establishment of a reliable reverse genetics system for FX2010-180P provides a foundation for future studies of DTMUV. PMID:27248497

  11. Rapid diagnosis of Argentine hemorrhagic fever by reverse transcriptase PCR-based assay.

    PubMed Central

    Lozano, M E; Enría, D; Maiztegui, J I; Grau, O; Romanowski, V

    1995-01-01

    Argentine hemorrhagic fever (AHF) is an endemo-epidemic disease caused by Junín virus. This report demonstrates that a reverse transcriptase (RT) PCR-based assay developed in our laboratory to detect Junín virus in whole blood samples is sensitive and specific. The experiments were conducted in a double-blinded manner using 94 clinical samples collected in the area in which AHF is endemic. The RT-PCR-based assay was compared with traditional methodologies, including enzyme-linked immunosorbent assay, plaque neutralization tests, and occasionally viral isolation. The calculated parameters for RT-PCR diagnosis, with seroconversion as the "gold standard," were 98% sensitivity and 76% specificity. It is noteworthy that 94% of the patients with putative false-positive results (RT-PCR positive and no seroconversion detected) exhibited febrile syndromes of undefined etiology. These results could be interpreted to mean that most of those patients with febrile syndromes were actually infected with Junín virus but did not develop a detectable immune response. Furthermore, 8 laboratory-fabricated samples and 25 blood samples of patients outside the area in which AHF is endemic tested in a similar way were disclosed correctly (100% match). The RT-PCR assay is the only laboratory test available currently for the early and rapid diagnosis of AHF. It is sensitive enough to detect the low viremia found during the period in which immune plasma therapy can be used effectively, reducing mortality rates from 30% to less than 1%. PMID:7542268

  12. Organic Substances Interfere with Reverse Transcription-Quantitative PCR-Based Virus Detection in Water Samples

    PubMed Central

    Katayama, Hiroyuki; Furumai, Hiroaki

    2014-01-01

    Reverse transcription (RT)-PCR-based virus detection from water samples is occasionally hampered by organic substances that are coconcentrated during virus concentration procedures. To characterize these organic substances, samples containing commercially available humic acid, which is known to inhibit RT-PCR, and river water samples were subjected to adsorption-elution-based virus concentration using an electronegative membrane. In this study, the samples before, during, and after the concentration were analyzed in terms of organic properties and virus detection efficiencies. Two out of the three humic acid solutions resulted in RT-quantitative PCR (qPCR) inhibition that caused >3-log10-unit underestimation of spiked poliovirus. Over 60% of the organics contained in the two solutions were recovered in the concentrate, while over 60% of the organics in the uninhibited solution were lost during the concentration process. River water concentrates also caused inhibition of RT-qPCR. Organic concentrations in the river water samples increased by 2.3 to 3.9 times after the virus concentration procedure. The inhibitory samples contained organic fractions in the 10- to 100-kDa size range, which are suspected to be RT-PCR inhibitors. According to excitation-emission matrices, humic acid-like and protein-like fractions were also recovered from river water concentrates, but these fractions did not seem to affect virus detection. Our findings reveal that detailed organic analyses are effective in characterizing inhibitory substances. PMID:25527552

  13. Detection of 22 common leukemic fusion genes using a single-step multiplex qRT-PCR-based assay.

    PubMed

    Lyu, Xiaodong; Wang, Xianwei; Zhang, Lina; Chen, Zhenzhu; Zhao, Yu; Hu, Jieying; Fan, Ruihua; Song, Yongping

    2017-07-25

    Fusion genes generated from chromosomal translocation play an important role in hematological malignancies. Detection of fusion genes currently employ use of either conventional RT-PCR methods or fluorescent in situ hybridization (FISH), where both methods involve tedious methodologies and require prior characterization of chromosomal translocation events as determined by cytogenetic analysis. In this study, we describe a real-time quantitative reverse transcription PCR (qRT-PCR)-based multi-fusion gene screening method with the capacity to detect 22 fusion genes commonly found in leukemia. This method does not require pre-characterization of gene translocation events, thereby facilitating immediate diagnosis and therapeutic management. We performed fluorescent qRT-PCR (F-qRT-PCR) using a commercially-available multi-fusion gene detection kit on a patient cohort of 345 individuals comprising 108 cases diagnosed with acute myeloid leukemia (AML) for initial evaluation; remaining patients within the cohort were assayed for confirmatory diagnosis. Results obtained by F-qRT-PCR were compared alongside patient analysis by cytogenetic characterization. Gene translocations detected by F-qRT-PCR in AML cases were diagnosed in 69.4% of the patient cohort, which was comparatively similar to 68.5% as diagnosed by cytogenetic analysis, thereby demonstrating 99.1% concordance. Overall gene fusion was detected in 53.7% of the overall patient population by F-qRT-PCR, 52.9% by cytogenetic prediction in leukemia, and 9.1% in non-leukemia patients by both methods. The overall concordance rate was calculated to be 99.0%. Fusion genes were detected by F-qRT-PCR in 97.3% of patients with CML, followed by 69.4% with AML, 33.3% with acute lymphoblastic leukemia (ALL), 9.1% with myelodysplastic syndromes (MDS), and 0% with chronic lymphocytic leukemia (CLL). We describe the use of a F-qRT-PCR-based multi-fusion gene screening method as an efficient one-step diagnostic procedure as an

  14. Reversible chemical reactions for single-color multiplexing microscopy.

    PubMed

    Brox, Dominik; Schwering, Michael; Engelhardt, Johann; Herten, Dirk-Peter

    2014-08-04

    Recent developments in biology demand an increasing number of simultaneously imaged structures with standard fluorescence microscopy. However, the number of multiplexed channels is limited for most multiplexing modalities, such as spectral multiplexing or fluorescence-lifetime imaging. We propose extending the number of imaging channels by using chemical reactions, controlling the emissive state of fluorescent dyes. As proof of concept, we reversibly switch a fluorescent copper sensor to enable successive imaging of two different structures in the same spectral channel. We also show that this chemical multiplexing is orthogonal to existing methods. By using two different dyes, we combine chemical with spectral multiplexing for the simultaneous imaging of four different structures with only two spectrally different channels. We characterize and discuss the approach and provide perspectives for extending imaging modalities in stimulated emission depletion microscopy, for which spectral multiplexing is technically demanding.

  15. An Efficient Multiplex PCR-Based Assay as a Novel Tool for Accurate Inter-Serovar Discrimination of Salmonella Enteritidis, S. Pullorum/Gallinarum and S. Dublin

    PubMed Central

    Xiong, Dan; Song, Li; Tao, Jing; Zheng, Huijuan; Zhou, Zihao; Geng, Shizhong; Pan, Zhiming; Jiao, Xinan

    2017-01-01

    Salmonella enterica serovars Enteritidis, Pullorum/Gallinarum, and Dublin are infectious pathogens causing serious problems for pig, chicken, and cattle production, respectively. Traditional serotyping for Salmonella is costly and labor-intensive. Here, we established a rapid multiplex PCR method to simultaneously identify three prevalent Salmonella serovars Enteritidis, Pullorum/Gallinarum, and Dublin individually for the first time. The multiplex PCR-based assay focuses on three genes tcpS, lygD, and flhB. Gene tcpS exists only in the three Salmonella serovars, and lygD exists only in S. Enteritidis, while a truncated region of flhB gene is only found in S. Pullorum/Gallinarum. The sensitivity and specificity of the multiplex PCR assay using three pairs of specific primers for these genes were evaluated. The results showed that this multiplex PCR method could accurately identify Salmonella Enteritidis, Pullorum/Gallinarum, and Dublin from eight non-Salmonella species and 27 Salmonella serovars. The least concentration of genomic DNA that could be detected was 58.5 pg/μL and the least number of cells was 100 CFU. Subsequently, this developed method was used to analyze clinical Salmonella isolates from one pig farm, one chicken farm, and one cattle farm. The results showed that blinded PCR testing of Salmonella isolates from the three farms were in concordance with the traditional serotyping tests, indicating the newly developed multiplex PCR system could be used as a novel tool to accurately distinguish the three specific Salmonella serovars individually, which is useful, especially in high-throughput screening. PMID:28360901

  16. A qPCR-based Multiplex Assay for Detection of Wuchereria bancrofti, Plasmodium falciparum, and Plasmodium vivax DNA

    PubMed Central

    Rao, Ramakrishna U.; Huang, Yuefang; Bockarie, Moses J.; Susapu, Melinda; Laney, Sandra J.; Weil, Gary J.

    2009-01-01

    Summary The purpose of this study was to develop multiplex qPCR assays for simultaneous detection of Wuchereria bancrofti (Wb), Plasmodium falciparum (Pf) and P. vivax (Pv) in mosquitoes. We optimized the assays with purified DNA samples and then used these assays to test DNA samples isolated from Anopheles punctulatus mosquitoes collected in villages in Papua New Guinea where these infections are co-endemic. Singleplex assays detected Wb, Pf, and Pv DNA in 32%, 19% and 15% of the mosquito pools, respectively, either alone or together with other parasites. Multiplex assay results agreed with singleplex results in most cases. Overall parasite DNA rates in mosquitoes (estimated by the Poolscreen2) for Wb, Pf, and Pv were 4.9%, 2.7%, and 2.1%, respectively. Parasite DNA rates were consistently higher in blood fed mosquitoes than in host seeking mosquitoes. Our results show that multiplex qPCR can be used to detect and estimate prevalence rates for multiple parasite species in arthropod vectors. We believe that multiplex molecular xenodiagnosis has great potential as a tool for non-invasively assessing the distribution and prevalence of vector-borne pathogens such as W. bancrofti and Plasmodium spp. in human populations and for assessing the impact of interventions aimed at controlling or eliminating these diseases. PMID:18801545

  17. Quantitative Reverse Transcription-qPCR-Based Gene Expression Analysis in Plants.

    PubMed

    Abdallah, Heithem Ben; Bauer, Petra

    2016-01-01

    The investigation of gene expression is an initial and essential step to understand the function of a gene in a physiological context. Reverse transcription-quantitative real-time PCR (RT-qPCR) assays are reproducible, quantitative, and fast. They can be adapted to study model and non-model plant species without the need to have whole genome or transcriptome sequence data available. Here, we provide a protocol for a reliable RT-qPCR assay, which can be easily adapted to any plant species of interest. We describe the design of the qPCR strategy and primer design, considerations for plant material generation, RNA preparation and cDNA synthesis, qPCR setup and run, and qPCR data analysis, interpretation, and final presentation.

  18. Partial and Full PCR-Based Reverse Genetics Strategy for Influenza Viruses

    PubMed Central

    Chen, Hongjun; Ye, Jianqiang; Xu, Kemin; Angel, Matthew; Shao, Hongxia; Ferrero, Andrea; Sutton, Troy; Perez, Daniel R.

    2012-01-01

    Since 1999, plasmid-based reverse genetics (RG) systems have revolutionized the way influenza viruses are studied. However, it is not unusual to encounter cloning difficulties for one or more influenza genes while attempting to recover virus de novo. To overcome some of these shortcomings we sought to develop partial or full plasmid-free RG systems. The influenza gene of choice is assembled into a RG competent unit by virtue of overlapping PCR reactions containing a cDNA copy of the viral gene segment under the control of RNA polymerase I promoter (pol1) and termination (t1) signals – herein referred to as Flu PCR amplicons. Transfection of tissue culture cells with either HA or NA Flu PCR amplicons and 7 plasmids encoding the remaining influenza RG units, resulted in efficient virus rescue. Likewise, transfections including both HA and NA Flu PCR amplicons and 6 RG plasmids also resulted in efficient virus rescue. In addition, influenza viruses were recovered from a full set of Flu PCR amplicons without the use of plasmids. PMID:23029501

  19. Multiplex PCR-based simultaneous amplification of selectable marker and reporter genes for the screening of genetically modified crops.

    PubMed

    Randhawa, Gurinder Jit; Chhabra, Rashmi; Singh, Monika

    2009-06-24

    The development and commercialization of genetically modified (GM) crops with enhanced insect and herbicide resistance, abiotic stress tolerance, and improved nutritional quality has expanded dramatically. Notwithstanding the huge potential benefits of GM crops, the perceived environmental risks associated with these crops need to be addressed in proper perspective. One critical concern is the adventitious presence or unintentional mixing of GM seed in non-GM seed lots, which can seriously affect the global seed market. It would therefore be necessary though a challenging task to develop reliable, efficient, and economical assays for GM detection, identification, and quantification in non-GM seed lots. This can be systematically undertaken by preliminary screening for control elements and selectable or scorable (reporter) marker genes. In this study, simplex and multiplex polymerase chain reaction (PCR) assays individually as well as simultaneously amplifying the commonly used selectable marker genes, i.e., aadA, bar, hpt, nptII, pat encoding, respectively, for aminoglycoside-3'-adenyltransferase, Streptococcus viridochromogenes phosphinothricin-N-acetyltransferase, hygromycin phosphotransferase, neomycin phosphotransferase, Streptococcus hygroscopicus phosphinothricin-N-acetyltransferase, and a reporter gene uidA encoding beta-d-glucuronidase, were developed as a reliable tool for qualitative screening of GM crops. The efficiency of the assays was also standardized in the test samples prepared by artificial mixing of transgenic seed samples in different proportions. The developed multiplex PCR assays will be useful in verifying the GM status of a sample irrespective of the crop and GM trait.

  20. [Detection of ABO genotype genetic polymorphism by multiplex-PCR based sequencing and application in forensic medicine].

    PubMed

    Chen, Feng; Chen, Teng; Yan, Chun-Xia; Dang, Yong-Hui; Mu, Hao-Fang; Yu, Xiao-Guang; Zhang, Bo; Deng, Ya-Jun

    2008-06-01

    Multiplex PCR-direct sequencing method was established to detect 9 different SNPs in exon 6 and exon 7 of ABO genes and could identify at least 28 different ABO genotypes. Population study was carried out in a sample of 80 unrelated Chinese Tibetan minority individual dwelled in Qinghai Province. The method was also applied to forensic cases. A variety of degeneration forensic samples, including blood stain, hair root, swab, bone and mixed stain were successfully identified by this efficient method and in conformance with serological typing. There were no significant deviations from Hardy-Weinberg equilibrium in ABO genotypes of Tibetan population. The heterozygosity, polymorphic information content, discrimination power, paternity of exclusion, and probability of genetic identity were 0.675, 0.672, 0.874, 0.391, and 0.126 respectively. The gene frequency of ABO was O>B>A. The multiplex PCR-directed sequencing method can accurately and reliably detect ABO genotypes in many kinds of samples, and it improves personal identification efficiency. The ABO genotype is high variance in Qinghai Tibetan minority group, and it can be applied in forensic medicine and population genetic study.

  1. Development and evaluation of a rapid multiplex-PCR based system for Mycobacterium tuberculosis diagnosis using sputum samples.

    PubMed

    Mutingwende, Isaac; Vermeulen, Urban; Steyn, Faans; Viljoen, Hendrik; Grobler, Anne

    2015-09-01

    Global tuberculosis (TB) control and eradication is hampered by the unavailability of simple, rapid and affordable diagnostic tests deployable at low infrastructure microscopy centers. We have developed and evaluated the performance of a nucleic acid amplification test for detection of Mycobacterium tuberculosis (MTB), the NWU-TB test, in clinical sputum specimens from 306 patients with suspected pulmonary tuberculosis. The test involves sputum sample processing using a Lyser device within 7 min, followed by rapid multiplex-PCR on a fast thermal cycler within 25 min, and amplicon resolution on agarose gel electrophoresis. Samples were also examined for presence of MTB using smear microscopy, GeneXpert and MGIT culture. Results were assessed in comparison to a MGIT culture as gold standard. Of the 306 patients, 174 had a previous TB history or already on treatment, and 132 were TB naïve cases. The NWU-TB system was found to have an overall sensitivity and specificity of 80.8% (95% CI: 75-85.7) and 75.6% (95% CI: 64.9-84.4) respectively, in comparison to 85.3% (95% CI: 79.9-89.6) and 73.2% (95% CI: 62.2-82.4) respectively for GeneXpert; and 62.1% (95% CI: 55.3-68.4) and 56.1% (95% CI: 44.7-67) respectively for smear microscopy. The study has shown that the NWU-TB system allows detection of TB in less than two hours and can be utilized at low infrastructure sites to provide quick and accurate diagnosis at a very low cost. Copyright © 2015. Published by Elsevier B.V.

  2. Multiplex PCR-Based Serogrouping and Serotyping of Salmonella enterica from Tonsil and Jejunum with Jejunal Lymph Nodes of Slaughtered Swine in Metro Manila, Philippines.

    PubMed

    Ng, Kamela Charmaine S; Rivera, Windell L

    2015-05-01

    Food poisoning outbreaks and livestock mortalities caused by Salmonella enterica are widespread in the Philippines, with hogs being the most commonly recognized carriers of the pathogen. To prevent and control the occurrence of S. enterica infection in the country, methods were used in this study to isolate and rapidly detect, differentiate, and characterize S. enterica in tonsils and jejuna with jejunal lymph nodes of swine slaughtered in four locally registered meat establishments (LRMEs) and four meat establishments accredited by the National Meat Inspection Services in Metro Manila. A total of 480 samples were collected from 240 animals (120 pigs from each type of meat establishment). A significantly higher proportion of pigs were positive for S. enterica in LRMEs (60 of 120) compared with meat establishments accredited by the National Meat Inspection Services (38 of 120). More S. enterica-positive samples were found in tonsils compared with jejuna with jejunal lymph nodes in LRMEs, but this difference was not significant. A PCR assay targeting the invA gene had sensitivity that was statistically similar to that of the culture method, detecting 93 of 98 culture-confirmed samples. Multiplex PCR-based O-serogrouping and H/Sdf I typing revealed four S. enterica serogroups (B, C1, D, and E) and six serotypes (Agona, Choleraesuis, Enteritidis, Heidelberg, Typhimurium, and Weltevreden), respectively, which was confirmed by DNA sequencing of the PCR products. This study was the first to report detection of S. enterica serotype Agona in the country.

  3. Development of conventional and real-time multiplex PCR-based assays for estimation of natural infection rates and Trypanosoma cruzi load in triatomine vectors.

    PubMed

    Moreira, Otacilio C; Verly, Thaiane; Finamore-Araujo, Paula; Gomes, Suzete A O; Lopes, Catarina M; de Sousa, Danielle M; Azevedo, Lívia R; da Mota, Fabio F; d'Avila-Levy, Claudia M; Santos-Mallet, Jacenir R; Britto, Constança

    2017-08-29

    %), followed by the Atlantic Rainforest and Cerrado with 7.1 and 6.1%, respectively. In addition, a wide range distribution of parasite load, varying from 8.05 × 10(-2) to 6.31 × 10(10) was observed with a median of 2.29 × 10(3) T. cruzi/intestine units. When parasite load was analyzed by triatomine species, a significantly higher median was found for Panstrongylus lutzi in comparison with Triatoma brasiliensis. Our results demonstrate highly sensitive PCR-based methodologies to monitor T. cruzi infection in triatomines. In addition, the qPCR assay offers the possibility of further evaluation parasite load, as a promising biomarker of the vectorial capacity of triatomines in Chagas disease endemic areas.

  4. Typing and Subtyping Influenza Virus Using DNA Microarrays and Multiplex Reverse Transcriptase PCR

    PubMed Central

    Li, Jiping; Chen, Shu; Evans, David H.

    2001-01-01

    A model DNA microarray has been prepared and shown to facilitate typing and subtyping of human influenza A and B viruses. Reverse transcriptase PCR was used to prepare cDNAs encoding ∼500-bp influenza virus gene fragments, which were then cloned, sequenced, reamplified, and spotted to form a glass-bound microarray. These target DNAs included multiple fragments of the hemagglutinin, neuraminidase, and matrix protein genes. Cy3- or Cy5-labeled fluorescent probes were then hybridized to these target DNAs, and the arrays were scanned to determine the probe binding site(s). The hybridization pattern agreed perfectly with the known grid location of each target, and the signal-to-background ratio varied from 5 to 30. No cross-hybridization could be detected beyond that expected from the limited degree of sequence overlap between different probes and targets. At least 100 to 150 bp of homology was required for hybridization under the conditions used in this study. Combinations of Cy3- and Cy5-labeled DNAs can also be hybridized to the same chip, permitting further differentiation of amplified molecules in complex mixtures. In a more realistic test of the technology, several sets of multiplex PCR primers that collectively target influenza A and B virus strains were identified and were used to type and subtype several previously unsequenced influenza virus isolates. The results show that DNA microarray technology provides a useful supplement to PCR-based diagnostic methods. PMID:11158130

  5. Single-Reaction Multiplex Reverse Transcription PCR for Detection of Zika, Chikungunya, and Dengue Viruses.

    PubMed

    Waggoner, Jesse J; Gresh, Lionel; Mohamed-Hadley, Alisha; Ballesteros, Gabriela; Davila, Maria Jose Vargas; Tellez, Yolanda; Sahoo, Malaya K; Balmaseda, Angel; Harris, Eva; Pinsky, Benjamin A

    2016-07-01

    Clinical manifestations of Zika virus, chikungunya virus, and dengue virus infections can be similar. To improve virus detection, streamline molecular workflow, and decrease test costs, we developed and evaluated a multiplex real-time reverse transcription PCR for these viruses.

  6. Development of a Rapid Identification Method for the Differentiation of Enterococcus Species Using a Species-Specific Multiplex PCR Based on Comparative Genomics.

    PubMed

    Park, Jongbin; Jin, Gwi-Deuk; Pak, Jae In; Won, Jihyun; Kim, Eun Bae

    2017-04-01

    Enterococci are lactic acid bacteria that are commonly found in food and in animal gut. Since 16 S ribosomal RNA (rRNA) sequences, genetic markers for bacterial identification, are similar among several Enterococcus species, it is very difficult to determine the correct species based on only 16 S rRNA sequences. Therefore, we developed a rapid method for the identification of different Enterococcus species using comparative genomics. We compared 38 genomes of 13 Enterococcus species retrieved from the National Center of Biotechnology Information database and identified 25,623 orthologs. Among the orthologs, four genes were specific to four Enterococcus species (Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, and Enterococcus durans). We designed species-specific primer sets targeting the genes and developed a multiplex PCR using primer sets that could distinguish the four Enterococcus species among the nine strains of Enterococcus species that were available locally. The multiplex PCR method also distinguished the four species isolated from various environments, such as feces of chicken and cow, meat of chicken, cow, and pigs, and fermented soybeans (Cheonggukjang and Doenjang). These results indicated that our novel multiplex PCR using species-specific primers could identify the four Enterococcus species in a rapid and easy way. This method will be useful to distinguish Enterococcus species in food, feed, and clinical settings.

  7. Evaluation of Curetis Unyvero, a multiplex PCR-based testing system, for rapid detection of bacteria and antibiotic resistance and impact of the assay on management of severe nosocomial pneumonia.

    PubMed

    Jamal, Wafaa; Al Roomi, Ebtehal; AbdulAziz, Lubna R; Rotimi, Vincent O

    2014-07-01

    Health care-associated pneumonia due to multidrug-resistant organisms represents a major therapeutic challenge. Unfortunately, treatment is dependent on empirical therapy, which often leads to improper and inadequate antimicrobial therapy. A rapid multiplex PCR-based Unyvero pneumonia application (UPA) assay that assists in timely decision-making has recently become available. In this study, we evaluated the performance of UPA in detecting etiological pathogens and resistance markers in patients with nosocomial pneumonia (NP). The impact of this assay on the management of severe nosocomial pneumonia was also assessed. Appropriate specimens were processed by UPA according to the manufacturer's protocol in parallel with conventional culture methods. Of the 56 patients recruited into the study, 49 (87.5%) were evaluable. Of these, 27 (55.1%) and 4 (8.2%) harbored multiple bacteria by the PCR assay and conventional culture, respectively. A single pathogen was detected in 8 (16.3%) and 4 (8.2%) patients, respectively. Thirteen different genes were detected from 38 patients, including the ermB gene (40.8%), the blaOXA-51-like gene (28.6%), the sul1 (28.6%) and int1 (20.4%) integrase genes, and the mecA and blaCTX-M genes (12.3% each). The time from sample testing to results was 4 h versus 48 to 96 h by UPA and culture, respectively. Initial empirical treatment was changed within 5 to 6 h in 33 (67.3%) patients based on the availability of UPA results. Thirty (62.2%) of the patients improved clinically. A total of 3 (6.1%) patients died, mainly from their comorbidities. These data demonstrate the potential of a multiplex PCR-based assay for accurate and timely detection of etiological agents of NP, multidrug-resistant (MDR) organisms, and resistance markers, which can guide clinicians in making early antibiotic adjustments. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  8. A multiplex PCR-based molecular identification of five morphologically related, medically important subgenus Stegomyia mosquitoes from the genus Aedes (Diptera: Culicidae) found in the Ryukyu Archipelago, Japan.

    PubMed

    Higa, Yukiko; Toma, Takako; Tsuda, Yoshio; Miyagi, Ichiro

    2010-09-01

    Internal transcribed spacer regions of ribosomal DNA were sequenced, and new species-specific primers were designed to simplify the molecular identification of five morphologically related subgenus Stegomyia mosquito species--Aedes aegypti, Ae. albopictus, Ae. riversi, Ae. flavopictus, and Ae. daitensis--found in the Ryukyu Archipelago, Japan. Each newly designed primer was able to amplify a species-specific fragment with a different size. Conditions for multiplex PCR were optimized to identify all five species in a single PCR. This method is a convenient tool for entomological field surveys, particularly in arbovirus endemic/epidemic areas where some of these species coexist.

  9. How well does physician selection of microbiologic tests identify Clostridium difficile and other pathogens in paediatric diarrhoea? Insights using multiplex PCR-based detection.

    PubMed

    Stockmann, C; Rogatcheva, M; Harrel, B; Vaughn, M; Crisp, R; Poritz, M; Thatcher, S; Korgenski, E K; Barney, T; Daly, J; Pavia, A T

    2015-02-01

    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 (GI) 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 three (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 Clostridium 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.

  10. Phylogenetic study and multiplex PCR-based detection of Burkholderia plantarii, Burkholderia glumae and Burkholderia gladioli using gyrB and rpoD sequences.

    PubMed

    Maeda, Yukiko; Shinohara, Hirosuke; Kiba, Akinori; Ohnishi, Kouhei; Furuya, Naruto; Kawamura, Yoshiaki; Ezaki, Takayuki; Vandamme, Peter; Tsushima, Seiya; Hikichi, Yasufumi

    2006-05-01

    In order to develop a detection method for the rice pathogens Burkholderia plantarii, Burkholderia glumae and Burkholderia gladioli, the phylogeny of six plant-pathogenic Burkholderia species was analysed using the combined nucleotide sequences of gyrB and rpoD. B. plantarii, B. glumae and B. gladioli formed tight monophyletic branches supported by high bootstrap probabilities. The high sequence similarity revealed a close phylogenetic relationship between B. glumae and B. plantarii. B. plantarii strains were divided into three subclusters comprising rice strains, whereas the single Vanda strain occupied a unique position in the phylogenetic tree. The gyrB and rpoD sequences of all B. glumae strains examined were highly conserved. In contrast, B. gladioli strains demonstrated a far greater sequence diversity, but this diversity did not correlate with pathovar, host plant or geographical origin of the strains. A multiplex-PCR protocol using specific primers from the gyrB sequences was designed that allowed the specific detection and identification of B. plantarii, B. glumae and B. gladioli in rice seeds infected with these pathogenic species.

  11. Comparison of multiplex PCR hybridization-based and singleplex real-time PCR-based assays for detection of low prevalence pathogens in spiked samples.

    PubMed

    Hockman, Donna; Dong, Ming; Zheng, Hong; Kumar, Sanjai; Huff, Matthew D; Grigorenko, Elena; Beanan, Maureen; Duncan, Robert

    2017-01-01

    Molecular diagnostic devices are increasingly finding utility in clinical laboratories. Demonstration of the effectiveness of these devices is dependent upon comparing results from clinical samples tested with the new device to an alternative testing method. The preparation of mock clinical specimens will be necessary for the validation of molecular diagnostic devices when a sufficient number of clinical specimens is unobtainable. Examples include rare pathogens, some of which are pathogens posing a biological weapon threat. Here we describe standardized steps for developers to follow for the culture and quantification of three organisms used to spike human whole blood to create mock specimens. The three organisms chosen for this study were the Live Vaccine Strain (LVS) of Francisella tularensis, surrogate for a potential biothreat pathogen, Escherichia coli, a representative Gram-negative bacterium and Babesia microti (Franca) Reichenow Peabody strain, representing a protozoan parasite. Mock specimens were prepared with blood from both healthy donors and donors with nonspecific symptoms including fever, malaise, and flu-like symptoms. There was no significant difference in detection results between the two groups for any pathogen. Testing of the mock samples was compared on two platforms, Target Enriched Multiplex-PCR (TEM-PCR™) and singleplex real-time PCR (RT-PCR). Results were reproducible on both platforms. The reproducibility demonstrated by obtaining the same results between two testing methods and between healthy and symptomatic mock specimens, indicates the standardized methods described for creating the mock specimens are valid and effective for evaluating diagnostic devices.

  12. Multiplex-PCR-Based Screening and Computational Modeling of Virulence Factors and T-Cell Mediated Immunity in Helicobacter pylori Infections for Accurate Clinical Diagnosis

    PubMed Central

    Oktem-Okullu, Sinem; Tiftikci, Arzu; Saruc, Murat; Cicek, Bahattin; Vardareli, Eser; Tozun, Nurdan; Kocagoz, Tanil; Sezerman, Ugur; Yavuz, Ahmet Sinan; Sayi-Yazgan, Ayca

    2015-01-01

    The outcome of H. pylori infection is closely related with bacteria's virulence factors and host immune response. The association between T cells and H. pylori infection has been identified, but the effects of the nine major H. pylori specific virulence factors; cagA, vacA, oipA, babA, hpaA, napA, dupA, ureA, ureB on T cell response in H. pylori infected patients have not been fully elucidated. We developed a multiplex- PCR assay to detect nine H. pylori virulence genes with in a three PCR reactions. Also, the expression levels of Th1, Th17 and Treg cell specific cytokines and transcription factors were detected by using qRT-PCR assays. Furthermore, a novel expert derived model is developed to identify set of factors and rules that can distinguish the ulcer patients from gastritis patients. Within all virulence factors that we tested, we identified a correlation between the presence of napA virulence gene and ulcer disease as a first data. Additionally, a positive correlation between the H. pylori dupA virulence factor and IFN-γ, and H. pylori babA virulence factor and IL-17 was detected in gastritis and ulcer patients respectively. By using computer-based models, clinical outcomes of a patients infected with H. pylori can be predicted by screening the patient's H. pylori vacA m1/m2, ureA and cagA status and IFN-γ (Th1), IL-17 (Th17), and FOXP3 (Treg) expression levels. Herein, we report, for the first time, the relationship between H. pylori virulence factors and host immune responses for diagnostic prediction of gastric diseases using computer—based models. PMID:26287606

  13. Single-Reaction Multiplex Reverse Transcription PCR for Detection of Zika, Chikungunya, and Dengue Viruses

    PubMed Central

    Waggoner, Jesse J.; Gresh, Lionel; Mohamed-Hadley, Alisha; Ballesteros, Gabriela; Davila, Maria Jose Vargas; Tellez, Yolanda; Sahoo, Malaya K.; Balmaseda, Angel; Harris, Eva

    2016-01-01

    Clinical manifestations of Zika virus, chikungunya virus, and dengue virus infections can be similar. To improve virus detection, streamline molecular workflow, and decrease test costs, we developed and evaluated a multiplex real-time reverse transcription PCR for these viruses. PMID:27184629

  14. A multiplex reverse transcription PCR assay for simultaneous detection of five tobacco viruses in tobacco plants.

    PubMed

    Dai, Jin; Cheng, Julong; Huang, Ting; Zheng, Xuan; Wu, Yunfeng

    2012-07-01

    Tobacco viruses including Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), Tobacco etch virus (TEV), Potato virus Y (PVY) and Tobacco vein banding mosaic virus (TVBMV) are major viruses infecting tobacco and can cause serious crop losses. A multiplex reverse transcription polymerase chain reaction assay was developed to detect simultaneously and differentiate all five viruses. The system used specific primer sets for each virus producing five distinct fragments 237, 273, 347, 456 and 547 bp, representing TMV, CMV subgroup I, TEV, PVY(O) and TVBMV, respectively. These primers were used for detection of the different viruses by single PCR and multiplex PCR and the results were confirmed by DNA sequencing analysis. The protocol was used to detect viruses from different parts of China. The simultaneous and sensitive detection of different viruses using the multiplex PCR is more efficient and economical than other conventional methods for tobacco virus detection. This multiplex PCR provides a rapid and reliable method for the detection and identification of major tobacco viruses, and will be useful for epidemiological studies.

  15. Reverse transcription PCR-based detection of Crimean-Congo hemorrhagic fever virus isolated from ticks of domestic ruminants in Kurdistan province of Iran.

    PubMed

    Fakoorziba, Mohammad Reza; Golmohammadi, Parvaneh; Moradzadeh, Rahmatollah; Moemenbellah-Fard, Mohammad Djaefar; Azizi, Kourosh; Davari, Behrooz; Alipour, Hamzeh; Ahmadnia, Sara; Chinikar, Sadegh

    2012-09-01

    Crimean-Congo hemorrhagic fever (CCHF) is a potentially fatal viral vector-borne zoonosis which has a mortality rate of up to 30% without treatment in humans. CCHF virus is transmitted to humans by ticks, predominantly from the Hyalomma genus. Following the report of two confirmed and one suspected death due to CCHF virus in Kurdistan province of Iran in 2007, this study was undertaken to determine the fauna of hard ticks on domestic ruminants (cattle, sheep, and goats) and their possible infection with CCHF virus using reverse transcription PCR technique. This is the first detection of CCHF virus in ticks from the Kurdistan province of Iran. Overall, 414 ixodid ticks were collected from two districts in this province. They represented four genera from which 10 separate species were identified. The Hyalomma genus was the most abundant tick genus (70%). It was the only genus shown to be infected with the CCHF virus using RT-PCR technique. The number of ticks positive for CCHF virus was 5 out of 90 (5.6%) adult ticks. The three remaining genera (Haemaphysalis, Rhipicephalus, and Dermacentor) were all negative following molecular survey. Four of the five virally-infected ticks were from cattle mainly in the Sanandaj district. We concluded that CCHF virus is present in the Hyalomma ticks on domestic ruminants (cattle) in Kurdistan province of Iran.

  16. Application of anti-listerial bacteriocins: monitoring enterocin expression by multiplex relative reverse transcription-PCR.

    PubMed

    Williams, D Ross; Chanos, Panagiotis

    2012-12-01

    Listeriosis is a deadly food-borne disease, and its incidence may be limited through the biotechnological exploitation of a number of anti-listerial biocontrol agents. The most widely used of these agents are bacteriocins and the Class II enterocins are characterized by their activity against Listeria. Enterocins are primarily produced by enterococci, particularly Enterococcus faecium and many strains have been described, often encoding multiple bacteriocins. The use of these strains in food will require that they are free of virulence functions and that they exhibit a high level expression of anti-listerial enterocins in fermentation conditions. Multiplex relative RT (reverse transcription)-PCR is a technique that is useful in the discovery of advantageous expression characteristics among enterocin-producing strains. It allows the levels of individual enterocin gene expression to be monitored and determination of how expression is altered under different growth conditions.

  17. Detection of circulating tumor cells in breast cancer patients using multiplex reverse transcription-polymerase chain reaction and specific primers for MGB, PTHRP and KRT19 correlation with clinicopathological features.

    PubMed

    Skondra, Maria; Gkioka, Eliona; Kostakis, Ioannis D; Pissimissis, Nikolaos; Lembessis, Panagiotis; Pectasides, Dimitrios; Koutsilieris, Michael

    2014-11-01

    The aim of this study was to correlate the clinicopathological features of breast cancer patients with the positive detection of parathyroid hormone-related protein (PTHRP), cytokeratin protein 19 (KRT19) and mammaglobin (MGB) using a multiplex reverse transcription polymerase chain reaction (RT-PCR) assay developed to detect circulating tumor cells (CTCs) in peripheral blood of patients with breast cancer. Peripheral blood samples were collected from 54 breast cancer patients and 20 healthy blood donors. Subsequently, the samples were processed for RNA extraction and analyzed for the expression of PTHRP, KRT19 and MGB using specific primers and multiplex RT-PCR. The positive detection rates in breast cancer patients for PTHRP, KRT19 and MGB were 68.5%, 63% and 22.2% and for healthy donors 10%, 0% and 10%, respectively. The statistical analysis revealed that PTHRP- and KRT19-positive detections correlated with the diagnosis of breast cancer while the combined positive detections of PTHRP-plus-KRT19 correlated with the presence of distant metastasis, especially with bone metastasis. Moreover, positive detections of KRT19 correlated with high proliferation rate of breast cancer tumors. MGB-positive detections did not add any diagnostic advantage in such analysis. Multiplex-PCR based detection of CTCs using PTHRP and KRT19 primers can provide useful information for the disease. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  18. Design of reversible multiplexer using electro-optic effect inside lithium niobate-based Mach-Zehnder interferometers

    NASA Astrophysics Data System (ADS)

    Kumar, Santosh; Chauhan, Chanderkanta

    2016-11-01

    With the demand of ultrahigh-speed logic, there has been an emphasis on low-power design techniques. Reversible computing has been proposed as a possible alternative to address the energy dissipation problem. Thus, the reversible circuit implementation in optical domain gives a new dimension in ultrahigh-speed, low-power consumption of quantum computing. In this study, a design of reversible multiplexer using electro-optic effect of lithium niobate-based Mach-Zehnder interferometer is proposed. It is verified using a beam propagation method along with MATLAB simulation.

  19. The prevalence of urogenital micro-organisms detected by a multiplex PCR-reverse line blot assay in women attending three sexual health clinics in Sydney, Australia.

    PubMed

    McKechnie, Michelle L; Hillman, Richard J; Jones, Rachel; Lowe, Penelope C; Couldwell, Deborah L; Davies, Stephen C; King, Fiona; Kong, Fanrong; Gilbert, Gwendolyn L

    2011-07-01

    This study used a previously described multiplex PCR-based reverse line blot (mPCR/RLB) assay to assess the prevalence and distribution of 14 urogenital pathogens or putative pathogens, namely Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Trichomonas vaginalis, Gardnerella vaginalis, Ureaplasma parvum, Ureaplasma urealyticum, Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae, herpes simplex virus types 1 and 2, and human adenovirus. First-voided urine specimens and endocervical and self-collected vaginal swabs from each of 216 women attending three sexual health clinics in Sydney, Australia, were tested and the results were compared with those of reference methods for each organism. One hundred and sixty-eight women (77.7 %) had at least one and 105 (48.6 %) had more than one target organism, most commonly G. vaginalis and Ureaplasma spp. The prevalence of each of the four known sexually transmissible pathogens was <5 %. Of the 216 women, 111 (51.4 %) reported at least one symptom consistent with genital or urethral infection, including discharge, pain or discomfort. Only G. vaginalis was detected more frequently in women with symptoms (P = 0.05). The specificity of the mPCR/RLB assay compared with that of the reference methods for each organism and for all specimen types was 100 %. The mean sensitivities of the mPCR/RLB assay compared with those of the reference methods for self-collected vaginal swabs, cervical swabs and first-voided urine specimens for all organisms were 99.3, 98.1 and 84.6 %, respectively; however, these differences were not significant. There were no differences in sensitivities between specimen types for C. trachomatis, N. gonorrhoeae, T. vaginalis and H. influenzae, although all were found infrequently. Overall, the mPCR/RLB platform was found to be an accurate testing platform in a sexual health clinic setting.

  20. Simultaneous detection of four garlic viruses by multiplex reverse transcription PCR and their distribution in Indian garlic accessions.

    PubMed

    Majumder, S; Baranwal, V K

    2014-06-01

    Indian garlic is infected with Onion yellow dwarf virus (OYDV), Shallot latent virus (SLV), Garlic common latent virus (GarCLV) and allexiviruses. Identity and distribution of garlic viruses in various garlic accessions from different geographical regions of India were investigated. OYDV and allexiviruses were observed in all the garlic accessions, while SLV and GarCLV were observed only in a few accessions. A multiplex reverse transcription (RT)-PCR method was developed for the simultaneous detection and identification of OYDV, SLV, GarCLV and Allexivirus infecting garlic accessions in India. This multiplex protocol standardized in this study will be useful in indexing of garlic viruses and production of virus free seed material.

  1. Development and Validation of a Multiplex Reverse Transcription PCR Assay for Simultaneous Detection of Three Papaya Viruses

    PubMed Central

    Tuo, Decai; Shen, Wentao; Yang, Yong; Yan, Pu; Li, Xiaoying; Zhou, Peng

    2014-01-01

    Papaya ringspot virus (PRSV), Papaya leaf distortion mosaic virus (PLDMV), and Papaya mosaic virus (PapMV) produce similar symptoms in papaya. Each threatens commercial production of papaya on Hainan Island, China. In this study, a multiplex reverse transcription PCR assay was developed to detect simultaneously these three viruses by screening combinations of mixed primer pairs and optimizing the multiplex RT-PCR reaction conditions. A mixture of three specific primer pairs was used to amplify three distinct fragments of 613 bp from the P3 gene of PRSV, 355 bp from the CP gene of PLDMV, and 205 bp from the CP gene of PapMV, demonstrating the assay’s specificity. The sensitivity of the multiplex RT-PCR was evaluated by showing plasmids containing each of the viral target genes with 1.44 × 103, 1.79 × 103, and 1.91 × 102 copies for the three viruses could be detected successfully. The multiplex RT-PCR was applied successfully for detection of three viruses from 341 field samples collected from 18 counties of Hainan Island, China. Rates of single infections were 186/341 (54.5%), 93/341 (27.3%), and 3/341 (0.9%), for PRSV, PLDMV, and PapMV, respectively; 59/341 (17.3%) of the samples were co-infected with PRSV and PLDMV, which is the first time being reported in Hainan Island. This multiplex RT-PCR assay is a simple, rapid, sensitive, and cost-effective method for detecting multiple viruses in papaya and can be used for routine molecular diagnosis and epidemiological studies in papaya. PMID:25337891

  2. Development and validation of a multiplex reverse transcription PCR assay for simultaneous detection of three papaya viruses.

    PubMed

    Tuo, Decai; Shen, Wentao; Yang, Yong; Yan, Pu; Li, Xiaoying; Zhou, Peng

    2014-10-21

    Papaya ringspot virus (PRSV), Papaya leaf distortion mosaic virus (PLDMV), and Papaya mosaic virus (PapMV) produce similar symptoms in papaya. Each threatens commercial production of papaya on Hainan Island, China. In this study, a multiplex reverse transcription PCR assay was developed to detect simultaneously these three viruses by screening combinations of mixed primer pairs and optimizing the multiplex RT-PCR reaction conditions. A mixture of three specific primer pairs was used to amplify three distinct fragments of 613 bp from the P3 gene of PRSV, 355 bp from the CP gene of PLDMV, and 205 bp from the CP gene of PapMV, demonstrating the assay's specificity. The sensitivity of the multiplex RT-PCR was evaluated by showing plasmids containing each of the viral target genes with 1.44 × 103, 1.79 × 103, and 1.91 × 102 copies for the three viruses could be detected successfully. The multiplex RT-PCR was applied successfully for detection of three viruses from 341 field samples collected from 18 counties of Hainan Island, China. Rates of single infections were 186/341 (54.5%), 93/341 (27.3%), and 3/341 (0.9%), for PRSV, PLDMV, and PapMV, respectively; 59/341 (17.3%) of the samples were co-infected with PRSV and PLDMV, which is the first time being reported in Hainan Island. This multiplex RT-PCR assay is a simple, rapid, sensitive, and cost-effective method for detecting multiple viruses in papaya and can be used for routine molecular diagnosis and epidemiological studies in papaya.

  3. Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses

    PubMed Central

    Naveca, Felipe Gomes; do Nascimento, Valdinete Alves; de Souza, Victor Costa; Nunes, Bruno Tardelli Diniz; Rodrigues, Daniela Sueli Guerreiro; Vasconcelos, Pedro Fernando da Costa

    2017-01-01

    ABSTRACT We describe a sensitive method for simultaneous detection of Oropouche and Oropouche-like viruses carrying the Oropouche S segment, as well as the Mayaro virus, using a multiplexed one-step reverse transcription real-time polymerase chain reaction (RT-qPCR). A chimeric plasmid containing both Mayaro and Oropouche targets was designed and evaluated for the in vitro production of transcribed RNA, which could be easily used as a non-infectious external control. To track false-negative results due to PCR inhibition or equipment malfunction, the MS2 bacteriophage was also included in the multiplex assay as an internal positive control. The specificity of the multiplex assay was evaluated by Primer-Blast analysis against the entire GenBank database, and further against a panel of 17 RNA arboviruses. The results indicated an accurate and highly sensitive assay with amplification efficiency greater than 98% for both targets, and a limit of detection between two and 20 copies per reaction. We believe that the assay described here will provide a tool for Mayaro and Oropouche virus detection, especially in areas where differential diagnosis of Dengue, Zika and Chikungunya viruses should be performed. PMID:28591313

  4. Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses.

    PubMed

    Naveca, Felipe Gomes; Nascimento, Valdinete Alves do; Souza, Victor Costa de; Nunes, Bruno Tardelli Diniz; Rodrigues, Daniela Sueli Guerreiro; Vasconcelos, Pedro Fernando da Costa

    2017-07-01

    We describe a sensitive method for simultaneous detection of Oropouche and Oropouche-like viruses carrying the Oropouche S segment, as well as the Mayaro virus, using a multiplexed one-step reverse transcription real-time polymerase chain reaction (RT-qPCR). A chimeric plasmid containing both Mayaro and Oropouche targets was designed and evaluated for the in vitro production of transcribed RNA, which could be easily used as a non-infectious external control. To track false-negative results due to PCR inhibition or equipment malfunction, the MS2 bacteriophage was also included in the multiplex assay as an internal positive control. The specificity of the multiplex assay was evaluated by Primer-Blast analysis against the entire GenBank database, and further against a panel of 17 RNA arboviruses. The results indicated an accurate and highly sensitive assay with amplification efficiency greater than 98% for both targets, and a limit of detection between two and 20 copies per reaction. We believe that the assay described here will provide a tool for Mayaro and Oropouche virus detection, especially in areas where differential diagnosis of Dengue, Zika and Chikungunya viruses should be performed.

  5. A pipeline with multiplex reverse transcription polymerase chain reaction and microarray for screening of chromosomal translocations in leukemia.

    PubMed

    Xiong, Fei-Fei; Li, Ben-Shang; Zhang, Chun-Xiu; Xiong, Hui; Shen, Shu-Hong; Zhang, Qing-Hua

    2013-01-01

    Chromosome rearrangements and fusion genes present major portion of leukemogenesis and contribute to leukemic subtypes. It is practical and helpful to detect the fusion genes in clinic diagnosis of leukemia. Present application of reverse transcription polymerase chain reaction (RT-PCR) method to detect the fusion gene transcripts is effective, but time- and labor-consuming. To set up a simple and rapid system, we established a method that combined multiplex RT-PCR and microarray. We selected 15 clinically most frequently observed chromosomal rearrangements generating more than 50 fusion gene variants. Chimeric reverse primers and chimeric PCR primers containing both gene-specific and universal sequences were applied in the procedure of multiplex RT-PCR, and then the PCR products hybridized with a designed microarray. With this approach, among 200 clinic samples, 63 samples were detected to have gene rearrangements. All the detected fusion genes positive and negative were validated with RT-PCR and Sanger sequencing. Our data suggested that the RT-PCR-microarray pipeline could screen 15 partner gene pairs simultaneously at the same accuracy of the fusion gene detection with regular RT-PCR. The pipeline showed effectiveness in multiple fusion genes screening in clinic samples.

  6. Simultaneous detection of papaya ringspot virus, papaya leaf distortion mosaic virus, and papaya mosaic virus by multiplex real-time reverse transcription PCR.

    PubMed

    Huo, P; Shen, W T; Yan, P; Tuo, D C; Li, X Y; Zhou, P

    2015-12-01

    Both the single infection of papaya ringspot virus (PRSV), papaya leaf distortion mosaic virus (PLDMV) or papaya mosaic virus (PapMV) and double infection of PRSV and PLDMV or PapMV which cause indistinguishable symptoms, threaten the papaya industry in Hainan Island, China. In this study, a multiplex real-time reverse transcription PCR (RT-PCR) was developed to detect simultaneously the three viruses based on their distinctive melting temperatures (Tms): 81.0±0.8°C for PRSV, 84.7±0.6°C for PLDMV, and 88.7±0.4°C for PapMV. The multiplex real-time RT-PCR method was specific and sensitive in detecting the three viruses, with a detection limit of 1.0×10(1), 1.0×10(2), and 1.0×10(2) copies for PRSV, PLDMV, and PapMV, respectively. Indeed, the reaction was 100 times more sensitive than the multiplex RT-PCR for PRSV, and 10 times more sensitive than multiplex RT-PCR for PLDMV. Field application of the multiplex real-time RT-PCR demonstrated that some non-symptomatic samples were positive for PLDMV by multiplex real-time RT-PCR but negative by multiplex RT-PCR, whereas some samples were positive for both PRSV and PLDMV by multiplex real-time RT-PCR assay but only positive for PLDMV by multiplex RT-PCR. Therefore, this multiplex real-time RT-PCR assay provides a more rapid, sensitive and reliable method for simultaneous detection of PRSV, PLDMV, PapMV and their mixed infections in papaya.

  7. Combining reverse-transcription multiplex PCR and microfluidic electrophoresis to simultaneously detect seven mosquito-transmitted zoonotic encephalomyelitis viruses.

    PubMed

    Wang, Yu; Ostlund, Eileen N; Jun, Yang; Nie, Fu-Ping; Li, Ying-Guo; Johnson, Donna J; Lin, Rui; Li, Zheng-Guo

    2016-06-01

    Several mosquito-transmitted viruses are causative agents for zoonotic encephalomyelitis. Rapid identification of these viruses in mosquito populations is an effective method for surveying these diseases. To detect multiple mosquito-transmitted viral agents, including West Nile virus, Saint Louis encephalitis virus, Venezuelan equine encephalomyelitis virus, Western equine encephalomyelitis virus, Eastern equine encephalomyelitis virus, Highlands J virus and Japanese encephalitis virus, an assay using multiplex reverse-transcription PCR combined with microfluidic electrophoresis was developed and evaluated. Tailed nested primers were used in the assay to amplify specific viral genomic segments, and products with specific length were further analyzed by using a microfluidic electrophoresis chip. The assay exhibited good specificity and analytical sensitivity (10(2) copies/µL). This technology can be helpful in the quarantine and surveillance of exotic encephalomyelitis viruses which are transmitted by mosquitoes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Development of a multiplex real-time reverse transcriptase-polymerase chain reaction for equine infectious anemia virus (EIAV).

    PubMed

    Cook, R Frank; Cook, S J; Li, F Li; Montelaro, R C; Issel, C J

    2002-08-01

    A single-tube reverse transcriptase-polymerase chain reaction (RT-PCR) using a fluorogenic real-time PCR detection method is described for the quantitation of equine infectious anemia virus (EIAV) RNA in the plasma of equids. To compensate for variations inherent in sample preparation a multiplex real-time RT-PCR system was developed that permitted the simultaneous calculation of the nucleic acid recovery rate along with the copy number of viral RNA molecules. Detection of EIAV RNA was linear from 10(9) to 10(1) molecules with intra- and inter-assay variability of less than 1% at 10(8), 10(6), 10(4) and 10(2) molecules.

  9. Detection of Staphylococcus aureus enterotoxigenic strains in bovine raw milk by reversed passive latex agglutination and multiplex polymerase chain reaction

    PubMed Central

    Mansour, Asmaa Samy; Wagih, Gad El-Said; Morgan, Sabry D.; Elhariri, Mahmoud; El-Shabrawy, Mona A.; Abuelnaga, Azza S. M.; Elgabry, E. A.

    2017-01-01

    Aim: This review gives an outline of the assessment of enterotoxigenic Staphylococcus aureus tainting levels in raw milk from different sources in Egypt and characterization of enterotoxigenic strains utilizing a technique in light of PCR to identify genes coding for the production of staphylococcal enterotoxin (SE). The obtained data were compared with results from the application of the reversed passive latex. Materials and Methods: Multiplex PCR and reversed passive latex agglutination (RPLA) were used. A total of 141 samples of raw milk (cow’s milk=33, buffalo’s milk=58, and bulk tank milk=50) were investigated for S. aureus contamination and tested for enterotoxin genes presence and toxin production. Results: S. aureus was detected in 23 (16.3%) samples phenotypically and genotypically by amplification of nuc gene. The S. aureus isolates were investigated for SEs genes (sea to see) by multiplex PCR and the toxin production by these isolates was screened by RPLA. SEs genes were detected in six isolates (26.1%) molecularly; see was the most observed gene where detected in all isolates, two isolates harbored seb, and two isolates harbored sec. According to RPLA, three isolates produced SEB and SEC. Conclusion: The study revealed the widespread of S. aureus strains caring genes coding for toxins. The real significance of the presence of these strains or its toxins in raw milk and their possible impact a potential hazard for staphylococcal food poisoning by raw milk consumption. Therefore, detection of enterotoxigenic S. aureus strains in raw milk is necessary for consumer safety. PMID:28919671

  10. Fast reversible learning based on neurons functioning as anisotropic multiplex hubs

    NASA Astrophysics Data System (ADS)

    Vardi, Roni; Goldental, Amir; Sheinin, Anton; Sardi, Shira; Kanter, Ido

    2017-05-01

    Neural networks are composed of neurons and synapses, which are responsible for learning in a slow adaptive dynamical process. Here we experimentally show that neurons act like independent anisotropic multiplex hubs, which relay and mute incoming signals following their input directions. Theoretically, the observed information routing enriches the computational capabilities of neurons by allowing, for instance, equalization among different information routes in the network, as well as high-frequency transmission of complex time-dependent signals constructed via several parallel routes. In addition, this kind of hubs adaptively eliminate very noisy neurons from the dynamics of the network, preventing masking of information transmission. The timescales for these features are several seconds at most, as opposed to the imprint of information by the synaptic plasticity, a process which exceeds minutes. Results open the horizon to the understanding of fast and adaptive learning realities in higher cognitive brain's functionalities.

  11. Detection and differentiation of genotype I and III Japanese encephalitis virus in mosquitoes by multiplex reverse transcriptase-polymerase chain reaction.

    PubMed

    Chen, Y Y; Lin, J W; Fan, Y C; Chiou, S S

    2014-02-01

    Japanese encephalitis (JE) is a disease that threatens both human and animal populations in Asian countries, and the causative agent of JE, Japanese encephalitis virus (JEV), has recently changed from genotype III (GIII) to genotype I (GI). However, a test for the rapid differentiation of GI and GIII JEV is still unavailable, especially one that can be used for mosquito-based surveillance. We have designed GI- and GIII-specific primer sets for the rapid detection and differentiation of GI and GIII JEV by multiplex reverse transcriptase-polymerase chain reaction (multiplex RT-PCR). The GI-specific and GIII-specific primer sets were able to specifically amplify the target gene from GI and GIII JEV, respectively. The limitations of detection were 0.00225 and 0.225 pfu for the GI-specific and GIII-specific primers, respectively. Using a mixture of GI-specific and GIII-specific primers, the multiplex RT-PCR was able to specifically detect and differentiate GI and GIII JEV. The multiplex RT-PCR was able to successfully differentiate GI and GIII virus in JEV-infected mosquitoes. Thus, a sensitive and specific multiplex RT-PCR system for the rapid detection and differentiation of GI and GIII JEV has been developed, and this test is likely to be valuable when carrying out mosquito-based JEV surveillance. © 2012 Blackwell Verlag GmbH.

  12. Development of a novel dengue virus serotype-specific multiplex real-time reverse transcription-polymerase chain reaction assay for blood screening.

    PubMed

    Tezuka, Kenta; Kuramitsu, Madoka; Okuma, Kazu; Nojima, Kiyoko; Araki, Kumiko; Shinohara, Naoya; Matsumoto, Chieko; Satake, Masahiro; Takasaki, Tomohiko; Saijo, Masayuki; Kurane, Ichiro; Hamaguchi, Isao

    2016-12-01

    Dengue fever is caused by four related RNA viruses of the genus Flavivirus, dengue virus (DENV)-1, -2, -3, and -4, which are transmitted to humans by mosquitoes. Although DENV is not endemic in Japan, an autochthonous dengue outbreak occurred in 2014. Several transfusion-transmitted cases have also been reported after the use of blood and plasma products in DENV-endemic countries. The aim of this study was to develop a novel multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay for DENV blood screening. Large-scale oligonucleotide screening was performed to obtain DENV-specific primers and probes using a variety of DENV clinical isolates. A multiplex RT-PCR assay was then developed using the identified oligonucleotides and the ability of this assay to detect DENV RNA was evaluated. A number of oligonucleotides suitable for DENV RNA detection were identified and a novel DENV serotype-specific multiplex RT-PCR assay was successfully established. Comparative analysis revealed that the multiplex assay could detect levels of viral contamination as low as 100 viral copies/mL. This established serotype-specific multiplex RT-PCR assay provides a simple, sensitive, and quantitative detection method for DENV, which could be applied in the screening of blood samples to prevent transfusion-transmitted DENV infection. © 2016 AABB.

  13. The genotyping of infectious bronchitis virus in Taiwan by a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction.

    PubMed

    Huang, Shr-Wei; Ho, Chia-Fang; Chan, Kun-Wei; Cheng, Min-Chung; Shien, Jui-Hung; Liu, Hung-Jen; Wang, Chi-Young

    2014-11-01

    Infectious bronchitis virus (IBV; Avian coronavirus) causes acute respiratory and reproductive and urogenital diseases in chickens. Following sequence alignment of IBV strains, a combination of selective primer sets was designed to individually amplify the IBV wild-type and vaccine strains using a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction (ARMS RT-PCR) approach. This system was shown to discriminate the IBV wild-type and vaccine strains. Moreover, an ARMS real-time RT-PCR (ARMS qRT-PCR) was combined with a high-resolution analysis (HRMA) to establish a melt curve analysis program. The specificity of the ARMS RT-PCR and the ARMS qRT-PCR was verified using unrelated avian viruses. Different melting temperatures and distinct normalized and shifted melting curve patterns for the IBV Mass, IBV H120, IBV TW-I, and IBV TW-II strains were detected. The new assays were used on samples of lung and trachea as well as virus from allantoic fluid and cell culture. In addition to being able to detect the presence of IBV vaccine and wild-type strains by ARMS RT-PCR, the IBV Mass, IBV H120, IBV TW-I, and IBV TW-II strains were distinguished using ARMS qRT-PCR by their melting temperatures and by HRMA. These approaches have acceptable sensitivities and specificities and therefore should be able to serve as options when carrying out differential diagnosis of IBV in Taiwan and China.

  14. Simultaneous detection and differentiation of three genotypes of Brassica yellows virus by multiplex reverse transcription-polymerase chain reaction.

    PubMed

    Zhang, Xiaoyan; Peng, Yanmei; Wang, Ying; Zhang, Zongying; Li, Dawei; Yu, Jialin; Han, Chenggui

    2016-11-22

    Brassica yellows virus (BrYV), proposed to be a new polerovirus species, three distinct genotypes (BrYV-A, BrYV-B and BrYV-C) have been described. This study was to develop a simple, rapid, sensitive, cost-effective method for simultaneous detection and differentiation of three genotypes of BrYV. In this study, a multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for simultaneous detection and differentiation of the three genotypes of BrYV. The three genotypes of BrYV and Tunip yellows virus (TuYV) could be differentiated simultaneously using six optimized specific oligonucleotide primers, including one universal primer for detecting BrYV, three BrYV genotype-specific primers, and a pair of primers for specific detection of TuYV. Primers were designed from conserved regions of each virus and their specificity was confirmed by sequencing PCR products. The mRT-PCR products were 278 bp for BrYV-A, 674 bp for BrYV-B, 505 bp for BrYV-C, and 205 bp for TuYV. Amplification of three target genotypes was optimized by increasing the PCR annealing temperatures to 62 °C. One to three fragments specific for the virus genotypes were simultaneously amplified from infected samples and identified by their specific molecular sizes in agarose gel electrophoresis. No specific products could be amplified from cDNAs of other viruses which could infect crucifer crops. Detection limits of the plasmids for multiplex PCR were 100 fg for BrYV-A and BrYV-B, 10 pg for BrYV-C, and 1 pg for TuYV, respectively. The mRT-PCR was applied successfully for detection of three BrYV genotypes from field samples collected in China. The simple, rapid, sensitive, and cost-effective mRT-PCR was developed successfully for detection and differentiation of the three genotypes of BrYV.

  15. Utilization of the respiratory virus multiplex reverse transcription-polymerase chain reaction test for adult patients at a Korean tertiary care center.

    PubMed

    Ahn, Mi Young; Choi, Seong-Ho; Chung, Jin-Won; Kim, Hye Ryoun

    2015-01-01

    Respiratory viruses (RVs) are considered to be important respiratory pathogens in adult patients, and the multiplex reverse transcription-polymerase chain reaction (RT-PCR) test is used frequently in adult patients with respiratory infections. However, clinical data regarding utilization of the multiplex RT-PCR test for RVs are lacking. We investigated the utilization of the multiplex RT-PCR test for RVs at Chung-Ang University Hospital in Seoul, Korea, between January 2012 and April 2013. During the study period, the multiplex RT-PCR test was performed for 291 adult patients. The test frequency was 4.9% of rapid influenza antigen detection tests and 0.8% of respiratory bacterial culture studies. A turnaround time of < 48 hours was observed in 25.9% of positive tests. Most of the tests were performed for admitted patients (97.9%) with a community-acquired infection (84.2%) during the flu season (82.5%). RVs were detected in 81 of 291 cases (27.8%). The RV positivity rates for community- and hospital-acquired infections did not differ (28.6% vs. 23.9%, p = 0.52). Of 166 patients with pneumonia, 44 (26.5%) had a viral infection. Among the patients with RV-associated pneumonia, an RV other than influenza was detected in 20 patients (45.4%). The multiplex RT-PCR test for RVs was infrequently performed at a tertiary care center, and the test results were often reported late. The test was most often performed for admitted adult patients with community-acquired infections during the flu season. The utilization of multiplex RT-PCR testing for RVs in current clinical practice should be improved.

  16. Multiplex real-time reverse transcription-PCR assay for determination of hepatitis C virus genotypes.

    PubMed

    Cook, Linda; Sullivan, KaWing; Krantz, Elizabeth M; Bagabag, Arthur; Jerome, Keith R

    2006-11-01

    A variety of methods have been used to determine hepatitis C virus (HCV) genotypes. Because therapeutic decisions for chronic HCV-related hepatitis are made on the basis of genotype, it is important that genotype be accurately determined by clinical laboratories. Existing methods are often subjective, inaccurate, manual, time-consuming, and contamination prone. We therefore evaluated real-time reverse transcription-PCR (RT-PCR) reagents that have recently become commercially available (Abbott HCV Genotype ASR). The assay developed by our laboratory starts with purified RNA and can be performed in 4 to 5 h. An initial evaluation of 479 samples was done with a restriction fragment length polymorphism (RFLP) method and the RT-PCR assay, and discrepant samples were sequenced. An additional 1,200 samples were then tested, and data from all assays were used to evaluate the efficiency and specificity of each genotype-specific reaction. Good correlation between results by the two methods was seen. Discrepant samples included those indeterminate by the RT-PCR assay (n = 110) and a subset that were incorrectly called 2a by the RFLP method (n = 75). The real-time RT-PCR assay performed well with genotype 1, 2, and 3 samples. Inadequate numbers of samples were available to evaluate fully genotypes 4, 5, and 6. Analysis of each primer-probe set demonstrated that weak cross-reactive amplifications were common but usually did not interfere with the genotype determination. However, in about 1% of samples, two or more genotypes amplified at roughly equivalent amounts. Further studies are necessary to determine whether these mixed-genotype samples are true mixtures or a reflection of occasional cross-reactive amplifications.

  17. Simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-PCR assays.

    PubMed

    Coiras, M T; Aguilar, J C; García, M L; Casas, I; Pérez-Breña, P

    2004-03-01

    There is a need for rapid, sensitive, and accurate diagnosis of lower respiratory tract infections in children, elderly, and immunocompromised patients, who are susceptible to serious complications. The multiplex RT-nested PCR assay has been used widely for simultaneous detection of non-related viruses involved in infectious diseases because of its high specificity and sensitivity. A new multiplex RT-PCR assay is described in this report. This approach includes nested primer sets targeted to conserve regions of human parainfluenza virus haemagglutinin, human coronavirus spike protein, and human enterovirus and rhinovirus polyprotein genes. It permits rapid, sensitive, and simultaneous detection and typing of the four types of parainfluenza viruses (1, 2, 3, 4AB), human coronavirus 229E and OC43, and the generic detection of enteroviruses and rhinoviruses. The testing of 201 clinical specimens with this multiplex assay along with other one formerly described by our group to simultaneously detect and type the influenza viruses, respiratory syncytial viruses, and a generic detection of all serotypes of adenovirus, covers the detection of most viruses causing respiratory infectious disease in humans. The results obtained were compared with conventional viral culture, immunofluorescence assay, and a third multiplex RT-PCR assay for all human parainfluenza viruses types described previously. In conclusion, both multiplex RT-PCR assays provide a system capable of detecting and identifying simultaneously 14 different respiratory viruses in clinical specimens with high sensitivity and specificity, being useful for routine diagnosis and survey of these viruses within the population.

  18. Simultaneous detection of the seven main tomato-infecting RNA viruses by two multiplex reverse transcription polymerase chain reactions.

    PubMed

    Panno, Stefano; Davino, Salvatore; Rubio, Luis; Rangel, Ezequiel; Davino, Mario; García-Hernández, Jorge; Olmos, Antonio

    2012-12-01

    Cucumber mosaic virus, Tomato spotted wilt virus, Tomato mosaic virus, Tomato chlorosis virus, Pepino mosaic virus, Torrado tomato virus and Tomato infectious chlorosis virus cause serious damage and significant economic losses in tomato crops worldwide. The early detection of these pathogens is essential for preventing the viruses from spreading and improving their control. In this study, a procedure based on two multiplex RT-PCRs was developed for the sensitive and reliable detection of these seven viruses. Serial dilutions of positive controls were analysed by this methodology, and the results were compared with those obtained by ELISA and singleplex versions of RT-PCR. The multiplex and singleplex RT-PCR assays were able to detect specific targets at the same dilution and were 100 times more sensitive than ELISA. The multiplex versions were able to detect composite samples containing different concentrations of specific targets at ratios from 1:1 to 1:1000. In addition, 45 symptomatic tomato samples collected in different tomato-growing areas of Sicily (Italy) were analysed by multiplex RT-PCR, singleplex RT-PCR and commercially available ELISA tests. Similar results were obtained using the RT-PCR techniques, with a higher sensitivity than ELISA, revealing a common occurrence of mixed infections and confirming the presence of these seven virus species in Italy.

  19. Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses

    PubMed Central

    Myers, Todd; Guevara, Carolina; Jungkind, Donald; Williams, Maya; Houng, Huo-Shu

    2016-01-01

    Dengue virus (DENV) and chikungunya virus (CHIKV) are important human pathogens with common transmission vectors and similar clinical presentations. Patient care may be impacted by the misdiagnosis of DENV and CHIKV in areas where both viruses cocirculate. In this study, we have developed and validated a one-step multiplex reverse transcriptase PCR (RT-PCR) to simultaneously detect, quantify, and differentiate between four DENV serotypes (pan-DENV) and chikungunya virus. The assay uses TaqMan technology, employing two forward primers, three reverse primers, and four fluorophore-labeled probes in a single-reaction format. Coextracted and coamplified RNA was used as an internal control (IC), and in vitro-transcribed DENV and CHIKV RNAs were used to generate standard curves for absolute quantification. The diagnostic 95% limits of detection (LOD) within the linear range were 50 and 60 RNA copies/reaction for DENV (serotypes 1 to 4) and CHIKV, respectively. Our assay was able to detect 53 different strains of DENV, representing four serotypes, and six strains of CHIKV. No cross-reactivity was observed with related flaviviruses and alphaviruses, To evaluate diagnostic sensitivity and specificity, 89 clinical samples positive or negative for DENV (serotypes 1 to 4) and CHIKV by the standard virus isolation method were tested in our assay. The multiplex RT-PCR assay showed 95% sensitivity and 100% specificity for DENV and 100% sensitivity and specificity for CHIKV. With an assay turnaround time of less than 2 h, including extraction of RNA, the multiplex quantitative RT-PCR assay provides rapid diagnosis for the differential detection of two clinically indistinguishable diseases, whose geographical occurrence is increasingly overlapping. PMID:27098955

  20. Detection of genetically modified crops using multiplex asymmetric polymerase chain reaction and asymmetric hyperbranched rolling circle amplification coupled with reverse dot blot.

    PubMed

    Wang, Xiumin; Teng, Da; Guan, Qingfeng; Tian, Fang; Wang, Jianhua

    2015-04-15

    To meet the ever-increasing demand for detection of genetically modified crops (GMCs), low-cost, high-throughput and high-accuracy detection assays are needed. The new multiplex asymmetric polymerase chain reaction and asymmetric hyper-branched rolling circle amplification coupled with reverse dot blot (RDB) systems were developed to detect GMCs. Thirteen oligonucleotide probes were designed to identify endogenous targets (Lec1, Hmg and Sad1), event-specific targets (RRS-5C, RRS-3C, Bt176-3C and MON810-3C), screening targets (35S promoter and NOS terminator), and control targets (18S and PLX). Optimised conditions were as follows: tailed hybridization probes (1-2 pmol/l) were immobilized on a membrane by baking for 2h, and a 10:1 ratio of forward to reverse primers was used. The detection limits were 0.1 μg/l of 2% RRS and 0.5 ng/l of DNA from genetically modified (GM) soybean. These results indicate that the RDB assay could be used to detect multiplex target genes of GMCs rapidly and inexpensively.

  1. A multiplexed reverse transcriptase PCR assay for identification of viral respiratory pathogens at point-of-care

    SciTech Connect

    Letant, S E; .Ortiz, J I; Tammero, L; Birch, J M; Derlet, R W; Cohen, S; Manning, D; McBride, M T

    2007-04-11

    We have developed a nucleic acid-based assay that is rapid, sensitive, specific, and can be used for the simultaneous detection of 5 common human respiratory pathogens including influenza A, influenza B, parainfluenza type 1 and 3, respiratory syncytial virus, and adenovirus group B, C, and E. Typically, diagnosis on an un-extracted clinical sample can be provided in less than 3 hours, including sample collection, preparation, and processing, as well as data analysis. Such a multiplexed panel would enable rapid broad-spectrum pathogen testing on nasal swabs, and therefore allow implementation of infection control measures, and timely administration of antiviral therapies. This article presents a summary of the assay performance in terms of sensitivity and specificity. Limits of detection are provided for each targeted respiratory pathogen, and result comparisons are performed on clinical samples, our goal being to compare the sensitivity and specificity of the multiplexed assay to the combination of immunofluorescence and shell vial culture currently implemented at the UCDMC hospital. Overall, the use of the multiplexed RT-PCR assay reduced the rate of false negatives by 4% and reduced the rate of false positives by up to 10%. The assay correctly identified 99.3% of the clinical negatives, 97% of adenovirus, 95% of RSV, 92% of influenza B, and 77% of influenza A without any extraction performed on the clinical samples. The data also showed that extraction will be needed for parainfluenza virus, which was only identified correctly 24% of the time on un-extracted samples.

  2. Diagnostic Evaluation of Multiplexed Reverse Transcription-PCR Microsphere Array Assay for Detection of Foot-and-Mouth and Look-Alike Disease Viruses▿

    PubMed Central

    Hindson, Benjamin J.; Reid, Scott M.; Baker, Brian R.; Ebert, Katja; Ferris, Nigel P.; Tammero, Lance F. Bentley; Lenhoff, Raymond J.; Naraghi-Arani, Pejman; Vitalis, Elizabeth A.; Slezak, Thomas R.; Hullinger, Pamela J.; King, Donald P.

    2008-01-01

    A high-throughput multiplexed assay was developed for the differential laboratory detection of foot-and-mouth disease virus (FMDV) from viruses that cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses by using multiplexed reverse transcription-PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the 17 primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR assay was evaluated using 287 field samples, including 247 samples (213 true-positive samples and 35 true-negative samples) from suspected cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true-negative samples collected from healthy animals. The mRT-PCR assay results were compared to those of two singleplex rRT-PCR assays, using virus isolation with antigen enzyme-linked immunosorbent assays as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% (95% confidence interval [CI], 89.8 to 96.4%), and the sensitivity was 98.1% (95% CI, 95.3 to 99.3%) for the two singleplex rRT-PCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses, such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n = 2) and bovine viral diarrhea virus (n = 2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized by using focused single-target rRT-PCR assays. PMID:18216216

  3. Detection of influenza viruses by coupling multiplex reverse-transcription loop-mediated isothermal amplification with cascade invasive reaction using nanoparticles as a sensor

    PubMed Central

    Ge, Yiyue; Zhou, Qiang; Zhao, Kangchen; Chi, Ying; Liu, Bin; Min, Xiaoyan; Shi, Zhiyang; Zou, Bingjie; Cui, Lunbiao

    2017-01-01

    Influenza virus infections represent a worldwide public health and economic problem due to the significant morbidity and mortality caused by seasonal epidemics and pandemics. Sensitive and convenient methodologies for detection of influenza viruses are essential for further disease control. Loop-mediated isothermal amplification (LAMP) is the most commonly used method of nucleic acid isothermal amplification. However, with regard to multiplex LAMP, differentiating the ladder-like LAMP products derived from multiple targets is still challenging today. The requirement of specialized instruments has further hindered the on-site application of multiplex LAMP. We have developed an integrated assay coupling multiplex reverse transcription LAMP with cascade invasive reaction using nanoparticles (mRT-LAMP-CIRN) as a sensor for the detection of three subtypes of influenza viruses: A/H1N1pdm09, A/H3 and influenza B. The analytic sensitivities of the mRT-LAMP-CIRN assay were 101 copies of RNA for both A/H1N1pdm09 and A/H3, and 102 copies of RNA for influenza B. This assay demonstrated highly specific detection of target viruses and could differentiate them from other genetically or clinically related viruses. Clinical specimen analysis showed the mRT-LAMP-CIRN assay had an overall sensitivity and specificity of 98.3% and 100%, respectively. In summary, the mRT-LAMP-CIRN assay is highly sensitive and specific, and can be used as a cost-saving and instrument-free method for the detection of influenza viruses, especially for on-site use. PMID:28435249

  4. Rapid and sensitive detection of 68 unique varicella zoster virus gene transcripts in five multiplex reverse transcription-polymerase chain reactions

    PubMed Central

    Nagel, Maria A.; Gilden, Don; Shade, Ted; Gao, Bifeng; Cohrs, Randall J.

    2010-01-01

    Varicella zoster virus (VZV) becomes latent in ganglionic neurons along the entire neuraxis. Although all predicted VZV open reading frames (ORFs) have been detected by macroarray and microarray analysis in virus-infected cells in culture where virus gene expression is abundant, array technology does not detect all VZV gene transcripts in latently-infected human ganglia, where the abundance of ganglionic RNA is low and VZV gene transcription is highly variable. Using reverse transcription-polymerase chain reaction (RT-PCR) and the GenomeLab Genetic Analysis System (GeXPS), transcripts mapping to all 68 predicted unique VZV ORFs were detected in VZV-infected MeWo cells. Oligonucleotide primers contained both VZV- and cell-specific sequences linked to universal DNA sequences such that PCR amplification products were of predetermined sizes. Amplification products were resolved by capillary gel electrophoresis and detected by fluorescence spectrophotometry. Serial dilutions of total RNA extracted from VZV-infected MeWo cells were analyzed in parallel by GeXPS multiplex RT-PCR and real-time RT-PCR. GeXPS technology detected as few as 20 copies of VZV gene-specific transcripts. Only five multiplex RT-PCR assays were needed to analyze the entire VZV transcriptome. This technology will allow rapid analysis of all VZV genes transcribed during latency in human ganglia. PMID:19109999

  5. Is real-time PCR-based diagnosis similar in performance to routine parasitological examination for the identification of Giardia intestinalis, Cryptosporidium parvum/Cryptosporidium hominis and Entamoeba histolytica from stool samples? Evaluation of a new commercial multiplex PCR assay and literature review.

    PubMed

    Laude, A; Valot, S; Desoubeaux, G; Argy, N; Nourrisson, C; Pomares, C; Machouart, M; Le Govic, Y; Dalle, F; Botterel, F; Bourgeois, N; Cateau, E; Leterrier, M; Le Pape, P; Morio, F

    2016-02-01

    Microscopy is the reference standard for routine laboratory diagnosis in faecal parasitology but there is growing interest in alternative methods to overcome the limitations of microscopic examination, which is time-consuming and highly dependent on an operator's skills and expertise. Compared with microscopy, DNA detection by PCR is simple and can offer a better turnaround time. However, PCR performances remain difficult to assess as most studies have been conducted on a limited number of positive clinical samples and used in-house PCR methods. Our aim was to evaluate a new multiplex PCR assay (G-DiaParaTrio; Diagenode Diagnostics), targeting Giardia intestinalis, Cryptosporidium parvum/Cryptosporidium hominis and Entamoeba histolytica. To minimize the turnaround time, PCR was coupled with automated DNA extraction (QiaSymphony; Qiagen). The PCR assay was evaluated using a reference panel of 185 samples established by routine microscopic examination using a standardized protocol including Ziehl-Neelsen staining and adhesin detection by ELISA (E. histolytica II; TechLab). This panel, collected from 12 French parasitology laboratories, included 135 positive samples for G. intestinalis (n = 38), C. parvum/C. hominis (n = 26), E. histolytica (n = 5), 21 other gastrointestinal parasites, together with 50 negative samples. In all, the G-DiaParaTrio multiplex PCR assay identified 38 G. intestinalis, 25 C. parvum/C. hominis and five E. histolytica leading to sensitivity/specificity of 92%/100%, 96%/100% and 100%/100% for G. intestinalis, C. parvum/C. hominis and E. histolytica, respectively. This new multiplex PCR assay offers fast and reliable results, similar to microscopy-driven diagnosis for the detection of these gastrointestinal protozoa, allowing its implementation in routine clinical practice.

  6. Optimized PCR-based detection of mycoplasma.

    PubMed

    Dobrovolny, Paige L; Bess, Dan

    2011-06-20

    The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research. The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination. PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per μl. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines. Given the extreme sensitivity of the kit, great

  7. Comparison of Viral Isolation and Multiplex Real-Time Reverse Transcription-PCR for Confirmation of Respiratory Syncytial Virus and Influenza Virus Detection by Antigen Immunoassays▿

    PubMed Central

    Liao, R. S.; Tomalty, L. L.; Majury, A.; Zoutman, D. E.

    2009-01-01

    We evaluated the Prodesse ProFlu-1 real-time reverse transcription-PCR multiplex assay with the SmartCycler instrument for the detection of human respiratory syncytial virus (RSV) and influenza A and B viruses in comparison to conventional cell culture and antigen immunoassays with the BD Directigen A+B and Binax NOW RSV assays over two successive respiratory virus seasons. Ninety-two percent of the 361 specimens tested were nasopharyngeal aspirates obtained from individual patients, of which 119 were positive for RSV and 59 were positive for influenza virus. The median age of the patients whose specimens were positive for RSV and influenza virus were 6.3 months and 42.4 years, respectively. The specificity of all of the methods tested was ≥99%, and the individual sensitivities of NOW RSV, RSV culture, Directigen A+B, influenza virus culture, and the Proflu-1 PCR for influenza/RSV were 82% (95% confidence interval [CI], 73 to 88), 57% (95% CI, 44 to 69), 59% (95% CI, 44 to 72), 54% (95% CI, 38 to 69), and 98% (95% CI, 93 to 100)/95% (95% CI, 85 to 99), respectively. In a clinical setting where viral isolation is performed to confirm rapid antigen immunoassay results for these common respiratory viruses, one-step real-time reverse transcriptase PCR testing can be a more sensitive and timely confirmatory method. PMID:19129410

  8. Multiplex detection of agricultural pathogens

    DOEpatents

    Siezak, Thomas R.; Gardner, Shea; Torres, Clinton; Vitalis, Elizabeth; Lenhoff, Raymond J.

    2013-01-15

    Described are kits and methods useful for detection of agricultural pathogens in a sample. Genomic sequence information from agricultural pathogens was analyzed to identify signature sequences, e.g., polynucleotide sequences useful for confirming the presence or absence of a pathogen in a sample. Primer and probe sets were designed and optimized for use in a PCR based, multiplexed Luminex assay and/or an array assay to successfully identify the presence or absence of pathogens in a sample.

  9. Multiplex detection of agricultural pathogens

    DOEpatents

    McBride, Mary Teresa; Slezak, Thomas Richard; Messenger, Sharon Lee

    2010-09-14

    Described are kits and methods useful for detection of seven agricultural pathogens (BPSV; BHV; BVD; FMDV; BTV; SVD; and VESV) in a sample. Genomic sequence information from 7 agricultural pathogens was analyzed to identify signature sequences, e.g., polynucleotide sequences useful for confirming the presence or absence of a pathogen in a sample. Primer and probe sets were designed and optimized for use in a PCR based, multiplexed Luminex assay to successfully identify the presence or absence of pathogens in a sample.

  10. design of multiplexed detection assays for identification of avian influenza a virus subtypes pathogenic to humans by SmartCycler real-time reverse transcription-PCR.

    PubMed

    Wang, Wei; Ren, Peijun; Mardi, Sek; Hou, Lili; Tsai, Cheguo; Chan, Kwok Hung; Cheng, Peter; Sheng, Jun; Buchy, Philippe; Sun, Bing; Toyoda, Tetsuya; Lim, Wilina; Peiris, J S Malik; Zhou, Paul; Deubel, Vincent

    2009-01-01

    Influenza A virus (IAV) epidemics are the result of human-to-human or poultry-to-human transmission. Tracking seasonal outbreaks of IAV and other avian influenza virus (AIV) subtypes that can infect humans, aquatic and migratory birds, poultry, and pigs is essential for epidemiological surveillance and outbreak alerts. In this study, we performed four real-time reverse transcription-PCR (rRT-PCR) assays for identification of the IAV M and hemagglutinin (HA) genes from six known AIVs infecting pigs, birds, and humans. IAV M1 gene-positive samples tested by single-step rRT-PCR and a fluorogenic Sybr green I detection system were further processed for H5 subtype identification by using two-primer-set multiplex and Sybr green I rRT-PCR assays. H5 subtype-negative samples were then tested with either a TaqMan assay for subtypes H1 and H3 or a TaqMan assay for subtypes H2, H7, and H9 and a beacon multiplex rRT-PCR identification assay. The four-tube strategy was able to detect 10 RNA copies of the HA genes of subtypes H1, H2, H3, H5, and H7 and 100 RNA copies of the HA gene of subtype H9. At least six H5 clades of H5N1 viruses isolated in Southeast Asia and China were detected by that test. Using rRT-PCR assays for the M1 and HA genes in 202 nasopharyngeal swab specimens from children with acute respiratory infections, we identified a total of 39 samples positive for the IAV M1 gene and subtypes H1 and H3. When performed with a portable SmartCycler instrument, the assays offer an efficient, flexible, and reliable platform for investigations of IAV and AIV in remote hospitals and in the field.

  11. Profiling viral infections in grapevine using a randomly primed reverse transcription-polymerase chain reaction/macroarray multiplex platform.

    PubMed

    Thompson, J R; Fuchs, Marc; McLane, Heather; Celebi-Toprak, Fevziye; Fischer, Kael F; Potter, Jamie L; Perry, Keith L

    2014-02-01

    Crop-specific diagnostics to simultaneously detect a large number of pathogens provides an invaluable platform for the screening of vegetative material prior to its propagation. Here we report the use of what is to-date the largest published example of a crop-specific macroarray for the detection of 38 of the most prevalent or emergent viruses to infect grapevine. The reusable array consists of 1,578 virus-specific 60 to 70mer oligonucleotide probes and 19 plant and internal control probes spotted onto an 18 × 7 cm nylon membrane. In a survey of 99 grapevines from the United States and Europe, virus infections were detected in 46 selections of Vitis vinifera, V. labrusca, and interspecific hybrids. The majority of infected vines (30) was singly infected, while 16 were mixed-infected with viruses from two or more families. Representatives of the four main virus families Betaflexiviridae, Closteroviridae, Secoviridae, and Tymoviridae present in grapevines were found alone and in combination, with a notable bias in representation by members of the family Tymoviridae. This work demonstrates the utility of the macroarray platform for the multiplex detection of viruses in a single crop, its potential for characterizing grapevine virus associations, and usefulness for rapid diagnostics of introduced material in quarantine centers or in certification programs.

  12. Evidence of presence of Mycobacterium tuberculosis in bovine tissue samples by multiplex PCR: possible relevance to reverse zoonosis.

    PubMed

    Mittal, M; Chakravarti, S; Sharma, V; Sanjeeth, B S; Churamani, C P; Kanwar, N S

    2014-04-01

    Bovine tuberculosis, caused by Mycobacterium bovis, remains one of the most important zoonotic health concerns worldwide. The transmission of Mycobacterium tuberculosis from humans to animals also occurs especially in countries where there is close interaction of humans with the animals. In the present study, thirty bovine lung tissue autopsy samples from an organized dairy farm located in North India were screened for the presence of Mycobacterium tuberculosis complex by smear microscopy, histopathological findings and PCR. Differential diagnosis of M. tuberculosis and M. bovis was made based on the deletion of mce-3 operon in M. bovis. The present study found eight of these samples positive for M. tuberculosis by multiplex PCR. Sequencing was performed on two PCR-positive representative samples and on annotation, and BLAST analysis confirmed the presence of gene fragment specific to Mycobacterium tuberculosis. The presence of M. tuberculosis in all the positive samples raises the possibility of human-to-cattle transmission and possible adaptation of this organism in bovine tissues. This study accentuates the importance of screening and differential diagnosis of Mycobacterium tuberculosis complex in humans and livestock for adopting effective TB control and eradication programmes. © 2014 Blackwell Verlag GmbH.

  13. A multiplex reverse transcription-nested polymerase chain reaction for detection and differentiation of wild-type and vaccine strains of canine distemper virus.

    PubMed

    Si, Wei; Zhou, Shun; Wang, Zhao; Cui, Shang-jin

    2010-05-01

    A multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR) method was developed for the detection and differentiation of wild-type and vaccine strains of canine distemper virus (CDV). A pair of primers (P1 and P4) specific for CDV corresponding to the highly conserved region of the CDV genome were used as a common primer pair in the first-round PCR of the nested PCR. Primers P2 specific for CDV wild-type strains, were used as the forward primer together with the common reverse primer P4 in the second round of nested PCR. Primers P3, P5 specific for CDV wild-type strain or vaccine strain, were used as the forward primer together with the common reverse primer P4+P6 in the second round of nested PCR. A fragment of 177 bp was amplified from vaccine strain genomic RNA, and a fragment of 247 bp from wild-type strain genomic RNA in the RT-nPCR, and two fragments of 247 bp and 177 bp were amplified from the mixed samples of vaccine and wild-type strains. No amplification was achieved for uninfected cells, or cells infected with Newcastle disease virus (NDV), canine parvovirus (CPV), canine coronavirus (CCV), rabies virus (RV), or canine adenovirus (CAV). The RT-nPCR method was used to detect 30 field samples suspected of canine distemper from Heilongjiang and Jilin Provinces, and 51 samples in Shandong province. As a result of 30 samples, were found to be wild-type-like, and 5 to be vaccine-strain-like. The RT-nPCR method can be used to effectively detect and differentiate wild-type CDV-infected dogs from dogs vaccinated with CDV vaccine, and thus can be used in clinical detection and epidemiological surveillance.

  14. Evaluation of PCR-based preimplantation genetic diagnosis applied to monogenic diseases: a collaborative ESHRE PGD consortium study.

    PubMed

    Dreesen, Jos; Destouni, Aspasia; Kourlaba, Georgia; Degn, Birte; Mette, Wulf Christensen; Carvalho, Filipa; Moutou, Celine; Sengupta, Sioban; Dhanjal, Seema; Renwick, Pamela; Davies, Steven; Kanavakis, Emmanouel; Harton, Gary; Traeger-Synodinos, Joanne

    2014-08-01

    Preimplantation genetic diagnosis (PGD) for monogenic disorders currently involves polymerase chain reaction (PCR)-based methods, which must be robust, sensitive and highly accurate, precluding misdiagnosis. Twelve adverse misdiagnoses reported to the ESHRE PGD-Consortium are likely an underestimate. This retrospective study, involving six PGD centres, assessed the validity of PCR-based PGD through reanalysis of untransferred embryos from monogenic-PGD cycles. Data were collected on the genotype concordance at PGD and follow-up from 940 untransferred embryos, including details on the parameters of PGD cycles: category of monogenic disease, embryo morphology, embryo biopsy and genotype assay strategy. To determine the validity of PCR-based PGD, the sensitivity (Se), specificity (Sp) and diagnostic accuracy were calculated. Stratified analyses were also conducted to assess the influence of the parameters above on the validity of PCR-based PGD. The analysis of overall data showed that 93.7% of embryos had been correctly classified at the time of PGD, with Se of 99.2% and Sp of 80.9%. The stratified analyses found that diagnostic accuracy is statistically significantly higher when PGD is performed on two cells versus one cell (P=0.001). Se was significantly higher when multiplex protocols versus singleplex protocols were applied (P=0.005), as well as for PGD applied on cells from good compared with poor morphology embryos (P=0.032). Morphology, however, did not affect diagnostic accuracy. Multiplex PCR-based methods on one cell, are as robust as those on two cells regarding false negative rate, which is the most important criteria for clinical PGD applications. Overall, this study demonstrates the validity, robustness and high diagnostic value of PCR-based PGD.

  15. Evaluation of PCR-based preimplantation genetic diagnosis applied to monogenic diseases: a collaborative ESHRE PGD consortium study

    PubMed Central

    Dreesen, Jos; Destouni, Aspasia; Kourlaba, Georgia; Degn, Birte; Mette, Wulf Christensen; Carvalho, Filipa; Moutou, Celine; Sengupta, Sioban; Dhanjal, Seema; Renwick, Pamela; Davies, Steven; Kanavakis, Emmanouel; Harton, Gary; Traeger-Synodinos, Joanne

    2014-01-01

    Preimplantation genetic diagnosis (PGD) for monogenic disorders currently involves polymerase chain reaction (PCR)-based methods, which must be robust, sensitive and highly accurate, precluding misdiagnosis. Twelve adverse misdiagnoses reported to the ESHRE PGD-Consortium are likely an underestimate. This retrospective study, involving six PGD centres, assessed the validity of PCR-based PGD through reanalysis of untransferred embryos from monogenic-PGD cycles. Data were collected on the genotype concordance at PGD and follow-up from 940 untransferred embryos, including details on the parameters of PGD cycles: category of monogenic disease, embryo morphology, embryo biopsy and genotype assay strategy. To determine the validity of PCR-based PGD, the sensitivity (Se), specificity (Sp) and diagnostic accuracy were calculated. Stratified analyses were also conducted to assess the influence of the parameters above on the validity of PCR-based PGD. The analysis of overall data showed that 93.7% of embryos had been correctly classified at the time of PGD, with Se of 99.2% and Sp of 80.9%. The stratified analyses found that diagnostic accuracy is statistically significantly higher when PGD is performed on two cells versus one cell (P=0.001). Se was significantly higher when multiplex protocols versus singleplex protocols were applied (P=0.005), as well as for PGD applied on cells from good compared with poor morphology embryos (P=0.032). Morphology, however, did not affect diagnostic accuracy. Multiplex PCR-based methods on one cell, are as robust as those on two cells regarding false negative rate, which is the most important criteria for clinical PGD applications. Overall, this study demonstrates the validity, robustness and high diagnostic value of PCR-based PGD. PMID:24301057

  16. Development of a Microsphere-Based Serologic Multiplexed Fluorescent Immunoassay and a Reverse Transcriptase PCR Assay To Detect Murine Norovirus 1 Infection in Mice

    PubMed Central

    Hsu, Charlie C.; Wobus, Christiane E.; Steffen, Earl K.; Riley, Lela K.; Livingston, Robert S.

    2005-01-01

    Murine norovirus 1 (MNV-1) is a newly recognized pathogen of mice that causes lethal infection in mice deficient in components of the innate immune response but not in wild-type 129 mice. In this study, in vitro-propagated MNV-1 was used as antigen to develop a multiplexed fluorescent immunoassay (MFI) to detect antibodies to MNV-1 in infected mice. The MNV-1 MFI was 100% specific and 100% sensitive in detecting anti-MNV-1 antibody in sera from experimentally infected mice. Testing of a large number of mouse serum samples (n = 12,639) submitted from contemporary laboratory mouse colonies in the United States and Canada revealed that 22.1% of these sera contained antibodies to MNV-1, indicating infection with MNV-1 is widespread in research mice. In addition, a reverse transcriptase PCR primer pair with a sensitivity of 25 virus copies was developed and used to demonstrate that MNV-1 RNA could be detected in the spleen, mesenteric lymph node, and jejunum from some experimentally infected mice 5 weeks postinoculation. These diagnostic assays provide the necessary tools to define the MNV-1 infection status of research mice and to aid in the establishment of laboratory mouse colonies free of MNV-1 infection. PMID:16210475

  17. Detection and identification of human parainfluenza viruses 1, 2, 3, and 4 in clinical samples of pediatric patients by multiplex reverse transcription-PCR.

    PubMed

    Aguilar, J C; Pérez-Breña, M P; García, M L; Cruz, N; Erdman, D D; Echevarría, J E

    2000-03-01

    We describe a multiplex reverse transcription-PCR (m-RT-PCR) assay that is able to detect and differentiate all known human parainfluenza viruses (HPIVs). Serial dilution experiments with reference strains that compared cell culture isolation and m-RT-PCR showed sensitivities ranging from 0.0004 50% tissue culture infective dose (TCID(50)) for HPIV type 4B (HPIV-4B) to 32 TCID(50)s for HPIV-3. As few as 10 plasmids containing HPIV PCR products could be detected in all cases. When 201 nasopharyngeal aspirate specimens from pediatric patients hospitalized for lower respiratory illness were tested, m-RT-PCR assay detected 64 HPIVs (24 HPIV-3, 23 HPIV-1, 10 HPIV-4, and 7 HPIV-2), while only 42 of them (21 HPIV-1, 14 HPIV-3, 6 HPIV-2, and 1 HPIV-4 isolates) grew in cell culture. Our m-RT-PCR assay was more sensitive than either cell culture isolation or indirect immunofluorescence with monoclonal antibodies for the detection of HPIV infections. Also, HPIV-4 was more frequently detected than HPIV-2 in this study, suggesting that it may have been underestimated as a lower respiratory tract pathogen because of the insensitivity of cell culture.

  18. Simultaneous Detection of Rift Valley Fever, Bluetongue, Rinderpest, and Peste des Petits Ruminants Viruses by a Single-Tube Multiplex Reverse Transcriptase-PCR Assay Using a Dual-Priming Oligonucleotide System▿

    PubMed Central

    Yeh, Jung-Yong; Lee, Ji-Hye; Seo, Hyun-Ji; Park, Jee-Yong; Moon, Jin-San; Cho, In-Soo; Choi, In-Soo; Park, Seung-Yong; Song, Chang-Seon; Lee, Joong-Bok

    2011-01-01

    The aim of this study was to develop a highly sensitive and specific one-step multiplex reverse transcriptase PCR assay for the simultaneous and differential detection of Rift Valley Fever virus (RVFV), bluetongue virus (BTV), rinderpest virus (RPV), and Peste des petits ruminants virus (PPRV). These viruses cause mucosal lesions in cattle, sheep, and goats, and they are difficult to differentiate from one another based solely on their clinical presentation in suspected disease cases. In this study, we developed a multiplex reverse transcriptase PCR to detect these viruses using a novel dual-priming oligonucleotide (DPO). The DPO contains two separate priming regions joined by a polydeoxyinosine linker, which blocks extension of nonspecifically primed templates and consistently allows high PCR specificity even under less-than-optimal PCR conditions. A total of 19 DPO primers were designed to detect and discriminate between RVFV, BTV, RPV, and PPRV by the generation of 205-, 440-, 115-, and 243-bp cDNA products, respectively. The multiplex reverse transcriptase PCR described here enables the early diagnosis of these four viruses and may also be useful as part of a testing regime for cattle, sheep, or goats exhibiting similar clinical signs, including mucosal lesions. PMID:21307219

  19. Simultaneous detection of Rift Valley Fever, bluetongue, rinderpest, and Peste des petits ruminants viruses by a single-tube multiplex reverse transcriptase-PCR assay using a dual-priming oligonucleotide system.

    PubMed

    Yeh, Jung-Yong; Lee, Ji-Hye; Seo, Hyun-Ji; Park, Jee-Yong; Moon, Jin-San; Cho, In-Soo; Choi, In-Soo; Park, Seung-Yong; Song, Chang-Seon; Lee, Joong-Bok

    2011-04-01

    The aim of this study was to develop a highly sensitive and specific one-step multiplex reverse transcriptase PCR assay for the simultaneous and differential detection of Rift Valley Fever virus (RVFV), bluetongue virus (BTV), rinderpest virus (RPV), and Peste des petits ruminants virus (PPRV). These viruses cause mucosal lesions in cattle, sheep, and goats, and they are difficult to differentiate from one another based solely on their clinical presentation in suspected disease cases. In this study, we developed a multiplex reverse transcriptase PCR to detect these viruses using a novel dual-priming oligonucleotide (DPO). The DPO contains two separate priming regions joined by a polydeoxyinosine linker, which blocks extension of nonspecifically primed templates and consistently allows high PCR specificity even under less-than-optimal PCR conditions. A total of 19 DPO primers were designed to detect and discriminate between RVFV, BTV, RPV, and PPRV by the generation of 205-, 440-, 115-, and 243-bp cDNA products, respectively. The multiplex reverse transcriptase PCR described here enables the early diagnosis of these four viruses and may also be useful as part of a testing regime for cattle, sheep, or goats exhibiting similar clinical signs, including mucosal lesions.

  20. Reversals.

    ERIC Educational Resources Information Center

    National Center on Educational Media and Materials for the Handicapped, Columbus, OH.

    Selected from the National Instructional Materials Information System (NIMIS)--a computer based on-line interactive retrieval system on special education materials--the bibliography covers nine materials for remediating reversals in handicapped students at the early childhood and elementary levels. Entries are presented in order of NIMIS accession…

  1. Development and validation of a multiplex reverse transcription quantitative PCR (RT-qPCR) assay for the rapid detection of Citrus tristeza virus, Citrus psorosis virus, and Citrus leaf blotch virus.

    PubMed

    Osman, Fatima; Hodzic, Emir; Kwon, Sun-Jung; Wang, Jinbo; Vidalakis, Georgios

    2015-08-01

    A single real-time multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay for the simultaneous detection of Citrus tristeza virus (CTV), Citrus psorosis virus (CPsV), and Citrus leaf blotch virus (CLBV) was developed and validated using three different fluorescently labeled minor groove binding qPCR probes. To increase the detection reliability, coat protein (CP) genes from large number of different isolates of CTV, CPsV and CLBV were sequenced and a multiple sequence alignment was generated with corresponding CP sequences from the GenBank and a robust multiplex RT-qPCR assay was designed. The capacity of the multiplex RT-qPCR assay in detecting the viruses was compared to singleplex RT-qPCR designed specifically for each virus and was assessed using multiple virus isolates from diverse geographical regions and citrus species as well as graft-inoculated citrus plants infected with various combination of the three viruses. No significant difference in detection limits was found and specificity was not affected by the inclusion of the three assays in a multiplex RT-qPCR reaction. Comparison of the viral load for each virus using singleplex and multiplex RT-qPCR assays, revealed no significant differences between the two assays in virus detection. No significant difference in Cq values was detected when using one-step and two-step multiplex RT-qPCR detection formats. Optimizing the RNA extraction technique for citrus tissues and testing the quality of the extracted RNA using RT-qPCR targeting the cytochrome oxidase citrus gene as an RNA specific internal control proved to generate better diagnostic assays. Results showed that the developed multiplex RT-qPCR can streamline viruses testing of citrus nursery stock by replacing three separate singleplex assays, thus reducing time and labor while retaining the same sensitivity and specificity. The three targeted RNA viruses are regulated pathogens for California's mandatory "Section 3701

  2. Simultaneous detection of influenza A, B, and C viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-PCR assay.

    PubMed

    Coiras, M T; Pérez-Breña, P; García, M L; Casas, I

    2003-01-01

    The clinical presentation of infections caused by the heterogeneous group of the respiratory viruses can be very similar. Thus, the implementation of virological assays that rapidly identify the most important viruses involved is of great interest. A new multiplex reverse transcription nested-polymerase chain reaction (RT-PCR) assay that is able to detect and type different respiratory viruses simultaneously is described. Primer sets were targeted to conserved regions of nucleoprotein genes of the influenza viruses, fusion protein genes of respiratory syncytial viruses (RSV), and hexon protein genes of adenoviruses. Individual influenza A, B, and C viruses, RSV (A and B), and a generic detection of the 48 serotypes of adenoviruses were identified and differentiated by the size of the PCR products. An internal amplification control was included in the reaction mixture to exclude false-negative results due to sample inhibitors and/or extraction failure. Detection levels of 0.1 and 0.01 TCID50 of influenza A and B viruses and 1-10 molecules of cloned amplified products of influenza C virus, RSV A and B, and adenovirus serotype 1 were achieved. The specificity was checked using specimens containing other respiratory viruses and no amplified products were detected in any case. A panel of 290 respiratory specimens from the 1999-2000 and 2000-2001 seasons was used to validate the assay. Accurately amplifying RNA from influenza and RSV prototype strains and DNA from all adenovirus serotypes demonstrates the use of this method for both laboratory routine diagnosis and surveillance of all these viruses.

  3. Simultaneously Typing Nine Serotypes of Enteroviruses Associated with Hand, Foot, and Mouth Disease by a GeXP Analyzer-Based Multiplex Reverse Transcription-PCR Assay

    PubMed Central

    Hu, Xiumei; Zhang, Yong; Zhou, Xiaomian; Xu, Banglao; Yang, Mengjie; Wang, Miao; Zhang, Chen; Li, Jin; Bai, Ruyin

    2012-01-01

    Hand, foot, and mouth disease (HFMD) is a contagious enteroviral disease occurring primarily in young children and caused by enterovirus 71 (EV71), coxsackievirus A16 (CVA16), and other serotypes of coxsackievirus and echovirus. In this study, a GeXP analyzer-based multiplex reverse transcription (RT)-PCR assay (GeXP assay) consisting of chimeric primer-based PCR amplification with fluorescent labeling and capillary electrophoresis separation was developed to simultaneously identify nine serotypes of enteroviruses associated with HFMD in China, including EV71, CVA16, CVA4, -5, -9, and -10, and CVB1, -3, and -5. The RNAs extracted from cell cultures of viral isolates and synthetic RNAs via in vitro transcription were used to analyze the specificity and sensitivity of the assay. The GeXP assay detected as little as 0.03 tissue culture infective dose (TCID50) of EV71 and CVA16, 10 copies of panenterovirus, EV71, CVA16, CVB1, and CVB5, and 100 copies of 10 (including panenterovirus) premixed RNA templates. A total of 180 stool specimens collected from HFMD patients and persons suspected of having HFMD were used to evaluate the clinical performance of this assay. In comparison with the results of conventional methods, the sensitivities of the GeXP assay for detection of panenterovirus, EV71, and CVA16 were 98.79% (163/165), 91.67% (44/48), and 91.67% (33/36), respectively, and the specificities were 80.00% (12/15), 98.48% (130/132), and 100% (144/144), respectively. The concordance of typing seven other serotypes of enteroviruses with the results of conventional methods was 92.59% (25/27). In conclusion, the GeXP assay is a rapid, cost-effective, and high-throughput method for typing nine serotypes of HFMD-associated enteroviruses. PMID:22116146

  4. Development of a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction assay for the differential diagnosis of Feline leukemia virus vaccine and wild strains.

    PubMed

    Ho, Chia-Fang; Chan, Kun-Wei; Yang, Wei-Cheng; Chiang, Yu-Chung; Chung, Yang-Tsung; Kuo, James; Wang, Chi-Young

    2014-07-01

    A multiplex amplification refractory mutation system reverse transcription polymerase chain reaction (ARMS RT-PCR) was developed for the differential diagnosis of Feline leukemia virus (FeLV) vaccine and wild-type strains based on a point mutation between the vaccine strain (S) and the wild-type strain (T) located in the p27 gene. This system was further upgraded to obtain a real-time ARMS RT-PCR (ARMS qRT-PCR) with a high-resolution melt analysis (HRMA) platform. The genotyping of various strains of FeLV was determined by comparing the HRMA curves with the defined wild-type FeLV (strain TW1), and the results were expressed as a percentage confidence. The detection limits of ARMS RT-PCR and ARMS qRT-PCR combined with HRMA were 100 and 1 copies of transcribed FeLV RNA per 0.5 ml of sample, respectively. No false-positive results were obtained with 6 unrelated pathogens and 1 feline cell line. Twelve FeLV Taiwan strains were correctly identified using ARMS qRT-PCR combined with HRMA. The genotypes of the strains matched the defined FeLV wild-type strain genotype with at least 91.17% confidence. A higher degree of sequence polymorphism was found throughout the p27 gene compared with the long terminal repeat region. In conclusion, the current study describes the phylogenetic relationship of the FeLV Taiwan strains and demonstrates that the developed ARMS RT-PCR assay is able to be used to detect the replication of a vaccine strain that has not been properly inactivated, thus acting as a safety check for the quality of FeLV vaccines.

  5. Simultaneously typing nine serotypes of enteroviruses associated with hand, foot, and mouth disease by a GeXP analyzer-based multiplex reverse transcription-PCR assay.

    PubMed

    Hu, Xiumei; Zhang, Yong; Zhou, Xiaomian; Xu, Banglao; Yang, Mengjie; Wang, Miao; Zhang, Chen; Li, Jin; Bai, Ruyin; Xu, Wenbo; Ma, Xuejun

    2012-02-01

    Hand, foot, and mouth disease (HFMD) is a contagious enteroviral disease occurring primarily in young children and caused by enterovirus 71 (EV71), coxsackievirus A16 (CVA16), and other serotypes of coxsackievirus and echovirus. In this study, a GeXP analyzer-based multiplex reverse transcription (RT)-PCR assay (GeXP assay) consisting of chimeric primer-based PCR amplification with fluorescent labeling and capillary electrophoresis separation was developed to simultaneously identify nine serotypes of enteroviruses associated with HFMD in China, including EV71, CVA16, CVA4, -5, -9, and -10, and CVB1, -3, and -5. The RNAs extracted from cell cultures of viral isolates and synthetic RNAs via in vitro transcription were used to analyze the specificity and sensitivity of the assay. The GeXP assay detected as little as 0.03 tissue culture infective dose (TCID(50)) of EV71 and CVA16, 10 copies of panenterovirus, EV71, CVA16, CVB1, and CVB5, and 100 copies of 10 (including panenterovirus) premixed RNA templates. A total of 180 stool specimens collected from HFMD patients and persons suspected of having HFMD were used to evaluate the clinical performance of this assay. In comparison with the results of conventional methods, the sensitivities of the GeXP assay for detection of panenterovirus, EV71, and CVA16 were 98.79% (163/165), 91.67% (44/48), and 91.67% (33/36), respectively, and the specificities were 80.00% (12/15), 98.48% (130/132), and 100% (144/144), respectively. The concordance of typing seven other serotypes of enteroviruses with the results of conventional methods was 92.59% (25/27). In conclusion, the GeXP assay is a rapid, cost-effective, and high-throughput method for typing nine serotypes of HFMD-associated enteroviruses.

  6. Development and application of a multiplex reverse-transcription polymerase chain reaction assay for screening a global collection of Citrus tristeza virus isolates.

    PubMed

    Roy, Avijit; Ananthakrishnan, G; Hartung, John S; Brlansky, R H

    2010-10-01

    The emerging diversity of Citrus tristeza virus (CTV) genotypes has complicated detection and diagnostic measures and prompted the search for new differentiation methods. To simplify the identification and differentiation of CTV genotypes, a multiplex reverse-transcription polymerase chain reaction (RT-PCR) technique for the screening of CTV isolates was developed. Variable regions within the open reading frame (ORF)-1a of diverse CTV genotypes were identified to develop first a simplex (S) and then a hexaplex (H) RT-PCR. CTV isolates have been grouped previously into five genotypes (namely, T3, T30, T36, VT, and B165) based on the nucleotide sequence comparisons and phylogenetic analyses. Nucleotide sequences from GenBank were used to design species and genotype-specific primers (GSPs). The GSPs were initially used for reliable detection of all CTV genotypes using S-RT-PCR. Furthermore, detection of all five recognized CTV genotypes was established using the H-RT-PCR. Six amplicons, one generic to all CTV isolates and one for each of the five recognized genotypes, were identified on the basis of their size and were confirmed by sequence analysis. In all, 175 CTV isolates from 29 citrus-growing countries were successfully analyzed by S- and H-RT-PCR. Of these, 97 isolates contained T36 genotypes, 95 contained T3 genotypes, 76 contained T30 genotypes, 71 contained VT genotypes, and 24 contained B165 genotype isolates. In total, 126 isolates contained mixed infections of 2 to 5 of the known CTV genotypes. Two of the CTV isolates could not be assigned to a known genotype. H-RT-PCR provides a sensitive, specific, reliable, and rapid way to screen for CTV genotypes compared with other methods for CTV genotype detection. Efficient identification of CTV genotypes will facilitate a better understanding of CTV isolates, including the possible interaction of different genotypes in causing or preventing diseases. The methods described can also be used in virus-free citrus

  7. Multiplex detection of respiratory pathogens

    DOEpatents

    McBride, Mary [Brentwood, CA; Slezak, Thomas [Livermore, CA; Birch, James M [Albany, CA

    2012-07-31

    Described are kits and methods useful for detection of respiratory pathogens (influenza A (including subtyping capability for H1, H3, H5 and H7 subtypes) influenza B, parainfluenza (type 2), respiratory syncytial virus, and adenovirus) in a sample. Genomic sequence information from the respiratory pathogens was analyzed to identify signature sequences, e.g., polynucleotide sequences useful for confirming the presence or absence of a pathogen in a sample. Primer and probe sets were designed and optimized for use in a PCR based, multiplexed Luminex assay to successfully identify the presence or absence of pathogens in a sample.

  8. Narcissus yellow stripe virus and Narcissus mosaic virus detection in Narcissus via multiplex TaqMan-based reverse transcription-PCR assay.

    PubMed

    Jin, J; Shen, J G; Cai, W; Xie, G H; Liao, F R; Gao, F L; Ma, J F; Chen, X H; Wu, Z J

    2017-05-01

    Development of a multiplex TaqMan RT-qPCR assay to simultaneously detect Narcissus yellow stripe virus (NYSV) and Narcissus mosaic virus (NMV), frequently causing mixed narcissus infection. Feasibility verification was confirmed in natural samples. Primers and probes were designed based on the conserved CP gene regions of NYSV or NMV and their suitability for singleplex and multiplex TaqMan RT-qPCR assays as well as for conventional RT-PCR. Conventional RT-PCR, singleplex and multiplex TaqMan RT-qPCR assays proved to be NYSV and NMV specific. P-values and coefficients of variation of TaqMan RT-qPCR assays indicated high reproducibility. Significantly increased sensitivity was achieved compared to conventional RT-PCR. The detection limit of both viruses was 10(3) copies with superior correlation coefficients and linear standard curve responses between plasmid concentrations and Ct values. NYSV and NMV infection of narcissus leaves, petals and bulbs could successfully be detected via our multiplex RT-qPCR method at 1·25 mg. Our multiplex TaqMan RT-qPCR assay provides rapid, specific, sensitive and reliable testing to simultaneously detect NYSV and NMV, supplying useful routine monitoring for different narcissus samples. Efficient identification and discrimination of the narcissus viruses provides reliable information for scientists and conventional growers. Furthermore, it enriches the information of NYSV, NMV and other narcissus viruses. © 2017 The Society for Applied Microbiology.

  9. Evaluation of the one-step multiplex real-time reverse transcription-PCR ProFlu-1 assay for detection of influenza A and influenza B viruses and respiratory syncytial viruses in children.

    PubMed

    Legoff, Jérôme; Kara, Rachid; Moulin, Florence; Si-Mohamed, Ali; Krivine, Anne; Bélec, Laurent; Lebon, Pierre

    2008-02-01

    We evaluated the one-step multiplex real-time reverse transcription-PCR ProFlu-1 assay for the detection of influenza A and influenza B viruses and respiratory syncytial viruses from 353 pediatric nasopharyngeal aspirates. As assessed by comparison with the results of immunofluorescence testing and cell culture, the specificity and the sensitivity of the ProFlu-1 assay ranged from 97% to 100%. In addition, the ProFlu-1 assay amplified 9% of samples not detected by conventional methods.

  10. Evaluation of the One-Step Multiplex Real-Time Reverse Transcription-PCR ProFlu-1 Assay for Detection of Influenza A and Influenza B Viruses and Respiratory Syncytial Viruses in Children▿

    PubMed Central

    LeGoff, Jérôme; Kara, Rachid; Moulin, Florence; Si-Mohamed, Ali; Krivine, Anne; Bélec, Laurent; Lebon, Pierre

    2008-01-01

    We evaluated the one-step multiplex real-time reverse transcription-PCR ProFlu-1 assay for the detection of influenza A and influenza B viruses and respiratory syncytial viruses from 353 pediatric nasopharyngeal aspirates. As assessed by comparison with the results of immunofluorescence testing and cell culture, the specificity and the sensitivity of the ProFlu-1 assay ranged from 97% to 100%. In addition, the ProFlu-1 assay amplified 9% of samples not detected by conventional methods. PMID:18057126

  11. PCR-Based Detection of DNA Copy Number Variation.

    PubMed

    Mehrotra, Meenakshi

    2016-01-01

    Copy number variations are important polymorphisms that can influence gene expression within and close to the rearranged region, and results in phenotypic variation. Techniques that detect abnormalities in DNA copy number are therefore useful for studying the associations between DNA aberrations and disease phenotype and for locating critical genes. PCR-based detection of copy number of target gene using TaqMan copy number assay offers a reliable method to measure copy number variation in human genome.

  12. Evaluation of a multiplex real-time reverse transcriptase PCR assay for detection and differentiation of influenza viruses A and B during the 2001-2002 influenza season in Israel.

    PubMed

    Hindiyeh, Musa; Levy, Virginia; Azar, Roberto; Varsano, Noemi; Regev, Liora; Shalev, Yael; Grossman, Zehava; Mendelson, Ella

    2005-02-01

    The ability to rapidly diagnose influenza virus infections is of the utmost importance in the evaluation of patients with upper respiratory tract infections. It is also important for the influenza surveillance activities performed by national influenza centers. In the present study we modified a multiplex real-time reverse transcriptase PCR (RT-PCR) assay (which uses TaqMan chemistry) and evaluated it for its ability to detect and concomitantly differentiate influenza viruses A and B in 370 patient samples collected during the 2001-2002 influenza season in Israel. The performance of the TaqMan assay was compared to those of a multiplex one-step RT-PCR with gel detection, a shell vial immunofluorescence assay, and virus isolation in tissue culture. The TaqMan assay had an excellent sensitivity for the detection of influenza viruses compared to that of tissue culture. The overall sensitivity and specificity of the TaqMan assay compared to the results of culture were 98.4 and 85.5%, respectively. The sensitivity and specificity of the TaqMan assay for the detection of influenza virus A alone were 100 and 91.1%, respectively. On the other hand, the sensitivity and specificity for the detection of influenza virus B alone were 95.7 and 98.7%, respectively. The rapid turnaround time for the performance of the TaqMan assay (4.5 h) and the relatively low direct cost encourage the routine use of this assay in place of tissue culture. We conclude that the multiplex TaqMan assay is highly suitable for the rapid diagnosis of influenza virus infections both in well-established molecular biology laboratories and in reference clinical laboratories.

  13. Comparison of multiplex real-time PCR and PCR-reverse blot hybridization assay for the direct and rapid detection of bacteria and antibiotic resistance determinants in positive culture bottles.

    PubMed

    Wang, Hye-Young; Kim, Seoyong; Kim, Jungho; Park, Soon Deok; Kim, Hyo Youl; Uh, Young; Lee, Hyeyoung

    2016-09-01

    The aim of this study was to evaluate the performance of a commercially available multiplex real-time PCR assay and a PCR-reverse blot hybridization assay (PCR-REBA) for the rapid detection of bacteria and identification of antibiotic resistance genes directly from blood culture bottles and to compare the results of these molecular assays with conventional culture methods. The molecular diagnostic methods were used to evaluate 593 blood culture bottles from patients with bloodstream infections. The detection positivity of multiplex real-time PCR assay for Gram-positive bacteria, Gram-negative bacteria and Candida spp. was equivalent to PCR-REBA as 99.6 %, 99.1 % and 100 %, respectively. Using conventional bacterial cultures as the gold standard, the sensitivity, specificity, positive predictive value and negative predictive value of these two molecular methods were 99.5 % [95 % confidence interval (CI), 0.980-1.000; P<0.001), 100 % (95 % CI, 0.983-1.000; P<0.001), 100 % and 99 %, respectively. However, positivity of the Real-methicillin-resistant Staphylococcusaureus multiplex real-time PCR assay targeting the mecA gene to detect methicillin resistance was lower than that of the PCR-REBA method, detecting an overall positivity of 98.4 % (n=182; 95 % CI, 0.964-1.000; P<0.009) and 99.5 % (n=184; 95 % CI, 0.985-1.000; P<0.0001), respectively. The entire two methods take about 3 h, while results from culture can take up to 48-72 h. Therefore, the use of these two molecular methods was rapid and reliable for the characterization of causative pathogens in bloodstream infections.

  14. Multiplex PCR identification of Taenia spp. in rodents and carnivores.

    PubMed

    Al-Sabi, Mohammad N S; Kapel, Christian M O

    2011-11-01

    The genus Taenia includes several species of veterinary and public health importance, but diagnosis of the etiological agent in definitive and intermediate hosts often relies on labor intensive and few specific morphometric criteria, especially in immature worms and underdeveloped metacestodes. In the present study, a multiplex PCR, based on five primers targeting the 18S rDNA and ITS2 sequences, produced a species-specific banding patterns for a range of Taenia spp. Species typing by the multiplex PCR was compared to morphological identification and sequencing of cox1 and/or 12S rDNA genes. As compared to sequencing, the multiplex PCR identified 31 of 32 Taenia metacestodes from rodents, whereas only 14 cysts were specifically identified by morphology. Likewise, the multiplex PCR identified 108 of 130 adult worms, while only 57 were identified to species by morphology. The tested multiplex PCR system may potentially be used for studies of Taenia spp. transmitted between rodents and carnivores.

  15. Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification Coupled with Cascade Invasive Reaction and Nanoparticle Hybridization for Subtyping of Influenza A Virus

    PubMed Central

    Chi, Ying; Ge, Yiyue; Zhao, Kangchen; Zou, Bingjie; Liu, Bin; Qi, Xian; Bian, Qian; Shi, Zhiyang; Zhu, Fengcai; Zhou, Minghao; Cui, Lunbiao; Su, Chuan

    2017-01-01

    Considering the fatal human victims and economic loss caused by influenza virus infection every year, methodologies for rapid and on-site detection of influenza viruses are urgently needed. LAMP is the most commonly used nucleic acid isothermal amplification technology suitable for on-site use. However, for multiplex LAMP, differentiation of the amplicons derived from multiple targets is still challengeable currently. Here we developed a multiplex RT-LAMP assay for simultaneous amplification of three prominent subtypes of influenza viruses (A/H5, A/H7 and 2009A/H1). The amplicons were further identified by cascade invasive reaction and nanoparticle hybridization in separate target-specific detection tubes (referred to as mRT-LAMP-IRNH). The analytic sensitivities of the assay are 10 copies of RNA for all the three HA subtypes, and the specificity reached 100%. Clinical specimen analysis showed this assay had a combined sensitivity and specificity of 98.1% and 100%, respectively. Overall, the mRT-LAMP-IRNH assay can be used as a cost-saving method that utilizes a simple instrument to detect A/H5, A/H7, and 2009A/H1 influenza viruses, especially in resource-limited settings. PMID:28322309

  16. TipMT: Identification of PCR-based taxon-specific markers.

    PubMed

    Rodrigues-Luiz, Gabriela F; Cardoso, Mariana S; Valdivia, Hugo O; Ayala, Edward V; Gontijo, Célia M F; Rodrigues, Thiago de S; Fujiwara, Ricardo T; Lopes, Robson S; Bartholomeu, Daniella C

    2017-02-11

    Molecular genetic markers are one of the most informative and widely used genome features in clinical and environmental diagnostic studies. A polymerase chain reaction (PCR)-based molecular marker is very attractive because it is suitable to high throughput automation and confers high specificity. However, the design of taxon-specific primers may be difficult and time consuming due to the need to identify appropriate genomic regions for annealing primers and to evaluate primer specificity. Here, we report the development of a Tool for Identification of Primers for Multiple Taxa (TipMT), which is a web application to search and design primers for genotyping based on genomic data. The tool identifies and targets single sequence repeats (SSR) or orthologous/taxa-specific genes for genotyping using Multiplex PCR. This pipeline was applied to the genomes of four species of Leishmania (L. amazonensis, L. braziliensis, L. infantum and L. major) and validated by PCR using artificial genomic DNA mixtures of the Leishmania species as templates. This experimental validation demonstrates the reliability of TipMT because amplification profiles showed discrimination of genomic DNA samples from Leishmania species. The TipMT web tool allows for large-scale identification and design of taxon-specific primers and is freely available to the scientific community at http://200.131.37.155/tipMT/ .

  17. Triplex PCR-based detection of enterotoxigenic Bacillus cereus ATCC 14579 in nonfat dry milk.

    PubMed

    Gracias, Kiev S; McKillip, John L

    2011-04-01

    Although many strains of Bacillaceae are considered nonpathogenic, Bacillus cereus is recognized worldwide as a bacterial pathogen in a variety of foods. The ability of B. cereus to cause gastroenteritis following ingestion of contaminated food is due to the production of enterotoxins. The ubiquity of this genus makes it a persistent problem for quality assurance in food processing environments. The primary objective of this study was to develop and apply a multiplex real-time PCR-based assay for rapid and sensitive detection of enterotoxigenic B. cereus. Template DNA was separately extracted from tryptic soy broth (TSB)-grown and 2.5% Nonfat Dry Milk (NFDM)-grown B. cereus using a commercial system. Three enterotoxin gene fragments (hblC, nheA, and hblA) were simultaneously amplified in real-time followed by melting curve analysis to confirm amplicon identity. Resolution of melting curves (characteristic T(m)) was achieved for each amplicon (hblC = 74.5 °C; nheA = 78 °C; and hblA = 85.5 °C in TSB and 84 °C in NFDM) with an assay sensitivities of 10(1) CFU/ml for both TSB and NFDM-grown B. cereus compared to 10(4) CFU/ml in either matrix using gel electrophoresis. The results demonstrate the potential sensitivity of real-time bacterial detection methods in a heterogenous food matrix using real-time PCR.

  18. A PCR-based linkage map of human chromosome 1

    SciTech Connect

    Engelstein, M.; Hudson, T.J.; Lane, J.M.; Lee, M.K.; Dracopoli, C. ); Leverone, B.; Landes, G.M. ); Peltonen, L. ); Weber, J.L. )

    1993-02-01

    A genetic linkage map of human chromosome 1 based entirely on PCR-typable markers has been developed using 38 simple sequence repeat (SSR) polymorphisms. These SSRs include 36 dinucleotide repeats and 2 tetranucleotide repeats. The average heterozygosity at these markers was 0.73 and ranged form 0.52 to 0.95. Multipoint linkage analysis was used to develop a map of these 38 markers in which the relative placement of each locus is supported by likelihood odds > 1000:1. This PCR-based map was anchored at the centromere by the D1Z5 [alpha]-satellite polymorphism, and the ends of the map were defined by D1Z2 and D1S68, which are the most distal loci in the CEPH consortium map of chromosome 1. The sex-averaged, male, and female maps extend for 328, 273, and 409 cM, respectively. The average distance between markers on the sex-averaged map is 8 cM, and the largest interval is 32 cM. This map of highly informative PCR-based markers will provide a rapid means of screening human chromosome 1 for the presence of disease genes. 36 refs., 4 figs., 4 tabs.

  19. 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

  20. PCR-based polymorphisms in neurofibromatosis type 1 (NFI)

    SciTech Connect

    Lai, P.S.; Chee, S.; Low, P.S.

    1994-09-01

    Neurofibromatosis type 1 (NF1) is one of the most common genetic disorders in humans with an incidence of 1 in 3,000. The NF1 gene is located on chromosome 17q 11.2 and encodes an ubiquitously expressed transcript of about 13kb. Direct mutation detection is difficult in this disorder due to the large gene size, high mutation rate and variety of mutations. We have studied the allele frequencies of seven PCR-based polymorphisms. Six of the probes used flank the NF1 gene, namely p11.3C4.2/Msp I (proximal), pEW206/Msp I (distal), p2.f9.8/Rsa I (distal), pEW207/Bgl II (distal), pEW207/Hind III (distal) and pHHH202/Rsa I (proximal). An intragenic RFLP, pEvi 2B-B/Eco R1 polymorphism in intron 27, was also analyzed by PCR. Allele frequencies for 48 normal unrelated individuals were obtained as follows: A1 = 0.40, A2 = 0.6 (p11.3C4.2/Msp I), A1 = 0.44, A2 = 0.56 (pEW206/Msp I), A1 = 0.17, A2 = 0.83 (p2.F9.8/Rsa I), A1 = 0.64, A2 = 0.36 (pEW207/Bgl I), A1 = 0.45, A2 = 0.55 (pEvi 2B-B/Eco RI). Heterozygosity rates of the alleles ranged from 20.8% to 51.7%. Using a combination of these markers, seven local families with NF1 were studied. Normal Mendelian segregation of alleles was observed in these families and no recombination was detected so far. These PCR-based markers were found to be useful for linkage analysis in our families.

  1. Accurate Classification of Germinal Center B-Cell-Like/Activated B-Cell-Like Diffuse Large B-Cell Lymphoma Using a Simple and Rapid Reverse Transcriptase-Multiplex Ligation-Dependent Probe Amplification Assay: A CALYM Study.

    PubMed

    Mareschal, Sylvain; Ruminy, Philippe; Bagacean, Cristina; Marchand, Vinciane; Cornic, Marie; Jais, Jean-Philippe; Figeac, Martin; Picquenot, Jean-Michel; Molina, Thierry Jo; Fest, Thierry; Salles, Gilles; Haioun, Corinne; Leroy, Karen; Tilly, Hervé; Jardin, Fabrice

    2015-04-09

    Diffuse large B-cell lymphoma, the most common non-Hodgkin lymphoma, is subdivided into germinal center B-cell-like and activated B-cell-like subtypes. Unfortunately, these lymphomas are difficult to differentiate in routine diagnosis, impeding the development of treatments. Patients with these lymphomas can benefit from specific therapies. We therefore developed a simple and rapid classifier based on a reverse transcriptase multiplex ligation-dependent probe amplification assay and 14 gene signatures. Compared with the Affymetrix U133+2 gold standard, all 46 samples (95% CI, 92%-100%) of a validation cohort classified by both techniques were attributed to the expected subtype. Similarly, 93% of the 55 samples (95% CI, 82%-98%) of a second independent series characterized with a mid-throughput gene expression profiling method were classified correctly. Unclassifiable sample proportions reached 13.2% and 13.8% in these cohorts, comparable with the frequency originally reported. The developed assay was also sensitive enough to obtain reliable results from formalin-fixed, paraffin-embedded samples and flexible enough to include prognostic factors such as MYC/BCL2 co-expression. Finally, in a series of 135 patients, both overall (P = 0.01) and progression-free (P = 0.004) survival differences between the two subtypes were confirmed. Because the multiplex ligation-dependent probe amplification method is already in use and requires only common instruments and reagents, it could easily be applied to clinical trial patient stratification to help in treatment decisions.

  2. Memory erasure using time-multiplexed potentials

    NASA Astrophysics Data System (ADS)

    Talukdar, Saurav; Bhaban, Shreyas; Salapaka, Murti V.

    2017-06-01

    We study the thermodynamics of a Brownian particle under the influence of a time-multiplexed harmonic potential of finite width. The memory storage mechanism and the erasure protocol based on time-multiplexed potentials are utilized to experimentally realize erasure with work performed close to Landauer's bound. We quantify the work performed on the system with respect to the duty ratio of time multiplexing, which also provides a handle for approaching reversible erasures. A Langevin dynamics based simulation model is developed for the proposed memory bit and the erasure protocol, which guides the experimental realization. The study also provides insight into transport on the microscale.

  3. PCR-based screening for cystic fibrosis carrier mutations in an ethnically diverse pregnant population.

    PubMed Central

    Grody, W W; Dunkel-Schetter, C; Tatsugawa, Z H; Fox, M A; Fang, C Y; Cantor, R M; Novak, J M; Bass, H N; Crandall, B F

    1997-01-01

    As the most common lethal autosomal recessive disorder in North America, cystic fibrosis (CF) is an obvious candidate for general population carrier screening. Although the identification of the causative gene has made detection of asymptomatic carriers possible, the extreme heterogeneity of its mutations has limited the sensitivity of the available DNA screening tests and has called into question their utility when they are applied to patients with no family history of the disease. The purpose of this study was to determine the technical feasibility, patient acceptance and understanding, and psychosocial impact of large-scale CF carrier screening in an ethnically diverse pregnant population. A total of 4,739 pregnant women attending prenatal clinics located in both an academic medical center and a large HMO were invited in person to participate. Of this group, 3,543 received CF instruction and assessments of knowledge and mood, and 3,192 underwent DNA testing for the six most common CF mutations, by means of a noninvasive PCR-based reverse-dot-blot method. Overall participation rates (ranging from 53% at the HMO to 77% at the academic center) and consent rates for DNA testing after CF instruction (>98%) exceeded those of most other American studies. The PCR-based screening method worked efficiently on large numbers of samples, and 55 carriers and one at-risk couple were identified. Understanding of residual risk, anxiety levels, and overall satisfaction with the program were acceptable across all ethnic groups. Our strategy of approaching a motivated pregnant population in person with a rapid and noninvasive testing method may provide a practical model for developing a larger CF screening program targeting appropriate high-risk groups at the national level, and may also serve as a paradigm for population-based screening of other genetically heterogeneous disorders in the future. Images Figure 1 PMID:9106541

  4. PCR-based identification of drowning: four case reports.

    PubMed

    Rácz, Evelin; Könczöl, Franciska; Tóth, Dénes; Patonai, Zoltán; Porpáczy, Zoltán; Kozma, Zsolt; Poór, Viktor S; Sipos, Katalin

    2016-09-01

    Proper diagnosis in drowning victims is often difficult due to the lack of signs specific to drowning. The diatom test is a widely used procedure for the diagnosis. Some types of water contain only minimal amounts of diatom cells which may provide false-negative results, while a negative diatom test result does not exclude drowning. In proving drowning, we used a polymerase chain reaction (PCR)-based biological method in addition to the conventional methods. DNA was extracted from postmortem spleen tissues and water of the drowning site. Samples were tested with algae (diatoms and small green algae)- and cyanobacteria (blue-green algae)-specific primers. We present here multiple drowning cases in which diatom tests of the postmortem tissue samples and the water were negative. In each case, the presence of phytoplanktonic DNA strengthened the autopsy diagnosis of drowning even in the absence of visible diatoms. In the future, the PCR method may be of consideration as a possible supplement of the diatom test in the examination of presumed drowning cases.

  5. Quenching of Unincorporated Amplification Signal Reporters in Reverse-Transcription Loop-Mediated Isothermal Amplification Enabling Bright, Single-Step, Closed-Tube, and Multiplexed Detection of RNA Viruses.

    PubMed

    Ball, Cameron S; Light, Yooli K; Koh, Chung-Yan; Wheeler, Sarah S; Coffey, Lark L; Meagher, Robert J

    2016-04-05

    Reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common detection techniques used for LAMP and RT-LAMP have drawbacks, including poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes postamplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of the reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for quenching of unincorporated amplification signal reporters, read "quasar"), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). Furthermore, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA.

  6. Quenching of unincorporated amplification signal reporters in reverse-transcription loop-mediated isothermal amplification enabling bright, single-step, closed-tube, and multiplexed detection of RNA viruses

    DOE PAGES

    Ball, Cameron S.; Light, Yooli K.; Koh, Chung -Yan; ...

    2016-03-16

    Reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common detection techniques used for LAMP and RT-LAMP have drawbacks, including poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes postamplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of themore » reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for quenching of unincorporated amplification signal reporters, read “quasar”), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). As a result, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA.« less

  7. A Ribeiroia spp. (Class: Trematoda) - Specific PCR-based diagnostic

    USGS Publications Warehouse

    Reinitz, D.M.; Yoshino, T.P.; Cole, R.A.

    2007-01-01

    Increased reporting of amphibian malformations in North America has been noted with concern in light of reports that amphibian numbers and species are declining worldwide. Ribeiroia ondatrae has been shown to cause a variety of types of malformations in amphibians. However, little is known about the prevalence of R. ondatrae in North America. To aid in conducting field studies of Ribeiroia spp., we have developed a polymerase chain reaction (PCR)-based diagnostic. Herein, we describe the development of an accurate, rapid, simple, and cost-effective diagnostic for detection of Ribeiroia spp. infection in snails (Planorbella trivolvis). Candidate oligonucleotide primers for PCR were designed via DNA sequence analyses of multiple ribosomal internal transcribed spacer-2 regions from Ribeiroia spp. and Echinostoma spp. Comparison of consensus sequences determined from both genera identified areas of sequence potentially unique to Ribeiroia spp. The PCR reliably produced a diagnostic 290-base pair (bp) product in the presence of a wide concentration range of snail or frog DNA. Sensitivity was examined with DNA extracted from single R. ondatrae cercaria. The single-tube PCR could routinely detect less than 1 cercariae equivalent, because DNA isolated from a single cercaria could be diluted at least 1:50 and still yield a positive result via gel electrophoresis. An even more sensitive nested PCR also was developed that routinely detected 100 fg of the 290-bp fragment. The assay did not detect furcocercous cercariae of certain Schistosomatidae, Echinostoma sp., or Sphaeridiotrema globulus nor adults of Clinostomum sp. or Cyathocotyle bushiensis. Field testing of 137 P. trivolvis identified 3 positives with no overt environmental cross-reactivity, and results concurred with microscopic examinations in all cases. ?? American Society of Parasitologists 2007.

  8. Detection of African swine fever, classical swine fever, and foot-and-mouth disease viruses in swine oral fluids by multiplex reverse transcription real-time polymerase chain reaction.

    PubMed

    Grau, Frederic R; Schroeder, Megan E; Mulhern, Erin L; McIntosh, Michael T; Bounpheng, Mangkey A

    2015-03-01

    African swine fever (ASF), classical swine fever (CSF), and foot-and-mouth disease (FMD) are highly contagious animal diseases of significant economic importance. Pigs infected with ASF and CSF viruses (ASFV and CSFV) develop clinical signs that may be indistinguishable from other diseases. Likewise, various causes of vesicular disease can mimic clinical signs caused by the FMD virus (FMDV). Early detection is critical to limiting the impact and spread of these disease outbreaks, and the ability to perform herd-level surveillance for all 3 diseases rapidly and cost effectively using a single diagnostic sample and test is highly desirable. This study assessed the feasibility of simultaneous ASFV, CSFV, and FMDV detection by multiplex reverse transcription real-time polymerase chain reaction (mRT-qPCR) in swine oral fluids collected through the use of chewing ropes. Animal groups were experimentally infected independently with each virus, observed for clinical signs, and oral fluids collected and tested throughout the course of infection. All animal groups chewed on the ropes readily before and after onset of clinical signs and before onset of lameness or serious clinical signs. ASFV was detected as early as 3 days postinoculation (dpi), 2-3 days before onset of clinical disease; CSFV was detected at 5 dpi, coincident with onset of clinical disease; and FMDV was detected as early as 1 dpi, 1 day before the onset of clinical disease. Equivalent results were observed in 4 independent studies and demonstrate the feasibility of oral fluids and mRT-qPCR for surveillance of ASF, CSF, and FMD in swine populations. © 2015 The Author(s).

  9. One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees.

    PubMed

    Malandraki, Ioanna; Varveri, Christina; Olmos, Antonio; Vassilakos, Nikon

    2015-03-01

    A one-step multiplex real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan chemistry was developed for the simultaneous detection of Pear blister canker viroid and Apple scar skin viroid along with universal detection of phytoplasmas, in pome trees. Total nucleic acids (TNAs) extraction was performed according to a modified CTAB protocol. Primers and TaqMan MGB probes for specific detection of the two viroids were designed in this study, whereas for phytoplasma detection published universal primers and probe were used, with the difference that the later was modified to carry a MGB quencher. The pathogens were detected simultaneously in 10-fold serial dilutions of TNAs from infected plant material into TNAs of healthy plant up to dilutions 10(-5) for viroids and 10(-4) for phytoplasmas. The multiplex real-time assay was at least 10 times more sensitive than conventional protocols for viroid and phytoplasma detection. Simultaneous detection of the three targets was achieved in composite samples at least up to a ratio of 1:100 triple-infected to healthy tissue, demonstrating that the developed assay has the potential to be used for rapid and massive screening of viroids and phytoplasmas of pome fruit trees in the frame of certification schemes and surveys.

  10. RT-PCR-based analysis of microRNA (miR-1 and -124) expression in mouse CNS.

    PubMed

    Mishima, Takuya; Mizuguchi, Yoshiaki; Kawahigashi, Yutaka; Takizawa, Takami; Takizawa, Toshihiro

    2007-02-02

    More than 700 microRNAs (miRNAs) have been cloned, and the functions of these molecules in developmental timing, cell proliferation, and cancer have been investigated widely. MiRNAs are analyzed with Northern blot and sequential colony evaluation; however, reverse transcription-polymerase chain reaction (RT-PCR)-based miRNA assay remains to be developed. In this report, we describe improved real-time RT-PCR methods using specific or non-specific RT primer for the semi-quantitative analysis of miRNA expression. The use of the new methods in a model study revealed differential expression of miRNA-1 (miR-1) and miR-124 in mouse organs. Specifically, our methods revealed that miR-124 concentrations in the mouse central nervous system (CNS; cerebral cortex, cerebellum, and spinal cord) were more than 100 times those in other organs. By contrast, miR-1 expression in the CNS was 100-1000 times lower than that in skeletal muscle and heart. Furthermore, we revealed anatomically regional differences in miR-124 expression within the CNS: expression ratios versus the cerebral cortex were 60.7% for the cerebellum and 35.4% for the spinal cord. These results suggest that our RT-PCR-based methods would be a powerful tool for studies of miRNA expression that is associated with various neural events.

  11. Comparison of two PCR-based human papillomavirus genotyping methods.

    PubMed

    Castle, Philip E; Porras, Carolina; Quint, Wim G; Rodriguez, Ana Cecilia; Schiffman, Mark; Gravitt, Patti E; González, Paula; Katki, Hormuzd A; Silva, Sandra; Freer, Enrique; Van Doorn, Leen-Jan; Jiménez, Silvia; Herrero, Rolando; Hildesheim, Allan

    2008-10-01

    We compared two consensus primer PCR human papillomavirus (HPV) genotyping methods for the detection of individual HPV genotypes and carcinogenic HPV genotypes as a group, using a stratified sample of enrollment cervical specimens from sexually active women participating in the NCI/Costa Rica HPV16/18 Vaccine Efficacy Trial. For the SPF(10) method, DNA was extracted from 0.1% of the cervical specimen by using a MagNA Pure LC instrument, a 65-bp region of the HPV L1 gene was targeted for PCR amplification by using SPF(10) primers, and 25 genotypes were detected by reverse-line blot hybridization of the amplicons. For the Linear Array (LA) method, DNA was extracted from 0.5% of the cervical specimen by using an MDx robot, a 450-bp region of the HPV L1 gene was targeted for PCR amplification by using PGMY09/11 L1 primers, and 37 genotypes were detected by reverse-line blot hybridization of the amplicons. Specimens (n = 1,427) for testing by the LA method were randomly selected from strata defined on the basis of enrollment test results from the SPF(10) method, cytology, and Hybrid Capture 2. LA results were extrapolated to the trial cohort (n = 5,659). The LA and SPF(10) methods detected 21 genotypes in common; HPV16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, -68, and -73 were considered the carcinogenic HPV genotypes. There was no difference in the overall results for grouped detection of carcinogenic HPV by the SPF(10) and LA methods (35.3% versus 35.9%, respectively; P = 0.5), with a 91.8% overall agreement and a kappa value of 0.82. In comparisons of individual HPV genotypes, the LA method detected significantly more HPV16, HPV18, HPV39, HPV58, HPV59, HPV66, and HPV68/73 and less HPV31 and HPV52 than the SPF(10) method; inclusion of genotype-specific testing for HPV16 and HPV18 for those specimens testing positive for HPV by the SPF(10) method but for which no individual HPV genotype was detected abrogated any differences between the LA and SPF

  12. Evaluation of Two PCR-Based Swine-Specific Fecal Source Tracking Assays (Poster)

    EPA Science Inventory

    Several PCR-based methods have been proposed to identify swine fecal pollution in environmental waters. However, the specificity and distribution of these targets have not been adequately assessed. Consequently, the utility of these assays in identifying swine fecal contamination...

  13. Evaluation of Two PCR-Based Swine-Specific Fecal Source Tracking Assays (Poster)

    EPA Science Inventory

    Several PCR-based methods have been proposed to identify swine fecal pollution in environmental waters. However, the specificity and distribution of these targets have not been adequately assessed. Consequently, the utility of these assays in identifying swine fecal contamination...

  14. Comparison of Two Widely Used Human Papillomavirus Detection and Genotyping Methods, GP5+/6+-Based PCR Followed by Reverse Line Blot Hybridization and Multiplex Type-Specific E7-Based PCR

    PubMed Central

    Vaccarella, Salvatore; Franceschi, Silvia; Tenet, Vanessa; Umulisa, M. Chantal; Tshomo, Ugyen; Dondog, Bolormaa; Vorsters, Alex; Tommasino, Massimo; Heideman, Daniëlle A. M.; Snijders, Peter J. F.; Gheit, Tarik

    2016-01-01

    GP5+/6+-based PCR followed by reverse line blot hybridization (GP5+/6+RLB) and multiplex type-specific PCR (E7-MPG) are two human papillomavirus (HPV) genotyping methodologies widely applied in epidemiological research. We investigated their relative analytical performance in 4,662 samples derived from five studies in Bhutan, Rwanda, and Mongolia coordinated by the International Agency for Research on Cancer (IARC). A total of 630 samples were positive by E7-MPG only (13.5%), 24 were positive by GP5+/6+RLB only (0.5%), and 1,014 were positive (21.8%) by both methods. Ratios of HPV type-specific positivity of the two tests (E7-MPG:GP5+/6+RLB ratio) were calculated among 1,668 samples that were HPV positive by one or both tests. E7-MPG:GP5+/6+RLB ratios were >1 for all types and highly reproducible across populations and sample types. E7-MPG:GP5+/6+RLB ratios were highest for HPV53 (7.5) and HPV68 (7.1). HPV16 (1.6) and HPV18 (1.7) had lower than average E7-MPG:GP5+/6+RLB ratios. Among E7-MPG positive infections, median mean fluorescence intensity (MFI; a semiquantitative measure of viral load) tended to be higher among samples positive for the same virus type by GP5+/6+RLB than for those negative for the same type by GP5+/6+RLB. Exceptions, however, included HPV53, -59, and -82, for which the chances of being undetected by GP5+/6+RLB appeared to be MFI independent. Furthermore, the probability of detecting an additional type by E7-MPG was higher when another type was already detected by GP5+/6+RLB, suggesting the existence of masking effects due to competition for GP5+/6+ PCR primers. In conclusion, this analysis is not an evaluation of clinical performance but may inform choices for HPV genotyping methods in epidemiological studies, when the relative merits and dangers of sensitivity versus specificity for individual types should be considered, as well as the potential to unmask nonvaccine types following HPV vaccination. PMID:27225411

  15. Comparison of Two Widely Used Human Papillomavirus Detection and Genotyping Methods, GP5+/6+-Based PCR Followed by Reverse Line Blot Hybridization and Multiplex Type-Specific E7-Based PCR.

    PubMed

    Clifford, Gary M; Vaccarella, Salvatore; Franceschi, Silvia; Tenet, Vanessa; Umulisa, M Chantal; Tshomo, Ugyen; Dondog, Bolormaa; Vorsters, Alex; Tommasino, Massimo; Heideman, Daniëlle A M; Snijders, Peter J F; Gheit, Tarik

    2016-08-01

    GP5+/6+-based PCR followed by reverse line blot hybridization (GP5+/6+RLB) and multiplex type-specific PCR (E7-MPG) are two human papillomavirus (HPV) genotyping methodologies widely applied in epidemiological research. We investigated their relative analytical performance in 4,662 samples derived from five studies in Bhutan, Rwanda, and Mongolia coordinated by the International Agency for Research on Cancer (IARC). A total of 630 samples were positive by E7-MPG only (13.5%), 24 were positive by GP5+/6+RLB only (0.5%), and 1,014 were positive (21.8%) by both methods. Ratios of HPV type-specific positivity of the two tests (E7-MPG:GP5+/6+RLB ratio) were calculated among 1,668 samples that were HPV positive by one or both tests. E7-MPG:GP5+/6+RLB ratios were >1 for all types and highly reproducible across populations and sample types. E7-MPG:GP5+/6+RLB ratios were highest for HPV53 (7.5) and HPV68 (7.1). HPV16 (1.6) and HPV18 (1.7) had lower than average E7-MPG:GP5+/6+RLB ratios. Among E7-MPG positive infections, median mean fluorescence intensity (MFI; a semiquantitative measure of viral load) tended to be higher among samples positive for the same virus type by GP5+/6+RLB than for those negative for the same type by GP5+/6+RLB. Exceptions, however, included HPV53, -59, and -82, for which the chances of being undetected by GP5+/6+RLB appeared to be MFI independent. Furthermore, the probability of detecting an additional type by E7-MPG was higher when another type was already detected by GP5+/6+RLB, suggesting the existence of masking effects due to competition for GP5+/6+ PCR primers. In conclusion, this analysis is not an evaluation of clinical performance but may inform choices for HPV genotyping methods in epidemiological studies, when the relative merits and dangers of sensitivity versus specificity for individual types should be considered, as well as the potential to unmask nonvaccine types following HPV vaccination. Copyright © 2016, American Society for

  16. [Clarification of a break-in theft crime by multiplex PCR analysis of cigarette butts].

    PubMed

    Hochmeister, M; Haberl, J; Borer, V; Rudin, O; Dirnhofer, R

    1995-01-01

    This paper describes the first use of multiplex PCR amplification kits for the analysis of DNA extracted from cigarette butts in a criminal case. Two suspects could be excluded as potential contributors to the samples, whereas the multi locus PCR-based DNa profile derived from the cigarette butts was consistent with a DNA profile derived from a third suspect. For identity testing in criminal cases where cigarette butts are involved, commercially available PCR amplification kits provide currently the most powerful tool. Furthermore this PCR-based analysis can be implemented into most application orientated laboratories.

  17. Weighted multiplex networks.

    PubMed

    Menichetti, Giulia; Remondini, Daniel; Panzarasa, Pietro; Mondragón, Raúl J; Bianconi, Ginestra

    2014-01-01

    One of the most important challenges in network science is to quantify the information encoded in complex network structures. Disentangling randomness from organizational principles is even more demanding when networks have a multiplex nature. Multiplex networks are multilayer systems of [Formula: see text] nodes that can be linked in multiple interacting and co-evolving layers. In these networks, relevant information might not be captured if the single layers were analyzed separately. Here we demonstrate that such partial analysis of layers fails to capture significant correlations between weights and topology of complex multiplex networks. To this end, we study two weighted multiplex co-authorship and citation networks involving the authors included in the American Physical Society. We show that in these networks weights are strongly correlated with multiplex structure, and provide empirical evidence in favor of the advantage of studying weighted measures of multiplex networks, such as multistrength and the inverse multiparticipation ratio. Finally, we introduce a theoretical framework based on the entropy of multiplex ensembles to quantify the information stored in multiplex networks that would remain undetected if the single layers were analyzed in isolation.

  18. Weighted Multiplex Networks

    PubMed Central

    Menichetti, Giulia; Remondini, Daniel; Panzarasa, Pietro; Mondragón, Raúl J.; Bianconi, Ginestra

    2014-01-01

    One of the most important challenges in network science is to quantify the information encoded in complex network structures. Disentangling randomness from organizational principles is even more demanding when networks have a multiplex nature. Multiplex networks are multilayer systems of nodes that can be linked in multiple interacting and co-evolving layers. In these networks, relevant information might not be captured if the single layers were analyzed separately. Here we demonstrate that such partial analysis of layers fails to capture significant correlations between weights and topology of complex multiplex networks. To this end, we study two weighted multiplex co-authorship and citation networks involving the authors included in the American Physical Society. We show that in these networks weights are strongly correlated with multiplex structure, and provide empirical evidence in favor of the advantage of studying weighted measures of multiplex networks, such as multistrength and the inverse multiparticipation ratio. Finally, we introduce a theoretical framework based on the entropy of multiplex ensembles to quantify the information stored in multiplex networks that would remain undetected if the single layers were analyzed in isolation. PMID:24906003

  19. A Targeted Q-PCR-Based Method for Point Mutation Testing by Analyzing Circulating DNA for Cancer Management Care.

    PubMed

    Thierry, Alain R

    2016-01-01

    Circulating cell-free DNA (cfDNA) is a valuable source of tumor material available with a simple blood sampling enabling a noninvasive quantitative and qualitative analysis of the tumor genome. cfDNA is released by tumor cells and exhibits the genetic and epigenetic alterations of the tumor of origin. Circulating cell-free DNA (cfDNA) analysis constitutes a hopeful approach to provide a noninvasive tumor molecular test for cancer patients. Based upon basic research on the origin and structure of cfDNA, new information on circulating cell-free DNA (cfDNA) structure, and specific determination of cfDNA fragmentation and size, we revisited Q-PCR-based method and recently developed a the allele-specific-Q-PCR-based method with blocker (termed as Intplex) which is the first multiplexed test for cfDNA. This technique, named Intplex(®) and based on a refined Q-PCR method, derived from critical observations made on the specific structure and size of cfDNA. It enables the simultaneous determination of five parameters: the cfDNA total concentration, the presence of a previously known point mutation, the mutant (tumor) cfDNA concentration (ctDNA), the proportion of mutant cfDNA, and the cfDNA fragmentation index. Intplex(®) has enabled the first clinical validation of ctDNA analysis in oncology by detecting KRAS and BRAF point mutations in mCRC patients and has demonstrated that a blood test could replace tumor section analysis for the detection of KRAS and BRAF mutations. The Intplex(®) test can be adapted to all mutations, genes, or cancers and enables rapid, highly sensitive, cost-effective, and repetitive analysis. As regards to the determination of mutations on cfDNA Intplex(®) is limited to the mutational status of known hotspot mutation; it is a "targeted approach." However, it offers the opportunity in detecting quantitatively and dynamically mutation and could constitute a noninvasive attractive tool potentially allowing diagnosis, prognosis, theranostics

  20. Brief communication: multiplex X/Y-PCR improves sex identification in aDNA analysis.

    PubMed

    Schmidt, Diane; Hummel, Susanne; Herrmann, Bernd

    2003-08-01

    This study introduces a polymerase chain reaction (PCR)-based multiplex approach to improve the certainty of molecular sex identification on archaeological skeletal material. We coamplified amelogenin, two X-chromosomal short tandem repeats (STRs) (DXS6789 and DXS9898), and two Y-specific STRs (DYS391 and DYS392). The amplification results of this multiplex approach back each other up, and enable a reliable sex identification. This coamplification of X- and Y-specific markers in a multiplex assay combines the added advantage of positive identification of both female and male individuals with raising the validity of the diagnosis by obtaining multiple data simultaneously. This multiplex system was successfully applied to 3,000-year-old bone material.

  1. Multiplex PageRank.

    PubMed

    Halu, Arda; Mondragón, Raúl J; Panzarasa, Pietro; Bianconi, Ginestra

    2013-01-01

    Many complex systems can be described as multiplex networks in which the same nodes can interact with one another in different layers, thus forming a set of interacting and co-evolving networks. Examples of such multiplex systems are social networks where people are involved in different types of relationships and interact through various forms of communication media. The ranking of nodes in multiplex networks is one of the most pressing and challenging tasks that research on complex networks is currently facing. When pairs of nodes can be connected through multiple links and in multiple layers, the ranking of nodes should necessarily reflect the importance of nodes in one layer as well as their importance in other interdependent layers. In this paper, we draw on the idea of biased random walks to define the Multiplex PageRank centrality measure in which the effects of the interplay between networks on the centrality of nodes are directly taken into account. In particular, depending on the intensity of the interaction between layers, we define the Additive, Multiplicative, Combined, and Neutral versions of Multiplex PageRank, and show how each version reflects the extent to which the importance of a node in one layer affects the importance the node can gain in another layer. We discuss these measures and apply them to an online multiplex social network. Findings indicate that taking the multiplex nature of the network into account helps uncover the emergence of rankings of nodes that differ from the rankings obtained from one single layer. Results provide support in favor of the salience of multiplex centrality measures, like Multiplex PageRank, for assessing the prominence of nodes embedded in multiple interacting networks, and for shedding a new light on structural properties that would otherwise remain undetected if each of the interacting networks were analyzed in isolation.

  2. Multiplexity and multireciprocity in directed multiplexes

    NASA Astrophysics Data System (ADS)

    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.

  3. A fast PCR-based method for the characterization of prophage profiles in strains of the Lactobacillus casei group.

    PubMed

    Zaburlin, Delfina; Mercanti, Diego J; Quiberoni, Andrea

    2017-10-01

    Lysogeny is widespread among Lactobacillus strains of the casei group (L. casei, L. paracasei and L. rhamnosus), and prophages account for most strain-specific DNA. Numerous PCR based methods have been developed to detect free phages of lactic acid bacteria, but they do not take in consideration prophages. In this study, a new PCR method for the detection of lysogeny was developed using genome sequences of L. casei group strains (including BL23) and bacteriophages. Nine pairs of primers were designed to selectively amplify the highly conserved prophage iA2 (pairs #1-#3) and fragments of two groups phages of temperate origin: CL1/CL2/iLp1308/iLp84 (pairs #4 and #5) and Lrm1/J-1/PL-1/A2/AT3/Lc-Nu (pairs #6 to #9). Forty-nine strains of the casei group were subjected to PCR. Strains containing remnants of lytic phages outnumbered those containing iA2-related prophages. The combination of pair #2, annealing on the terminase large subunit (TLS), and pair #3, annealing on the helicase (forward) and a non-coding region (reverse), showed the best diagnostic performance for iA2-like prophages. For the assessment of remnants of phages CL1/CL2/iLp1308/iLp84, pair #4 (annealing on the TLS) was preferred over pair #5 (portal protein). Detection of phages Lrm1/J-1/PL-1/A2/AT3/Lc-Nu was optimal with primers of pair #6, designed on non-coding regions of phage genomes; pair #6 also evidenced a high conservation of certain prophage remnants. Overall, our PCR-based method successfully detected and discriminated groups of prophages or remnants in L. casei group strains. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. A high-throughput multiplex method adapted for GMO detection.

    PubMed

    Chaouachi, Maher; Chupeau, Gaëlle; Berard, Aurélie; McKhann, Heather; Romaniuk, Marcel; Giancola, Sandra; Laval, Valérie; Bertheau, Yves; Brunel, Dominique

    2008-12-24

    A high-throughput multiplex assay for the detection of genetically modified organisms (GMO) was developed on the basis of the existing SNPlex method designed for SNP genotyping. This SNPlex assay allows the simultaneous detection of up to 48 short DNA sequences (approximately 70 bp; "signature sequences") from taxa endogenous reference genes, from GMO constructions, screening targets, construct-specific, and event-specific targets, and finally from donor organisms. This assay avoids certain shortcomings of multiplex PCR-based methods already in widespread use for GMO detection. The assay demonstrated high specificity and sensitivity. The results suggest that this assay is reliable, flexible, and cost- and time-effective for high-throughput GMO detection.

  5. MIG-seq: an effective PCR-based method for genome-wide single-nucleotide polymorphism genotyping using the next-generation sequencing platform

    PubMed Central

    Suyama, Yoshihisa; Matsuki, Yu

    2015-01-01

    Restriction-enzyme (RE)-based next-generation sequencing methods have revolutionized marker-assisted genetic studies; however, the use of REs has limited their widespread adoption, especially in field samples with low-quality DNA and/or small quantities of DNA. Here, we developed a PCR-based procedure to construct reduced representation libraries without RE digestion steps, representing de novo single-nucleotide polymorphism discovery, and its genotyping using next-generation sequencing. Using multiplexed inter-simple sequence repeat (ISSR) primers, thousands of genome-wide regions were amplified effectively from a wide variety of genomes, without prior genetic information. We demonstrated: 1) Mendelian gametic segregation of the discovered variants; 2) reproducibility of genotyping by checking its applicability for individual identification; and 3) applicability in a wide variety of species by checking standard population genetic analysis. This approach, called multiplexed ISSR genotyping by sequencing, should be applicable to many marker-assisted genetic studies with a wide range of DNA qualities and quantities. PMID:26593239

  6. MIG-seq: an effective PCR-based method for genome-wide single-nucleotide polymorphism genotyping using the next-generation sequencing platform.

    PubMed

    Suyama, Yoshihisa; Matsuki, Yu

    2015-11-23

    Restriction-enzyme (RE)-based next-generation sequencing methods have revolutionized marker-assisted genetic studies; however, the use of REs has limited their widespread adoption, especially in field samples with low-quality DNA and/or small quantities of DNA. Here, we developed a PCR-based procedure to construct reduced representation libraries without RE digestion steps, representing de novo single-nucleotide polymorphism discovery, and its genotyping using next-generation sequencing. Using multiplexed inter-simple sequence repeat (ISSR) primers, thousands of genome-wide regions were amplified effectively from a wide variety of genomes, without prior genetic information. We demonstrated: 1) Mendelian gametic segregation of the discovered variants; 2) reproducibility of genotyping by checking its applicability for individual identification; and 3) applicability in a wide variety of species by checking standard population genetic analysis. This approach, called multiplexed ISSR genotyping by sequencing, should be applicable to many marker-assisted genetic studies with a wide range of DNA qualities and quantities.

  7. Benefits of Adding a Rapid PCR-Based Blood Culture Identification Panel to an Established Antimicrobial Stewardship Program

    PubMed Central

    2016-01-01

    Studies have demonstrated that the combination of antimicrobial stewardship programs (ASP) and rapid organism identification improves outcomes in bloodstream infections (BSI) but have not controlled for the incremental contribution of the individual components. Hospitalized adult patients with blood culture pathogens on a rapid, multiplex PCR-based blood culture identification panel (BCID) that included 19 bacterial species, 5 Candida spp., and 4 antimicrobial resistance genes were studied over sequential time periods in a pre-post quasiexperimental study in 3 groups in the following categories: conventional organism identification (controls), conventional organism identification with ASP (AS), and BCID with ASP (BCID). Clinical and economic outcomes were compared between groups. There were 783 patients with positive blood cultures; of those patients, 364 (115 control, 104 AS, and 145 BCID) met inclusion criteria. The time from blood culture collection to organism identification was shorter in the BCID group (17 h; P < 0.001) than in the control group (57 h) or the AS group (54 h). The BCID group had a shorter time to effective therapy (5 h; P < 0.001) than the control group (15 h) or AS group (13 h). The AS (57%) and BCID (52%) groups had higher rates of antimicrobial de-escalation than the control group (34%), with de-escalation occurring sooner in the BCID group (48 h; P = 0.034) than in the AS group (61 h) or the control group (63 h). No difference between the control group, AS group, and BCID group was seen with respect to mortality, 30-day readmission, intensive care unit length of stay (LOS), postculture LOS, or costs. In patients with BSI, ASP alone improved antimicrobial utilization. Addition of BCID to an established ASP shortened the time to effective therapy and further improved antimicrobial use compared to ASP alone, even in a setting of low antimicrobial resistance rates. PMID:27487951

  8. Proposal of a quantitative PCR-based protocol for an optimal Pseudomonas aeruginosa detection in patients with cystic fibrosis.

    PubMed

    Le Gall, Florence; Le Berre, Rozenn; Rosec, Sylvain; Hardy, Jeanne; Gouriou, Stéphanie; Boisramé-Gastrin, Sylvie; Vallet, Sophie; Rault, Gilles; Payan, Christopher; Héry-Arnaud, Geneviève

    2013-06-21

    The lung of patients with cystic fibrosis (CF) is particularly sensitive to Pseudomonas aeruginosa. This bacterium plays an important role in the poor outcome of CF patients. During the disease progress, first acquisition of P. aeruginosa is the key-step in the management of CF patients. Quantitative PCR (qPCR) offers an opportunity to detect earlier the first acquisition of P. aeruginosa by CF patients. Given the lack of a validated protocol, our goal was to find an optimal molecular protocol for detection of P. aeruginosa in CF patients. We compared two formerly described qPCR formats in early detection of P. aeruginosa in CF sputum samples: a qPCR targeting oprL gene, and a multiplex PCR targeting gyrB and ecfX genes. Tested in vitro on a large panel of P. aeruginosa isolates and others gram-negative bacilli, oprL qPCR exhibited a better sensitivity (threshold of 10 CFU/mL versus 730 CFU/mL), whereas the gyrB/ecfX qPCR exhibited a better specificity (90% versus 73%). These results were validated ex vivo on 46 CF sputum samples positive for P. aeruginosa in culture. Ex vivo assays revealed that qPCR detected 100 times more bacterial cells than culture-based method did. Based on these results, we proposed a reference molecular protocol combining the two qPCRs, which offers a sensitivity of 100% with a threshold of 10 CFU/mL and a specificity of 100%. This combined qPCR-based protocol can be adapted and used for other future prospective studies.

  9. Multiplex gas chromatography

    NASA Technical Reports Server (NTRS)

    Valentin, Jose R.

    1990-01-01

    The principles of the multiplex gas chromatography (GC) technique, which is a possible candidate for chemical analysis of planetary atmospheres, are discussed. Particular attention is given to the chemical modulators developed by present investigators for multiplex GC, namely, the thermal-desorption, thermal-decomposition, and catalytic modulators, as well as to mechanical modulators. The basic technique of multiplex GC using chemical modulators and a mechanical modulator is demonstrated. It is shown that, with the chemical modulators, only one gas stream consisting of the carrier in combination with the components is being analyzed, resulting in a simplified instrument that requires relatively few consumables. The mechanical modulator demonstrated a direct application of multiplex GC for the analysis of gases in atmosphere of Titan at very low pressures.

  10. Multiplexed Engineering in Biology.

    PubMed

    Rogers, Jameson K; Church, George M

    2016-03-01

    Biotechnology is the manufacturing technology of the future. However, engineering biology is complex, and many possible genetic designs must be evaluated to find cells that produce high levels of a desired drug or chemical. Recent advances have enabled the design and construction of billions of genetic variants per day, but evaluation capacity remains limited to thousands of variants per day. Here we evaluate biological engineering through the lens of the design–build–test cycle framework and highlight the role that multiplexing has had in transforming the design and build steps. We describe a multiplexed solution to the ‘test’ step that is enabled by new research. Achieving a multiplexed test step will permit a fully multiplexed engineering cycle and boost the throughput of biobased product development by up to a millionfold.

  11. Microfluidic multiplexing in bioanalyses.

    PubMed

    Araz, M Kursad; Tentori, Augusto M; Herr, Amy E

    2013-10-01

    The importance of biological assays spans from clinical diagnostics to environmental monitoring. Simultaneous detection of multiple analytes enhances the efficacy of bioassays by providing more data per assay under standardized conditions. Nevertheless, simultaneous handling and assaying of multiple samples, targets, and experimental conditions can be laborious, reagent consuming, and time intensive. Given these demands, microfluidic platforms have emerged over the past two decades as well-suited approaches for multiplexed assays. Microfluidic design supports integration of assay steps and reproducible sample manipulation across large sets of conditions--all relevant to multiplexed assays. Taken together, reduced reagent consumption, faster assay times, and potential for automation stemming from microfluidic assay design are attractive and needed multiplexed assay performance attributes. This review highlights recent advances in multiplexed bioanalyses benefitting from microfluidic integration.

  12. Multiplex gas chromatography

    NASA Technical Reports Server (NTRS)

    Valentin, Jose R.

    1990-01-01

    The principles of the multiplex gas chromatography (GC) technique, which is a possible candidate for chemical analysis of planetary atmospheres, are discussed. Particular attention is given to the chemical modulators developed by present investigators for multiplex GC, namely, the thermal-desorption, thermal-decomposition, and catalytic modulators, as well as to mechanical modulators. The basic technique of multiplex GC using chemical modulators and a mechanical modulator is demonstrated. It is shown that, with the chemical modulators, only one gas stream consisting of the carrier in combination with the components is being analyzed, resulting in a simplified instrument that requires relatively few consumables. The mechanical modulator demonstrated a direct application of multiplex GC for the analysis of gases in atmosphere of Titan at very low pressures.

  13. Multiplex television transmission system

    NASA Technical Reports Server (NTRS)

    Reed, W. R.

    1967-01-01

    Time-multiplexing system enables several cameras to share a single commercial television transmission channel. This system is useful in industries for visually monitoring several operating areas or instrument panels from a remote location.

  14. Downlink data multiplexer

    NASA Technical Reports Server (NTRS)

    Holland, S. Douglas (Inventor); Steele, Glen F. (Inventor); Romero, Denise M. (Inventor); Koudelka, Robert David (Inventor)

    2008-01-01

    A data multiplexer that accommodates both industry standard CCSDS data packets and bits streams and standard IEEE 1394 data is described. The multiplexer provides a statistical allotment of bandwidth to the channels in turn, preferably four, but expandable in increments of four up to sixteen. A microcontroller determines bandwidth requested by the plurality of channels, as well as the bandwidth available, and meters out the available bandwidth on a statistical basis employing flow control to the input channels.

  15. Multiplexed chirp waveform synthesizer

    DOEpatents

    Dudley, Peter A.; Tise, Bert L.

    2003-09-02

    A synthesizer for generating a desired chirp signal has M parallel channels, where M is an integer greater than 1, each channel including a chirp waveform synthesizer generating at an output a portion of a digital representation of the desired chirp signal; and a multiplexer for multiplexing the M outputs to create a digital representation of the desired chirp signal. Preferably, each channel receives input information that is a function of information representing the desired chirp signal.

  16. DEVELOPMENT OF AN IMPROVED PCR-BASED TECHNIQUE FOR DETECTION OF PHYTOPHTHORA CACTORUM IN STRAWBERRY PLANTS

    USDA-ARS?s Scientific Manuscript database

    Specific and rapid plant pathogen detection methods can aid in strawberry disease management decisions. PCR-based diagnostics for Phytophthora cactorum and other strawberry pathogens are hindered by PCR inhibitors and lack of species-specific PCR primers. We developed a DNA extraction and purificati...

  17. USE OF BACTEROIDES PCR-BASED METHODS TO EXAMINE FECAL CONTAMINATION SOURCES IN TROPICAL COASTAL WATERS

    EPA Science Inventory

    Several library independent Microbial Source Tracking methods have been developed to rapidly determine the source of fecal contamination. Thus far, none of these methods have been tested in tropical marine waters. In this study, we used a Bacteroides 16S rDNA PCR-based...

  18. Evaluation of Two PCR-based Swine-specific Fecal Source Tracking Assays (Abstract)

    EPA Science Inventory

    Several PCR-based methods have been proposed to identify swine fecal pollution in environmental waters. However, the utility of these assays in identifying swine fecal contamination on a broad geographic scale is largely unknown. In this study, we evaluated the specificity, distr...

  19. Development and evaluation of new primers for PCR-based identification of Prevotella intermedia.

    PubMed

    Zhou, Yanbin; Liu, Dali; Wang, Yiwei; Zhu, Cailian; Liang, Jingping; Shu, Rong

    2014-08-01

    The aim of this study was to develop new Prevotella intermedia-specific PCR primers based on the 16S rRNA. The new primer set, Pi-192 and Pi-468, increased the accuracy of PCR-based P. intermedia identification and could be useful in the detection of P. intermedia as well as epidemiological studies on periodontal disease.

  20. Evaluation of Two PCR-based Swine-specific Fecal Source Tracking Assays (Abstract)

    EPA Science Inventory

    Several PCR-based methods have been proposed to identify swine fecal pollution in environmental waters. However, the utility of these assays in identifying swine fecal contamination on a broad geographic scale is largely unknown. In this study, we evaluated the specificity, distr...

  1. Quantitative PCR based expression analysis on a nanoliter scale using polymer nano-well chips.

    PubMed

    Dahl, Andreas; Sultan, Marc; Jung, Alexander; Schwartz, Regine; Lange, Matthias; Steinwand, Michael; Livak, Kenneth J; Lehrach, Hans; Nyarsik, Lajos

    2007-06-01

    The analysis of gene expression is an essential element of functional genomics. Expression analysis is mainly based on DNA microarrays due to highly parallel readout and high throughput. Quantitative PCR (qPCR) based expression profiling is the gold standard for the precise monitoring of selected genes, and therefore used for validation of microarray data. Doing qPCR-based expression analysis in an array-like format can combine the higher sensitivity and accuracy of the qPCR methodology with a high data density at relatively low costs. This paper describes the development of an open-well based miniaturized platform for liquid PCR-based assays on the nanoliter scale using cost-effective polypropylene micro reactors (microPCR Chip). We show the quantification ability and reliability of qPCR in 200 nl with the microPCR chip down to 5 starting target molecules using TaqMan chemistry. An RNA expression analysis of four genes in mouse brain, liver and kidney tissues showed similar results in 200 nl as compared to standard 10 microl assays. The high sensitivity and quantification capability of the microPCR chip platform developed herein makes it a promising technology for performing high-throughput qPCR-based analysis in the nanoliter volume range.

  2. USE OF BACTEROIDES PCR-BASED METHODS TO EXAMINE FECAL CONTAMINATION SOURCES IN TROPICAL COASTAL WATERS

    EPA Science Inventory

    Several library independent Microbial Source Tracking methods have been developed to rapidly determine the source of fecal contamination. Thus far, none of these methods have been tested in tropical marine waters. In this study, we used a Bacteroides 16S rDNA PCR-based...

  3. Compact spatial multiplexers for mode division multiplexing.

    PubMed

    Chen, Haoshuo; van Uden, Roy; Okonkwo, Chigo; Koonen, Ton

    2014-12-29

    Spatial multiplexer (SMUX) for mode division multiplexing (MDM) has evolved from mode-selective excitation, multiple-spot and photonic-lantern based solutions in order to minimize both mode-dependent loss (MDL) and coupler insertion loss (CIL). This paper discusses the implementation of all the three solutions by compact components in a small footprint. Moreover, the compact SMUX can be manufactured in mass production and packaged to assure high reliability. First, push-pull scheme and center launch based SMUXes are demonstrated on two mostly-popular photonic integration platforms: Silicon-on-insulator (SOI) and Indium Phosphide (InP) for selectively exciting LP01 and LP11 modes. 2-dimensional (2D) top-coupling by using vertical emitters is explored to provide a coupling interface between a few-mode fiber (FMF) and the photonic integrated SMUX. SOI-based grating couplers and InP-based 45° vertical mirrors are proposed and researched as vertical emitters in each platform. Second, a 3-spot SMUX is realized on an InP-based circuit through employing 45° vertical mirrors. Third, as a newly-emerging photonic integration platform, laser-inscribed 3D waveguide (3DW) technology is applied for a fully-packaged dual-channel 6-mode SMUX including two 6-core photonic lantern structures as mode multiplexer and demultiplexer, respectively.

  4. Genetics Home Reference: steatocystoma multiplex

    MedlinePlus

    ... Genetic Changes Steatocystoma multiplex can be caused by mutations in the KRT17 gene. This gene provides instructions ... skin, nails, and other tissues. The KRT17 gene mutations that cause steatocystoma multiplex alter the structure of ...

  5. A multiplex PCR assay that separates Rhodnius prolixus from members of the Rhodnius robustus cryptic species complex (Hemiptera: Reduviidae).

    PubMed

    Pavan, M G; Monteiro, F A

    2007-06-01

    Rhodnius prolixus is one of the most important primary vectors of human Chagas disease in Latin America. Its morphology is, however, identical to that of the members of the Rhodnius robustus cryptic species complex, which includes secondary vectors. The correct identification of these taxa with differential vector competence is, therefore, of great epidemiological relevance. We used the alignment of 26 mitochondrial cytochrome b haplotypes (663 bp) to select for PCR-amplifiable species-specific regions. We designed one forward primer on a region conserved across all haplotypes, and three reverse primers that anneal to species-specific regions and amplify fragments of different lengths for R. prolixus (285 bp) and for members of the two major R. robustus lineages: group I (349 bp) and groups II-IV (239 bp). These fragments were easily identifiable on regular 1.5% agarose gels. This multiplex PCR assay was successfully tested on 81 specimens from six Latin American countries, and used to determine the phylogeographic boundaries for each species. It is a simple, objective, and cost-effective assay. Its PCR-based nature makes it applicable to any insect developmental stage, as well as to dried specimens, and insect remains. It should be particularly useful in areas where representatives of these Rhodnius species occur in sympatry.

  6. PCR-based bioprospecting for homing endonucleases in fungal mitochondrial rRNA genes.

    PubMed

    Hafez, Mohamed; Guha, Tuhin Kumar; Shen, Chen; Sethuraman, Jyothi; Hausner, Georg

    2014-01-01

    Fungal mitochondrial genomes act as "reservoirs" for homing endonucleases. These enzymes with their DNA site-specific cleavage activities are attractive tools for genome editing and gene therapy applications. Bioprospecting and characterization of naturally occurring homing endonucleases offers an alternative to synthesizing artificial endonucleases. Here, we describe methods for PCR-based screening of fungal mitochondrial rRNA genes for homing endonuclease encoding sequences, and we also provide protocols for the purification and biochemical characterization of putative native homing endonucleases.

  7. Performance of PCR-based and Bioluminescent assays for mycoplasma detection.

    PubMed

    Falagan-Lotsch, Priscila; Lopes, Talíria Silva; Ferreira, Nívea; Balthazar, Nathália; Monteiro, Antônio M; Borojevic, Radovan; Granjeiro, José Mauro

    2015-11-01

    Contaminated eukaryotic cell cultures are frequently responsible for unreliable results. Regulatory entities request that cell cultures must be mycoplasma-free. Mycoplasma contamination remains a significant problem for cell cultures and may have an impact on biological analysis since they affect many cell parameters. The gold standard microbiological assay for mycoplasma detection involves laborious and time-consuming protocols. PCR-based and Bioluminescent assays have been considered for routine cell culture screening in research laboratories since they are fast, easy and sensitive. Thus, the aim of this work is to compare the performance of two popular commercial assays, PCR-based and Bioluminescent assays, by assessing the level of mycoplasma contamination in cell cultures from Rio de Janeiro Cell Bank (RJCB) and also from customers' laboratories. The results obtained by both performed assays were confirmed by scanning electron microscopy. In addition, we evaluated the limit of detection of the PCR kit under our laboratory conditions and the storage effects on mycoplasma detection in frozen cell culture supernatants. The performance of both assays for mycoplasma detection was not significantly different and they showed very good agreement. The Bioluminescent assay for mycoplasma detection was slightly more dependable than PCR-based due to the lack of inconclusive results produced by the first technique, especially considering the ability to detect mycoplasma contamination in frozen cell culture supernatants. However, cell lines should be precultured for four days or more without antibiotics to obtain safe results. On the other hand, a false negative result was obtained by using this biochemical approach. The implementation of fast and reliable mycoplasma testing methods is an important technical and regulatory issue and PCR-based and Bioluminescent assays may be good candidates. However, validation studies are needed.

  8. Computer assisted multiplex sequencing

    SciTech Connect

    Church, G.M.

    1992-08-01

    The objectives of this project are automation and optimization of multiplex sequencing. This year we have integrated direct transfer electrophoresis, automated multiplex hybridizations and automated film reading and applied this toward sequencing of three contiguous E. coli cosmids. Primers for the directed dideoxy sequence walking and sequence confirmation steps were synthesized with a 15 base tag complimentary to an alkaline phosphatase conjugate. A higher throughput synthesis device is well along in testing as are new automated hybridization devices. We have developed software for automatically annotating ORFs and databases of precise termini of proteis and RNA.

  9. Adaptive Telemetry Multiplexer

    NASA Technical Reports Server (NTRS)

    Sinderson, R. L.; Salazar, G. A.; Haddick, C. M., Jr.; Spahn, C. J.; Venkatesh, C. N.

    1989-01-01

    Telemetry-data-acquisition unit adjusted remotely to produce changes in sampling rate, sampling channels, measurement scale, and output-bias level. Functional configuration adapted to changing conditions or new requirements by distant operator over telemetry link. Reconfiguration done in real time, without removing equipment from service. Bus-interface unit accepts reprogramming commands and translates them for low-rate adaptive multiplexer. Reprogrammable equipment reduces need for spare parts, since not necessary to stock variety of hardware with fixed characteristics. Adaptive multiplexer performs well in tests, amplitude errors less than 0.5 percent, distortion less than 0.25 percent, and crosstalk and common-mode rejection indiscernible.

  10. Monitoring Babesia bovis infections in cattle by using PCR-based tests.

    PubMed Central

    Calder, J A; Reddy, G R; Chieves, L; Courtney, C H; Littell, R; Livengood, J R; Norval, R A; Smith, C; Dame, J B

    1996-01-01

    The sensitivity and specificity of PCR tests based on the small-subunit rRNA gene sequence of Babesia bovis were compared in a blind study of experimentally infected cattle with the corresponding parameters of the complement fixation (CF) test currently used in the United States to screen for bovine babesiosis. Cattle were experimentally infected with a single inoculum of a cloned laboratory strain of B. bovis. Blood samples were collected and tested over a period covering from the day of infection to 10 months postinfection. The level of parasitemia (percent infected erythrocytes) present in each sample was estimated from test results and was plotted as a function of time postinfection. These data are the first describing the course of infection by methods capable of detecting parasitemias in the range of 10(-7)%, which frequently occur in the carrier state. Parasitemias in the samples tested strongly influenced the sensitivity and negative predictive value of the PCR-based tests which varied with time postinfection. The average sensitivities of the three PCR-based tests for B. bovis ranged from 58 to 70% for a single determination, while the sensitivity of the CF test was only 6%. Both PCR-based and CF tests for B. bovis had high specificity values ranging from 96 to 100%. PMID:8897177

  11. Molecular sexing of birds: A comparative review of polymerase chain reaction (PCR)-based methods.

    PubMed

    Morinha, F; Cabral, J A; Bastos, E

    2012-09-01

    Accurate identification of sex in birds is important for the management and conservation of avian wildlife in several ways, namely in the development of population, behavioral and ecological studies, as well as in the improvement of ex situ captive breeding programs. In general, nestlings, juveniles and adult birds of a wide number of sexually monomorphic species cannot be sexed based on phenotypic traits. The development of molecular methodologies for avian sexing overcame these difficulties, allowing a reliable gender differentiation for these species. The polymerase chain reaction (PCR)-based methods have been widely applied in molecular sexing of birds, using a large diversity of sex-linked markers. During the last 15 yrs, there was a continuous improvement in the PCR-based protocols for bird sexing, increasing the accuracy, speed and high-throughput applicability of these techniques. The recent advances in real-time PCR platforms and whole genome analysis methods provided new resources for the detection and analysis of novel specific markers and protocols. This review presents a comparative guide of classical and recent advances in PCR-based methods for avian molecular sexing, highlighting its strengths and limitations. Future research opportunities in this field are also addressed. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Essential genes in the infection model of Pseudomonas aeruginosa-PCR-based signature-tagged mutagenesis.

    PubMed

    Kukavica-Ibrulj, Irena; Levesque, Roger C

    2015-01-01

    PCR-based signature tagged mutagenesis is an "en masse" screening technique based upon unique oligonucleotide tags (molecular barcodes) for identification of genes that will diminish or enhance maintenance of an organism in a specific ecological niche or environment. PCR-based STM applied to Pseudomonas aeruginosa permitted the identification of genes essential for in vivo maintenance by transposon insertion and negative selection in a mixed population of bacterial mutants. The innovative adaptations and refinement of the technology presented here with P. aeruginosa STM mutants selected in the rat model of chronic lung infection have given critical information about genes essential for causing a chronic infection and a wealth of information about biological processes in vivo. The additional use of competitive index analysis for measurement of the level of virulence in vivo, microarray-based screening of selected prioritized STM mutants coupled to metabolomics analysis can now be attempted systematically on a genomic scale. PCR-based STM and combined whole-genome methods can also be applied to any organism having selectable phenotypes for screening.

  13. A PCR-based tool for the cultivation-independent monitoring of Pandora neoaphidis.

    PubMed

    Fournier, A; Enkerli, J; Keller, S; Widmer, F

    2008-09-01

    Pandora neoaphidis is one of the most important fungal pathogens of aphids and has a great potential for use in biocontrol. Little is known on how this fungus persists in an area and in particular on its overwintering strategies. It is hypothesized that natural areas play an important role for survival and that soil may serve as a source of inoculum for new aphid populations in spring. To test these hypotheses, a cultivation-independent PCR-based diagnostic tool was developed, that allows the detection of P. neoaphidis in the environment. Two P. neoaphidis specific PCR primer pairs were designed, targeting sequences in the ribosomal RNA gene cluster. Specificity of both primer pairs was demonstrated with P. neoaphidis and non-target close entomophthoralean relatives. Moreover, single amplicons of expected sizes were obtained with both primer pairs from various environmental sample types, including aphid cadavers, plant material, and soil. The PCR-based diagnostic tool was applied to investigate the persistence of P. neoaphidis in soil samples obtained in 2004/2005 from a nettle field harboring infected aphids in fall 2004. P. neoaphidis was detected in every sample collected in November 2004 and March 2005, suggesting an overwintering stage of P. neoaphidis in top soil layers. The developed cultivation-independent PCR-based tool will be valuable for further investigation of the ecology of P. neoaphidis and for the development and future implementation of management strategies against aphids involving conservation biocontrol.

  14. Dual phase multiplex polymerase chain reaction

    DOEpatents

    Pemov, Alexander; Bavykin, Sergei

    2008-10-07

    Highly specific and sensitive methods were developed for multiplex amplification of nucleic acids on supports such as microarrays. Based on a specific primer design, methods include five types of amplification that proceed in a reaction chamber simultaneously. These relate to four types of multiplex amplification of a target DNA on a solid support, directed by forward and reverse complex primers immobilized to the support and a fifth type--pseudo-monoplex polymerase chain reaction (PCR) of multiple targets in solution, directed by a single pair of unbound universal primers. The addition of the universal primers in the reaction mixture increases the yield over the traditional "bridge" amplification on a solid support by approximately ten times. Methods that provide multitarget amplification and detection of as little as 0.45-4.5.times.10.sup.-12 g (equivalent to 10.sup.2-10.sup.3 genomes) of a bacterial genomic DNA are disclosed.

  15. High-throughput real-time PCR-based genotyping without DNA purification

    PubMed Central

    2012-01-01

    Background While improvements in genotyping technology have allowed for increased throughput and reduced time and expense, protocols remain hindered by the slow upstream steps of isolating, purifying, and normalizing DNA. Various methods exist for genotyping samples directly through blood, without having to purify the DNA first. These procedures were designed to be used on smaller throughput systems, however, and have not yet been tested for use on current high-throughput real-time (q)PCR based genotyping platforms. In this paper, a method of quantitative qPCR-based genotyping on blood without DNA purification was developed using a high-throughput qPCR platform. Findings The performances of either DNA purified from blood or the same blood samples without DNA purification were evaluated through qPCR-based genotyping. First, 60 different mutations prevalent in the Ashkenazi Jewish population were genotyped in 12 Ashkenazi Jewish individuals using the QuantStudio™12K Flex Real-Time PCR System. Genotyping directly from blood gave a call rate of 99.21%, and an accuracy of 100%, while the purified DNA gave a call rate of 92.49%, and an accuracy of 99.74%. Although no statistical difference was found for these parameters, an F test comparing the standard deviations of the wild type clusters for the two different methods indicated significantly less variation when genotyping directly from blood instead of after DNA purification. To further establish the ability to perform high-throughput qPCR based genotyping directly from blood, 96 individuals of Ashkenazi Jewish decent were genotyped for the same 60 mutations (5,760 genotypes in 5 hours) and resulted in a call rate of 98.38% and a diagnostic accuracy of 99.77%. Conclusion This study shows that accurate qPCR-based high-throughput genotyping can be performed without DNA purification. The direct use of blood may further expedite the entire genotyping process, reduce costs, and avoid tracking errors which can occur during

  16. Time-division SQUID multiplexers

    NASA Astrophysics Data System (ADS)

    Irwin, K. D.; Vale, L. R.; Bergren, N. E.; Deiker, S.; Grossman, E. N.; Hilton, G. C.; Nam, S. W.; Reintsema, C. D.; Rudman, D. A.; Huber, M. E.

    2002-02-01

    SQUID multiplexers make it possible to build arrays of thousands of low-temperature bolometers and microcalorimeters based on superconducting transition-edge sensors with a manageable number of readout channels. We discuss the technical tradeoffs between proposed time-division multiplexer and frequency-division multiplexer schemes and motivate our choice of time division. Our first-generation SQUID multiplexer is now in use in an astronomical instrument. We describe our second-generation SQUID multiplexer, which is based on a new architecture that significantly reduces the dissipation of power at the first stage, allowing thousands of SQUIDs to be operated at the base temperature of a cryostat. .

  17. Multiplex detection of food allergens and gluten.

    PubMed

    Cho, Chung Y; Nowatzke, William; Oliver, Kerry; Garber, Eric A E

    2015-05-01

    To help safeguard the food supply and detect the presence of undeclared food allergens and gluten, most producers and regulatory agencies rely on commercial test kits. Most of these are ELISAs with a few being PCR-based. These methods are very sensitive and analyte specific, requiring different assays to detect each of the different food allergens. Mass spectrometry offers an alternative approach whereby multiple allergens may be detected simultaneously. However, mass spectrometry requires expensive equipment, highly trained analysts, and several years before a quantitative approach can be achieved. Using multianalyte profiling (xMAP®) technology, a commercial multiplex test kit based on the use of established antibodies was developed for the simultaneous detection of up to 14 different food allergens plus gluten. The assay simultaneously detects crustacean seafood, egg, gluten, milk, peanut, soy, and nine tree nuts (almond, Brazil nut, cashew, coconut, hazelnut, macadamia, pine nut, pistachio, and walnut). By simultaneously performing multiple tests (typically two) for each analyte, this magnetic bead-based assay offers built-in confirmatory analyses without the need for additional resources. Twenty-five of the assays were performed on buffer extracted samples, while five were conducted on samples extracted using reduced-denatured conditions. Thus, complete analysis for all 14 allergens and gluten requires only two wells of a 96-well microtiter plate. This makes it possible to include in a single analytical run up to 48 samples. All 30 bead sets in this multiplex assay detected 5 ng/mL of food allergen and gluten with responses greater than background. In addition, 26 of the bead sets displayed signal/noise ratios of five or greater. The bead-based design makes this 30-plex assay expandable to incorporate new antibodies and capture/detector methodologies by ascribing these new detectors to any of the unassigned bead sets that are commercially available.

  18. Validation of a Short Tandem Repeat Multiplex Typing System for Genetic Individualization of Domestic Cat Samples

    PubMed Central

    Coomber, Nikia; David, Victor A.; O’Brien, Stephen J.; Menotti-Raymond, Marilyn

    2007-01-01

    Aim To conduct developmental validation studies on a polymerase chain reaction (PCR) based short tandem repeat (STR) multiplex typing system, developed for the purpose of genetic individualization and parentage testing in domestic cat samples. Methods To evaluate reproducibility of the typing system, the multiplex was amplified using DNA extracted from hair, blood, and buccal samples obtained from the same individual (n = 13). Additional studies were performed to evaluate the system’s species’ specificity, using 26 North American mammalian species and two prokaryotes Sacchromyces and Escherichia coli, sensitivity, and ability to identify DNA mixtures. Patterns of Mendelian inheritance and mutation rates for the 11 loci were directly examined in a large multi-generation domestic cat pedigree (n = 263). Results Our studies confirm that the multiplex system was species-specific for feline DNA and amplified robustly with as little as 125 picograms of genomic template DNA, demonstrating good product balance. The multiplex generated all components of a two DNA mixture when the minor component was one tenth of the major component at a threshold of 50 relative fluorescence units. The multiplex was reproducible in multiple tissue types of the same individual. Mutation rates for the 11 STR were within the range of sex averaged rates observed for Combined DNA Index System (CODIS) loci. Conclusion The cat STR multiplex typing system is a robust and reliable tool for the use of forensic DNA analysis of domestic cat samples. PMID:17696310

  19. 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.

  20. Extracting information from multiplex networks.

    PubMed

    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 Θ̃(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.

  1. Downlink Data Multiplexer

    NASA Technical Reports Server (NTRS)

    Holland, Douglas; Steele, Glen F.; Romero, Denise M.; Koudelka, Robert David

    2004-01-01

    A multiplexer/demultiplexer system has been developed to enable the transmission, over a single channel, of four data streams generated by a variety of sources at different (including variable) bit rates. In the original intended application, replicas of this multiplexer/demultiplexer system would be incorporated into the spacecraft-to-ground communication systems of the space shuttles. The multiplexer of each system would be installed in the spacecraft, where it would acquire and process data from such sources as commercial digital camcorders, video tape recorders, and the spacecraft telemetry system. The demultiplexer of each system would be installed in a ground station. Purely terrestrial systems of similar design could be attractive for use in situations in which there are requirements to transmit multiple streams of high-quality video data and possibly other data over single channels. The figure is a block diagram of the multiplexer as configured to process data received via three fiber-optic channels like those of the International Space Station and one electrical-cable channel that conforms to the Institute of Electrical and Electronic Engineers (IEEE) 1394 standard. (This standard consists of specifications of a high-speed serial data interface, the physical layer of which includes a cable known in the art as "FireWire." An IEEE 1394 interface can also transfer power between the components to which it is connected.) The fiber-optic channels carry packet and/or bit-stream signals that conform to the standards of the Consultative Committee for Space Data Systems (CCSDS). The IEEE 1394 interface accepts an isochronous signal like that from a digital camcorder or a video tape recorder. The processing of the four input data streams to combine them into one output stream is governed by a statistical multiplexing algorithm that features a flow-control capability and makes it possible to utilize the transmission channel with nearly 100-percent efficiency. This

  2. Single-step multiplex reverse transcription-polymerase chain reaction assay for detection and differentiation of the 2009 (H1N1) influenza A virus pandemic in Thai swine populations

    USDA-ARS?s Scientific Manuscript database

    A recently emerged H1N1 Influenza A virus (pandemic 1 H1N1: pH1N1) with a Swine influenza virus (SIV) genetic background spread globally from human-to-human causing the first influenza virus pandemic of the 21st century. In a short period reverse zoonotic cases in pigs followed by a wide spread of t...

  3. Application of a PCR-based approach to identify sex in Hawaiian honeycreepers (Drepanidinae)

    USGS Publications Warehouse

    Jarvi, S.I.; Banko, P.C.

    2000-01-01

    The application of molecular techniques to conservation genetics issues can provide important guidance criteria for management of endangered species. The results from this study establish that PCR-based approaches for sex determination developed in other bird species (Griffiths and Tiwari 1995; Griffiths et al. 1996, 1998; Ellegren 1996) can be applied with a high degree of confidence to at least four species of Hawaiian honeycreepers. This provides a rapid, reliable method with which population managers can optimize sex ratios within populations of endangered species that are subject to artificial manipulation through captive breeding programmes or geographic translocation.

  4. A PCR-based method to identify Entomophaga spp. infections in North American grasshoppers.

    PubMed

    Casique-Valdes, Rebeca; Sanchez-Peña, Sergio; Ivonne Torres-Acosta, R; Bidochka, Michael J

    2012-01-01

    A PCR-based method was developed for the detection and identification of two species of grasshopper-specific pathogens belonging to the genus Entomophaga in North America, Entomophaga calopteni and Entomophaga macleodii. Two separate sets of primers specific for amplification of a DNA product from each species of Entomophaga as well as a positive control were utilized. Grasshoppers were collected from two sites in Mexico during an epizootic with grasshoppers found in "summit disease", typical of Entomophaga infections. There was a preponderance of Melanopline grasshoppers infected by E. calopteni. The described method is an accurate tool for identification of North American grasshopper infections by Entomophaga species.

  5. Frequency Hopping Transceiver Multiplexer

    DTIC Science & Technology

    1983-03-01

    8217 block number) frequency hopping, quadrature coupler, bandpass filter, coupling circuit, filter, helical resonator, matching network, PIN diode switch...which investigated the concept and feasibility of a 30MHz to 88MHz frequency hopping transceiver multiplexer. An approach which uses helical resonator...and Analysis 90 5.9.1 Helical Resonator 90 5.9.2 Shunt Capacitance Binary Bus Discussion 94 5.9.3 Resonator Design Decisions 97 5.9.4 Results and

  6. Performance of PCR-based assays targeting Bacteroidales genetic markers of human fecal pollution in sewage and fecal samples

    EPA Science Inventory

    There are numerous PCR-based methods available to characterize human fecal pollution in ambient waters. Each assay employs distinct oligonucleotides and many target different genes and microorganisms leading to potential variations in method performance. Laboratory comparisons ...

  7. Improved detection of episomal Banana streak viruses by multiplex immunocapture PCR.

    PubMed

    Le Provost, Grégoire; Iskra-Caruana, Marie-Line; Acina, Isabelle; Teycheney, Pierre-Yves

    2006-10-01

    Banana streak viruses (BSV) are currently the main viral constraint to Musa germplasm movement, genetic improvement and mass propagation. Therefore, it is necessary to develop and implement BSV detection strategies that are both reliable and sensitive, such as PCR-based techniques. Unfortunately, BSV endogenous pararetrovirus sequences (BSV EPRVs) are present in the genome of Musa balbisiana. They interfere with PCR-based detection of episomal BSV in infected banana and plantain, such as immunocapture PCR. Therefore, a multiplex, immunocapture PCR (M-IC-PCR) was developed for the detection of BSV. Musa sequence tagged microsatellite site (STMS) primers were selected and used in combination with BSV species-specific primers in order to monitor possible contamination by Musa genomic DNA, using multiplex PCR. Furthermore, immunocapture conditions were optimized in order to prevent Musa DNA from interfering with episomal BSV DNA during the PCR step. This improved detection method successfully allowed the accurate, specific and sensitive detection of episomal DNA only from distinct BSV species. Its implementation should benefit PCR-based detection of viruses for which homologous sequences are present in the genome of their hosts, including transgenic plants expressing viral sequences.

  8. Evaluation of DNA extraction methods suitable for PCR-based detection and genotyping of Clostridium botulinum.

    PubMed

    Auricchio, Bruna; Anniballi, Fabrizio; Fiore, Alfonsina; Skiby, Jeffrey E; De Medici, Dario

    2013-09-01

    Sufficient quality and quantity of extracted DNA is critical to detecting and performing genotyping of Clostridium botulinum by means of PCR-based methods. An ideal extraction method has to optimize DNA yield, minimize DNA degradation, allow multiple samples to be extracted, and be efficient in terms of cost, time, labor, and supplies. Eleven botulinum toxin-producing clostridia strains and 25 samples (10 food, 13 clinical, and 2 environmental samples) naturally contaminated with botulinum toxin-producing clostridia were used to compare 4 DNA extraction procedures: Chelex(®) 100 matrix, Phenol-Cloroform-Isoamyl alcohol, NucliSENS(®) magnetic extraction kit, and DNeasy(®) Blood & Tissue kit. Integrity, purity, and amount of amplifiable DNA were evaluated. The results show that the DNeasy(®) Blood & Tissue kit is the best extraction method evaluated because it provided the most pure, intact, and amplifiable DNA. However, Chelex(®) 100 matrix seems to be suitable for PCR-based methods intended for laboratory diagnosis of suspected outbreaks of botulism, because it is faster and cheaper compared to DNeasy(®) Blood & Tissue kit, and for samples in which the mean of Ct values obtained are statistically different (P>0.05) with respect to the best method, no lack of PCR amplification was shown. In addition, molecular methods for laboratory diagnosis currently are based on a microbial enrichment step prior to PCR, and so the differences in amplification seem to not influence the analytical results.

  9. A Novel PCR-Based Approach for Accurate Identification of Vibrio parahaemolyticus

    PubMed Central

    Li, Ruichao; Chiou, Jiachi; Chan, Edward Wai-Chi; Chen, Sheng

    2016-01-01

    A PCR-based assay was developed for more accurate identification of Vibrio parahaemolyticus through targeting the blaCARB-17 like element, an intrinsic β-lactamase gene that may also be regarded as a novel species-specific genetic marker of this organism. Homologous analysis showed that blaCARB-17 like genes were more conservative than the tlh, toxR and atpA genes, the genetic markers commonly used as detection targets in identification of V. parahaemolyticus. Our data showed that this blaCARB-17-specific PCR-based detection approach consistently achieved 100% specificity, whereas PCR targeting the tlh and atpA genes occasionally produced false positive results. Furthermore, a positive result of this test is consistently associated with an intrinsic ampicillin resistance phenotype of the test organism, presumably conferred by the products of blaCARB-17 like genes. We envision that combined analysis of the unique genetic and phenotypic characteristics conferred by blaCARB-17 shall further enhance the detection specificity of this novel yet easy-to-use detection approach to a level superior to the conventional methods used in V. parahaemolyticus detection and identification. PMID:26858713

  10. Identification of probiotic microorganisms in South African products using PCR-based DGGE analysis.

    PubMed

    Theunissen, J; Britz, T J; Torriani, S; Witthuhn, R C

    2005-01-15

    Probiotic microorganisms in commercial yoghurts and other food products are currently identified by traditional methods such as growth on selective media, morphological and biochemical characteristics. In this study, PCR-based DGGE analysis was used for the rapid and accurate identification of probiotic microorganisms from South African yoghurts and lyophilized preparations in capsule and tablet form. To identify the microorganisms present in these products, the DGGE profiles obtained were compared to two reference markers (A and B) composed of five lactobacilli and seven Bifidobacterium species, respectively. The results obtained were confirmed by species-specific PCR, as well as sequence analyses of unknown bands not present in the reference markers. It was found that only 54.5% of the probiotic yoghurts contained the microorganisms stated on the label compared to only a third (33.3%) of the lyophilized probiotic products. Some Bifidobacterium species were incorrectly identified and various microorganisms were detected that were not listed on the label. Sequence analyses confirmed the presence of Streptococcus spp. other than the yoghurt starter, Streptococcus thermophilus, in some of these products and in some instances label information was vague and non-scientific. PCR-based DGGE analyses proved to be a valuable culture-independent approach for the rapid and specific identification of the microbial species present in South African probiotic products.

  11. Genetic analysis of Giardia and Cryptosporidium from people in Northern Australia using PCR-based tools.

    PubMed

    Ebner, Janine; Koehler, Anson V; Robertson, Gemma; Bradbury, Richard S; Jex, Aaron R; Haydon, Shane R; Stevens, Melita A; Norton, Robert; Joachim, Anja; Gasser, Robin B

    2015-12-01

    To date, there has been limited genetic study of the gastrointestinal pathogens Giardia and Cryptosporidium in northern parts of Australia. Here, PCR-based methods were used for the genetic characterization of Giardia and Cryptosporidium from 695 people with histories of gastrointestinal disorders from the tropical North of Australia. Genomic DNAs from fecal samples were subjected to PCR-based analyses of regions from the triose phosphate isomerase (tpi), small subunit (SSU) of the nuclear ribosomal RNA and/or the glycoprotein (gp60) genes. Giardia and Cryptosporidium were detected in 13 and four of the 695 samples, respectively. Giardia duodenalis assemblages A and B were found in 4 (31%) and 9 (69%) of the 13 samples in persons of <9 years of age. Cryptosporidium hominis (subgenotype IdA18), Cryptosporidium mink genotype (subgenotype IIA16R1) and C. felis were also identified in single patients of 11-21 years of age. Future studies might focus on a comparative study of these and other protists in rural communities in Northern Australia.

  12. Development of PCR-based technique for detection of purity of Pashmina fiber from textile materials.

    PubMed

    Kumar, Rajiv; Shakyawar, D B; Pareek, P K; Raja, A S M; Prince, L L L; Kumar, Satish; Naqvi, S M K

    2015-04-01

    Pashmina fiber is one of major specialty animal fiber in India. The quality of Pashmina obtained from Changthangi and Chegu goats in India is very good. Due to restricted availability and high prices, adulteration of natural prized fibers is becoming a common practice by the manufacturers. Sheep wool is a cheap substitute, which is usually used for adulteration and false declaration of Pashmina-based products. Presently, there is lack of cost-effective and readily available methodology to identify the adulteration of Pashmina products from other similar looking substitutes like sheep wool. Polymerase chain reaction (PCR)-based detection method can be used to identify origin of animal fiber. Extraction of quality DNA from dyed and processed animal fiber and textile materials is a limiting factor in the development of such detection methods. In the present study, quality DNA was extracted from textile materials, and PCR-based technique using mitochondrial gene (12S rRNA) specific primers was developed for detection of the Pashmina in textile blends. This technique has been used for detection of the adulteration of the Pashmina products with sheep wool. The technique can detect adulteration level up to 10 % of sheep/goat fibers in textile blends.

  13. A PCR-based genetic linkage map of human chromosome 16

    SciTech Connect

    Shen, Y.; Kozman, H.M.; Thompson, A.

    1994-07-01

    A high-resolution cytogenetic-based physical map and a genetic linkage map of human chromosome 16 have been developed based on 79 PCR-typable genetic markers and 2 Southern-based RFLP markers. The PCR-based markers were previously-characterized polymorphic (AC){sub n} repeats. Two approaches have led to the characterization of 47 highly informative genetic markers spread along chromosome 16, some of which are closely linked to disease loci. In addition, 22 markers (D16S401-423) previously genetically mapped were also physically mapped. Ten markers characterized by other laboratories were physically mapped and genotyped on the CEPH families. These 32 markers were incorporated into the PCR-based map. Seventy-two markers have heterozygosities >0.50 and 51 of these markers >0.70. By multipoint linkage analysis a framework genetic map and a comprehensive genetic map were constructed. The length of the sex-averaged framework genetic map if 152.1 cM. The average distance and the median distance between markers on this map are 3.2 and 2.7 cM, respectively, and the largest gap is 15.9 cM. These maps were anchored to the high-resolution cytogenetic map (on average 1.5 Mb per interval). Together these integrated genetic and physical maps of human chromosome 16 provide the basis for the localization and ultimately the isolation of disease genes that map to this chromosome. 1 fig., 3 tabs.

  14. Comparison of PCR-based detection of Plasmodium falciparum infections based on single and multicopy genes

    PubMed Central

    Oyedeji, Segun I; Awobode, Henrietta O; Monday, Gamaliel C; Kendjo, Eric; Kremsner, Peter G; Kun, Jürgen F

    2007-01-01

    PCR-based assays are the most sensitive and specific methods to detect malaria parasites. This study compared the diagnostic accuracy of three PCR-based assays that do not only differ in their sequence target, but also in the number of copies of their target region, for the detection of Plasmodium falciparum in 401 individuals living in a malaria-endemic area in Nigeria. Compared to a composite reference generated from results of all the 3 PCR assays, the stevor gene amplification had a sensitivity of 100% (Kappa = 1; 95% CI = 1.000–1.000), 83% (Kappa = 0.718; 95% CI = 0.648–0.788) by SSUrRNA gene PCR and 71% (Kappa = 0.552; 95% CI = 0.478–0.627) by the msa-2 gene amplification. Results from this study indicate that the stevor gene amplification is the most sensitive technique for the detection of P. falciparum. This assay may be an important reference standard, especially when a confirmatory technique with high sensitivity and specificity is needed for ruling out P. falciparum infection. PMID:17705826

  15. Genotyping of PCR-based polymorphisms and linkage-disequilibrium analysis at the NF1 locus

    SciTech Connect

    Purandare, S.M.; Viskochil, D.H.; Cawthon, R.

    1996-07-01

    Six polymorphism across the NF1 gene have been adapted for genotyping through application of PCR-based assays. Three exon-based polymorphisms - at positions 702, 2034, and 10647 in the NF1 cDNA - were genotyped by mutagenically separated PCR (MS-PCR). A fourth polymorphism, DV1.9, is an L1 insertion element in intron 30, and the other two polymorphisms, GXAlu and EVI-20, are short tandem repeats in intron 27b. All the polymorphisms were evaluated in a cohort of 110 CEPH individuals who previously had been analyzed by use of eight RFLPs at the NF1 locus. Pairwise linkage-disequilibrium analyses with the six PCR-based polymorphisms and their flanking markers demonstrated disequilibrium between all tested loci. Genotypes of the four diallelic polymorphisms (702, 2034, 10647, and DV1.9) were also evaluated in cohorts from the CEPH, African, and Japanese populations. The CEPH and Japanese cohorts showed similar heterozygosities and linkage-disequilibrium coefficients. The African cohort showed a higher degree of heterozygosity and lower linkage-disequilibrium values, compared with the CEPH and Japanese cohorts. 36 refs., 2 figs., 3 tabs.

  16. A PCR-Based Molecular Detection of Strongyloides stercoralisin Human Stool Samples from Tabriz City, Iran.

    PubMed

    Ghasemikhah, Reza; Tabatabaiefar, Mohammad Amin; Shariatzadeh, Seyed Ali; Shahbazi, Abbas; Hazratian, Teymour

    2017-03-27

    Strongyloides stercoralis is a nematode causing serious infections in immunocompromised patients. In chronically infected patients, the low parasitic content as well as the resemblance of the larvae to several other species make diagnosis basedonmorphology difficult. In the present study, a PCR-based method targeting the internal transcribed sequence 2 (ITS2) of the rDNA region was examined for the molecular detection of S. stercoralis infection from the stool samples. A total of 1800 patients were included. Three fresh stool samples were collected per patient, and S. stercoralis isolates were identified by the morphological method. A subset of isolates was later used in the PCR-based method as positive controls. Additionally, negative and no-template controls were included. Data analysis was accomplished using an x² test. Ap-value less than 0.05 was considered significant. In total, fivestool samples were found to be infected with S. stercoralis using the morphology method. PCR method detected S. stercoralis DNA target from all of the fiveDNA samples extracted from positive fecal samples.

  17. Detection of microalgal resting cysts in European coastal sediments using a PCR-based assay

    NASA Astrophysics Data System (ADS)

    Penna, Antonella; Battocchi, Cecilia; Garcés, Esther; Anglès, Silvia; Cucchiari, Emellina; Totti, Cecilia; Kremp, Anke; Satta, Cecilia; Grazia Giacobbe, Maria; Bravo, Isabel; Bastianini, Mauro

    2010-02-01

    A PCR-based assay was developed and applied to sediment and sediment trap samples for the detection of different cysts belonging to dinoflagellates and raphidophytes in European coastal areas. Oligonucleotide primers were designed based on the ITS-5.8S and LSU ribosomal gene sequences. The specificity and sensitivity of the PCR assay were assessed using genomic DNA from clonal cultures, plasmid copy number of cloned target sequences, as well as from sediment samples. Qualitative PCR determinations of different cysts in sediment and sediment trap samples were compared to taxonomic examinations by light microscopy. This molecular methodology permitted a fast and specific detection of target cysts in sediment samples. We also detected dinoflagellate and raphidophyte cysts at concentrations not detectable by microscopic methods or that are difficult to identify. The results given by molecular and microscopic methods were comparable. However, higher values of positive detection for target cysts were obtained by PCR than with microscopy. Some taxa were detected in 100% of the samples using PCR, while others were only found in 10% of the samples. The data obtained in this study showed that the PCR-based method is a valid tool for cyst identification in marine sediments.

  18. 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.

  19. Polarization-multiplexed plasmonic phase generation with distributed nanoslits.

    PubMed

    Lee, Seung-Yeol; Kim, Kyuho; Lee, Gun-Yeal; Lee, Byoungho

    2015-06-15

    Methods for multiplexing surface plasmon polaritons (SPPs) have been attracting much attention due to their potentials for plasmonic integrated systems, plasmonic holography, and optical tweezing. Here, using closely-distanced distributed nanoslits, we propose a method for generating polarization-multiplexed SPP phase profiles which can be applied for implementing general SPP phase distributions. Two independent types of SPP phase generation mechanisms - polarization-independent and polarization-reversible ones - are combined to generate fully arbitrary phase profiles for each optical handedness. As a simple verification of the proposed scheme, we experimentally demonstrate that the location of plasmonic focus can be arbitrary designed, and switched by the change of optical handedness.

  20. Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Cylindrocladium scoparium on Eucalyptus

    PubMed Central

    Qiao, Tian-Min; Zhang, Jing; Li, Shu-Jiang; Han, Shan; Zhu, Tian-Hui

    2016-01-01

    Eucalyptus dieback disease, caused by Cylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium. The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products. PMID:27721691

  1. Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Cylindrocladium scoparium on Eucalyptus.

    PubMed

    Qiao, Tian-Min; Zhang, Jing; Li, Shu-Jiang; Han, Shan; Zhu, Tian-Hui

    2016-10-01

    Eucalyptus dieback disease, caused by Cylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium. The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products.

  2. Chopped molecular beam multiplexing system

    NASA Technical Reports Server (NTRS)

    Adams, Billy R. (Inventor)

    1986-01-01

    The integration of a chopped molecular beam mass spectrometer with a time multiplexing system is described. The chopping of the molecular beam is synchronized with the time intervals by a phase detector and a synchronous motor. Arithmetic means are generated for phase shifting the chopper with respect to the multiplexer. A four channel amplifier provides the capacity to independently vary the baseline and amplitude in each channel of the multiplexing system.

  3. Layer Communities in Multiplex Networks

    NASA Astrophysics Data System (ADS)

    Kao, Ta-Chu; Porter, Mason A.

    2017-08-01

    Multiplex networks are a type of multilayer network in which entities are connected to each other via multiple types of connections. We propose a method, based on computing pairwise similarities between layers and then doing community detection, for grouping structurally similar layers in multiplex networks. We illustrate our approach using both synthetic and empirical networks, and we are able to find meaningful groups of layers in both cases. For example, we find that airlines that are based in similar geographic locations tend to be grouped together in a multiplex airline network and that related research areas in physics tend to be grouped together in a multiplex collaboration network.

  4. Functional Multiplex PageRank

    NASA Astrophysics Data System (ADS)

    Iacovacci, Jacopo; Rahmede, Christoph; Arenas, Alex; Bianconi, Ginestra

    2016-10-01

    Recently it has been recognized that many complex social, technological and biological networks have a multilayer nature and can be described by multiplex networks. Multiplex networks are formed by a set of nodes connected by links having different connotations forming the different layers of the multiplex. Characterizing the centrality of the nodes in a multiplex network is a challenging task since the centrality of the node naturally depends on the importance associated to links of a certain type. Here we propose to assign to each node of a multiplex network a centrality called Functional Multiplex PageRank that is a function of the weights given to every different pattern of connections (multilinks) existent in the multiplex network between any two nodes. Since multilinks distinguish all the possible ways in which the links in different layers can overlap, the Functional Multiplex PageRank can describe important non-linear effects when large relevance or small relevance is assigned to multilinks with overlap. Here we apply the Functional Page Rank to the multiplex airport networks, to the neuronal network of the nematode C. elegans, and to social collaboration and citation networks between scientists. This analysis reveals important differences existing between the most central nodes of these networks, and the correlations between their so-called pattern to success.

  5. Multiplex Flow Assays

    PubMed Central

    2016-01-01

    Lateral flow or dipstick assays (e.g., home pregnancy tests), where an analyte solution is drawn through a porous membrane and is detected by localization onto a capture probe residing at a specific site on the flow strip, are the most commonly and extensively used type of diagnostic assay. However, after over 30 years of use, these assays are constrained to measuring one or a few analytes at a time. Here, we describe a completely general method, in which any single-plex lateral flow assay is transformed into a multiplex assay capable of measuring an arbitrarily large number of analytes simultaneously. Instead of identifying the analyte by its localization onto a specific geometric location in the flow medium, the analyte-specific capture probe is identified by its association with a specific optically encoded region within the flow medium. The capture probes for nucleic acids, antigens, or antibodies are attached to highly porous agarose beads, which have been encoded using multiple lanthanide emitters to create a unique optical signature for each capture probe. The optically encoded capture probe-derivatized beads are placed in contact with the analyte-containing porous flow medium and the analytes are captured onto the encoded regions as the solution flows through the porous medium. To perform a multiplex diagnostic assay, a solution comprising multiple analytes is passed through the flow medium containing the capture probe-derivatized beads, and the captured analyte is treated with a suitable fluorescent reporter. We demonstrate this multiplex analysis technique by simultaneously measuring DNA samples, antigen–antibody pairs, and mixtures of multiple nucleic acids and antibodies. PMID:27819063

  6. Rapid detection and identification of Clostridium chauvoei by PCR based on flagellin gene sequence.

    PubMed

    Kojima, A; Uchida, I; Sekizaki, T; Sasaki, Y; Ogikubo, Y; Tamura, Y

    2001-02-26

    We developed a one-step polymerase chain reaction (PCR) system that specifically detects Clostridium chauvoei. Oligonucleotide primers were designed to amplify a 516-bp fragment of the structural flagellin gene. The specificity of the PCR was investigated by analyzing 59 strains of clostridia, and seven strain of other genera. A 516-bp fragment could be amplified from all the C. chauvoei strains tested, and no amplification was observed by using DNAs from the other strains tested, including Clostridium septicum. Similarly, this PCR-based method specifically detected C. chauvoei DNA sequences in samples of muscle and exudate of obtained from mice within 12h of inoculation. In tests using samples of muscle or liver, the limit of detection was about 200 organisms per reaction. These results suggest that the one-step PCR system may be useful for direct detection and identification of C. chauvoei in clinical specimens.

  7. PCR-based assessment of shellfish traceability and sustainability in international Mediterranean seafood markets.

    PubMed

    Galal-Khallaf, Asmaa; Ardura, Alba; Borrell, Yaisel J; Garcia-Vazquez, Eva

    2016-07-01

    Two mitochondrial markers (cytochrome oxidase COI and 16S rDNA) were employed for species identification of commercial shellfish from two Mediterranean countries. New COI Barcodes were generated for six species: Pleoticus robustus, Metapenaeopsis barbata, Parapenaeus fissuroides, Hymenopenaeus debilis, Metapenaeus affinis and Sepia aculeata. Biodiversity of the seafood species analyzed was greater in Egypt, with nine crustacean and two cephalopod species found compared with only three crustaceans and three cephalopods in Spain. In total, 17.2% and 15.2% products were mislabeled in Egypt and Spain, respectively. Population decline is a problem for some of the substitute species. Others were exotic and/or invasive in exporters' regions. This study offers the first comparable study of shellfish traceability in these Mediterranean markets. The PCR-based method used in this study proved to be reliable, effective and, therefore, could be employed for routine seafood analysis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. PCR-based analysis of mitochondrial DNA copy number, mitochondrial DNA damage, and nuclear DNA damage

    PubMed Central

    Gonzalez-Hunt, Claudia P.; Rooney, John P.; Ryde, Ian T.; Anbalagan, Charumathi; Joglekar, Rashmi

    2016-01-01

    Because of the role DNA damage and depletion play in human disease, it is important to develop and improve tools to assess these endpoints. This unit describes PCR-based methods to measure nuclear and mitochondrial DNA damage and copy number. Long amplicon quantitative polymerase chain reaction (LA-QPCR) is used to detect DNA damage by measuring the number of polymerase-inhibiting lesions present based on the amount of PCR amplification; real-time PCR (RT-PCR) is used to calculate genome content. In this unit we provide step-by-step instructions to perform these assays in Homo sapiens, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Oryzias latipes, Fundulus grandis, and Fundulus heteroclitus, and discuss the advantages and disadvantages of these assays. PMID:26828332

  9. Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques.

    PubMed

    Aljanabi, S M; Martinez, I

    1997-11-15

    A very simple, fast, universally applicable and reproducible method to extract high quality megabase genomic DNA from different organisms is described. We applied the same method to extract high quality complex genomic DNA from different tissues (wheat, barley, potato, beans, pear and almond leaves as well as fungi, insects and shrimps' fresh tissue) without any modification. The method does not require expensive and environmentally hazardous reagents and equipment. It can be performed even in low technology laboratories. The amount of tissue required by this method is approximately 50-100 mg. The quantity and the quality of the DNA extracted by this method is high enough to perform hundreds of PCR-based reactions and also to be used in other DNA manipulation techniques such as restriction digestion, Southern blot and cloning.

  10. Epidemiological investigation of Pseudomonas aeruginosa nosocomial bacteraemia isolates by PCR-based DNA fingerprinting analysis.

    PubMed

    Liu, Y; Davin-Regli, A; Bosi, C; Charrel, R N; Bollet, C

    1996-11-01

    Between July 1994 and March 1995, 64 isolates of Pseudomonas aeruginosa were implicated in bacteraemia in 25 cancer patients in five wards of two hospitals. These, together with 24 environmental isolates and one isolate from a bacteraemia in a non-cancer patient were examined by three PCR-based DNA fingerprinting methods: random amplified polymorphic DNA (RAPD), enterobacterial-repetitive intergenic consensus (ERIC)-PCR, and 16S-23S spacer region-based RAPD. These methods were reproducible, discriminatory and showed close agreement; all indicated that 47 isolates that had caused bacteraemia in 19 cancer patients were indistinguishable. Seventeen other isolates that had caused bacteraemia in 10 cancer patients were discriminated into eight further groups, and the 24 environmental and non-cancer patient isolates into further distinct groups. No environmental source of the epidemic strain was found, but it was suspected that the outbreak was related to infusion implants.

  11. Multiplexed capillary electrophoresis system

    DOEpatents

    Yeung, Edward S.; Li, Qingbo; Lu, Xiandan

    1998-04-21

    The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification ("base calling") is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations.

  12. Multiplexed capillary electrophoresis system

    DOEpatents

    Yeung, Edward S.; Chang, Huan-Tsang; Fung, Eliza N.; Li, Qingbo; Lu, Xiandan

    1996-12-10

    The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification ("base calling") is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations.

  13. Multiplexed capillary electrophoresis system

    DOEpatents

    Yeung, E.S.; Li, Q.; Lu, X.

    1998-04-21

    The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification (``base calling``) is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations. 19 figs.

  14. Multiplexed capillary electrophoresis system

    DOEpatents

    Yeung, E.S.; Chang, H.T.; Fung, E.N.; Li, Q.; Lu, X.

    1996-12-10

    The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification (``base calling``) is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations. 19 figs.

  15. Television multiplexing system

    NASA Technical Reports Server (NTRS)

    Simpkins, L. G. (Inventor)

    1973-01-01

    A television multiplexing system which includes a circuit that inserts a digital codes sync signal and a digital code into a video signal for identifying the channel is described. The digital sync signal and the digital coded signals are generated by a single crystal controlled clock so that they are always in synchronism with each other. In demultiplexing the signals are utilized for shifting the digital coded signals into a shift register. The shift register, in turn, activates a decoder according to the code stored in the shift register for selecting the proper recording disk or receiver for storing the video signal.

  16. Utility, reliability and reproducibility of immunoassay multiplex kits.

    PubMed

    Tighe, Paddy; Negm, Ola; Todd, Ian; Fairclough, Lucy

    2013-05-15

    Multiplex technologies are becoming increasingly important in biomarker studies as they enable patterns of biomolecules to be examined, which provide a more comprehensive depiction of disease than individual biomarkers. They are crucial in deciphering these patterns, but it is essential that they are endorsed for reliability, reproducibility and precision. Here we outline the theoretical basis of a variety of multiplex technologies: Bead-based multiplex immunoassays (i.e. Cytometric Bead Arrays, Luminex™ and Bio-Plex Pro™), microtitre plate-based arrays (i.e. Mesoscale Discovery (MSD) and Quantsys BioSciences QPlex), Slide-based Arrays (i.e. FastQuant™) and reverse phase protein arrays. Their utility, reliability and reproducibility are discussed.

  17. Comparative PCR-based fingerprinting of Vibrio cholerae isolated in Malaysia.

    PubMed

    Shuan Ju Teh, Cindy; Thong, Kwai Lin; Osawa, Ro; Heng Chua, Kek

    2011-01-01

    Vibrio cholerae, the causative agent of cholera, is endemic in many parts of the world, especially in countries poor in resources. Molecular subtyping of V. cholerae is useful to trace the regional spread of a clone or multidrug-resistant strains during outbreaks of cholera. Current available PCR-based fingerprinting methods such as Random Amplified Polymorphic DNA (RAPD)-PCR, Enterobacterial Repetitive Intergenic Consensus Sequence (ERIC)-PCR, and Repetitive Extragenic Palindromic (REP)-PCR were used to subtype V. cholerae. However, there are problems for inter-laboratory comparison as these PCR methods have their own limitations especially when different PCR methods have been used for molecular typing. In this study, a Vibrio cholerae Repeats-PCR (VCR-PCR) approach which targets the genetic polymorphism of the integron island of Vibrios was used and compared with other PCR-based fingerprinting methods in subtyping. Forty-three V. cholerae of different serogroups from various sources were tested. The PCR-fingerprinting approaches were evaluated on typeability, reproducibility, stability and discriminatory power. Overall, Malaysian non-O1/non-O139 V. cholerae were more diverse than O1 strains. Four non-O1/non-O139 strains were closely related with O1 strains. The O139 strain in this study shared similarity with strains of both O1 and non-O1/non-O139 serogroups. ERIC-PCR was the most discriminative approach (D value = 0.996). VCR-PCR was useful in discriminating non-O1/non-O139 strains. RAPD-PCR and REP-PCR were less suitable for efficient subtyping purposes as they were not reproducible and lacked stability. The combination of the ERIC-PCR and VCR-PCR may overcome the inadequacy of any one approach and hence provide more informative data.

  18. PCR based detection of mycobacteria in paraffin wax embedded material routinely processed for morphological examination.

    PubMed Central

    Frevel, T; Schäfer, K L; Tötsch, M; Böcker, W; Dockhorn-Dworniczak, B

    1999-01-01

    BACKGROUND: The incidence of mycobacterial infections has increased during the past five years. A prompt diagnosis is indispensable for initiating appropriate treatment. Because culturing of mycobacteria takes three to six weeks and sensitivity of microscopic detection of acid fast bacilli is low, amplification methods provide promising possibilities. Recently, the polymerase chain reaction (PCR) has been shown to be useful for confirming a mycobacterial infection, especially in cases with unexpected histological findings or lack of suitable material for culturing. AIMS: To evaluate the impact of PCR based techniques in the detection of mycobacterial infections in uncultured routine histological specimens as an alternative to surgical pathology. METHODS: Two hundred and twenty nine formalin fixed and paraffin wax embedded samples from 141 patients with clinical or histological suspicion of a mycobacterial infection were investigated using three different PCR assays and Southern blotting. PCR results were compared with histology and culture and the patients' clinical findings. RESULTS: When using culture as the reference method, the sensitivity for the detection of mycobacteria of the tuberculosis complex was 90%, specificity was 92%, the positive predictive value was 81%, and the negative predictive value was 96%. The sensitivity for the detection of nontuberculous mycobacteria was 100% and specificity was 78%, the positive predictive value was 26%, and the negative predictive value was 100%. The patients' clinical findings supported the PCR positive results, indicating a mycobacterial infection in 11 of 18 initially culture negative cases and in 21 of 35 PCR positive cases without culture results. CONCLUSIONS: These results indicate that PCR based techniques are sensitive, specific, and rapid methods for the detection of mycobacteria in routinely processed paraffin wax embedded and formalin fixed histological samples. PMID:10748878

  19. [Development of uncompetitive exogenous internal amplification control for real-time PCR based on UFA method].

    PubMed

    Ivanov, M K; Bragin, A G; Prasolova, M A; Vedernikov, V E; Dymshits, G M

    2009-01-01

    An uncompetitive exogenous internal amplification control method (EIAC) was developed on the basis of short synthetic DNA segment, whose amplification can be detected in real time by UFA spectroscopy principle. The EIAC was shown to be useful as internal control in diagnostic test systems based on DNA or RNA detection by multiplex real-time PCR. It can be applied to assess the quality of extracted DNA or RNA, and also to detect and study the factors causing PCR inhibition and earlier plateau effect.

  20. Quantitative multiplexed quantum dot immunohistochemistry

    SciTech Connect

    Sweeney, E.; Ward, T.H.; Gray, N.; Womack, C.; Jayson, G.; Hughes, A.; Dive, C.; Byers, R.

    2008-09-19

    Quantum dots are photostable fluorescent semiconductor nanocrystals possessing wide excitation and bright narrow, symmetrical, emission spectra. These characteristics have engendered considerable interest in their application in multiplex immunohistochemistry for biomarker quantification and co-localisation in clinical samples. Robust quantitation allows biomarker validation, and there is growing need for multiplex staining due to limited quantity of clinical samples. Most reported multiplexed quantum dot staining used sequential methods that are laborious and impractical in a high-throughput setting. Problems associated with sequential multiplex staining have been investigated and a method developed using QDs conjugated to biotinylated primary antibodies, enabling simultaneous multiplex staining with three antibodies. CD34, Cytokeratin 18 and cleaved Caspase 3 were triplexed in tonsillar tissue using an 8 h protocol, each localised to separate cellular compartments. This demonstrates utility of the method for biomarker measurement enabling rapid measurement of multiple co-localised biomarkers on single paraffin tissue sections, of importance for clinical trial studies.

  1. Multiplex Recurrence Networks

    NASA Astrophysics Data System (ADS)

    Eroglu, Deniz; Marwan, Norbert

    2017-04-01

    The complex nature of a variety of phenomena in physical, biological, or earth sciences is driven by a large number of degrees of freedom which are strongly interconnected. Although the evolution of such systems is described by multivariate time series (MTS), so far research mostly focuses on analyzing these components one by one. Recurrence based analyses are powerful methods to understand the underlying dynamics of a dynamical system and have been used for many successful applications including examples from earth science, economics, or chemical reactions. The backbone of these techniques is creating the phase space of the system. However, increasing the dimension of a system requires increasing the length of the time series in order get significant and reliable results. This requirement is one of the challenges in many disciplines, in particular in palaeoclimate, thus, it is not easy to create a phase space from measured MTS due to the limited number of available obervations (samples). To overcome this problem, we suggest to create recurrence networks from each component of the system and combine them into a multiplex network structure, the multiplex recurrence network (MRN). We test the MRN by using prototypical mathematical models and demonstrate its use by studying high-dimensional palaeoclimate dynamics derived from pollen data from the Bear Lake (Utah, US). By using the MRN, we can distinguish typical climate transition events, e.g., such between Marine Isotope Stages.

  2. The effect of sampling technique on PCR-based bacteriological results of bovine milk samples.

    PubMed

    Hiitiö, Heidi; Simojoki, Heli; Kalmus, Piret; Holopainen, Jani; Pyörälä, Satu; Taponen, Suvi

    2016-08-01

    The aim of the study was to evaluate the effect of sampling technique on the microbiological results of bovine milk samples using multiplex real-time PCR. Comparison was made between a technique where the milk sample was taken directly from the udder cistern of the udder quarter using a needle and vacuum tube and conventional sampling. The effect of different cycle threshold (Ct) cutoff limits on the results was also tested to estimate the amount of amplified DNA in the samples. A total of 113 quarters from 53 cows were tested pairwise using both techniques, and each sample was studied with real-time PCR. Sampling from the udder cistern reduced the number of species per sample compared with conventional sampling. In conventional samples, the number of positive Staphylococcus spp. results was over twice that of samples taken with the needle technique, indicating that most of the Staphylococcus spp. originated from the teat or environmental sources. The Ct values also showed that Staphylococcus spp. were present in most samples only in low numbers. Routine use of multiplex real-time PCR in mastitis diagnostics could benefit from critical evaluation of positive Staphylococcus spp. results with Ct values between 34.0 and 37.0. Our results emphasize the importance of a careful aseptic milk sampling technique and a microbiologically positive result for a milk sample should not be automatically interpreted as an intramammary infection or mastitis. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  3. Using Multiplex PCR for Assessing the Quality of Whole Genome Amplified DNA.

    PubMed

    El-Heliebi, Amin; Chen, Shukun; Kroneis, Thomas

    2015-01-01

    This chapter describes a simple and inexpensive multiplex PCR-based method to assess the quality of whole genome amplification (WGA) products generated from heat-induced random fragmented DNA. A set of four primer pairs is used to amplify DNA sequences of WGA products in and downstream of GAPDH gene in yielding 100, 200, 300, and 400 bp fragments. PCR products are analyzed by agarose gel electrophoresis and the respective WGA quality is classified according to the number of obtained PCR bands. WGA products that yield three or four PCR bands are considered to be of high quality and yield good results when analyzed by means of array comparative genome hybridization (CGH).

  4. Detection of 11 common viral and bacterial pathogens causing community-acquired pneumonia or sepsis in asymptomatic patients by using a multiplex reverse transcription-PCR assay with manual (enzyme hybridization) or automated (electronic microarray) detection.

    PubMed

    Kumar, Swati; Wang, Lihua; Fan, Jiang; Kraft, Andrea; Bose, Michael E; Tiwari, Sagarika; Van Dyke, Meredith; Haigis, Robert; Luo, Tingquo; Ghosh, Madhushree; Tang, Huong; Haghnia, Marjan; Mather, Elizabeth L; Weisburg, William G; Henrickson, Kelly J

    2008-09-01

    Community-acquired pneumonia (CAP) and sepsis are important causes of morbidity and mortality. We describe the development of two molecular assays for the detection of 11 common viral and bacterial agents of CAP and sepsis: influenza virus A, influenza virus B, respiratory syncytial virus A (RSV A), RSV B, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, Legionella micdadei, Bordetella pertussis, Staphylococcus aureus, and Streptococcus pneumoniae. Further, we report the prevalence of carriage of these pathogens in respiratory, skin, and serum specimens from 243 asymptomatic children and adults. The detection of pathogens was done using both a manual enzyme hybridization assay and an automated electronic microarray following reverse transcription and PCR amplification. The analytical sensitivities ranged between 0.01 and 100 50% tissue culture infective doses, cells, or CFU per ml for both detection methods. Analytical specificity testing demonstrated no significant cross-reactivity among 19 other common respiratory organisms. One hundred spiked "surrogate" clinical specimens were all correctly identified with 100% specificity (95% confidence interval, 100%). Overall, 28 (21.7%) of 129 nasopharyngeal specimens, 11 of 100 skin specimens, and 2 of 100 serum specimens from asymptomatic subjects tested positive for one or more pathogens, with S. pneumoniae and S. aureus giving 89% of the positive results. Our data suggest that asymptomatic carriage makes the use of molecular assays problematic for the detection of S. pneumoniae or S. aureus in upper respiratory tract secretions; however, the specimens tested showed virtually no carriage of the other nine viral and bacterial pathogens, and the detection of these pathogens should not be a significant diagnostic problem. In addition, slightly less sensitive molecular assays may have better correlation with clinical disease in the case of CAP.

  5. Development and evaluation of a novel RT-qPCR based test for the quantification of HER2 gene expression in breast cancer.

    PubMed

    El Hadi, Hicham; Abdellaoui-Maane, Imane; Kottwitz, Denise; El Amrani, Manal; Bouchoutrouch, Nadia; Qmichou, Zineb; Karkouri, Mehdi; ElAttar, Hicham; Errihani, Hassan; Fernandez, Pedro L; Bakri, Youssef; Sefrioui, Hassan; Moumen, Abdeladim

    2017-03-20

    Accurate measurement of Human epidermal growth factor receptor (HER2) gene expression is central for breast or stomach cancer therapy orientation and prognosis. The current standards testing methods for HER2 expression are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). In the current study, we explored the use of quantitative real time reverse transcription-PCR (RT-qPCR) as a potential method for the accurate relative quantification of the HER2 gene using formalin fixed paraffin embedded (FFPE) breast cancer biopsy samples. The main aim of the current study is to measure the level of concordance of RT-qPCR based quantification of HER2 overexpression with both IHC and FISH. Accordingly, an endogenous control gene (ECG) is required for this relative quantification and should ideally be expressed equivalently across tested samples. Stably expressed ECGs have been selected from a panel of seven genes using GenEx V6 software which is based on geNorm and NormFinder and statistical methods. Quantification of HER2 gene expression was performed by our RT-qPCR-based test and compared to the results obtained by both IHC and FISH methods. HER2 gene quantification using RT-qPCR test was normalized using the two ECGs (RPL30 and RPL37A) that were successfully identified and selected from a panel of seven genes as the most stable and reliable ECGs. We evaluated a total of 216 FFPE tissue samples from breast cancer patients. The results obtained with RT-qPCR in the current study were compared to both IHC and FISH data collected for the same patients. In addition to an internal evaluation, an external evaluation of this assay was also performed in a recognized pathology center in Europe (Clinic Barcelona Hospital Universitari, Spain) using 116 FFPE breast cancer tissue samples. The results demonstrated a high concordance between RT-qPCR and either IHC (98%) or FISH (72%) methods. Accordantly, the overall concordance was 85%. To our knowledge, this is the

  6. Multiplexed Elispot Assay

    PubMed Central

    Harriman, William D.; Collarini, Ellen J.; Cromer, Remy G.; Dutta, April; Strandh, Magnus; Zhang, Fen; Kauvar, Lawrence M.

    2009-01-01

    Micron scale latex beads are well established as highly biocompatible reagents. Imbibing two fluorescent dyes into the interior of the beads enables the creation of a family of combinatorially colored labels. Previous use of such beads, in flow cytometry for example, has focused on beads of ~5μm diameter. We show here that 280 nm combinatorially labeled particles can be used to create ELISA-style assays in 200 μm scale virtual wells, using digital microscopy as the readout. The utility of this technique is illustrated by profiling the secreted cytokine footprints of peripheral blood mononuclear cells in a multiparametric version of the popular Elispot assay. Doing so reveals noncanonical classes of T lymphocytes. We further show that the secreting cell type can be concurrently identified by surface staining with a cell type specific antibody conjugated to the same multiplexed beads. PMID:19084532

  7. Multiplex biomarkers in blood

    PubMed Central

    2013-01-01

    Advances in the field of blood biomarker discovery will help in identifying Alzheimer's disease in its preclinical stage, allowing treatment to be initiated before irreversible damage occurs. This review discusses some recent past and current approaches being taken by researchers in the field. Individual blood biomarkers have been unsuccessful in defining the disease pathology, progression and thus diagnosis. This directs to the need for discovering a multiplex panel of blood biomarkers as a promising approach with high sensitivity and specificity for early diagnosis. However, it is a great challenge to standardize a worldwide blood biomarker panel due to the innate differences in the population tested, nature of the samples and methods utilised in different studies across the globe. We highlight several issues that result in the lack of reproducibility in this field of research currently faced by researchers. Several important measures are summarized towards the end of the review that can be taken to minimize the variability among various centres. PMID:23795953

  8. Arthrogryposis multiplex congenita

    PubMed Central

    Bharucha, E. P.; Pandya, S. S.; Dastur, Darab K.

    1972-01-01

    Sixteen cases with arthrogryposis multiplex congenita were examined clinically and electromyographically; three of them were re-examined later. Joint deformities were present in all extremities in 13 of the cases; in eight there was some degree of mental retardation. In two cases, there was clinical and electromyographic evidence of a myopathic disorder. In the majority, the appearances of the shoulder-neck region suggested a developmental defect. At the same time, selective weakness of muscles innervated by C5-C6 segments suggested a neuropathic disturbance. EMG revealed, in eight of 13 cases, clear evidence of denervation of muscles, but without any regenerative activity. The non-progressive nature of this disorder and capacity for improvement in muscle bulk and power suggest that denervation alone cannot explain the process. Re-examination of three patients after two to three years revealed persistence of the major deformities and muscle weakness noted earlier, with no appreciable deterioration. Images PMID:5049804

  9. Multiplex families with epilepsy

    PubMed Central

    Afawi, Zaid; Oliver, Karen L.; Kivity, Sara; Mazarib, Aziz; Blatt, Ilan; Neufeld, Miriam Y.; Helbig, Katherine L.; Goldberg-Stern, Hadassa; Misk, Adel J.; Straussberg, Rachel; Walid, Simri; Mahajnah, Muhammad; Lerman-Sagie, Tally; Ben-Zeev, Bruria; Kahana, Esther; Masalha, Rafik; Kramer, Uri; Ekstein, Dana; Shorer, Zamir; Wallace, Robyn H.; Mangelsdorf, Marie; MacPherson, James N.; Carvill, Gemma L.; Mefford, Heather C.; Jackson, Graeme D.; Scheffer, Ingrid E.; Bahlo, Melanie; Gecz, Jozef; Heron, Sarah E.; Corbett, Mark; Mulley, John C.; Dibbens, Leanne M.; Korczyn, Amos D.

    2016-01-01

    Objective: To analyze the clinical syndromes and inheritance patterns of multiplex families with epilepsy toward the ultimate aim of uncovering the underlying molecular genetic basis. Methods: Following the referral of families with 2 or more relatives with epilepsy, individuals were classified into epilepsy syndromes. Families were classified into syndromes where at least 2 family members had a specific diagnosis. Pedigrees were analyzed and molecular genetic studies were performed as appropriate. Results: A total of 211 families were ascertained over an 11-year period in Israel. A total of 169 were classified into broad familial epilepsy syndrome groups: 61 generalized, 22 focal, 24 febrile seizure syndromes, 33 special syndromes, and 29 mixed. A total of 42 families remained unclassified. Pathogenic variants were identified in 49/211 families (23%). The majority were found in established epilepsy genes (e.g., SCN1A, KCNQ2, CSTB), but in 11 families, this cohort contributed to the initial discovery (e.g., KCNT1, PCDH19, TBC1D24). We expand the phenotypic spectrum of established epilepsy genes by reporting a familial LAMC3 homozygous variant, where the predominant phenotype was epilepsy with myoclonic-atonic seizures, and a pathogenic SCN1A variant in a family where in 5 siblings the phenotype was broadly consistent with Dravet syndrome, a disorder that usually occurs sporadically. Conclusion: A total of 80% of families were successfully classified, with pathogenic variants identified in 23%. The successful characterization of familial electroclinical and inheritance patterns has highlighted the value of studying multiplex families and their contribution towards uncovering the genetic basis of the epilepsies. PMID:26802095

  10. Portable Multiplex Pathogen Detector

    SciTech Connect

    Visuri, S; McBride, M T; Matthews, D; Rao, R

    2002-07-15

    Tumor marker concentrations in serum provide useful information regarding clinical stage and prognosis of cancer and can thus be used for presymptomatic diagnostic purposes. Currently, detection and identification of soluble analytes in biological fluids is conducted by methods including bioassays, ELISA, PCR, DNA chip or strip tests. While these technologies are generally sensitive and specific, they are time consuming, labor intensive and cannot be multiplexed. Our goal is to develop a simple, point-of-care, portable, liquid array-based immunoassay device capable of simultaneous detection of a variety of cancer markers. Here we describe the development of assays for the detection of Serum Prostate Specific Antigen, and Ovalbumin from a single sample. The multiplexed immunoassays utilize polystyrene microbeads. The beads are imbedded with precise ratios of red and orange fluorescent dyes yielding an array of 100 beads, each with a unique spectral address (Figure 1). Each bead can be coated with capture antibodies specific for a given antigen. After antigen capture, secondary antibodies sandwich the bound antigen and are indirectly labeled by the fluorescent reporter phycoerythrin (PE). Each optically encoded and fluorescently-labeled microbead is then individually interrogated. A red laser excites the dye molecules imbedded inside the bead and classifies the bead to its unique bead set, and a green laser quantifies the assay at the bead surface. This technology has been proven to be comparable to the ELISA in terms of sensitivity and specificity. We also describe the laser-based instrumentation used to acquire fluorescent bead images Following the assay, droplets of bead suspension containing a mixture of bead classes were deposited onto filters held in place by a disposable plexiglass device and the resultant arrays viewed under the fluorescent imaging setup. Using the appropriate filter sets to extract the necessary red, orange and green fluorescence from the

  11. PCR based diagnosis of trypanosomiasis exploring invariant surface glycoprotein (ISG) 75 gene.

    PubMed

    Rudramurthy, G R; Sengupta, P P; Balamurugan, V; Prabhudas, K; Rahman, H

    2013-03-31

    The invariant surface glycoprotein (ISG-75) gene of Trypanosoma evansi buffalo isolate from Karnataka state in India was sequenced and analyzed to elucidate its relationship with other isolates/species. The sequenced ISG-75 gene was also explored to device a polymerase chain reaction (PCR) strategy for the diagnosis of trypanosomiasis in carrier animals. The six cloned ISG gene sequences revealed the open reading frame (ORF) of 1572 and 1527 nucleotide (nt) encoding a polypeptide of 523 and 508 amino acids (aa) respectively and belongs ISG-75 gene family. Sequence analysis revealed 91-100% and 65-99% similarity at nt and aa levels, respectively with other isolates/species and belongs to the RoTat 1.2 strain. The diagnostic PCR based on ISG-75 sequence amplifies a 407 bp product specifically from the different T. evansi isolates and could detect 0.04 pg and 1.2 ng of DNA from purified trypanosomes and T. evansi infected rat blood samples respectively. Subsequently the PCR detected 0.02 and 0.27 trypanosomes ml(-1) respectively, from purified trypanosomes and T. evansi (buffalo isolate) infected rat blood. By the developed PCR assay trypanosomal nucleic acid was detected in experimental rats and buffalo on 24 h post infection (p.i.) and 3rd day post infection (d.p.i.), respectively. The developed ISG-75 gene based PCR assay could be useful in detection of carrier status of trypanosomiasis in animals.

  12. Rapid and Robust PCR-Based All-Recombinant Cloning Methodology.

    PubMed

    Dubey, Abhishek Anil; Singh, Manika Indrajit; Jain, Vikas

    2016-01-01

    We report here a PCR-based cloning methodology that requires no post-PCR modifications such as restriction digestion and phosphorylation of the amplified DNA. The advantage of the present method is that it yields only recombinant clones thus eliminating the need for screening. Two DNA amplification reactions by PCR are performed wherein the first reaction amplifies the gene of interest from a source template, and the second reaction fuses it with the designed expression vector fragments. These vector fragments carry the essential elements that are required for the fusion product selection. The entire process can be completed in less than 8 hours. Furthermore, ligation of the amplified DNA by a DNA ligase is not required before transformation, although the procedure yields more number of colonies upon transformation if ligation is carried out. As a proof-of-concept, we show the cloning and expression of GFP, adh, and rho genes. Using GFP production as an example, we further demonstrate that the E. coli T7 express strain can directly be used in our methodology for the protein expression immediately after PCR. The expressed protein is without or with 6xHistidine tag at either terminus, depending upon the chosen vector fragments. We believe that our method will find tremendous use in molecular and structural biology.

  13. Rapid and Robust PCR-Based All-Recombinant Cloning Methodology

    PubMed Central

    Jain, Vikas

    2016-01-01

    We report here a PCR-based cloning methodology that requires no post-PCR modifications such as restriction digestion and phosphorylation of the amplified DNA. The advantage of the present method is that it yields only recombinant clones thus eliminating the need for screening. Two DNA amplification reactions by PCR are performed wherein the first reaction amplifies the gene of interest from a source template, and the second reaction fuses it with the designed expression vector fragments. These vector fragments carry the essential elements that are required for the fusion product selection. The entire process can be completed in less than 8 hours. Furthermore, ligation of the amplified DNA by a DNA ligase is not required before transformation, although the procedure yields more number of colonies upon transformation if ligation is carried out. As a proof-of-concept, we show the cloning and expression of GFP, adh, and rho genes. Using GFP production as an example, we further demonstrate that the E. coli T7 express strain can directly be used in our methodology for the protein expression immediately after PCR. The expressed protein is without or with 6xHistidine tag at either terminus, depending upon the chosen vector fragments. We believe that our method will find tremendous use in molecular and structural biology. PMID:27007922

  14. Molecular characterization of CTNS deletions in nephropathic cystinosis: development of a PCR-based detection assay.

    PubMed

    Forestier, L; Jean, G; Attard, M; Cherqui, S; Lewis, C; van't Hoff, W; Broyer, M; Town, M; Antignac, C

    1999-08-01

    Nephropathic cystinosis is an autosomal recessive disorder that is characterized by accumulation of intralysosomal cystine and is caused by a defect in the transport of cystine across the lysosomal membrane. Using a positional cloning strategy, we recently cloned the causative gene, CTNS, and identified pathogenic mutations, including deletions, that span the cystinosis locus. Two types of deletions were detected-one of 9.5-16 kb, which was seen in a single family, and one of approximately 65 kb, which is the most frequent mutation found in the homozygous state in nearly one-third of cystinotic individuals. We present here characterization of the deletion breakpoints and demonstrate that, although both deletions occur in regions of repetitive sequences, they are the result of nonhomologous recombination. This type of mechanism suggests that the approximately 65-kb deletion is not a recurrent mutation, and our results confirm that it is identical in all patients. Haplotype analysis shows that this large deletion is due to a founder effect that occurred in a white individual and that probably arose in the middle of the first millenium. We also describe a rapid PCR-based assay that will accurately detect both homozygous and heterozygous deletions, and we use it to show that the approximately 65-kb deletion is present in either the homozygous or the heterozygous state in 76% of cystinotic patients of European origin.

  15. Adaptive PCR Based on Hybridization Sensing of Mirror-Image l-DNA.

    PubMed

    Adams, Nicholas M; Gabella, William E; Hardcastle, Austin N; Haselton, Frederick R

    2017-01-03

    Polymerase chain reaction (PCR) is dependent on two key hybridization events during each cycle of amplification, primer annealing and product melting. To ensure that these hybridization events occur, current PCR approaches rely on temperature set points and reaction contents that are optimized and maintained using rigid thermal cycling programs and stringent sample preparation procedures. This report describes a fundamentally simpler and more robust PCR design that dynamically controls thermal cycling by more directly monitoring the two key hybridization events during the reaction. This is achieved by optically sensing the annealing and melting of mirror-image l-DNA analogs of the reaction's primers and targets. Because the properties of l-DNA enantiomers parallel those of natural d-DNAs, the l-DNA reagents indicate the cycling conditions required for effective primer annealing and product melting during each cycle without interfering with the reaction. This hybridization-sensing approach adapts in real time to variations in reaction contents and conditions that impact primer annealing and product melting and eliminates the requirement for thermal calibrations and cycling programs. Adaptive PCR is demonstrated to amplify DNA targets with high efficiency and specificity under both controlled conditions and conditions that are known to cause traditional PCR to fail. The advantages of this approach promise to make PCR-based nucleic acid analysis simpler, more robust, and more accessible outside of well-controlled laboratory settings.

  16. Multitarget real-time PCR-based system: monitoring for unauthorized genetically modified events in India.

    PubMed

    Randhawa, Gurinder Jit; Singh, Monika; Sood, Payal; Bhoge, Rajesh K

    2014-07-23

    A multitarget TaqMan real-time PCR (RTi-PCR) based system was developed to monitor unauthorized genetically modified (GM) events in India. Most of the GM events included in this study are either authorized for commercial cultivation or field trials, which were indigenously developed or imported for research purposes. The developed system consists of a 96-well prespotted plate with lyophilized primers and probes, for simultaneous detection of 47 targets in duplicate, including 21 event-specific sequences, 5 construct regions, 15 for transgenic elements, and 6 taxon-specific targets for cotton, eggplant, maize, potato, rice, and soybean. Limit of detection (LOD) of assays ranged from 0.1 to 0.01% GM content for different targets. Applicability, robustness, and practical utility of the developed system were verified with stacked GM cotton event, powdered samples of proficiency testing and two unknown test samples. This user-friendly multitarget approach can be efficiently utilized for monitoring the unauthorized GM events in an Indian context.

  17. Development of PCR-based codominant markers flanking the Alt3 gene in rye.

    PubMed

    Miftahudin; Scoles, G J; Gustafson, J P

    2004-04-01

    Aluminum (Al) toxicity is considered to be a major problem for crop growth and production on acid soils. The ability of crops to overcome Al toxicity varies among crop species and cultivars. Rye (Secale cereale L.) is the most Al-tolerant species among the Triticeae. Our previous study showed that Al tolerance in a rye F6 recombinant inbred line (RIL) population was controlled by a single gene designated as the aluminum tolerance (Alt3) gene on chromosome 4RL. Based on the DNA sequence of a rice (Oryza sativa L.) BAC clone suspected to be syntenic to the Alt3 gene region, we developed two PCR-based codominant markers flanking the gene. These two markers, a sequence-tagged site (STS) marker and a cleaved amplified polymorphic sequence (CAPS) marker, each flanked the Alt3 gene at an approximate distance of 0.4 cM and can be used to facilitate high-resolution mapping of the gene. The markers might also be used for marker-assisted selection in rye or wheat (Triticum aestivum L.) breeding programs to obtain Al-tolerant lines and (or) cultivars.

  18. Optimization of a quantitative PCR based method for plasmid copy number determination in human cell lines.

    PubMed

    Fliedl, Lukas; Kast, Florian; Grillari, Johannes; Wieser, Matthias; Grillari-Voglauer, Regina

    2015-12-25

    Transient gene expression (TGE) is an essential tool for the production of recombinant proteins, especially in early drug discovery and development phases of biopharmaceuticals. The need for fast production of sufficient recombinant protein for initial tests has dramatically increased with increase in the identification of potential novel pharmaceutical targets. One of the critical factors for transient transfection is plasmid copy number (PCN), for which we here provide an optimized qPCR based protocol. Thereby, we show the loss of PCN during a typical batch process of HEK293 cells after transfection from 606,000 to 4560 copies per cell within 5 days. Finally two novel human kidney cell lines, RS and RPTEC/TERT1 were compared to HEK293 and proved competitive in terms of PCN and specific productivity. In conclusion, since trafficking and degradation of plasmid DNA is not fully understood yet, improved methods for analysis of PCN may contribute to design specific and more stable plasmids for high yield transient gene expression systems. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. A polyplex qPCR-based binding assay for protein-DNA interactions.

    PubMed

    Moreau, Morgane J J; Schaeffer, Patrick M

    2012-09-21

    The measurement of protein-DNA interactions is difficult and often involves radioisotope-labelled DNA to obtain the desired assay sensitivity. More recently, high-throughput proteomic approaches were developed but they generally lack sensitivity. For these methods, the level of technical difficulties involved is high due to the need for specialised facilities or equipment and training. The new qPCR-based DNA-binding assay involves immunoprecipitation of a GFP-tagged DNA-binding protein in complex with various DNA targets (Ter sites) followed by qPCR quantification, affording a very sensitive and quantitative method that can be performed in polyplex. Using a single binding reaction, the binding specificity of the DNA replication terminator protein Tus for ten termination sites TerA-J could be obtained for the first time in just a few hours. This new qPCR DNA-binding assay can easily be adapted to determine the binding specificity of virtually any soluble and functional epitope-tagged DNA-binding protein.

  20. PCR-based molecular discrimination between Maritrema eroliae and Probolocoryphe uca (Digenea: Microphallidae) in Kuwait Bay.

    PubMed

    Al-Kandari, W Y; Al-Bustan, S A; Alnaqeeb, M; Isaac, A M

    2014-06-01

    Microphallid trematodes are common parasites in marine snails and crustacean hosts at Kuwait Bay. The larval stages of two microphallids, Maritrema eroliae and Probolocoryphe uca, are difficult to differentiate morphologically. In this study, two PCR-based techniques were established for quick and accurate discrimination between the larval stages of the two microphallid species, employing restriction fragment length polymorphism (PCR-RFLP) and species-specific primers. Both techniques utilized nucleotide differences in the second internal transcribed region (ITS2) of the ribosomal DNA (rDNA) in the two species. For the PCR-RFLP technique, restriction enzyme AvaII was selected and it generated different restriction profiles among the two microphallids. In addition, species-specific primers were prepared for each microphallid species that amplified distinctive fragments. Both techniques showed that the larval stages of the two microphallid species can be identified accurately. However, direct PCR amplification using species-specific primers was more advantageous than the PCR-RFLP technique since it allowed rapid and specific discrimination between the two species. This technique provides a useful tool that can be used in future studies for the study of the distribution of microphallid species and their definitive hosts at different localities of Kuwait Bay.

  1. PCR-based Approaches for the Detection of Clinical Methicillin-resistant Staphylococcus aureus

    PubMed Central

    Liu, Ying; Zhang, Jiang; Ji, Yinduo

    2016-01-01

    Staphylococcus aureus is an important pathogen that can cause a variety of infections, including superficial and systematic infections, in humans and animals. The persistent emergence of multidrug resistant S. aureus, particularly methicillin-resistant S. aureus, has caused dramatically economic burden and concerns in the public health due to limited options of treatment of MRSA infections. In order to make a correct choice of treatment for physicians and understand the prevalence of MRSA, it is extremely critical to precisely and timely diagnose the pathogen that induces a specific infection of patients and to reveal the antibiotic resistant profile of the pathogen. In this review, we outlined different PCR-based approaches that have been successfully utilized for the rapid detection of S. aureus, including MRSA and MSSA, directly from various clinical specimens. The sensitivity and specificity of detections were pointed out. Both advantages and disadvantages of listed approaches were discussed. Importantly, an alternative approach is necessary to further confirm the detection results from the molecular diagnostic assays. PMID:27335617

  2. A Digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells

    PubMed Central

    Berman, Jennifer R.; Postovit, Lynne-Marie

    2016-01-01

    The rapid adoption of gene editing tools such as CRISPRs and TALENs for research and eventually therapeutics necessitates assays that can rapidly detect and quantitate the desired alterations. Currently, the most commonly used assay employs “mismatch nucleases” T7E1 or “Surveyor” that recognize and cleave heteroduplexed DNA amplicons containing mismatched base-pairs. However, this assay is prone to false positives due to cancer-associated mutations and/or SNPs and requires large amounts of starting material. Here we describe a powerful alternative wherein droplet digital PCR (ddPCR) can be used to decipher homozygous from heterozygous mutations with superior levels of both precision and sensitivity. We use this assay to detect knockout inducing alterations to stem cell associated proteins, NODAL and SFRP1, generated using either TALENs or an “all-in-one” CRISPR/Cas plasmid that we have modified for one-step cloning and blue/white screening of transformants. Moreover, we highlight how ddPCR can be used to assess the efficiency of varying TALEN-based strategies. Collectively, this work highlights how ddPCR-based screening can be paired with CRISPR and TALEN technologies to enable sensitive, specific, and streamlined approaches to gene editing and validation. PMID:27089539

  3. Determination of allele frequency in pooled DNA: comparison of three PCR-based methods.

    PubMed

    Wilkening, Stefan; Hemminki, Kari; Thirumaran, Ranjit Kumar; Bermejo, Justo Lorenzo; Bonn, Stefan; Försti, Asta; Kumar, Rajiv

    2005-12-01

    Determination of allele frequency in pooled DNA samples is a powerful and efficient tool for large-scale association studies. In this study, we tested and compared three PCR-based methods for accuracy, reproducibility, cost, and convenience. The methods compared were: (i) real-time PCR with allele-specific primers, (ii) real-time PCR with allele-specific TaqMan probes, and (iii) quantitative sequencing. Allele frequencies of three single nucleotide polymorphisms in three different genes were estimated from pooled DNA. The pools were made of genomic DNA samples from 96 cases with basal cell carcinoma of the skin and 96 healthy controls with known genotypes. In this study, the allele frequency estimation made by real-time PCR with allele-specific primers had the smallest median deviation (MD) from the real allele frequency with 1.12% (absolute percentage points) and was also the cheapest method. However; this method required the most time for optimization and showed the highest variation between replicates (SD = 6.47%). Quantitative sequencing, the simplest method, was found to have intermediate accuracies (MD = 1.44%, SD = 4.2%). Real-time PCR with TaqMan probes, a convenient but very expensive method, had an MD of 1.47% and the lowest variation between replicates (SD = 3.18%).

  4. PCR-based diversity estimates of artificial and environmental 18S rRNA gene libraries.

    PubMed

    Potvin, Marianne; Lovejoy, Connie

    2009-01-01

    Environmental clone libraries constructed using small subunit ribosomal RNA (rRNA) or other gene-specific primers have become the standard molecular approach for identifying microorganisms directly from their environment. This technique includes an initial polymerase chain reaction (PCR) amplification step of a phylogenetically useful marker gene using universal primers. Although it is acknowledged that such primers introduce biases, there have been few studies if any to date systematically examining such bias in eukaryotic microbes. We investigated some implications of such bias by constructing clone libraries using several universal primer pairs targeting rRNA genes. Firstly, we constructed artificial libraries using a known mix of small cultured pelagic arctic algae with representatives from five major lineages and secondly we investigated environmental samples using several primer pairs. No primer pair retrieved all of the original algae in the artificial clone libraries and all showed a favorable bias toward the dinoflagellate Polarella glacialis and a bias against the prasinophyte Micromonas and a pennate diatom. Several other species were retrieved by only one primer pair tested. Despite this, sequences from nine environmental libraries were diverse and contained representatives from all major eukaryotic clades expected in marine samples. Further, libraries from the same sample grouped together using Bray-Curtis clustering, irrespective of primer pairs. We conclude that environmental PCR-based techniques are sufficient to compare samples, but the total diversity will probably always be underestimated and relative abundance estimates should be treated with caution.

  5. Evaluation of a rapid PCR-based method for the detection of animal material.

    PubMed

    Yancy, Haile F; Mohla, Anuja; Farrell, Dorothy E; Myers, Michael J

    2005-12-01

    A rapid PCR-based analytical method for detection of animal-derived materials in complete feed was developed. Using a commercially available DNA forensic kit for the extraction of DNA from animal feed, a sensitive method was developed that was capable of detecting as little as 0.03% bovine meat and bone meal in complete feed in under 8 h of total assay time. The reduction in assay time was accomplished by reducing the DNA extraction time to 2 h and using the simpler cleanup procedure of the kit. Assay sensitivity can be increased to 0.006% by increasing the DNA extraction time to an overnight incubation of approximately 16 h. Examination of dairy feed samples containing either bovine meat and bone meal, porcine meat and bone meal, or lamb meal at a level of 0.1% (wt/wt basis) suggested that this method may be suitable for regulatory uses. The adoption of this commercially available kit for use with animal feeds yields an assay that is quicker and simpler to perform than a previously validated assay for the detection of animal proteins in animal feed.

  6. PCR-based detection of bioluminescent microbial populations in Tyrrhenian Sea

    NASA Astrophysics Data System (ADS)

    Gentile, Gabriela; De Luca, Massimo; Denaro, Renata; La Cono, Violetta; Smedile, Francesco; Scarfì, Simona; De Domenico, Emilio; De Domenico, Maria; Yakimov, Michail M.

    2009-05-01

    The present study is focused on the development of a cultivation-independent molecular approach for specific detection of bioluminescent bacteria within microbial communities by direct amplification of luxA gene from environmental DNA. A new set of primers, specifically targeting free-living bioluminescent bacteria, was designed on the base of l uxA sequences available from the public database. Meso- and bathypelagic seawater samples were collected from two stations in Tyrrhenian Sea at the depths of 500 and 2750 m. The same seawater samples also were used to isolate bioluminescent bacteria that were further subjected to luxA and 16S rRNA gene sequencing. PCR products obtained by amplification with designed primers were cloned, and the phylogenetic affiliation of 40 clones was determined. All of them were clustered into three groups, only distantly related to the Photobacterium phosphoreum and Photobacterium kishitanii clades. The half of all clones formed a tight monophyletic clade, while the rest of clones were organized in "compartment"-specific, meso- and bathypelagic ecotypes. No matches with luxA gene sequences of four bioluminescent strains, isolated from the same seawater samples, were observed. These findings indicate that the PCR-based approach developed in present manuscript, allowed us to detect the novel, "yet to be cultivated" lineages of bioluminescent bacteria, which are likely specific for distinct warm bathypelagic realms of Mediterranean Sea.

  7. Experimental factors affecting PCR-based estimates of microbial species richness and evenness.

    PubMed

    Engelbrektson, Anna; Kunin, Victor; Wrighton, Kelly C; Zvenigorodsky, Natasha; Chen, Feng; Ochman, Howard; Hugenholtz, Philip

    2010-05-01

    Pyrosequencing of 16S rRNA gene amplicons for microbial community profiling can, for equivalent costs, yield more than two orders of magnitude more sensitivity than traditional PCR cloning and Sanger sequencing. With this increased sensitivity and the ability to analyze multiple samples in parallel, it has become possible to evaluate several technical aspects of PCR-based community structure profiling methods. We tested the effect of amplicon length and primer pair on estimates of species richness (number of species) and evenness (relative abundance of species) by assessing the potentially tractable microbial community residing in the termite hindgut. Two regions of the 16S rRNA gene were sequenced from one of two common priming sites, spanning the V1-V2 or V8 regions, using amplicons ranging in length from 352 to 1443 bp. Our results show that both amplicon length and primer pair markedly influence estimates of richness and evenness. However, estimates of species evenness are consistent among different primer pairs targeting the same region. These results highlight the importance of experimental methodology when comparing diversity estimates across communities.

  8. Application of PCR-based methods for diagnosis of intestinal parasitic infections in the clinical laboratory.

    PubMed

    Verweij, Jaco J

    2014-12-01

    For many years PCR- and other DNA-based methods of pathogen detection have been available in most clinical microbiology laboratories; however, until recently these tools were not routinely exploited for the diagnosis of parasitic infections. Laboratories were initially reluctant to implement PCR as incorporation of such assays within the algorithm of tools available for the most accurate diagnosis of a large variety of parasites was unclear. With regard to diagnosis of intestinal parasitic infections, the diversity of parasites that one can expect in most settings is far less than the parasitological textbooks would have you believe, hence developing a simplified diagnostic triage is feasible. Therefore the classical algorithm based on population, patient groups, use of immuno-suppressive drugs, travel history etc. is also applicable to decide when to perform and which additional techniques are to be used, if a multiplex PCR panel is used as a first-line screening diagnostic.

  9. Real-time PCR-based identification of bacterial and fungal pathogens from blood samples.

    PubMed

    Mai, Madeleine; Müller, Iris; Maneg, Daniela; Lohr, Benedikt; Haecker, Achim; Haberhausen, Gerd; Hunfeld, Klaus-Peter

    2015-01-01

    Latest major contributions in the field of sepsis diagnostics result from advances in PCR technologies permitting new standards in speed and quality, given the fact that a timely diagnosis is the decisive factor to the survival of patients with bloodstream infections.Multiplex real-time PCR is a quantitative method for simultaneous amplification and detection of different targeted DNA molecules within hours. Nevertheless, various studies have shown a number of technical shortcomings as well as a high heterogeneity in sensitivity.The present method allows the standardized and rapid detection and identification of 25 common bacteria and fungi responsible for bloodstream infections from whole blood samples by using LightCycler(®) SeptiFast (LC-SF) test, based on real-time PCR.

  10. A 128 Multiplexing Factor Time-Domain SQUID Multiplexer

    NASA Astrophysics Data System (ADS)

    Prêle, D.; Voisin, F.; Piat, M.; Decourcelle, T.; Perbost, C.; Chapron, C.; Rambaud, D.; Maestre, S.; Marty, W.; Montier, L.

    2016-07-01

    A cryogenic 128:1 Time-Domain Multiplexer (TDM) has been developed for the readout of kilo-pixel Transition Edge Sensor (TES) arrays dedicated to the Q&U Bolometric Interferometer for Cosmology (QUBIC) instrument which aims to measure the B-mode polarization of the Cosmic Microwave Background. Superconducting QUantum Interference Devices (SQUIDs) are usually used to read out TESs. Moreover, SQUIDs are used to build TDM by biasing sequentially the SQUIDs connected together—one for each TES. In addition to this common technique which allows a typical 32 multiplexing factor, a cryogenic integrated circuit provides a 4:1 second multiplexing stage. This cryogenic integrated circuit is one of the original part of our TDM achieving an unprecedented 128 multiplexing factor. We present these two dimension TDM stages: topology of the SQUID multiplexer, operation of the cryogenic integrated circuit, and integration of the full system to read out a TES array dedicated to the QUBIC instrument. Flux-locked loop operation in multiplexed mode is also discussed.

  11. 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.

  12. Percolation in real multiplex networks

    NASA Astrophysics Data System (ADS)

    Bianconi, Ginestra; Radicchi, Filippo

    2016-12-01

    We present an exact mathematical framework able to describe site-percolation transitions in real multiplex networks. Specifically, we consider the average percolation diagram valid over an infinite number of random configurations where nodes are present in the system with given probability. The approach relies on the locally treelike ansatz, so that it is expected to accurately reproduce the true percolation diagram of sparse multiplex networks with negligible number of short loops. The performance of our theory is tested in social, biological, and transportation multiplex graphs. When compared against previously introduced methods, we observe improvements in the prediction of the percolation diagrams in all networks analyzed. Results from our method confirm previous claims about the robustness of real multiplex networks, in the sense that the average connectedness of the system does not exhibit any significant abrupt change as its individual components are randomly destroyed.

  13. Combining Watershed Variables with PCR-based Methods for Better Characterization and Management of Fecal Pollution in Small Streams

    EPA Science Inventory

    Ability to distinguish between human and animal fecal pollution is important for risk assessment and watershed management, particularly in bodies of water used as sources of drinking water or for recreation. PCR-based methods were used to determine the source of fecal pollution ...

  14. Development of PCR-based assays for detecting and differentiating three species of botrytis infecting broad bean

    USDA-ARS?s Scientific Manuscript database

    Botrytis cinerea, B. fabae and B. fabiopsis are known to cause chocolate spot on broad bean. This study was conducted to develop PCR-based assays to detect and differentiate this three species. Two sets of primers, Bc-f/Bc-r for B. cinerea and Bfab-f/Bfab-r for B. fabiopsis, were designed based on t...

  15. Combining Watershed Variables with PCR-based Methods for Better Characterization and Management of Fecal Pollution in Small Streams

    EPA Science Inventory

    Culture- and PCR-based measurements of fecal pollution were determined and compared to hydrologic and land use indicators. Stream water samples (n = 235) were collected monthly over a two year period from ten streams draining headwatersheds with different land use intensities ra...

  16. A Reliable and Inexpensive Method of Nucleic Acid Extraction for the PCR-Based Detection of Diverse Plant Pathogens

    USDA-ARS?s Scientific Manuscript database

    A reliable extraction method is described for the preparation of total nucleic acids from several plant genera for subsequent detection of plant pathogens by PCR-based techniques. By the combined use of a modified CTAB (cetyltrimethylammonium bromide) extraction protocol and a semi-automatic homogen...

  17. Combining Watershed Variables with PCR-based Methods for Better Characterization and Management of Fecal Pollution in Small Streams

    EPA Science Inventory

    Culture- and PCR-based measurements of fecal pollution were determined and compared to hydrologic and land use indicators. Stream water samples (n = 235) were collected monthly over a two year period from ten streams draining headwatersheds with different land use intensities ra...

  18. Performance of human fecal anaerobe-associated PCR-based assays in a multi-laboratory method evaluation study

    EPA Science Inventory

    A number of PCR-based methods for detecting human fecal material in environmental waters have been developed over the past decade, but these methods have rarely received independent comparative testing. Here, we evaluated ten of these methods (BacH, BacHum-UCD, B. thetaiotaomic...

  19. Evaluation of the repeatability and reproducibility of a suite of qPCR based microbial source tracking methods

    EPA Science Inventory

    Many PCR-based methods for microbial source tracking (MST) have been developed and validated within individual research laboratories. Inter-laboratory validation of these methods, however, has been minimal, and the effects of protocol standardization regimes have not been thor...

  20. Evaluation of the repeatability and reproducibility of a suite of qPCR based microbial source tracking methods

    EPA Science Inventory

    Many PCR-based methods for microbial source tracking (MST) have been developed and validated within individual research laboratories. Inter-laboratory validation of these methods, however, has been minimal, and the effects of protocol standardization regimes have not been thor...

  1. Performance of human fecal anaerobe-associated PCR-based assays in a multi-laboratory method evaluation study

    EPA Science Inventory

    A number of PCR-based methods for detecting human fecal material in environmental waters have been developed over the past decade, but these methods have rarely received independent comparative testing. Here, we evaluated ten of these methods (BacH, BacHum-UCD, B. thetaiotaomic...

  2. Navigability of multiplex temporal network

    NASA Astrophysics Data System (ADS)

    Wang, Yan; Song, Qiao-Zhen

    2017-01-01

    Real world complex systems have multiple levels of relationships and in many cases, they need to be modeled as multiplex networks where the same nodes can interact with each other in different layers, such as social networks. However, social relationships only appear at prescribed times so the temporal structures of edge activations can also affect the dynamical processes located above them. To consider both factors are simultaneously, we introduce multiplex temporal networks and propose three different walk strategies to investigate the concurrent dynamics of random walks and the temporal structure of multiplex networks. Thus, we derive analytical results for the multiplex centrality and coverage function in multiplex temporal networks. By comparing them with the numerical results, we show how the underlying topology of the layers and the walk strategy affect the efficiency when exploring the networks. In particular, the most interesting result is the emergence of a super-diffusion process, where the time scale of the multiplex is faster than that of both layers acting separately.

  3. MPprimer: a program for reliable multiplex PCR primer design.

    PubMed

    Shen, Zhiyong; Qu, Wubin; Wang, Wen; Lu, Yiming; Wu, Yonghong; Li, Zhifeng; Hang, Xingyi; Wang, Xiaolei; Zhao, Dongsheng; Zhang, Chenggang

    2010-03-18

    Multiplex PCR, defined as the simultaneous amplification of multiple regions of a DNA template or multiple DNA templates using more than one primer set (comprising a forward primer and a reverse primer) in one tube, has been widely used in diagnostic applications of clinical and environmental microbiology studies. However, primer design for multiplex PCR is still a challenging problem and several factors need to be considered. These problems include mis-priming due to nonspecific binding to non-target DNA templates, primer dimerization, and the inability to separate and purify DNA amplicons with similar electrophoretic mobility. A program named MPprimer was developed to help users for reliable multiplex PCR primer design. It employs the widely used primer design program Primer3 and the primer specificity evaluation program MFEprimer to design and evaluate the candidate primers based on genomic or transcript DNA database, followed by careful examination to avoid primer dimerization. The graph-expanding algorithm derived from the greedy algorithm was used to determine the optimal primer set combinations (PSCs) for multiplex PCR assay. In addition, MPprimer provides a virtual electrophotogram to help users choose the best PSC. The experimental validation from 2x to 5x plex PCR demonstrates the reliability of MPprimer. As another example, MPprimer is able to design the multiplex PCR primers for DMD (dystrophin gene which caused Duchenne Muscular Dystrophy), which has 79 exons, for 20x, 20x, 20x, 14x, and 5x plex PCR reactions in five tubes to detect underlying exon deletions. MPprimer is a valuable tool for designing specific, non-dimerizing primer set combinations with constrained amplicons size for multiplex PCR assays.

  4. Detection of aflatoxin-producing molds in Korean fermented foods and grains by multiplex PCR.

    PubMed

    Yang, Zheng-You; Shim, Won-Bo; Kim, Ji-Hun; Park, Seon-Ja; Kang, Sung-Jo; Nam, Baik-Sang; Chung, Duck-Hwa

    2004-11-01

    An assay based on multiplex PCR was applied for the detection of potential aflatoxin-producing molds in Korean fermented foods and grains. Three genes, avfA, omtA, and ver-1, coding for key enzymes in aflatoxin biosynthesis, were used as aflatoxin-detecting target genes in multiplex PCR. DNA extracted from Aspergillus flavus, Aspergillus parasiticus, Aspergillus oryzae, Aspergillus niger, Aspergillus terreus, Penicillium expansum, and Fusarium verticillioides was used as PCR template to test specificity of the multiplex PCR assay. Positive results were achieved only with DNA that was extracted from the aflatoxigenic molds A. flavus and A. parasiticus in all three primer pairs. This result was supported by aflatoxin detection with direct competitive enzyme-linked immunosorbent assay (DC-ELISA). The PCR assay required just a few hours, enabling rapid and simultaneous detection of many samples at a low cost. A total of 22 Meju samples, 24 Doenjang samples, and 10 barley samples commercially obtained in Korea were analyzed. The DC-ELISA assay for aflatoxin detection gave negative results for all samples, whereas the PCR-based method gave positive results for 1 of 22 Meju samples and 2 of 10 barley samples. After incubation of the positive samples with malt extract agar, DC-ELISA also gave positive results for aflatoxin detection. All Doenjang samples were negative by multiplex PCR and DC-ELISA assay, suggesting that aflatoxin contamination and the presence of aflatoxin-producing molds in Doenjang are probably low.

  5. 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

  6. Strategies to develop strain-specific PCR based assays for probiotics.

    PubMed

    Treven, P

    2015-01-01

    Since health benefits conferred by probiotics are strain-specific, identification to the strain level is mandatory to allow the monitoring of the presence and the abundance of specific probiotic in a product or in a gastrointestinal tract. Compared to standard plate counts, the reduced duration of the assays and higher specificity makes PCR-based methods (standard PCR and quantitative PCR) very appropriate for detection or quantification of probiotics. Development of strain-specific assay consists of 4 main stages: (1) strain-specific marker identification; (2) construction of potential strain-specific primers; (3) validation on DNA from pure cultures of target and related strains; and (4) validation on spiked samples. The most important and also the most challenging step is the identification of strain-specific sequences, which can be subsequently targeted by specific primers or probes. Such regions can be identified on sequences derived from 16S-23S internally transcribed spacers, randomly amplified polymorphic DNA, representational difference analysis and suppression subtractive hybridisation. Already known phenotypic or genotypic characteristics of the target strain can also be used to develop the strain-specific assay. However, the initial stage of strain-specific assay development can be replaced by comparative genomics analysis of target genome with related genomes in public databases. Advances in whole genome sequencing (WGS) have resulted in a cost reduction for bacterial genome sequencing and consequently have made this approach available to most laboratories. In the present paper I reviewed the available literature on PCR and qPCR assays developed for detection of a specific probiotic strain and discussed future WGS and comparative genomics-based approaches.

  7. quantGenius: implementation of a decision support system for qPCR-based gene quantification.

    PubMed

    Baebler, Špela; Svalina, Miha; Petek, Marko; Stare, Katja; Rotter, Ana; Pompe-Novak, Maruša; Gruden, Kristina

    2017-05-25

    Quantitative molecular biology remains a challenge for researchers due to inconsistent approaches for control of errors in the final results. Due to several factors that can influence the final result, quantitative analysis and interpretation of qPCR data are still not trivial. Together with the development of high-throughput qPCR platforms, there is a need for a tool allowing for robust, reliable and fast nucleic acid quantification. We have developed "quantGenius" ( http://quantgenius.nib.si ), an open-access web application for a reliable qPCR-based quantification of nucleic acids. The quantGenius workflow interactively guides the user through data import, quality control (QC) and calculation steps. The input is machine- and chemistry-independent. Quantification is performed using the standard curve approach, with normalization to one or several reference genes. The special feature of the application is the implementation of user-guided QC-based decision support system, based on qPCR standards, that takes into account pipetting errors, assay amplification efficiencies, limits of detection and quantification of the assays as well as the control of PCR inhibition in individual samples. The intermediate calculations and final results are exportable in a data matrix suitable for further statistical analysis or visualization. We additionally compare the most important features of quantGenius with similar advanced software tools and illustrate the importance of proper QC system in the analysis of qPCR data in two use cases. To our knowledge, quantGenius is the only qPCR data analysis tool that integrates QC-based decision support and will help scientists to obtain reliable results which are the basis for biologically meaningful data interpretation.

  8. Fluorescent detection of Southern blots and PCR-based genetic typing tests

    SciTech Connect

    Mansfield, E.S.; Worley, J.M.; Zimmerman, P.A.

    1994-09-01

    The Southern blot is used to study gene organization, to identify disease-causing genomic rearrangements, or for typing RFLP markers in forensic, paternity, or prenatal diagnostic testing. Fluorescence offers a much greater dynamic range and a more linear response than film used in radioactive or chemiluminescent detection of RFLPs. We therefore investigated using the Fluorimager{trademark} 575 (Molecular Dynamics, Inc.) for analyzing Southern blots. Using a single-locus probe to D2S44 (YNH24) (Promega Corp.), we detect as little as 100 ng (0.05 attomole) genomic DNA. The alkaline phosphatase-labeled probe is detected using AttoPhos (JBL Scientific), and the developed membrane is scanned with the Fluorimager. Biotinylated hybridization probes can also be developed using a streptavidin-alkaline phosphatase conjugate and AttoPhos. The instrument scan parameters can be adjusted to prevent overexposure and accompanying loss of resolution in images of blots, gels, or 96-well microplates. We have used these other sample formats in PCR-based genetic typing assays. We use FluorKit DQS (Molecular Dynamics) to accurately quantify PCR template DNA (1-500 ng) in 96-well microplates scanned using the same instrument. Mutation detection assays run include heteroduplex gels (5% polyacrylamide, 2.7 M urea), short tandem repeat (STR) markers, amplified fragment length polymorphisms (AmpFLP), competitive priming PCR, and allele-specific oligotyping. These assays are run using either 1- or 2-color labeling. We detect unlabeled PCR products, such as the AmpFLP marker D1S80 (Perkin-Elmer) by post-staining gels for 10 minutes with SYBR Green 1 (Molecular Probes) and scanning the wet gel. The Fluorimager scans a 20 x 25 cm sample within three minutes, allowing rapid optimization of fluorescent protocols and high sample throughput.

  9. PCR based RFLP genotyping of bovine lymphocyte antigen DRB3.2 in Iranian Holstein population.

    PubMed

    Pashmi, Morteza; Qanbari, Saber; Ghorashi, Seyed Ali; Salehi, Abdolreza

    2007-02-01

    Major Histocompatibility Complex (MHC) class II locus DRB3 was investigated by PCR based restriction fragment length polymorphism (PCR-RFLP) assay. A total of 262 Holstein cows participating in the national recording system were sampled from 10 herds. A two-step polymerase chain reaction was carried out in order to amplify a 284 base-pair fragment of exon 2 of the target gene. Second PCR products were treated with three restriction endonucleas enzymes RsaI, BstYI and HaeIII. Digested fragments were analyzed by polyacrylamid gel electrophoresis. Twenty-eight BoLA-DRB3 alleles were identified. Identified alleles are: BoLA-DRB3.2 *3, *6, *7, *8, *9, *10, *11, *12, *13, *14, *15, *16,20, *21, *22, *23, *24, *25, *26, *27, *28, *32, *36, *37, *40, *51, *iaa and *ibb. The BoLA-DRB3.2*40 allele that was observed in this study has not been reported previously. The calculated frequencies were as follows: 2.29, 1.34, 0.19, 14.5, 0.38, 3.05, 12.21, 1.34, 2.29, 1.34, 2.48, 9.16, 0.95, 0.77, 6.68, 9.16, 17.94, 1.15, 0.57, 1.15, 0.95, 0.57, 0.38, 1.91, 0.38, 5.73, 0.19 and 0.95% respectively. The six most frequently observed alleles (BoLA-DRB3.2 *8, *11, *16, *22, *23 and *24) accounted for 69.65% of the alleles in these 10 herds. The results of this study confirm the allelic distribution of six most frequent alleles in Holstein population's worldwide.

  10. PCR-Based Techniques for Leprosy Diagnosis: From the Laboratory to the Clinic

    PubMed Central

    Martinez, Alejandra Nóbrega; Talhari, Carolina; Moraes, Milton Ozório; Talhari, Sinésio

    2014-01-01

    In leprosy, classic diagnostic tools based on bacillary counts and histopathology have been facing hurdles, especially in distinguishing latent infection from active disease and diagnosing paucibacillary clinical forms. Serological tests and IFN-gamma releasing assays (IGRA) that employ humoral and cellular immune parameters, respectively, are also being used, but recent results indicate that quantitative PCR (qPCR) is a key technique due to its higher sensitivity and specificity. In fact, advances concerning the structure and function of the Mycobacterium leprae genome led to the development of specific PCR-based gene amplification assays for leprosy diagnosis and monitoring of household contacts. Also, based on the validation of point-of-care technologies for M. tuberculosis DNA detection, it is clear that the same advantages of rapid DNA detection could be observed in respect to leprosy. So far, PCR has proven useful in the determination of transmission routes, M. leprae viability, and drug resistance in leprosy. However, PCR has been ascertained to be especially valuable in diagnosing difficult cases like pure neural leprosy (PNL), paucibacillary (PB), and patients with atypical clinical presentation and histopathological features compatible with leprosy. Also, the detection of M. leprae DNA in different samples of the household contacts of leprosy patients is very promising. Although a positive PCR result is not sufficient to establish a causal relationship with disease outcome, quantitation provided by qPCR is clearly capable of indicating increased risk of developing the disease and could alert clinicians to follow these contacts more closely or even define rules for chemoprophylaxis. PMID:24722358

  11. Comparison of Two Different PCR-based Methods for Detection of GAA Expansions in Frataxin Gene.

    PubMed

    Entezam, Mona; Amirfiroozi, Akbar; Togha, Mansoureh; Keramatipour, Mohammad

    2017-02-01

    Expansion of GAA trinucleotide repeats is the molecular basis of Friedreich's ataxia (FRDA). Precise detection of the GAA expansion repeat in frataxin gene has always been a challenge. Different molecular methods have been suggested for detection of GAA expansion, including; short-PCR, long-PCR, Triplet repeat primed-PCR (TP-PCR) and southern blotting. The aim of study was to evaluate two PCR-based methods, TP-PCR and long-PCR, and to explore the use of TP-PCR accompanying with long-PCR for accurate genotyping of FRDA patients. Blood samples were collected from six Iranian patients suspected to FRDA, who referred to the Department of Medical Genetics at Tehran University of Medical Sciences during the year 2014. For one of these patients' four asymptomatic members of the family were also recruited for the analysis. DNA extraction was performed by two different methods. TP-PCR and long-PCR were carried out in all samples. The type of this study is assessment / investigation of methods. Using a combination of the above methods, the genotypes of all samples were confirmed as five homozygous mutants (expanded GAA repeats), two heterozygous and three homozygous normal (normal repeat size). The results obtained by TP-PCR are consistent with long-PCR results. The presence or absence of expanded alleles can be identified correctly by TP-PCR. Performing long-PCR and Fluorescent-long-PCR enables accurate genotyping in all samples. This approach is highly reliable. It could be successfully used for detection of GAA expansion repeats.

  12. PCR-Based Multiple Species Cell Counting for In Vitro Mixed Culture.

    PubMed

    Huang, Ruijie; Zhang, Junjie; Yang, X Frank; Gregory, Richard L

    2015-01-01

    Changes of bacterial profiles in microbial communities are strongly associated with human health. There is an increasing need for multiple species research in vitro. To avoid high cost or measurement of a limited number of species, PCR-based multiple species cell counting (PCR-MSCC) has been conceived. Species-specific sequence is defined as a unique sequence of one species in a multiple species mixed culture. This sequence is identified by comparing a random 1000 bp genomic sequence of one species with the whole genome sequences of the other species in the same artificial mixed culture. If absent in the other genomes, it is the species-specific sequence. Species-specific primers were designed based on the species-specific sequences. In the present study, ten different oral bacterial species were mixed and grown in Brain Heart Infusion Yeast Extract with 1% sucrose for 24 hours. Biofilm was harvested and processed for DNA extraction and q-PCR amplification with the species-specific primers. By comparing the q-PCR data of each species in the unknown culture with reference cultures, in which the cell number of each species was determined by colony forming units on agar plate, the cell number of that strain in the unknown mixed culture was calculated. This technique is reliable to count microorganism numbers that are less than 100,000 fold different from other species within the same culture. Theoretically, it can be used in detecting a species in a mixed culture of over 200 species. Currently PCR-MSCC is one of the most economic methods for quantifying single species cell numbers, especially for the low abundant species, in a multiple artificial mixed culture in vitro.

  13. Multiplexed lasing in tissues

    NASA Astrophysics Data System (ADS)

    Chen, Yu-Cheng; Chen, Qiushu; Fan, Xudong

    2017-02-01

    Biolasers are an emerging technology for next generation biochemical detection and clinical applications. Progress has recently been made to achieve lasing from biomolecules and single living cells. Tissues, which consist of cells embedded in extracellular matrix, mimic more closely the actual complex biological environment in a living body and therefore are of more practical significance. Here, we developed a highly versatile tissue laser platform, in which tissues stained with fluorophores are sandwiched in a high-Q Fabry-Pérot microcavity. Distinct lasing emissions from muscle and adipose tissues stained respectively with fluorescein isothiocyanate (FITC) and boron-dipyrromethene (BODIPY), and hybrid muscle/adipose tissue with dual-staining were achieved with a threshold of only 10 μJ/mm2. Additionally, we investigated how tissue structure/geometry, tissue thickness, and staining dye concentration affect the tissue laser. It is further found that, despite large fluorescence spectral overlap between FITC and BODIPY in tissues, their lasing emissions could be clearly distinguished and controlled due to their narrow lasing bands and different lasing thresholds, thus enabling highly multiplexed detection. Our tissue laser platform can be broadly applicable to various types of tissues/diseases. It provides a new tool for a wide range of biological and biomedical applications, such as diagnostics/screening of tissues and identification/monitoring of biological transformations in tissue engineering.

  14. Development of a Multiplex PCR-Ligase Detection Reaction Assay for Diagnosis of Infection by the Four Parasite Species Causing Malaria in Humans

    PubMed Central

    McNamara, David T.; Thomson, Jodi M.; Kasehagen, Laurin J.; Zimmerman, Peter A.

    2004-01-01

    The diagnosis of infections caused by Plasmodium species is critical for understanding the nature of malarial disease, treatment efficacy, malaria control, and public health. The demands of field-based epidemiological studies of malaria will require faster and more sensitive diagnostic methods as new antimalarial drugs and vaccines are explored. We have developed a multiplex PCR-ligase detection reaction (LDR) assay that allows the simultaneous diagnosis of infection by all four parasite species causing malaria in humans. This assay exhibits sensitivity and specificity equal to those of other PCR-based assays, identifying all four human malaria parasite species at levels of parasitemias equal to 1 parasitized erythrocyte/μl of blood. The multiplex PCR-LDR assay goes beyond other PCR-based assays by reducing technical procedures and by detecting intraindividual differences in species-specific levels of parasitemia. Application of the multiplex PCR-LDR assay will provide the sensitivity and specificity expected of PCR-based diagnostic assays and will contribute new insight regarding relationships between the human malaria parasite species and the human host in future epidemiological studies. PMID:15184411

  15. Development of a multiplex PCR-ligase detection reaction assay for diagnosis of infection by the four parasite species causing malaria in humans.

    PubMed

    McNamara, David T; Thomson, Jodi M; Kasehagen, Laurin J; Zimmerman, Peter A

    2004-06-01

    The diagnosis of infections caused by Plasmodium species is critical for understanding the nature of malarial disease, treatment efficacy, malaria control, and public health. The demands of field-based epidemiological studies of malaria will require faster and more sensitive diagnostic methods as new antimalarial drugs and vaccines are explored. We have developed a multiplex PCR-ligase detection reaction (LDR) assay that allows the simultaneous diagnosis of infection by all four parasite species causing malaria in humans. This assay exhibits sensitivity and specificity equal to those of other PCR-based assays, identifying all four human malaria parasite species at levels of parasitemias equal to 1 parasitized erythrocyte/microl of blood. The multiplex PCR-LDR assay goes beyond other PCR-based assays by reducing technical procedures and by detecting intraindividual differences in species-specific levels of parasitemia. Application of the multiplex PCR-LDR assay will provide the sensitivity and specificity expected of PCR-based diagnostic assays and will contribute new insight regarding relationships between the human malaria parasite species and the human host in future epidemiological studies.

  16. Efficient exploration of multiplex networks

    NASA Astrophysics Data System (ADS)

    Battiston, Federico; Nicosia, Vincenzo; Latora, Vito

    2016-04-01

    Efficient techniques to navigate networks with local information are fundamental to sample large-scale online social systems and to retrieve resources in peer-to-peer systems. Biased random walks, i.e. walks whose motion is biased on properties of neighbouring nodes, have been largely exploited to design smart local strategies to explore a network, for instance by constructing maximally mixing trajectories or by allowing an almost uniform sampling of the nodes. Here we introduce and study biased random walks on multiplex networks, graphs where the nodes are related through different types of links organised in distinct and interacting layers, and we provide analytical solutions for their long-time properties, including the stationary occupation probability distribution and the entropy rate. We focus on degree-biased random walks and distinguish between two classes of walks, namely those whose transition probability depends on a number of parameters which is extensive in the number of layers, and those whose motion depends on intrinsically multiplex properties of the neighbouring nodes. We analyse the effect of the structure of the multiplex network on the steady-state behaviour of the walkers, and we find that heterogeneous degree distributions as well as the presence of inter-layer degree correlations and edge overlap determine the extent to which a multiplex can be efficiently explored by a biased walk. Finally we show that, in real-world multiplex transportation networks, the trade-off between efficient navigation and resilience to link failure has resulted into systems whose diffusion properties are qualitatively different from those of appropriately randomised multiplex graphs. This fact suggests that multiplexity is an important ingredient to include in the modelling of real-world systems.

  17. Exact simulation and sensitivity analysis of multiplexing networks

    NASA Astrophysics Data System (ADS)

    Bandler, J. W.; Daijavad, S.; Zhang, Q.-J.

    1986-01-01

    This paper presents a novel approach to the simulation and sensitivity analysis of multiplexing networks. All computations are performed efficiently, utilizing the concept of forward and reverse analysis, which is elegant and effective in cascaded circuit analysis. Formulas are derived for such responses as input or output reflection coefficient, common port and channel output port return losses, insertion loss, gain slope, and group delay. Exact sensitivities with respect to all variables of interest, including frequency, are evaluated. The fundamental assumption is that the transmission matrices for the individual components of the network and their sensitivities with respect to possible variables inside them are available. An explicit algorithm is provided describing the details of the computational aspects of the theory. The formulas are applied to the optimal design of practical contiguous or noncontiguous band multiplexers consisting of multicavity filters distributed along a waveguide manifold. An example of optimizing a practical 12-channel, 12-GHz contiguous band multiplexer without dummy channels, which is the state-of-the-art structure used as the output multiplexer in satellite transponders, is presented.

  18. Bond Percolation on Multiplex Networks

    NASA Astrophysics Data System (ADS)

    Hackett, A.; Cellai, D.; Gómez, S.; Arenas, A.; Gleeson, J. P.

    2016-04-01

    We present an analytical approach for bond percolation on multiplex networks and use it to determine the expected size of the giant connected component and the value of the critical bond occupation probability in these networks. We advocate the relevance of these tools to the modeling of multilayer robustness and contribute to the debate on whether any benefit is to be yielded from studying a full multiplex structure as opposed to its monoplex projection, especially in the seemingly irrelevant case of a bond occupation probability that does not depend on the layer. Although we find that in many cases the predictions of our theory for multiplex networks coincide with previously derived results for monoplex networks, we also uncover the remarkable result that for a certain class of multiplex networks, well described by our theory, new critical phenomena occur as multiple percolation phase transitions are present. We provide an instance of this phenomenon in a multiplex network constructed from London rail and European air transportation data sets.

  19. Thermally multiplexed polymerase chain reaction

    PubMed Central

    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.

    2015-01-01

    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. PMID:26339317

  20. Structural measures for multiplex networks

    NASA Astrophysics Data System (ADS)

    Battiston, Federico; Nicosia, Vincenzo; Latora, Vito

    2014-03-01

    Many real-world complex systems consist of a set of elementary units connected by relationships of different kinds. All such systems are better described in terms of multiplex networks, where the links at each layer represent a different type of interaction between the same set of nodes rather than in terms of (single-layer) networks. In this paper we present a general framework to describe and study multiplex networks, whose links are either unweighted or weighted. In particular, we propose a series of measures to characterize the multiplexicity of the systems in terms of (i) basic node and link properties such as the node degree, and the edge overlap and reinforcement, (ii) local properties such as the clustering coefficient and the transitivity, and (iii) global properties related to the navigability of the multiplex across the different layers. The measures we introduce are validated on a genuinely multiplex data set of Indonesian terrorists, where information among 78 individuals are recorded with respect to mutual trust, common operations, exchanged communications, and business relationships.

  1. Development of multiplex PCR for simultaneous detection of six swine DNA and RNA viruses.

    PubMed

    Xu, Xin-Gang; Chen, Guang-Da; Huang, Yong; Ding, Li; Li, Zhao-Cai; Chang, Ching-Dong; Wang, Chi-Young; Tong, De-Wen; Liu, Hung-Jen

    2012-07-01

    Uniplex and multiplex reverse transcription-polymerase chain reaction (RT-PCR) and PCR protocols were developed and evaluated subsequently for its effectiveness in detecting simultaneously single and mixed infections in swine. Specific primers for three DNA viruses and three RNA viruses, including classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), Japanese encephalitis virus (JEV), porcine circovirus type 2 (PCV2), porcine pseudorabies virus (PRV) and porcine parvovirus (PPV) were used for testing procedure. A single nucleic acid extraction protocol was adopted for the simultaneous extraction of both RNA and DNA viruses. The multiplex PCR consisted with two-step procedure which included reverse transcription of RNA virus and multiplex PCR of viral cDNA and DNA. The multiplex PCR assay was shown to be sensitive detecting at least 450pg of viral genomic DNA or RNA from a mixture of six viruses in a reaction. The assay was also highly specific in detecting one or more of the same viruses in various combinations in specimens. Thirty clinical samples and aborted fetuses collected from 4- to 12-week-old piglets were detected among 39 samples tested by both uniplex and multiplex PCR, showing highly identification. Because of the sensitivity and specificity, the multiplex PCR is a useful approach for clinical diagnosis of mixed infections of DNA and RNA viruses in swine.

  2. Development of the 19 X-STR loci multiplex system and genetic analysis of a Zhejiang Han population in China.

    PubMed

    Yang, XingYi; Wu, WeiWei; Chen, LinLi; Liu, ChangHui; Zhang, XiaoFang; Chen, Ling; Feng, XingLin; Wang, HuiJun; Liu, Chao

    2016-08-01

    The 19 X-STRs multiplex system is a PCR-based amplification kit that facilitates simultaneous amplification of 19 X-chromosomal STR loci (i.e. DXS7423, DXS10148, DXS10159, DXS6809, DXS7424, DXS8378, DXS10164, DXS10162, DXS7132, DXS10079, DXS6789, DXS101, DXS10103,DXS10101, HPTRB, DXS10075, DXS10074, DXS10135, and DXS10134). Eleven loci were extensively used in an Investigator Qiagen Argus X-12 (DXS7423, DXS10148, DXS8378, DXS10162, DXS7132, DXS10079, DXS10103, DXS10101, HPTRB, DXS10074, and DXS10135). In this research, the multiplex system was tested for detection sensitivity, DNA mixtures, inhibitor tolerance and species specificity; SWGDAM Validation Guidelines - Approved December 2012 were followed for the human fluorescent STR multiplex PCR reagent. Samples from 181 unrelated Zhejiang Han individuals (121 males and 60 females) were typed using this multiplex system. The results show that this 19X-STRs multiplex system is a robust and reliable amplification means to facilitate forensic and human identification testing.

  3. PCR Based Microbial Monitor for Analysis of Recycled Water Aboard the ISSA: Issues and Prospects

    NASA Technical Reports Server (NTRS)

    Cassell, Gail H.; Lefkowitz, Elliot J.; Glass, John I.

    1995-01-01

    The monitoring of spacecraft life support systems for the presence of health threatening microorganisms is paramount for crew well being and successful completion of missions. Development of technology to monitor spacecraft recycled water based on detection and identification of the genetic material of contaminating microorganisms and viruses would be a substantial improvement over current NASA plans to monitor recycled water samples that call for the use of conventional microbiology techniques which are slow, insensitive, and labor intensive. The union of the molecular biology techniques of DNA probe hybridization and polymerase chain reaction (PCR) offers a powerful method for the detection, identification, and quantification of microorganisms and viruses. This technology is theoretically capable of assaying samples in as little as two hours with specificity and sensitivity unmatched by any other method. A major advance in probe-hybridization/PCR has come about in a technology called TaqMan(TM), which was invented by Perkin Elmer. Instrumentation using TaqMan concepts is evolving towards devices that could meet NASA's needs of size, low power use, and simplicity of operation. The chemistry and molecular biology needed to utilize these probe-hybridization/PCR instruments must evolve in parallel with the hardware. The following issues of chemistry and biology must be addressed in developing a monitor: Early in the development of a PCR-based microbial monitor it will be necessary to decide how many and which organisms does the system need the capacity to detect. We propose a set of 17 different tests that would detect groups of bacteria and fungus, as well as specific eukaryotic parasites and viruses; In order to use the great sensitivity of PCR it will be necessary to concentrate water samples using filtration. If a lower limit of detection of 1 microorganism per 100 ml is required then the microbes in a 100 ml sample must be concentrated into a volume that can be

  4. PCR-based method for the rapid identification of astaxanthin-accumulating yeasts (Phaffia spp.).

    PubMed

    Colabella, Fernando; Libkind, Diego

    2016-01-01

    It has been recently found that the natural distribution, habitat, and genetic diversity of astaxanthin-producing yeasts (i.e. Phaffia rhodozyma, synonym Xanthophyllomyces dendrorhous) is much greater than previously thought. P. rhodozyma is biotechnologically exploited due to its ability to produce the carotenoid pigment astaxanthin and thus, it is used as a natural source of this pigment for aquaculture. P. rhodozyma was also capable of synthesizing the potent UVB sunscreen mycosporine-glutaminol-glucoside (MGG). Therefore, further environmental studies are needed to elucidate its ecological aspects and detect new potential strains for the production of astaxanthin and MGG. However, obtaining new isolates of P. rhodozyma and related species is not always easy due to its low abundance and the presence of other sympatric and pigmented yeasts. In this work we report a successful development of a species-specific primer which has the ability to quickly and accurately detecting isolates representing all known lineages of the genus Phaffia (including novel species of the genus) and excluding closely related taxa. For this purpose, a primer of 20 nucleotides (called PhR) was designed to be used in combination with universal primers ITS3 and NL4 in a multiplex amplification. The proposed method has the sensitivity and specificity required for the precise detection of new isolates, and therefore represents an important tool for the environmental search for novel astaxanthin-producing yeasts. Copyright © 2015 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  5. Usefulness of blood vessels as a DNA source for PCR-based genotyping based on two cases of corpse dismemberment.

    PubMed

    Shintani-Ishida, Kaori; Harada, Kazuki; Nakajima, Makoto; Yoshida, Ken-ichi

    2010-01-01

    The success of PCR-based genotyping of decomposed remains depends on the quality of extracted DNA. Hard tissues and muscles are preferred because of their DNA stability. However, in dismembered corpses the choice of a suitable DNA source is more limited. In short tandem repeat (STR) analysis in two cases of dismembered corpses, we found an advantage of using blood vessels over muscles. To confirm that blood vessels are better for STR typing compared to muscle, we collected nine sets of blood vessels and the adjacent muscle from six other decomposed remains and compared the STR profiles between the blood vessel and muscle samples. Better results for STR typing were obtained in blood vessels. Based on these results, we recommend use of blood vessels as material for PCR-based genotyping in identification of dismembered human remains with heavy postmortem changes.

  6. Clinical performance of multiplex high-risk e6 mrna expression in comparison with hpv dna subtypes for the identification of women at risk of cervical cancer.

    PubMed

    Ho, Chih-Ming; Pan, Kui-You; Chen, Yun-Yuan; Huang, Chia-Yen; Chen, Yu-Li; Chang, Shwu-Fen

    2015-08-01

    We compared multiplex E6 messenger ribonucleic acid (mRNA) tests using real-time quantitative reverse transcriptase polymerase chain reactions (PCR) with human papillomavirus (HPV) DNA subtypes using a MY11/GP6+ PCR-based reverse-blot assay to identify cervical intraepithelial neoplasias of grade 2 or worse (CIN2+). In total, 684 women were studied, of whom 377 (55%) were diagnosed with CIN2+ histologically. The specificity of HPV mRNA to predict histological CIN2+ was higher than that of HPV DNA (81.3% vs. 44.2%). The odds ratios (ORs) to predict histological CIN2+ in women with positive for type 16, 18, 31, and 45 E6 mRNA or by HPV DNA detection were 7.1 (95% confidence interval [CI] 3.9-13.1) and 2.5 (95%CI 1.9-3.5), respectively, compared to those with negative for E6 mRNA or HPV DNA. The OR to predict histological CIN2+ in women with a cytological grade multiplex HPV E6 mRNA detection can be used as a triage for women with cytological grade

  7. Universal multiplexable matK primers for DNA barcoding of angiosperms1

    PubMed Central

    Heckenhauer, Jacqueline; Barfuss, Michael H. J.; Samuel, Rosabelle

    2016-01-01

    Premise of the study: PCR amplification of the matK barcoding region is often difficult when dealing with multiple angiosperm families. We developed a primer cocktail to amplify this region efficiently across angiosperm diversity. Methods and Results: We developed 14 matK primers (seven forward, seven reverse) for multiplex PCR, using sequences available in GenBank for 178 taxa belonging to 123 genera in 41 families and 18 orders. Universality of these new multiplexed primers was tested with 53 specimens from 44 representative angiosperm families in 23 different orders. Our primers showed high PCR amplification and sequencing success. Conclusions: These results show that our newly developed primers are highly effective for multiplex PCR and can be employed in future barcode projects involving taxonomically diverse samples across angiosperms. Using multiplex primers for barcoding will reduce the cost and time needed for PCR amplification. PMID:27347449

  8. Universal multiplexable matK primers for DNA barcoding of angiosperms.

    PubMed

    Heckenhauer, Jacqueline; Barfuss, Michael H J; Samuel, Rosabelle

    2016-06-01

    PCR amplification of the matK barcoding region is often difficult when dealing with multiple angiosperm families. We developed a primer cocktail to amplify this region efficiently across angiosperm diversity. We developed 14 matK primers (seven forward, seven reverse) for multiplex PCR, using sequences available in GenBank for 178 taxa belonging to 123 genera in 41 families and 18 orders. Universality of these new multiplexed primers was tested with 53 specimens from 44 representative angiosperm families in 23 different orders. Our primers showed high PCR amplification and sequencing success. These results show that our newly developed primers are highly effective for multiplex PCR and can be employed in future barcode projects involving taxonomically diverse samples across angiosperms. Using multiplex primers for barcoding will reduce the cost and time needed for PCR amplification.

  9. Helicity multiplexed broadband metasurface holograms

    PubMed Central

    Wen, Dandan; Yue, Fuyong; Li, Guixin; Zheng, Guoxing; Chan, Kinlong; Chen, Shumei; Chen, Ming; Li, King Fai; Wong, Polis Wing Han; Cheah, Kok Wai; Yue Bun Pun, Edwin; Zhang, Shuang; Chen, Xianzhong

    2015-01-01

    Metasurfaces are engineered interfaces that contain a thin layer of plasmonic or dielectric nanostructures capable of manipulating light in a desirable manner. Advances in metasurfaces have led to various practical applications ranging from lensing to holography. Metasurface holograms that can be switched by the polarization state of incident light have been demonstrated for achieving polarization multiplexed functionalities. However, practical application of these devices has been limited by their capability for achieving high efficiency and high image quality. Here we experimentally demonstrate a helicity multiplexed metasurface hologram with high efficiency and good image fidelity over a broad range of frequencies. The metasurface hologram features the combination of two sets of hologram patterns operating with opposite incident helicities. Two symmetrically distributed off-axis images are interchangeable by controlling the helicity of the input light. The demonstrated helicity multiplexed metasurface hologram with its high performance opens avenues for future applications with functionality switchable optical devices. PMID:26354497

  10. A PCR-based genotyping method to distinguish between wild-type and ornamental varieties of Imperata cylindrica.

    PubMed

    Cseke, Leland J; Talley, Sharon M

    2012-02-20

    viable wind-dispersed seeds that spread cogongrass over wide distances(5-7). JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass(8-10), and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential(3,7,11). The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

  11. Improved PCR-Based Detection of Soil Transmitted Helminth Infections Using a Next-Generation Sequencing Approach to Assay Design

    PubMed Central

    Pilotte, Nils; Papaiakovou, Marina; Grant, Jessica R.; Bierwert, Lou Ann; Llewellyn, Stacey; McCarthy, James S.; Williams, Steven A.

    2016-01-01

    Background The soil transmitted helminths are a group of parasitic worms responsible for extensive morbidity in many of the world’s most economically depressed locations. With growing emphasis on disease mapping and eradication, the availability of accurate and cost-effective diagnostic measures is of paramount importance to global control and elimination efforts. While real-time PCR-based molecular detection assays have shown great promise, to date, these assays have utilized sub-optimal targets. By performing next-generation sequencing-based repeat analyses, we have identified high copy-number, non-coding DNA sequences from a series of soil transmitted pathogens. We have used these repetitive DNA elements as targets in the development of novel, multi-parallel, PCR-based diagnostic assays. Methodology/Principal Findings Utilizing next-generation sequencing and the Galaxy-based RepeatExplorer web server, we performed repeat DNA analysis on five species of soil transmitted helminths (Necator americanus, Ancylostoma duodenale, Trichuris trichiura, Ascaris lumbricoides, and Strongyloides stercoralis). Employing high copy-number, non-coding repeat DNA sequences as targets, novel real-time PCR assays were designed, and assays were tested against established molecular detection methods. Each assay provided consistent detection of genomic DNA at quantities of 2 fg or less, demonstrated species-specificity, and showed an improved limit of detection over the existing, proven PCR-based assay. Conclusions/Significance The utilization of next-generation sequencing-based repeat DNA analysis methodologies for the identification of molecular diagnostic targets has the ability to improve assay species-specificity and limits of detection. By exploiting such high copy-number repeat sequences, the assays described here will facilitate soil transmitted helminth diagnostic efforts. We recommend similar analyses when designing PCR-based diagnostic tests for the detection of other

  12. Rapid detection and quantification of tyrosine decarboxylase gene (tdc) and its expression in gram-positive bacteria associated with fermented foods using PCR-based methods.

    PubMed

    Torriani, Sandra; Gatto, Veronica; Sembeni, Silvia; Tofalo, Rosanna; Suzzi, Giovanna; Belletti, Nicoletta; Gardini, Fausto; Bover-Cid, Sara

    2008-01-01

    In this study, PCR-based procedures were developed to detect the occurrence and quantify the expression of the tyrosine decarboxylase gene (tdc) in gram-positive bacteria associated with fermented foods. Consensus primers were used in conventional and reverse transcription PCR to analyze a collection of 87 pure cultures of lactic acid bacteria and staphylococci. All enterococci, Staphylococcus epidermidis, Lactobacillus brevis, Lactobacillus curvatus, and Lactobacillus fermentum strains and 1 of 10 Staphylococcus xylosus strains produced amplification products with the primers DEC5 and DEC3 in accordance with results of the screening plate method and with previously reported result obtained with high-performance liquid chromatography. No amplicons were obtained for tyramine-negative strains, confirming the high specificity of these new primers. A novel quantitative real-time PCR assay was successfully applied to quantify tdc and its transcript in pure cultures and in meat and meat products. This assay allowed estimation of the influence of different variables (pH, temperature, and NaCl concentration) on the tdc expression of the tyraminogenic strain Enterococcus faecalis EF37 after 72 h of growth in M17 medium. Data obtained suggest that stressful conditions could induce greater tyrosine decarboxylase activity. The culture-independent PCR procedures developed here may be used for reliable and fast detection and quantification of bacterial tyraminogenic activity without the limitations of conventional techniques.

  13. Generation of a reliable full-length cDNA of infectiousTembusu virus using a PCR-based protocol.

    PubMed

    Liang, Te; Liu, Xiaoxiao; Cui, Shulin; Qu, Shenghua; Wang, Dan; Liu, Ning; Wang, Fumin; Ning, Kang; Zhang, Bing; Zhang, Dabing

    2016-02-02

    Full-length cDNA of Tembusu virus (TMUV) cloned in a plasmid has been found instable in bacterial hosts. Using a PCR-based protocol, we generated a stable full-length cDNA of TMUV. Different cDNA fragments of TMUV were amplified by reverse transcription (RT)-PCR, and cloned into plasmids. Fragmented cDNAs were amplified and assembled by fusion PCR to produce a full-length cDNA using the recombinant plasmids as templates. Subsequently, a full-length RNA was transcribed from the full-length cDNA in vitro and transfected into BHK-21 cells; infectious viral particles were rescued successfully. Following several passages in BKH-21 cells, the rescued virus was compared with the parental virus by genetic marker checks, growth curve determinations and animal experiments. These assays clearly demonstrated the genetic and biological stabilities of the rescued virus. The present work will be useful for future investigations on the molecular mechanisms involved in replication and pathogenesis of TMUV. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Size-selective separation and overall-amplification of cell-free fetal DNA fragments using PCR-based enrichment.

    PubMed

    Yang, Qiwei; Du, Zhenwu; Song, Yang; Gao, Sujie; Yu, Shan; Zhu, He; Ren, Ming; Zhang, Guizhen

    2017-01-19

    This study aimed to establish a method for the selective amplification of cell-free fetal DNA (cffDNA) in maternal plasma and preserve the integrity of DNA fragments during amplification, thereby providing a sufficient amount of cffDNA to meet the requirement of routine non-invasive prenatal testing. We amplified DNA molecules in a one-reaction system without considering their particular sequences and lengths (overall amplification) by using PCR-based enrichment. We then modified PCR conditions to verify the effect of denaturation temperature on DNA amplification on various lengths of DNA (selective overall amplification). Finally, we used an optimum temperature range to amplify cffDNA selectively. Amplification results were validated by electrophoresis and real-time quantitative PCR. Our PCR-based enrichment efficiently amplified all DNA fragments with differing lengths within a single reaction system, as well as preserving the integrity of the DNA fragments. cffDNA was significantly amplified along with the selective amplification of small fragment maternal plasma DNA in an appropriate range of denaturation temperatures. We have established a PCR-based method for the simultaneous enrichment and amplification of cffDNA in order to meet the requirements of high cffDNA quantity for routine non-invasive prenatal testing.

  15. Comparison between automated system and PCR-based method for identification and antimicrobial susceptibility profile of clinical Enterococcus spp.

    PubMed

    Furlaneto-Maia, Luciana; Rocha, Kátia Real; Siqueira, Vera Lúcia Dias; Furlaneto, Márcia Cristina

    2014-01-01

    Enterococci are increasingly responsible for nosocomial infections worldwide. This study was undertaken to compare the identification and susceptibility profile using an automated MicrosScan system, PCR-based assay and disk diffusion assay of Enterococcus spp. We evaluated 30 clinical isolates of Enterococcus spp. Isolates were identified by MicrosScan system and PCR-based assay. The detection of antibiotic resistance genes (vancomycin, gentamicin, tetracycline and erythromycin) was also determined by PCR. Antimicrobial susceptibilities to vancomycin (30 µg), gentamicin (120 µg), tetracycline (30 µg) and erythromycin (15 µg) were tested by the automated system and disk diffusion method, and were interpreted according to the criteria recommended in CLSI guidelines. Concerning Enterococcus identification the general agreement between data obtained by the PCR method and by the automatic system was 90.0% (27/30). For all isolates of E. faecium and E. faecalis we observed 100% agreement. Resistance frequencies were higher in E. faecium than E. faecalis. The resistance rates obtained were higher for erythromycin (86.7%), vancomycin (80.0%), tetracycline (43.35) and gentamicin (33.3%). The correlation between disk diffusion and automation revealed an agreement for the majority of the antibiotics with category agreement rates of > 80%. The PCR-based assay, the van(A) gene was detected in 100% of vancomycin resistant enterococci. This assay is simple to conduct and reliable in the identification of clinically relevant enterococci. The data obtained reinforced the need for an improvement of the automated system to identify some enterococci.

  16. Size-selective separation and overall-amplification of cell-free fetal DNA fragments using PCR-based enrichment

    PubMed Central

    Yang, Qiwei; Du, Zhenwu; Song, Yang; Gao, Sujie; Yu, Shan; Zhu, He; Ren, Ming; Zhang, Guizhen

    2017-01-01

    This study aimed to establish a method for the selective amplification of cell-free fetal DNA (cffDNA) in maternal plasma and preserve the integrity of DNA fragments during amplification, thereby providing a sufficient amount of cffDNA to meet the requirement of routine non-invasive prenatal testing. We amplified DNA molecules in a one-reaction system without considering their particular sequences and lengths (overall amplification) by using PCR-based enrichment. We then modified PCR conditions to verify the effect of denaturation temperature on DNA amplification on various lengths of DNA (selective overall amplification). Finally, we used an optimum temperature range to amplify cffDNA selectively. Amplification results were validated by electrophoresis and real-time quantitative PCR. Our PCR-based enrichment efficiently amplified all DNA fragments with differing lengths within a single reaction system, as well as preserving the integrity of the DNA fragments. cffDNA was significantly amplified along with the selective amplification of small fragment maternal plasma DNA in an appropriate range of denaturation temperatures. We have established a PCR-based method for the simultaneous enrichment and amplification of cffDNA in order to meet the requirements of high cffDNA quantity for routine non-invasive prenatal testing. PMID:28102322

  17. Accuracy and Sensitivity of Commercial PCR-Based Methods for Detection of Salmonella enterica in Feed ▿

    PubMed Central

    Koyuncu, Sevinc; Andersson, M. Gunnar; Häggblom, Per

    2010-01-01

    The present study compared the performance of commercial PCR-based Salmonella enterica detection methods (BAX System Q7, the iQ-Check Salmonella II kit, and the TaqMan Salmonella enterica detection kit) with culture-based methods (modified semisolid Rappaport-Vassiliadis [MSRV] and NMKL71) in spiked and naturally contaminated samples of feed mill scrapings (FMS), palm kernel meal (PKM), pelleted feed (PF), rape seed meal (RSM), soybean meal (SM), and wheat grain (WG). When results from the various feeds were compared, the number of Salmonella enterica CFU/25 g required to produce a positive were as follows: PKM > FMS = WG > RSM = SM = PF. These data are similar to those developed in earlier studies with culture-based Salmonella detection methods. PCR-based methods were performed similarly to culture-based methods, with respect to sensitivity and specificity. However, many PCR positives could not be confirmed by Salmonella isolation and for that reason the evaluated methods were found to be suitable only when rapid results were paramount. Nevertheless, PCR-based methods cannot presently replace culture-based methods when typing information is required for tracing studies or epidemiological investigations. The observed difference in detection levels is a potential problem when prevalence data are compared as well as when feed ingredients are tested for conformance with microbiological criteria. This paper also presents a statistical model that describes the detection probability when different levels (CFU) of Salmonella contamination are present in feed materials. PMID:20228106

  18. A competitive PCR-based method for the detection and quantification of Erwinia carotovora subsp. atroseptica On potato tubers.

    PubMed

    Hyman, L J; Birch, P R; Dellagi, A; Avrova, A O; Toth, I K

    2000-04-01

    A PCR-based method was developed for the simultaneous detection and quantification of the potato pathogen Erwinia carotovora subsp. atroseptica (Eca) on potato tubers. The method incorporates a competitor PCR template cloned into Escherichia coli in vector pGEM-T (E. coli 4R l/l). Predetermined numbers of E. coli 4R were added to potato peel extract, either pre-inoculated with Eca or from naturally contaminated tubers, and Eca numbers estimated by comparing the ratio of products generated from Eca target DNA and competitor template DNA following PCR. Estimates of Eca numbers were consistent with counts obtained on crystal violet pectate medium and immunofluorescence colony staining. Unlike these methods, however, the PCR-based method is not affected by the presence of other erwinias and saprophytes and is able to detect all serogroups of Eca. Based on this method, a key was produced relating product ratios, obtained following PCR from contaminated tuber stocks, to the likelihood of blackleg disease incidence. This is the first quantitative PCR-based detection method described for Eca and is the first for any bacterial plant pathogen to incorporate a DNA extraction control.

  19. 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.

  20. Multiplex Fabry-Perot interferometer

    NASA Technical Reports Server (NTRS)

    Hays, Paul B.; Snell, Hilary E.

    1991-01-01

    Attention is given to a Fabry-Perot interferometer (FPI) technique in which one of the etalon plates is moved over a large optical distance while the other remains fixed, thus exploiting the multiplex advantage of the instrument. This technique involves the application of Fourier-transform spectrometer to the multiple harmonics passing through the FPI etalon. It is shown that the multiplex FPI acts as several Michelson interferometers working at the same time, over the same spectral interval, and at different spectral resolutions. A high spectral resolution has been obtained over a large wavenumber interval, while the advantage of a reasonable scan length has been retained.

  1. Multiplex Fabry-Perot interferometer

    NASA Technical Reports Server (NTRS)

    Hays, Paul B.; Snell, Hilary E.

    1991-01-01

    Attention is given to a Fabry-Perot interferometer (FPI) technique in which one of the etalon plates is moved over a large optical distance while the other remains fixed, thus exploiting the multiplex advantage of the instrument. This technique involves the application of Fourier-transform spectrometer to the multiple harmonics passing through the FPI etalon. It is shown that the multiplex FPI acts as several Michelson interferometers working at the same time, over the same spectral interval, and at different spectral resolutions. A high spectral resolution has been obtained over a large wavenumber interval, while the advantage of a reasonable scan length has been retained.

  2. Superresolved spatially multiplexed interferometric microscopy.

    PubMed

    Picazo-Bueno, José Ángel; Zalevsky, Zeev; García, Javier; Micó, Vicente

    2017-03-01

    Superresolution capability by angular and time multiplexing is implemented onto a regular microscope. The technique, named superresolved spatially multiplexed interferometric microscopy (S2MIM), follows our previously reported SMIM technique [Opt. Express22, 14929 (2014)OPEXFF1094-408710.1364/OE.22.014929, J. Biomed. Opt.21, 106007 (2016)JBOPFO1083-366810.1117/1.JBO.21.10.106007] improved with superresolved imaging. All together, S2MIM updates a commercially available non-holographic microscope into a superresolved holographic one. Validation is presented for an Olympus BX-60 upright microscope with resolution test targets.

  3. Rapid multiplex reverse transcription-PCR typing of influenza A and B virus, and subtyping of influenza A virus into H1, 2, 3, 5, 7, 9, N1 (human), N1 (animal), N2, and N7, including typing of novel swine origin influenza A (H1N1) virus, during the 2009 outbreak in Milwaukee, Wisconsin.

    PubMed

    He, Jie; Bose, Michael E; Beck, Eric T; Fan, Jiang; Tiwari, Sagarika; Metallo, Jacob; Jurgens, Lisa A; Kehl, Sue C; Ledeboer, Nathan; Kumar, Swati; Weisburg, William; Henrickson, Kelly J

    2009-09-01

    A large outbreak of novel influenza A (H1N1) virus (swine origin influenza virus [S-OIV]) infection in Milwaukee, WI, occurred in late April 2009. We had recently developed a rapid multiplex reverse transcription-PCR enzyme hybridization assay (FluPlex) to determine the type (A or B) and subtype (H1, H2, H3, H5, H7, H9, N1 [human], N1 [animal], N2, or N7) of influenza viruses, and this assay was used to confirm the diagnoses for the first infected patients in the state. The analytical sensitivity was excellent at 1.5 to 116 copies/reaction, or 10(-3) to 10(-1) 50% tissue culture infective doses/ml. The testing of all existing hemagglutinin and neuraminidase subtypes of influenza A virus and influenza B virus (41 influenza virus strains) and 24 common respiratory pathogens showed only one low-level H3 cross-reaction with an H10N7 avian strain and only at 5.2 x 10(6) copies/reaction, not at lower concentrations. Comparisons of the FluPlex results with results from multiple validated in-house molecular assays, CDC-validated FDA-approved assays, and gene sequencing demonstrated 100% positive agreement for the typing of 179 influenza A viruses and 3 influenza B viruses, the subtyping of 110 H1N1 (S-OIV; N1 [animal]), 62 H1N1 (human), and 6 H3N2 (human) viruses, and the identification of 24 negative clinical samples and 100% negative agreement for all viruses tested except H1N1 (human) (97.7%). The small number of false-positive H1N1 (human) samples most likely represent increased sensitivity over that of other in-house assays, with four of four results confirmed by the CDC's influenza virus subtyping assay. The FluPlex is a rapid, inexpensive, sensitive, and specific method for the typing and subtyping of influenza viruses and demonstrated outstanding utility during the first 2 weeks of an S-OIV infection outbreak. Methods for rapid detection and broad subtyping of influenza viruses, including animal subtypes, are needed to address public concern over the emergence of

  4. Rapid Multiplex Reverse Transcription-PCR Typing of Influenza A and B Virus, and Subtyping of Influenza A Virus into H1, 2, 3, 5, 7, 9, N1 (Human), N1 (Animal), N2, and N7, Including Typing of Novel Swine Origin Influenza A (H1N1) Virus, during the 2009 Outbreak in Milwaukee, Wisconsin▿

    PubMed Central

    He, Jie; Bose, Michael E.; Beck, Eric T.; Fan, Jiang; Tiwari, Sagarika; Metallo, Jacob; Jurgens, Lisa A.; Kehl, Sue C.; Ledeboer, Nathan; Kumar, Swati; Weisburg, William; Henrickson, Kelly J.

    2009-01-01

    A large outbreak of novel influenza A (H1N1) virus (swine origin influenza virus [S-OIV]) infection in Milwaukee, WI, occurred in late April 2009. We had recently developed a rapid multiplex reverse transcription-PCR enzyme hybridization assay (FluPlex) to determine the type (A or B) and subtype (H1, H2, H3, H5, H7, H9, N1 [human], N1 [animal], N2, or N7) of influenza viruses, and this assay was used to confirm the diagnoses for the first infected patients in the state. The analytical sensitivity was excellent at 1.5 to 116 copies/reaction, or 10−3 to 10−1 50% tissue culture infective doses/ml. The testing of all existing hemagglutinin and neuraminidase subtypes of influenza A virus and influenza B virus (41 influenza virus strains) and 24 common respiratory pathogens showed only one low-level H3 cross-reaction with an H10N7 avian strain and only at 5.2 × 106 copies/reaction, not at lower concentrations. Comparisons of the FluPlex results with results from multiple validated in-house molecular assays, CDC-validated FDA-approved assays, and gene sequencing demonstrated 100% positive agreement for the typing of 179 influenza A viruses and 3 influenza B viruses, the subtyping of 110 H1N1 (S-OIV; N1 [animal]), 62 H1N1 (human), and 6 H3N2 (human) viruses, and the identification of 24 negative clinical samples and 100% negative agreement for all viruses tested except H1N1 (human) (97.7%). The small number of false-positive H1N1 (human) samples most likely represent increased sensitivity over that of other in-house assays, with four of four results confirmed by the CDC's influenza virus subtyping assay. The FluPlex is a rapid, inexpensive, sensitive, and specific method for the typing and subtyping of influenza viruses and demonstrated outstanding utility during the first 2 weeks of an S-OIV infection outbreak. Methods for rapid detection and broad subtyping of influenza viruses, including animal subtypes, are needed to address public concern over the emergence of

  5. Novel Quaternary Quantum Decoder, Multiplexer and Demultiplexer Circuits

    NASA Astrophysics Data System (ADS)

    Haghparast, Majid; Monfared, Asma Taheri

    2017-05-01

    Multiple valued logic is a promising approach to reduce the width of the reversible or quantum circuits, moreover, quaternary logic is considered as being a good choice for future quantum computing technology hence it is very suitable for the encoded realization of binary logic functions through its grouping of 2-bits together into quaternary values. The Quaternary decoder, multiplexer, and demultiplexer are essential units of quaternary digital systems. In this paper, we have initially designed a quantum realization of the quaternary decoder circuit using quaternary 1-qudit gates and quaternary Muthukrishnan-Stroud gates. Then we have presented quantum realization of quaternary multiplexer and demultiplexer circuits using the constructed quaternary decoder circuit and quaternary controlled Feynman gates. The suggested circuits in this paper have a lower quantum cost and hardware complexity than the existing designs that are currently used in quaternary digital systems. All the scales applied in this paper are based on Nanometric area.

  6. Novel Quaternary Quantum Decoder, Multiplexer and Demultiplexer Circuits

    NASA Astrophysics Data System (ADS)

    Haghparast, Majid; Monfared, Asma Taheri

    2017-02-01

    Multiple valued logic is a promising approach to reduce the width of the reversible or quantum circuits, moreover, quaternary logic is considered as being a good choice for future quantum computing technology hence it is very suitable for the encoded realization of binary logic functions through its grouping of 2-bits together into quaternary values. The Quaternary decoder, multiplexer, and demultiplexer are essential units of quaternary digital systems. In this paper, we have initially designed a quantum realization of the quaternary decoder circuit using quaternary 1-qudit gates and quaternary Muthukrishnan-Stroud gates. Then we have presented quantum realization of quaternary multiplexer and demultiplexer circuits using the constructed quaternary decoder circuit and quaternary controlled Feynman gates. The suggested circuits in this paper have a lower quantum cost and hardware complexity than the existing designs that are currently used in quaternary digital systems. All the scales applied in this paper are based on Nanometric area.

  7. Holographic data storage system combining shift-multiplexing with peristrophic-multiplexing

    NASA Astrophysics Data System (ADS)

    Yoshikawa, Kengo; Tsukamoto, Yu; Okubo, Kaito; Yamamoto, Manabu

    2014-02-01

    Holographic data storage (HDS) is a next-generation optical storage that uses the principles of holography. The multiplex holographic recording method is an important factor that affects the recording capacity of this storage. Various multiplex recording methods have been proposed so far. In this study, we focus on shift multiplexing with spherical waves and propose a method of shift multiplex recording that combines the peristrophic multiplexed recording. Simulation and experimental verification shows that the proposed method is effective in principle.

  8. Multiplex quantification of 12 European Union authorized genetically modified maize lines with droplet digital polymerase chain reaction.

    PubMed

    Dobnik, David; Spilsberg, Bjørn; Bogožalec Košir, Alexandra; Holst-Jensen, Arne; Žel, Jana

    2015-08-18

    Presence of genetically modified organisms (GMO) in food and feed products is regulated in many countries. The European Union (EU) has implemented a threshold for labeling of products containing more than 0.9% of authorized GMOs per ingredient. As the number of GMOs has increased over time, standard-curve based simplex quantitative polymerase chain reaction (qPCR) analyses are no longer sufficiently cost-effective, despite widespread use of initial PCR based screenings. Newly developed GMO detection methods, also multiplex methods, are mostly focused on screening and detection but not quantification. On the basis of droplet digital PCR (ddPCR) technology, multiplex assays for quantification of all 12 EU authorized GM maize lines (per April first 2015) were developed. Because of high sequence similarity of some of the 12 GM targets, two separate multiplex assays were needed. In both assays (4-plex and 10-plex), the transgenes were labeled with one fluorescence reporter and the endogene with another (GMO concentration = transgene/endogene ratio). It was shown that both multiplex assays produce specific results and that performance parameters such as limit of quantification, repeatability, and trueness comply with international recommendations for GMO quantification methods. Moreover, for samples containing GMOs, the throughput and cost-effectiveness is significantly improved compared to qPCR. Thus, it was concluded that the multiplex ddPCR assays could be applied for routine quantification of 12 EU authorized GM maize lines. In case of new authorizations, the events can easily be added to the existing multiplex assays. The presented principle of quantitative multiplexing can be applied to any other domain.

  9. Weak percolation on multiplex networks

    NASA Astrophysics Data System (ADS)

    Baxter, Gareth J.; Dorogovtsev, Sergey N.; Mendes, José F. F.; Cellai, Davide

    2014-04-01

    Bootstrap percolation is a simple but nontrivial model. It has applications in many areas of science and has been explored on random networks for several decades. In single-layer (simplex) networks, it has been recently observed that bootstrap percolation, which is defined as an incremental process, can be seen as the opposite of pruning percolation, where nodes are removed according to a connectivity rule. Here we propose models of both bootstrap and pruning percolation for multiplex networks. We collectively refer to these two models with the concept of "weak" percolation, to distinguish them from the somewhat classical concept of ordinary ("strong") percolation. While the two models coincide in simplex networks, we show that they decouple when considering multiplexes, giving rise to a wealth of critical phenomena. Our bootstrap model constitutes the simplest example of a contagion process on a multiplex network and has potential applications in critical infrastructure recovery and information security. Moreover, we show that our pruning percolation model may provide a way to diagnose missing layers in a multiplex network. Finally, our analytical approach allows us to calculate critical behavior and characterize critical clusters.

  10. A High Resolution CCD Multiplexer

    NASA Astrophysics Data System (ADS)

    Sheu, Larry S.; Kadekod i, Narayan; Nugroho, Yohanes; Lo, Mike; Mortz, Margaret; Ibrahim, Ali

    1983-11-01

    This paper describes a high resolution CCD multiplexer for focal plane imaging systems. The multiplexer incorporates quadrilinear readout registers to achieve two times the resolution of conventional bilinear structure while using the same design rules. Complete parallel charge transfer are ensured by a novel buried channel poly gate isolation scheme. A monolithic silicon photodiode array of 8 Am pitch, 3533 elements was designed with the multi-plexer. Video preprocessing circuits of high speed four to one channel stitching, compensated sample and hold and bad pixel deletion were integrated on chip for improved performance. The modulation transfer functions due to the geometry and the transfer inefficiency are discussed. The theoretically calculated total MTF agrees with the experimental result. At Nyquist frequency of 62.5 c/mm the total MTF is better than 0.6 in the absence of the diffusion MTF degradation. The noise spectrum of the CCD and the output amplifier are presented. The RMS noise of the CCD in dark is approximately 0.35 my over 1 MHz bandwidth. The CCD noise increases with light input attributed primarily to the shot noise. The low noise nature of the multiplexer makes it ideal for the high resolution low light level detection applications.

  11. Multiplex enrichment quantitative PCR (ME-qPCR): a high-throughput, highly sensitive detection method for GMO identification.

    PubMed

    Fu, Wei; Zhu, Pengyu; Wei, Shuang; Zhixin, Du; Wang, Chenguang; Wu, Xiyang; Li, Feiwu; Zhu, Shuifang

    2017-04-01

    Among all of the high-throughput detection methods, PCR-based methodologies are regarded as the most cost-efficient and feasible methodologies compared with the next-generation sequencing or ChIP-based methods. However, the PCR-based methods can only achieve multiplex detection up to 15-plex due to limitations imposed by the multiplex primer interactions. The detection throughput cannot meet the demands of high-throughput detection, such as SNP or gene expression analysis. Therefore, in our study, we have developed a new high-throughput PCR-based detection method, multiplex enrichment quantitative PCR (ME-qPCR), which is a combination of qPCR and nested PCR. The GMO content detection results in our study showed that ME-qPCR could achieve high-throughput detection up to 26-plex. Compared to the original qPCR, the Ct values of ME-qPCR were lower for the same group, which showed that ME-qPCR sensitivity is higher than the original qPCR. The absolute limit of detection for ME-qPCR could achieve levels as low as a single copy of the plant genome. Moreover, the specificity results showed that no cross-amplification occurred for irrelevant GMO events. After evaluation of all of the parameters, a practical evaluation was performed with different foods. The more stable amplification results, compared to qPCR, showed that ME-qPCR was suitable for GMO detection in foods. In conclusion, ME-qPCR achieved sensitive, high-throughput GMO detection in complex substrates, such as crops or food samples. In the future, ME-qPCR-based GMO content identification may positively impact SNP analysis or multiplex gene expression of food or agricultural samples. Graphical abstract For the first-step amplification, four primers (A, B, C, and D) have been added into the reaction volume. In this manner, four kinds of amplicons have been generated. All of these four amplicons could be regarded as the target of second-step PCR. For the second-step amplification, three parallels have been taken for

  12. ROVER variant caller: read-pair overlap considerate variant-calling software applied to PCR-based massively parallel sequencing datasets

    PubMed Central

    2014-01-01

    Background We recently described Hi-Plex, a highly multiplexed PCR-based target-enrichment system for massively parallel sequencing (MPS), which allows the uniform definition of library size so that subsequent paired-end sequencing can achieve complete overlap of read pairs. Variant calling from Hi-Plex-derived datasets can thus rely on the identification of variants appearing in both reads of read-pairs, permitting stringent filtering of sequencing chemistry-induced errors. These principles underly ROVER software (derived from Read Overlap PCR-MPS variant caller), which we have recently used to report the screening for genetic mutations in the breast cancer predisposition gene PALB2. Here, we describe the algorithms underlying ROVER and its usage. Results ROVER enables users to quickly and accurately identify genetic variants from PCR-targeted, overlapping paired-end MPS datasets. The open-source availability of the software and threshold tailorability enables broad access for a range of PCR-MPS users. Methods ROVER is implemented in Python and runs on all popular POSIX-like operating systems (Linux, OS X). The software accepts a tab-delimited text file listing the coordinates of the target-specific primers used for targeted enrichment based on a specified genome-build. It also accepts aligned sequence files resulting from mapping to the same genome-build. ROVER identifies the amplicon a given read-pair represents and removes the primer sequences by using the mapping co-ordinates and primer co-ordinates. It considers overlapping read-pairs with respect to primer-intervening sequence. Only when a variant is observed in both reads of a read-pair does the signal contribute to a tally of read-pairs containing or not containing the variant. A user-defined threshold informs the minimum number of, and proportion of, read-pairs a variant must be observed in for a ‘call’ to be made. ROVER also reports the depth of coverage across amplicons to facilitate the

  13. ROVER variant caller: read-pair overlap considerate variant-calling software applied to PCR-based massively parallel sequencing datasets.

    PubMed

    Pope, Bernard J; Nguyen-Dumont, Tú; Hammet, Fleur; Park, Daniel J

    2014-01-24

    We recently described Hi-Plex, a highly multiplexed PCR-based target-enrichment system for massively parallel sequencing (MPS), which allows the uniform definition of library size so that subsequent paired-end sequencing can achieve complete overlap of read pairs. Variant calling from Hi-Plex-derived datasets can thus rely on the identification of variants appearing in both reads of read-pairs, permitting stringent filtering of sequencing chemistry-induced errors. These principles underly ROVER software (derived from Read Overlap PCR-MPS variant caller), which we have recently used to report the screening for genetic mutations in the breast cancer predisposition gene PALB2. Here, we describe the algorithms underlying ROVER and its usage. ROVER enables users to quickly and accurately identify genetic variants from PCR-targeted, overlapping paired-end MPS datasets. The open-source availability of the software and threshold tailorability enables broad access for a range of PCR-MPS users. ROVER is implemented in Python and runs on all popular POSIX-like operating systems (Linux, OS X). The software accepts a tab-delimited text file listing the coordinates of the target-specific primers used for targeted enrichment based on a specified genome-build. It also accepts aligned sequence files resulting from mapping to the same genome-build. ROVER identifies the amplicon a given read-pair represents and removes the primer sequences by using the mapping co-ordinates and primer co-ordinates. It considers overlapping read-pairs with respect to primer-intervening sequence. Only when a variant is observed in both reads of a read-pair does the signal contribute to a tally of read-pairs containing or not containing the variant. A user-defined threshold informs the minimum number of, and proportion of, read-pairs a variant must be observed in for a 'call' to be made. ROVER also reports the depth of coverage across amplicons to facilitate the identification of any regions that may

  14. PCR-based detection of arboviral RNA from mosquitoes homogenized in detergent.

    PubMed

    Vodkin, M H; Streit, T; Mitchell, C J; McLaughlin, G L; Novak, R J

    1994-07-01

    An improved method for the extraction of viral RNAs was developed to facilitate the reverse transcription (RT)-PCR detection of mosquitoes infected with Western equine encephalitis virus or La Crosse virus. The solubilization method, which uses only EDTA and sodium dodecyl sulfate (SDS) followed by dilution of sample, allows accurate viral detection through the use of random hexamers for the RT followed by specific primers for the PCR. Identities of the reaction products were confirmed either by sequencing or restriction endonuclease digestion. Previous methods for the extraction of RNA for the coupled RT-PCR depended on combinations of guanidinium isothiocyanate, acid phenol, detergents and multiple centrifugations. Ideally, routine detection of viral RNAs for diagnostic purposes should bypass many of the above steps, while still providing a sensitive assay. Our level of detection is 1 infected mosquito in a group of 100.

  15. Fluorescent multiplex linkage analysis and carrier detection for Duchenne/Becker muscular dystrophy

    SciTech Connect

    Schwartz, L.S.; Hoffman, E.P. ); Tarleton, J. ); Popovich, B. ); Seltzer, W.K. )

    1992-10-01

    The authors have developed a fast and accurate PCR-based linkage and carrier detection protocol for families of Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD) patients with or without detectable deletions of the dystrophin gene, using fluorescent PCR products analyzed on an automated sequencer. When a deletion is found in the affected male DMD/BMD patient by standard multiplex PCR, fluorescently labeled primers specific for the deleted and nondeleted exon(s) are used to amplify the DNA of at-risk female relatives by using multiplex PCR at low cycle number (20 cycles). The products are then quantitatively analyzed on an automatic sequencer to determine whether they are heterozygous for the deletion and thus are carriers. As a confirmation of the deletion data, and in cases in which a deletion is not found in the proband, fluorescent multiplex PCR linkage is done by using four previously described polymorphic dinucleotide sequences. The four (CA)[sub n] repeats are located throughout the dystrophin gene, making the analysis highly informative and accurate. The authors present the successful application of this protocol in families who proved refractory to more traditional analyses. 22 refs., 3 figs.

  16. Simultaneous Detection of Genetically Modified Organisms in a Mixture by Multiplex PCR-Chip Capillary Electrophoresis.

    PubMed

    Patwardhan, Supriya; Dasari, Srikanth; Bhagavatula, Krishna; Mueller, Steffen; Deepak, Saligrama Adavigowda; Ghosh, Sudip; Basak, Sanjay

    2015-01-01

    An efficient PCR-based method to trace genetically modified food and feed products is in demand due to regulatory requirements and contaminant issues in India. However, post-PCR detection with conventional methods has limited sensitivity in amplicon separation that is crucial in multiplexing. The study aimed to develop a sensitive post-PCR detection method by using PCR-chip capillary electrophoresis (PCR-CCE) to detect and identify specific genetically modified organisms in their genomic DNA mixture by targeting event-specific nucleotide sequences. Using the PCR-CCE approach, novel multiplex methods were developed to detect MON531 cotton, EH 92-527-1 potato, Bt176 maize, GT73 canola, or GA21 maize simultaneously when their genomic DNAs in mixtures were amplified using their primer mixture. The repeatability RSD (RSDr) of the peak migration time was 0.06 and 3.88% for the MON531 and Bt176, respectively. The RSD (RSDR) of the Cry1Ac peak ranged from 0.12 to 0.40% in multiplex methods. The method was sensitive in resolving amplicon of size difference up to 4 bp. The PCR-CCE method is suitable to detect multiple genetically modified events in a composite DNA sample by tagging their event specific sequences.

  17. A novel multiplex PCR for the simultaneous detection of Salmonella enterica and Shigella species.

    PubMed

    Radhika, M; Saugata, Majumder; Murali, H S; Batra, H V

    2014-01-01

    Salmonella enterica and Shigella species are commonly associated with food and water borne infections leading to gastrointestinal diseases. The present work was undertaken to develop a sensitive and reliable PCR based detection system for simultaneous detection of Salmonella enterica and Shigella at species level. For this the conserved regions of specific genes namely ipaH1, ipaH, wbgZ, wzy and invA were targeted for detection of Shigella genus, S. flexneri, S. sonnei, S. boydii and Salmonella enterica respectively along with an internal amplification control (IAC). The results showed that twenty Salmonella and eleven Shigella spp., were accurately identified by the assay without showing non-specificity against closely related other Enterobacteriaceae organisms and also against other pathogens. Further evaluation of multiplex PCR was undertaken on 50 natural samples of chicken, eggs and poultry litter and results compared with conventional culture isolation and identification procedure. The multiplex PCR identified the presence of Salmonella and Shigella strains with a short pre-enrichment step of 5 h in peptone water and the same samples were processed by conventional procedures for comparison. Therefore, this reported multiplex PCR can serve as an alternative to the tedious time-consuming procedure of culture and identification in food safety laboratories.

  18. High performance of a new PCR-based urine assay for HPV-DNA detection and genotyping.

    PubMed

    Tanzi, Elisabetta; Bianchi, Silvia; Fasolo, Maria Michela; Frati, Elena R; Mazza, Francesca; Martinelli, Marianna; Colzani, Daniela; Beretta, Rosangela; Zappa, Alessandra; Orlando, Giovanna

    2013-01-01

    Human papillomavirus (HPV) testing has been proposed as a means of replacing or supporting conventional cervical screening (Pap test). However, both methods require the collection of cervical samples. Urine sample is easier and more acceptable to collect and could be helpful in facilitating cervical cancer screening. The aim of this study was to evaluate the sensitivity and specificity of urine testing compared to conventional cervical smear testing using a PCR-based method with a new, designed specifically primer set. Paired cervical and first voided urine samples collected from 107 women infected with HIV were subjected to HPV-DNA detection and genotyping using a PCR-based assay and a restriction fragment length polymorphism method. Sensitivity, specificity, Positive Predictive Value (PPV), and Negative Predictive Value (NPV) were calculated using the McNemar's test for differences. Concordance between tests was assessed using the Cohen's unweighted Kappa (k). HPV DNA was detected in 64.5% (95% CI: 55.1-73.1%) of both cytobrush and urine samples. High concordance rates of HPV-DNA detection (k = 0.96; 95% CI: 0.90-1.0) and of high risk-clade and low-risk genotyping in paired samples (k = 0.80; 95% CI: 0.67-0.92 and k = 0.74; 95% CI: 0.60-0.88, respectively) were observed. HPV-DNA detection in urine versus cervix testing revealed a sensitivity of 98.6% (95% CI: 93.1-99.9%) and a specificity of 97.4% (95% CI: 87.7-99.9%), with a very high NPV (97.4%; 95% CI: 87.7-99.9%). The PCR-based assay utilized in this study proved highly sensitive and specific for HPV-DNA detection and genotyping in urine samples. These data suggest that a urine-based assay would be a suitable and effective tool for epidemiological surveillance and, most of all, screening programs.

  19. Comparison of cell-based and PCR-based assays as methods for measuring infectivity of Tulane virus.

    PubMed

    Shan, Lei; Yang, David; Wang, Dapeng; Tian, Peng

    2016-05-01

    In this study, we used Tulane virus (TV) as a surrogate for HuNoV to evaluate for correlation between two cell-based assays and three PCR-based assays. Specifically, the cell-based plaque and TCID50 assays measure for infectious virus particles, while the PCR-based RNase exposure, porcine gastric mucin in-situ-capture qRT-PCR (PGM-ISC-qRT-PCR), and antibody in-situ-capture qRT-PCR (Ab-ISC-qRT-PCR) assays measure for an amplicon within encapsidated viral genome. Ten batches of viral stocks ranging from 3.41 × 10(5) to 6.67 × 10(6) plaque forming units (PFUs) were used for side by side comparison with PFU as a reference. The results indicate that one PFU was equivalent to 6.69 ± 2.34 TCID50 units, 9.75 ± 10.87 RNase-untreated genomic copies (GCs), 2.87 ± 3.05 RNase-treated GCs, 0.07 ± 0.07 PGM-ISC-qRT-PCR GCs, and 0.52 ± 0.39 Ab-ISC-qRT-PCR GCs. We observed that while the cell-based assays were consistent with each other, the TCID50 assay was more sensitive than the plaque assay. In contrast, the PCR-based assays were not always consistent with the cell-based assays. The very high variations in GCs as measured by both ISC-RT-qPCR assays made them difficult to correlate against the relatively small variations (<20-fold) in the PFUs or TCID50 units as measured by the cell-based assays.

  20. European validation of a real-time PCR-based method for detection of Listeria monocytogenes in soft cheese.

    PubMed

    Gianfranceschi, Monica Virginia; Rodriguez-Lazaro, David; Hernandez, Marta; González-García, Patricia; Comin, Damiano; Gattuso, Antonietta; Delibato, Elisabetta; Sonnessa, Michele; Pasquali, Frederique; Prencipe, Vincenza; Sreter-Lancz, Zuzsanna; Saiz-Abajo, María-José; Pérez-De-Juan, Javier; Butrón, Javier; Kozačinski, Lidija; Tomic, Danijela Horvatek; Zdolec, Nevijo; Johannessen, Gro S; Jakočiūnė, Džiuginta; Olsen, John Elmerdahl; De Santis, Paola; Lovari, Sarah; Bertasi, Barbara; Pavoni, Enrico; Paiusco, Antonella; De Cesare, Alessandra; Manfreda, Gerardo; De Medici, Dario

    2014-08-01

    The classical microbiological method for detection of Listeria monocytogenes requires around 7 days for final confirmation, and due to perishable nature of RTE food products, there is a clear need for an alternative methodology for detection of this pathogen. This study presents an international (at European level) ISO 16140-based validation trial of a non-proprietary real-time PCR-based methodology that can generate final results in the following day of the analysis. This methodology is based on an ISO compatible enrichment coupled to a bacterial DNA extraction and a consolidated real-time PCR assay. Twelve laboratories from six European countries participated in this trial, and soft cheese was selected as food model since it can represent a difficult matrix for the bacterial DNA extraction and real-time PCR amplification. The limit of detection observed was down to 10 CFU per 25 of sample, showing excellent concordance and accordance values between samples and laboratories (>75%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (82.75%, 96.70% and 97.62%, respectively) when the results obtained for the real-time PCR-based methods were compared to those of the ISO 11290-1 standard method. An interesting observation was that the L. monocytogenes detection by the real-time PCR method was less affected in the presence of Listeria innocua in the contaminated samples, proving therefore to be more reliable than the reference method. The results of this international trial demonstrate that the evaluated real-time PCR-based method represents an excellent alterative to the ISO standard since it shows a higher performance as well as reduce the extent of the analytical process, and can be easily implemented routinely by the competent authorities and food industry laboratories. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Evaluation of a novel PCR-based diagnostic assay for detection of Mycobacterium tuberculosis in sputum samples.

    PubMed Central

    Maher, M; Glennon, M; Martinazzo, G; Turchetti, E; Marcolini, S; Smith, T; Dawson, M T

    1996-01-01

    We report on a PCR-based assay we have developed for the detection of Mycobacterium tuberculosis in sputum samples. One hundred sputum specimens, which included 34 culture-positive and 66 culture-negative specimens, were evaluated with this system. Of the 34 culture-positive specimens, 31 were PCR positive, and 60 of the culture-negative specimens were PCR negative. An internal standard has been included in the assay system to monitor PCR inhibition and to confirm the reliability of the PCR assay. PMID:8862607

  2. Long-PCR based next generation sequencing of the whole mitochondrial genome of the peacock skate Pavoraja nitida (Elasmobranchii: Arhynchobatidae).

    PubMed

    Yang, Lei; Naylor, Gavin J P

    2016-01-01

    We determined the complete mitochondrial genome sequence (16,760 bp) of the peacock skate Pavoraja nitida using a long-PCR based next generation sequencing method. It has 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 control region in the typical vertebrate arrangement. Primers, protocols, and procedures used to obtain this mitogenome are provided. We anticipate that this approach will facilitate rapid collection of mitogenome sequences for studies on phylogenetic relationships, population genetics, and conservation of cartilaginous fishes.

  3. PCR-based retrospective evaluation of diagnostic samples for emergence of porcine deltacoronavirus in US swine.

    PubMed

    Sinha, Avanti; Gauger, Phillip; Zhang, Jianqiang; Yoon, Kyoung-Jin; Harmon, Karen

    2015-09-30

    Porcine deltacoronavirus (PDCoV) was first identified in Hong Kong in a regional surveillance study for Coronaviruses in 2012 and was detected for the first time in United States (US) swine in February 2014. However, it remains unknown if PDCoV had been introduced into the US prior to that time period. In the present study, 1734 clinical samples (903 cases) submitted to the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) for enteric disease diagnosis between October 2012 and December 2013 were tested retrospectively for PDCoV using a virus-specific real-time reverse transcription (RT) PCR targeting conserved region of the membrane gene. PDCoV genome was first detected in a fecal sample collected on August 19th 2013 from Minnesota. Subsequently, PDCoV was observed in samples collected on August 20th and August 27th from Iowa and on August 29th from Illinois. Therefore, with available samples submitted to the ISU VDL, it can be inferred that PDCoV has been present in US swine at least since August 2013.

  4. RT real-time PCR-based quantification of Uromyces fabae in planta.

    PubMed

    Voegele, Ralf T; Schmid, Annette

    2011-09-01

    Quantification of obligate biotrophic parasites has been a long-standing problem in plant pathology. Many attempts have been made to determine how much of a pathogen is present in infected plant tissue. Methods of quantification included scoring disease symptoms, microscopic evaluation, determination of specific compounds like Ergosterol, and lately nucleic acid-based technologies. All of these methods have their drawbacks, and even real-time PCR may not be quantitative if for example the organism of interest has specific and differing numbers of nuclei in different infection structures. We applied reverse transcription (RT) real-time PCR to quantify Uromyces fabae within its host plant Vicia faba. We used three different genes, which have been shown to be constitutively expressed. Our analyses show an exponential increase of fungal material between 4 and 9 days post inoculation and thereafter reaching a steady state of around 45% of total RNA. We also used haustorium-specific genes to determine the amount of haustoria present at each time point. These analyses parallel the development of the whole fungus with the exception of the steady-state level, which is only around 5% of the total RNA. This indicates that RT real-time PCR is a suitable method for quantification of obligate biotrophic parasites, and also for the differentiation of developmental stages. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  5. Survey and RT-PCR Based Detection of Cardamom mosaic virus Affecting Small Cardamom in India.

    PubMed

    Biju, C N; Siljo, A; Bhat, A I

    2010-10-01

    Mosaic or marble or katte disease caused by Cardamom mosaic virus (CdMV) is an important production constraint in all cardamom growing regions of the world. In the present study, 84 cardamom plantations in 44 locations of Karnataka and Kerala were surveyed. The incidence of the disease ranged from 0 to 85%. The incidence was highest in Madikeri (Karnataka) while no incidence was recorded in Peermade (Kerala). In general, incidence and severity of the disease was higher in cardamom plantations of Karnataka. A procedure for total RNA isolation from cardamom and detection of CdMV through reverse transcription-polymerase chain reaction (RT-PCR) using primers targeting the conserved region of coat protein was standardized and subsequently validated by testing more than 50 field cardamom samples originating from Karnataka and Kerala states. The method can be used for indexing the planting material and identifying resistant lines/cultivars before either they are further multiplied in large scale or incorporated in breeding.

  6. Novel extraction strategy of ribosomal RNA and genomic DNA from cheese for PCR-based investigations.

    PubMed

    Bonaïti, Catherine; Parayre, Sandrine; Irlinger, Françoise

    2006-03-15

    Cheese microorganisms, such as bacteria and fungi, constitute a complex ecosystem that plays a central role in cheeses ripening. The molecular study of cheese microbial diversity and activity is essential but the extraction of high quality nucleic acid may be problematic: the cheese samples are characterised by a strong buffering capacity which negatively influenced the yield of the extracted rRNA. The objective of this study is to develop an effective method for the direct and simultaneous isolation of yeast and bacterial ribosomal RNA and genomic DNA from the same cheese samples. DNA isolation was based on a protocol used for nucleic acids isolation from anaerobic digestor, without preliminary washing step with the combined use of the action of chaotropic agent (acid guanidinium thiocyanate), detergents (SDS, N-lauroylsarcosine), chelating agent (EDTA) and a mechanical method (bead beating system). The DNA purification was carried out by two washing steps of phenol-chloroform. RNA was isolated successfully after the second acid extraction step by recovering it from the phenolic phase of the first acid extraction. The novel method yielded pure preparation of undegraded RNA accessible for reverse transcription-PCR. The extraction protocol of genomic DNA and rRNA was applicable to complex ecosystem of different cheese matrices.

  7. Analysis of spatial domain multiplexing/space division multiplexing (SDM) based hybrid architectures operating in tandem with wavelength division multiplexing

    NASA Astrophysics Data System (ADS)

    Murshid, Syed; Lovell, Greg; Chowdhury, Bilas; Hridoy, Arnob; Parhar, Gurinder; Chakravarty, Abhijit; Alanzi, Saud

    2014-09-01

    Spatial domain multiplexing (SDM) also known as space division multiplexing adds a new degree of photon freedom to existing optical fiber multiplexing techniques by allocating separate radial locations to different MIMO channels as a function of the input launch angle. These independent MIMO channels remain confined to the designated location while traversing the length of the carrier fiber, due to helical propagation of light inside the fiber core. The SDM technique can be used in tandem with other multiplexing techniques, such as time division multiplexing (TDM), and wavelength division multiplexing in hybrid optical communication schemes, to achieve higher optical fiber bandwidth by increasing the photon efficiency due to added degrees of photon freedom. This paper presents the feasibility of a novel hybrid optical fiber communications architecture and shows that SDM channels of different operating wavelengths continue to follow the input launch angle based radial distribution pattern.

  8. Parallel multiplex laser feedback interferometry

    SciTech Connect

    Zhang, Song; Tan, Yidong; Zhang, Shulian

    2013-12-15

    We present a parallel multiplex laser feedback interferometer based on spatial multiplexing which avoids the signal crosstalk in the former feedback interferometer. The interferometer outputs two close parallel laser beams, whose frequencies are shifted by two acousto-optic modulators by 2Ω simultaneously. A static reference mirror is inserted into one of the optical paths as the reference optical path. The other beam impinges on the target as the measurement optical path. Phase variations of the two feedback laser beams are simultaneously measured through heterodyne demodulation with two different detectors. Their subtraction accurately reflects the target displacement. Under typical room conditions, experimental results show a resolution of 1.6 nm and accuracy of 7.8 nm within the range of 100 μm.

  9. Multiplexed DNA-modified electrodes.

    PubMed

    Slinker, Jason D; Muren, Natalie B; Gorodetsky, Alon A; Barton, Jacqueline K

    2010-03-03

    We report the use of silicon chips with 16 DNA-modified electrodes (DME chips) utilizing DNA-mediated charge transport for multiplexed detection of DNA and DNA-binding protein targets. Four DNA sequences were simultaneously distinguished on a single DME chip with 4-fold redundancy, including one incorporating a single base mismatch. These chips also enabled investigation of the sequence-specific activity of the restriction enzyme Alu1. DME chips supported dense DNA monolayer formation with high reproducibility, as confirmed by statistical comparison to commercially available rod electrodes. The working electrode areas on the chips were reduced to 10 microm in diameter, revealing microelectrode behavior that is beneficial for high sensitivity and rapid kinetic analysis. These results illustrate how DME chips facilitate sensitive and selective detection of DNA and DNA-binding protein targets in a robust and internally standardized multiplexed format.

  10. Multiplexed DNA-Modified Electrodes

    PubMed Central

    Slinker, Jason D.; Muren, Natalie B.; Gorodetsky, Alon A.; Barton, Jacqueline K.

    2011-01-01

    We report the use of silicon chips with 16 DNA-modified electrodes (DME chips) utilizing DNA-mediated charge transport for multiplexed detection of DNA and DNA-binding protein targets. Four DNA sequences were simultaneously distinguished on a single DME chip with fourfold redundancy, including one incorporating a single base mismatch. These chips also enabled investigation of the sequence-specific activity of the restriction enzyme Alu1. DME chips supported dense DNA monolayer formation with high reproducibility, as confirmed by statistical comparison to commercially available rod electrodes. The working electrode areas on the chips were reduced to 10 µm in diameter, revealing microelectrode behavior that is beneficial for high sensitivity and rapid kinetic analysis. These results illustrate how DME chips facilitate sensitive and selective detection of DNA and DNA-binding protein targets in a robust and internally standardized multiplexed format. PMID:20131780

  11. Comparison of Two PCR-Based Human Papillomavirus Genotyping Methods▿ †

    PubMed Central

    Castle, Philip E.; Porras, Carolina; Quint, Wim G.; Rodriguez, Ana Cecilia; Schiffman, Mark; Gravitt, Patti E.; González, Paula; Katki, Hormuzd A.; Silva, Sandra; Freer, Enrique; Van Doorn, Leen-Jan; Jiménez, Silvia; Herrero, Rolando; Hildesheim, Allan

    2008-01-01

    We compared two consensus primer PCR human papillomavirus (HPV) genotyping methods for the detection of individual HPV genotypes and carcinogenic HPV genotypes as a group, using a stratified sample of enrollment cervical specimens from sexually active women participating in the NCI/Costa Rica HPV16/18 Vaccine Efficacy Trial. For the SPF10 method, DNA was extracted from 0.1% of the cervical specimen by using a MagNA Pure LC instrument, a 65-bp region of the HPV L1 gene was targeted for PCR amplification by using SPF10 primers, and 25 genotypes were detected by reverse-line blot hybridization of the amplicons. For the Linear Array (LA) method, DNA was extracted from 0.5% of the cervical specimen by using an MDx robot, a 450-bp region of the HPV L1 gene was targeted for PCR amplification by using PGMY09/11 L1 primers, and 37 genotypes were detected by reverse-line blot hybridization of the amplicons. Specimens (n = 1,427) for testing by the LA method were randomly selected from strata defined on the basis of enrollment test results from the SPF10 method, cytology, and Hybrid Capture 2. LA results were extrapolated to the trial cohort (n = 5,659). The LA and SPF10 methods detected 21 genotypes in common; HPV16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, -68, and -73 were considered the carcinogenic HPV genotypes. There was no difference in the overall results for grouped detection of carcinogenic HPV by the SPF10 and LA methods (35.3% versus 35.9%, respectively; P = 0.5), with a 91.8% overall agreement and a kappa value of 0.82. In comparisons of individual HPV genotypes, the LA method detected significantly more HPV16, HPV18, HPV39, HPV58, HPV59, HPV66, and HPV68/73 and less HPV31 and HPV52 than the SPF10 method; inclusion of genotype-specific testing for HPV16 and HPV18 for those specimens testing positive for HPV by the SPF10 method but for which no individual HPV genotype was detected abrogated any differences between the LA and SPF10 methods. The

  12. Atypical Steatocystoma Multiplex with Calcification

    PubMed Central

    Rahman, Muhammad Hasibur; Islam, Muhammad Saiful; Ansari, Nazma Parvin

    2011-01-01

    A 60-year-old male reported to us with an atypical case of giant steatocystoma multiplex in the scrotum with calcification. There was no family history of similar lesions. Yellowish, creamy material was expressed from a nodule during punch biopsy. The diagnosis was based on clinical as well as histological findings. Successful surgical excision was done to cure the case without any complications. PMID:22363850

  13. Catch and release: integrated system for multiplexed detection of bacteria.

    PubMed

    Verbarg, Jasenka; Plath, William D; Shriver-Lake, Lisa C; Howell, Peter B; Erickson, Jeffrey S; Golden, Joel P; Ligler, Frances S

    2013-05-21

    An integrated system with automated immunomagnetic separation and processing of fluidic samples was demonstrated for multiplexed optical detection of bacterial targets. Mixtures of target-specific magnetic bead sets were processed in the NRL MagTrap with the aid of rotating magnet arrays that entrapped and moved the beads within the channel during reagent processing. Processing was performed in buffer and human serum matrixes with 10-fold dilutions in the range of 10(2)-10(6) cells/mL of target bacteria. Reversal of magnets' rotation post-processing released the beads back into the flow and moved them into the microflow cytometer for optical interrogation. Identification of the beads and the detection of PE fluorescence were performed simultaneously for multiplexed detection. Multiplexing was performed with specifically targeted bead sets to detect E. coli 0157.H7, Salmonella Common Structural Antigen, Listeria sp., and Shigella sp., dose-response curves were obtained, and limits of detection were calculated for each target in the buffer and clinical matrix. Additional tests demonstrated the potential for using the MagTrap to concentrate target from larger volumes of sample prior to the addition of assay reagents.

  14. Development of a multiplex real-time PCR assay for the rapid diagnosis of neonatal late onset sepsis.

    PubMed

    van den Brand, Marre; Peters, Remco P H; Catsburg, Arnold; Rubenjan, Anna; Broeke, Ferdi J; van den Dungen, Frank A M; van Weissenbruch, Mirjam M; van Furth, A Marceline; Kõressaar, Triinu; Remm, Maido; Savelkoul, Paul H M; Bos, Martine P

    2014-11-01

    The diagnosis of late onset sepsis (LOS), a severe condition with high prevalence in preterm infants, is hampered by the suboptimal sensitivity and long turnaround time of blood culture. Detection of the infecting pathogen directly in blood by PCR would provide a much more timely result. Unfortunately, PCR-based assays reported so far are labor intensive and often lack direct species identification. Therefore we developed a real-time multiplex PCR assay tailored to LOS diagnosis which is easy-to-use, is applicable on small blood volumes and provides species-specific results within 4h. Species-specific PCR assays were selected from literature or developed using bioinformatic tools for the detection of the most prevalent etiologic pathogens: Enterococcus faecalis, Staphylococcus aureus, Staphylococcus spp., Streptococcus agalactiae, Escherichia coli, Pseudomonas aeruginosa, Klebsiella spp. and Serratia marcescens. The PCR assays showed 100% specificity, full coverage of the target pathogens and a limit of detection (LOD) of ≤10CFUeq./reaction. These LOD values were maintained in the multiplex format or when bacterial DNA was isolated from blood. Clinical evaluation showed high concordance between the multiplex PCR and blood culture. In conclusion, we developed a multiplex PCR that allows the direct detection of the most important bacterial pathogens causing LOS in preterm infants.

  15. Rapid sample preparation method for PCR-based detection of Escherichia coli O157:H7 in ground beef.

    PubMed

    Cui, S; Schroeder, C M; Zhang, D Y; Meng, J

    2003-01-01

    To develop an improved, rapid and sensitive sample preparation method for PCR-based detection of Escherichia coli O157:H7 in ground beef. Fresh ground beef samples were experimentally inoculated with varying concentrations of E. coli O157:H7. PCR inhibitors were removed and bacterial cells were concentrated by filtration and centrifugation, and lysed using enzymatic digestion and successive freeze/thaw cycles. DNA was purified and concentrated via phenol/chloroform extraction and the Shiga toxin 1 gene (stx1) was amplified using PCR to evaluate the sample preparation method. Without prior enrichment of cells in broth media, the detection limit was 103 CFU g-1 beef. When a 6 h enrichment step was incorporated, the detection limit was 1 CFU g-1 beef. The total time required from beginning to end of the procedure was 12 h. The sample preparation method developed here enabled substantially improved sensitivity in the PCR-based detection of E. coli O157:H7 in ground beef, as compared to previous reports. Superb sensitivity, coupled with quick turn-around time, relative ease of use and cost-effectiveness, makes this a useful method for detecting E. coli O157:H7 in ground beef.

  16. Real-time PCR based analysis of metal resistance genes in metal resistant Pseudomonas aeruginosa strain J007.

    PubMed

    Choudhary, Sangeeta; Sar, Pinaki

    2016-07-01

    A uranium (U)-resistant and -accumulating Pseudomonas aeruginosa strain was characterized to assess the response of toxic metals toward its growth and expression of metal resistance determinants. The bacterium showed MIC (minimum inhibitory concentration) values of 6, 3, and 2 mM for Zn, Cu, and Cd, respectively; with resistance phenotype conferred by periplasmic Cu sequestering copA and RND type heavy metal efflux czcA genes. Real-time PCR-based expression analysis revealed significant upregulation of both these genes upon exposure to low concentrations of metals for short duration, whereas the global stress response gene sodA encoding superoxide dismutase enzyme was upregulated only at higher metal concentrations or longer exposure time. It could also be inferred that copA and czcA are involved in providing resistance only at low metal concentrations, whereas involvement of "global stress response" phenomenon (expression of sodA) at higher metal concentration or increased exposure was evident. This study provides significant understanding of the adaptive response of bacteria surviving in metal and radionuclide contaminated environments along with the development of real-time PCR-based quantification method of using metal resistance genes as biomarker for monitoring relevant bacteria in such habitats. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Mitochondrial DNA diversity and PCR-based sex determination of Irrawaddy dolphin (Orcaella brevirostris) from Chilika Lagoon, India.

    PubMed

    Jayasankar, P; Patel, A; Khan, M; Das, P; Panda, S

    2011-03-01

    Of the only known two Lagoon populations of Irrawaddy dolphins (Orcaella) in the world, one is residing in the Chilika Lagoon in Orissa state, India. In addition to accidental deaths in gill net fishery and mechanized boat operations, there has been exploitation of the species for their oil. Extreme patchy distribution and vulnerability to becoming entangled in fishing gear has made it a focus of conservation concern. Information on genetic diversity of populations has considerable potential for informing conservation plans. The present paper reports the first genetic study of O. brevirostris from Chilika Lagoon based on mtDNA sequencing and PCR-based sex identification from 11 individuals. Control region sequence comparison showed two haplotypes and cytochrome b a single haplotype in the Chilika population of the species. Phylogenetic analysis indicated distinct clades within the Asian samples, with the Indian population showing closest genetic proximity to the haplotypes from Thailand. Sex of the animal was determined by PCR-based method. It is important to continue to examine the population discreteness and genetic variation of Irrawaddy dolphin in Chilika Lagoon vis-à-vis its global geographic distribution for formulating the conservation plans of the species.

  18. A reliable and inexpensive method of nucleic acid extraction for the PCR-based detection of diverse plant pathogens.

    PubMed

    Li, R; Mock, R; Huang, Q; Abad, J; Hartung, J; Kinard, G

    2008-12-01

    A reliable extraction method is described for the preparation of total nucleic acids from at least ten plant genera for subsequent detection of plant pathogens by PCR-based techniques. The method combined a modified CTAB (cetyltrimethylammonium bromide) extraction protocol with a semi-automatic homogenizer (FastPrep) instrument) for rapid sample processing and low potential of cross contamination. The method was applied to sample preparation for PCR-based detection of 28 different RNA and DNA viruses, six viroids, two phytoplasmas and two bacterial pathogens from a range of infected host plants including sweet potato, small fruits and fruit trees. The procedure is cost-effective and the qualities of the nucleic acid preparations are comparable to those prepared by commonly used commercial kits. The efficiency of the procedure permits processing of numerous samples and the use of a single nucleic acid preparation for testing both RNA and DNA genomes by PCR, making this an appealing method for testing multiple pathogens in certification and quarantine programs.

  19. PCR-based method for sex identification of Eastern sarus crane (Grus antigone sharpii): implications for reintroduction programs in Thailand.

    PubMed

    Insee, Jiranan; Kamolnorranath, Sumate; Baicharoen, Sudarat; Chumpadang, Sriphapai; Sawasu, Wanchai; Wajjwalku, Worawidh

    2014-02-01

    Due to human activity and a reduction in the size and quality of wetland habitats, populations of the Eastern sarus crane (Grus antigone sharpii) have declined dramatically across their range in Southeast Asia. Conservation efforts in Thailand have focused on reintroduction of the founders harboring the highest genetic diversity. One of the most important requirements to ensure the persistence of the reintroduced populations is a balanced sex ratio. In this study we tested three simple PCR-based methods which may be used for reliable sex identification in G. a. sharpii. The first method employs two combined primer sets based on a 0.6 kb EcoRI fragment (EE0.6). The second method is based on the intronic length polymorphism of the chromo-helicase DNA binding protein (CHD). The last technique relies on PCR-RFLP technique. The sex of six known and 24 unknown cranes were successfully identified by all three methods. These PCR-based sex identification methods are also useful for captive breeding management of G. a. sharpii.

  20. [Laboratory diagnosis of Treponema pallidum infection in patients with early syphilis and neurosyphilis through a PCR-based test].

    PubMed

    García, Patricia; Grassi, Bruno; Fich, Félix; Salvo, Aurelio; Araya, Luis; Abarzúa, Fernando; Soto, Julia; Poggi, Helena; Lagos, Marcela; Vásquez, Patricia; León, Eugenia P; Pérez, Carlos; Wozniak, Aniela

    2011-08-01

    Syphilis is a sexually transmitted disease caused by Treponema pallidum. The diagnosis is based mainly in clinical presentation and non-specific assays. PCR-based diagnosis has been suggested as an attractive alternative method. The aim of this study was the validation of a PCR-based test for the diagnosis of early syphilis (ES) and neurosyphilis (NS). Clinical samples of mucocutaneous lesions and cerebrospinal fluid (CSF) specimens from patients previously diagnosed for ES and NS respectively using an enlarged gold standard, were tested by PCR. The reaction was done using primers targeting the tpN47 gene. Twenty out of 21 mucocutaneous samples from patients diagnosed with ES were positive by PCR, with a clinical sensitivity of 95%. Four out of 8 CSF samples from patients previously diagnosed with NS were positive by PCR, with a clinical sensitivity of 50%. The clinical specificity for both ES and NS was 100%. The PCR sensitivity and specificity for mucocutaneous samples allowed us to implement this assay in our laboratory for routine diagnosis. Although the sensitivity of the PCR in CSF was low, it may be useful to support clinical diagnosis.

  1. Detection and characterization of recombinant DNA expressing vip3A-type insecticidal gene in GMOs--standard single, multiplex and construct-specific PCR assays.

    PubMed

    Singh, Chandra K; Ojha, Abhishek; Bhatanagar, Raj K; Kachru, Devendra N

    2008-01-01

    Vegetative insecticidal protein (Vip), a unique class of insecticidal protein, is now part of transgenic plants for conferring resistance against lepidopteron pests. In order to address the imminent regulatory need for detection and labeling of vip3A carrying genetically modified (GM) products, we have developed a standard single PCR and a multiplex PCR assay. As far as we are aware, this is the first report on PCR-based detection of a vip3A-type gene (vip-s) in transgenic cotton and tobacco. Our assay involves amplification of a 284-bp region of the vip-s gene. This assay can possibly detect as many as 20 natural wild-type isolates bearing a vip3A-like gene and two synthetic genes of vip3A in transgenic plants. The limit of detection as established by our assay for GM trait (vip-s) is 0.1%. Spiking with nontarget DNA originating from diverse plant sources had no inhibitory effect on vip-s detection. Since autoclaving of vip-s bearing GM leaf samples showed no deterioration/interference in detection efficacy, the assay seems to be suitable for processed food products as well. The vip-s amplicon identity was reconfirmed by restriction endonuclease assay. The primer set for vip-s was equally effective in a multiplex PCR assay format (duplex, triplex and quadruplex), used in conjunction with the primer sets for the npt-II selectable marker gene, Cauliflower mosaic virus 35S promoter and nopaline synthetase terminator, enabling concurrent detection of the transgene, regulatory sequences and marker gene. Further, the entire transgene construct was amplified using the forward primer of the promoter and the reverse primer of the terminator. The resultant amplicon served as a template for nested PCR to confirm the construct integrity. The method is suitable for screening any vip3A-carrying GM plant and food. The availability of a reliable PCR assay method prior to commercial release of vip3A-based transgenic crops and food would facilitate rapid and efficient regulatory

  2. Large-scale fibre-array multiplexing

    SciTech Connect

    Cheremiskin, I V; Chekhlova, T K

    2001-05-31

    The possibility of creating a fibre multiplexer/demultiplexer with large-scale multiplexing without any basic restrictions on the number of channels and the spectral spacing between them is shown. The operating capacity of a fibre multiplexer based on a four-fibre array ensuring a spectral spacing of 0.7 pm ({approx} 10 GHz) between channels is demonstrated. (laser applications and other topics in quantum electronics)

  3. Multiplexing of encrypted data using fractal masks.

    PubMed

    Barrera, John F; Tebaldi, Myrian; Amaya, Dafne; Furlan, Walter D; Monsoriu, Juan A; Bolognini, Néstor; Torroba, Roberto

    2012-07-15

    In this Letter, we present to the best of our knowledge a new all-optical technique for multiple-image encryption and multiplexing, based on fractal encrypting masks. The optical architecture is a joint transform correlator. The multiplexed encrypted data are stored in a photorefractive crystal. The fractal parameters of the key can be easily tuned to lead to a multiplexing operation without cross talk effects. Experimental results that support the potential of the method are presented.

  4. Measuring and modeling correlations in multiplex networks

    NASA Astrophysics Data System (ADS)

    Nicosia, Vincenzo; Latora, Vito

    2015-09-01

    The interactions among the elementary components of many complex systems can be qualitatively different. Such systems are therefore naturally described in terms of multiplex or multilayer networks, i.e., networks where each layer stands for a different type of interaction between the same set of nodes. There is today a growing interest in understanding when and why a description in terms of a multiplex network is necessary and more informative than a single-layer projection. Here we contribute to this debate by presenting a comprehensive study of correlations in multiplex networks. Correlations in node properties, especially degree-degree correlations, have been thoroughly studied in single-layer networks. Here we extend this idea to investigate and characterize correlations between the different layers of a multiplex network. Such correlations are intrinsically multiplex, and we first study them empirically by constructing and analyzing several multiplex networks from the real world. In particular, we introduce various measures to characterize correlations in the activity of the nodes and in their degree at the different layers and between activities and degrees. We show that real-world networks exhibit indeed nontrivial multiplex correlations. For instance, we find cases where two layers of the same multiplex network are positively correlated in terms of node degrees, while other two layers are negatively correlated. We then focus on constructing synthetic multiplex networks, proposing a series of models to reproduce the correlations observed empirically and/or to assess their relevance.

  5. Measuring and modeling correlations in multiplex networks.

    PubMed

    Nicosia, Vincenzo; Latora, Vito

    2015-09-01

    The interactions among the elementary components of many complex systems can be qualitatively different. Such systems are therefore naturally described in terms of multiplex or multilayer networks, i.e., networks where each layer stands for a different type of interaction between the same set of nodes. There is today a growing interest in understanding when and why a description in terms of a multiplex network is necessary and more informative than a single-layer projection. Here we contribute to this debate by presenting a comprehensive study of correlations in multiplex networks. Correlations in node properties, especially degree-degree correlations, have been thoroughly studied in single-layer networks. Here we extend this idea to investigate and characterize correlations between the different layers of a multiplex network. Such correlations are intrinsically multiplex, and we first study them empirically by constructing and analyzing several multiplex networks from the real world. In particular, we introduce various measures to characterize correlations in the activity of the nodes and in their degree at the different layers and between activities and degrees. We show that real-world networks exhibit indeed nontrivial multiplex correlations. For instance, we find cases where two layers of the same multiplex network are positively correlated in terms of node degrees, while other two layers are negatively correlated. We then focus on constructing synthetic multiplex networks, proposing a series of models to reproduce the correlations observed empirically and/or to assess their relevance.

  6. Reversible dementias

    PubMed Central

    Tripathi, Manjari; Vibha, Deepti

    2009-01-01

    In recent years, more attention has been given to the early diagnostic evaluation of patients with dementia which is essential to identify patients with cognitive symptoms who may have treatable conditions. Guidelines suggest that all patients presenting with dementia or cognitive symptoms should be evaluated with a range of laboratory tests, and with structural brain imaging with computed tomography (CT) or magnetic resonance imaging (MRI). While many of the disorders reported as ‘reversible dementias’ are conditions that may well be associated with cognitive or behavioral symptoms, these symptoms are not always sufficiently severe to fulfill the clinical criteria for dementia. Thus, while the etiology of a condition may be treatable it should not be assumed that the associated dementia is fully reversible. Potentially reversible dementias should be identified and treatment considered, even if the symptoms are not sufficiently severe to meet the clinical criteria for dementia, and even if partial or full reversal of the cognitive symptoms cannot be guaranteed. In the literature, the most frequently observed potentially reversible conditions identified in patients with cognitive impairment or dementia are depression, adverse effects of drugs, drug or alcohol abuse, space-occupying lesions, normal pressure hydrocephalus, and metabolic conditions land endocrinal conditions like hypothyroidism and nutritional conditions like vitamin B-12 deficiency. Depression is by far the most common of the potentially reversible conditions. The review, hence addresses the common causes of reversible dementia and the studies published so far. PMID:21416018

  7. Multiplex Nested PCR for Detection of Xanthomonas axonopodis pv. allii from Onion Seeds▿ †

    PubMed Central

    Robène-Soustrade, Isabelle; Legrand, Delphine; Gagnevin, Lionel; Chiroleu, Frédéric; Laurent, Annie; Pruvost, Olivier

    2010-01-01

    Bacterial blight of onion (BBO) is an emerging disease that is present in many onion-producing areas. The causal agent, Xanthomonas axonopodis pv. allii, is seed transmitted. A reliable and sensitive diagnostic tool for testing seed health is needed. Detection of X. axonopodis pv. allii was achieved using a multiplex nested PCR assay developed using two randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) sequences corresponding to pilus assembly genes (pilW and pilX) and the avrRxv gene, respectively. The multiplex nested PCR was used with a large collection of X. axonopodis pv. allii strains pathogenic to onion and/or other Allium species isolated in different regions of the world. The internal primers used in the multiplex PCR assay directed amplification for all 86 X. axonopodis pv. allii strains tested, resulting in a 401-bp amplicon, a 444- to 447-bp amplicon, or both amplicons, depending on the strain. No amplification was obtained for 41 unrelated phytopathogenic bacteria and for 14 saprophytic bacteria commonly isolated from onion leaves and seeds. Most Xanthomonas strains also did not produce amplicons, except for nine strains classified in X. axonopodis genetic subgroup 9.1 or 9.2 and not pathogenic to onion. Nevertheless, sequence signatures distinguished most of these strains from X. axonopodis pv. allii. The assay detected X. axonopodis pv. allii in seed lots with contamination levels of 5 × 102 CFU g−1 or higher. The sensitivity threshold of the multiplex nested PCR assay was found to be 1 infected seed in 27,340 seeds. This PCR-based assay should be useful for certifying that commercial seed lots are free of this important seed-borne pathogen. PMID:20208024

  8. Reversible Sterilization

    ERIC Educational Resources Information Center

    Largey, Gale

    1977-01-01

    Notes that difficult questions arise concerning the use of sterilization for alleged eugenic and euthenic purposes. Thus, how reversible sterilization will be used with relation to the poor, mentally ill, mentally retarded, criminals, and minors, is questioned. (Author/AM)

  9. Reversible Sterilization

    ERIC Educational Resources Information Center

    Largey, Gale

    1977-01-01

    Notes that difficult questions arise concerning the use of sterilization for alleged eugenic and euthenic purposes. Thus, how reversible sterilization will be used with relation to the poor, mentally ill, mentally retarded, criminals, and minors, is questioned. (Author/AM)

  10. Selection and evaluation of novel reference genes for quantitative reverse transcription PCR (qRT-PCR) based on genome and transcriptome data in Brassica napus L.

    PubMed

    Yang, Hongli; Liu, Jing; Huang, Shunmou; Guo, Tingting; Deng, Linbin; Hua, Wei

    2014-03-15

    Selection of reference genes in Brassica napus, a tetraploid (4×) species, is a very difficult task without information on genome and transcriptome. By now, only several traditional reference genes which show significant expression differentiation under different conditions are used in B. napus. In the present study, based on genome and transcriptome data of the rapeseed Zhongshuang-11 cultivar, 14 candidate reference genes were screened for investigation in different tissues, cultivars, and treated conditions of B. napus. These genes were as follows: ELF5, ENTH, F-BOX7, F-BOX2, FYPP1, GDI1, GYF, MCP2d, OTP80, PPR, SPOC, Unknown1, Unknown2 and UBA. Among them, excluding GYF and FYPP1, another 12 genes, were identified to perform better than traditional reference genes ACTIN7 and GAPDH. To further validate the accuracy of the newly developed reference genes in normalization, expression levels of BnCAT1 (B. napus catalase 1) in different rapeseed tissues and seedlings under stress conditions were normalized by the three most stable reference genes PPR, GDI1, and ENTH and little difference existed in normalization results. To the best of our knowledge, this is the first time B. napus reference genes have been provided with the help of complete genome and transcriptome information. The new reference genes provided in this study are more accurate than previously reported reference genes in quantifying expression levels of B. napus genes.

  11. In-vitro Cell Culture and Real-time Reverse Transcriptase PCR-based Assays to Detect Infective Toxoplas gondii Oocysts

    EPA Science Inventory

    Toxoplasma gondii is an obligate intracellular, apicomplexan parasite that infects humans. It is ubiquitous in nature and seroprevalence in the United States and in Europe ranges from 25->70%. Although typically associated with causing foodborne outbreaks, recent studies in Canad...

  12. In-vitro Cell Culture and Real-time Reverse Transcriptase PCR-based Assays to Detect Infective Toxoplas gondii Oocysts

    EPA Science Inventory

    Toxoplasma gondii is an obligate intracellular, apicomplexan parasite that infects humans. It is ubiquitous in nature and seroprevalence in the United States and in Europe ranges from 25->70%. Although typically associated with causing foodborne outbreaks, recent studies in Canad...

  13. Reversible Cardiomyopathies

    PubMed Central

    Patel, Harsh; Madanieh, Raef; Kosmas, Constantine E; Vatti, Satya K; Vittorio, Timothy J

    2015-01-01

    Cardiomyopathies (CMs) have many etiological factors that can result in severe structural and functional dysregulation. Fortunately, there are several potentially reversible CMs that are known to improve when the root etiological factor is addressed. In this article, we discuss several of these reversible CMs, including tachycardia-induced, peripartum, inflammatory, hyperthyroidism, Takotsubo, and chronic illness–induced CMs. Our discussion also includes a review on their respective pathophysiology, as well as possible management solutions. PMID:26052233

  14. Reusable holographic velocimetry system based on polarization multiplexing in bacteriorhodopsin

    NASA Astrophysics Data System (ADS)

    Koek, W. D.; Chan, V. S. S.; Ooms, T. A.; Bhattacharya, N.; Westerweel, J.; Braat, J. J. M.

    2005-04-01

    We present a novel holographic particle image velocimetry (HPIV) system using a reversible holographic material as the recording medium. In HPIV the three-dimensional flow field throughout a volume is detected by adding small tracer particles to a normally transparent medium. By recording the particle distribution twice with a known time shift the displacement and the velocity of the tracer particles can be retrieved. From this information the instantaneous three-dimensional flow field can be found. Our measurement system records double exposure particle holograms in a film based on the photo-chromic protein bacteriorhodopsin (BR). Polarization multiplexing is used to separate the two constituent holograms. We believe it is the first time that this type of multiplexing is used in (particle) velocimetry measurements. By using a polarization sensitive material we are able to simplify our setup and increase the storage capacity of our holographic medium. BR is a fully reversible recording material that does not require any chemical processing. This allows for fast experiments that require minimal operator involvement. A full measurement cycle can typically be completed within several minutes. We present our experimental system in detail and we will discuss how the material and optical properties of BR affect the holographic recording system. We will point out the advantages, disadvantages, and practical issues involved when working with BR.

  15. Quantitative and qualitative validations of a sonication-based DNA extraction approach for PCR-based molecular biological analyses.

    PubMed

    Dai, Xiaohu; Chen, Sisi; Li, Ning; Yan, Han

    2016-05-15

    The aim of this study was to comprehensively validate the sonication-based DNA extraction method, in hope of the replacement of the so-called 'standard DNA extraction method' - the commercial kit method. Microbial cells in the digested sludge sample, containing relatively high amount of PCR-inhibitory substances, such as humic acid and protein, were applied as the experimental alternatives. The procedure involving solid/liquid separation of sludge sample and dilution of both DNA templates and inhibitors, the minimum templates for PCR-based analyses, and the in-depth understanding from the bias analysis by pyrosequencing technology were obtained and confirmed the availability of the sonication-based DNA extraction method. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Rapid PCR-based method for detection and differentiation of Didymiaceae and Physaraceae (myxomycetes) in environmental samples.

    PubMed

    Kamono, Akiko; Fukui, Manabu

    2006-12-01

    Ecological studies of myxomycetes have been limited by the absence of universal cultivation techniques and the lack of life stage independent identification methods. We designed a novel PCR primer pair for the specific amplification of small subunit ribosomal RNA gene of Didymiaceae and Physaraceae. The primers produced amplicons from 192 fruiting body samples belonging to 10 genera. Twenty-four samples yielded longer fragments and sequence analysis revealed the presence of intron(s). As for the exonic regions, while sequence heterogeneities within a single species/varietas/forma were frequently observed, identical sequences were obtained only from identical species/varietas. The effectiveness of this primer pair in the analysis of morphologically unidentifiable samples was confirmed with the applications to samples of environmental plasmodium/sclerotium and soil. Denaturing gradient gel electrophoresis analysis was also tested with the soil samples. The results presented here demonstrate this PCR-based method can facilitate further ecological studies of Physaraceae and Didymiaceae in the environment.

  17. Survey for protozoan parasites in Eastern oysters (Crassostrea virginica) from the Gulf of Maine using PCR-based assays.

    PubMed

    Marquis, Nicholas D; Record, Nicholas R; Robledo, José A Fernández

    2015-10-01

    Protozoan pathogens represent a serious threat to oyster aquaculture, since they can lead to significant production loses. Moreover, oysters can concentrate human pathogens through filter feeding, thus putting at risk raw oyster consumers' health. Using PCR-based assays in oysters (Crassostrea virginica) from Maine, we expand the Northeast range in the USA for the protozoans Perkinsus marinus, Perkinsus chesapeaki, and Haplosporidium nelsoni, and report for the first time the detection of the human pathogens Toxoplasma gondii and Cryptosporidium parvum. Oysters hosting both P. marinus and P. chesapeaki were more than three times as likely to be infected by a non-Perkinsus than those free of Perkinsus infections. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Usefulness of PCR-based assays to assess drug efficacy in Chagas disease chemotherapy: value and limitations.

    PubMed

    Britto, Constança Carvalho

    2009-07-01

    One major goal of research on Chagas disease is the development of effective chemotherapy to eliminate the infection from individuals who have not yet developed cardiac and/or digestive disease manifestations. Cure evaluation is the more complex aspect of its treatment, often leading to diverse and controversial results. The absence of reliable methods or a diagnostic gold standard to assess etiologic treatment efficacy still constitutes a major challenge. In an effort to develop more sensitive tools, polymerase chain reaction (PCR)-based assays were introduced to detect low amounts of Trypanosoma cruzi DNA in blood samples from chagasic patients, thus improving the diagnosis and follow-up evaluation after chemotherapy. In this article, I review the main problems concerning drug efficacy and criteria used for cure estimation in treated chagasic patients, and the work conducted by different groups on developing PCR methodologies to monitor treatment outcome of congenital infections as well as recent and late chronic T. cruzi infections.

  19. Real-Time PCR-Based Quantitation Method for the Genetically Modified Soybean Line GTS 40-3-2.

    PubMed

    Kitta, Kazumi; Takabatake, Reona; Mano, Junichi

    2016-01-01

    This chapter describes a real-time PCR-based method for quantitation of the relative amount of genetically modified (GM) soybean line GTS 40-3-2 [Roundup Ready(®) soybean (RRS)] contained in a batch. The method targets a taxon-specific soybean gene (lectin gene, Le1) and the specific DNA construct junction region between the Petunia hybrida chloroplast transit peptide sequence and the Agrobacterium 5-enolpyruvylshikimate-3-phosphate synthase gene (epsps) sequence present in GTS 40-3-2. The method employs plasmid pMulSL2 as a reference material in order to quantify the relative amount of GTS 40-3-2 in soybean samples using a conversion factor (Cf) equal to the ratio of the RRS-specific DNA to the taxon-specific DNA in representative genuine GTS 40-3-2 seeds.

  20. The detection of inherent homologous recombination between repeat sequences in H. pylori 26695 by the PCR-based method.

    PubMed

    Fu, Yajuan; Zepeda-Gurrola, Reyna Cristina; Aguilar-Gutiérrez, Germán Rubén; Lara-Ramírez, Edgar E; De Luna-Santillana, Erick J; Rodríguez-Luna, Isabel Cristina; Sánchez-Varela, Alejandro; Carreño-López, Ricardo; Moreno-Medina, Víctor Ricardo; Rodríguez-Pérez, Mario A; López-Vidal, Yolanda; Guo, Xianwu

    2014-02-01

    Helicobacter pylori infects more than half of the world's population, making it the most widespread infection of bacteria. It has high genetic diversity and has been considered as one of the most variable bacterial species. In the present study, a PCR-based method was used to detect the presence and the relative frequency of homologous recombination between repeat sequences (>500 bp) in H. pylori 26695. All the recombinant structures have been confirmed by sequencing. The inversion generated between inverted repeats showed distinct features from the recombination for duplication or deletion between direct repeats. Meanwhile, we gave the mathematic reasoning of a general formula for the calculation of relative recombination frequency and indicated the conditions for its application. This formula could be extensively applied to detect the frequency of homologous recombination, site-specific recombination, and other types of predictable recombination. Our results should be helpful for better understanding the genome evolution and adaptation of bacteria.

  1. Investigation of angular multiplexing and de-multiplexing of digital holograms recorded in microscope configuration.

    PubMed

    Paturzo, M; Memmolo, P; Tulino, A; Finizio, A; Ferraro, P

    2009-05-25

    We investigated a method for the angular multiplexing and de-multiplexing of digital holograms recorded in microscope off-axis configuration. The multiplexing has been performed rotating numerically one hologram at different angles and adding all the rotated holograms to obtain a single synthetic digital hologram. Then the digital holograms were de-multiplexed thanks to the unique property of the digital holography to manage numerically the complex wavefields at different image planes. We show that it is possible to retrieve correctly quantitative information about the amplitude and phase maps. The obtained results can be useful to employ the multiplexing technique during the recording process by rotating the CCD array.

  2. PCR-based method for targeting 16S-23S rRNA intergenic spacer regions among Vibrio species

    PubMed Central

    2010-01-01

    Background The genus Vibrio is a diverse group of Gram-negative bacteria comprised of 74 species. Furthermore, the genus has and is expected to continue expanding with the addition of several new species annually. Consequently, it is of paramount importance to have a method which is able to reliably and efficiently differentiate the numerous Vibrio species. Results In this study, a novel and rapid polymerase chain reaction (PCR)-based intergenic spacer (IGS)-typing system for vibrios was developed that is based on the well-known IGS regions located between the 16S and 23S rRNA genes on the bacterial chromosome. The system was optimized to resolve heteroduplex formation as well as to take advantage of capillary gel electrophoresis technology such that reproducible analyses could be achieved in a rapid manner. System validation was achieved through testing of 69 archetypal Vibrio strains, representing 48 Vibrio species, from which an 'IGS-type' profile database was generated. These data, presented here in several cluster analyses, demonstrated successful differentiation of the 69 type strains showing that this PCR-based fingerprinting method easily discriminates bacterial strains at the species level among Vibrio. Furthermore, testing 36 strains each of V. parahaemolyticus and V. vulnificus, important food borne pathogens, isolated from a variety of geographical locations with the IGS-typing method demonstrated distinct IGS-typing patterns indicative of subspecies divergence in both populations making this technique equally useful for intraspecies differentiation, as well. Conclusion This rapid, reliable and efficient IGS-typing system, especially in combination with 16S rRNA gene sequencing, has the capacity to not only discern and identify vibrios at the species level but, in some cases, at the sub-species level, as well. This procedure is particularly well-suited for preliminary species identification and, lends itself nicely to epidemiological investigations

  3. Flexible Multiplexed Surface Temperature Sensor

    NASA Technical Reports Server (NTRS)

    Daryabeigi, Kamran; Dillon-Townes, L. A.; Johnson, Preston B.; Ash, Robert L.

    1995-01-01

    Unitary array of sensors measures temperatures at points distributed over designated area on surface. Useful in measuring surface temperatures of aerodynamic models and thermally controlled objects. Made of combination of integrated-circuit microchips and film circuitry. Temperature-sensing chips scanned at speeds approaching 10 kHz. Operating range minus 40 degrees C to 120 degrees C. Flexibility of array conforms to curved surfaces. Multiplexer eliminates numerous monitoring cables. Control of acquisition and recording of data effected by connecting array to microcomputers via suitable interface circuitry.

  4. Flexible Multiplexed Surface Temperature Sensor

    NASA Technical Reports Server (NTRS)

    Daryabeigi, Kamran; Dillon-Townes, L. A.; Johnson, Preston B.; Ash, Robert L.

    1995-01-01

    Unitary array of sensors measures temperatures at points distributed over designated area on surface. Useful in measuring surface temperatures of aerodynamic models and thermally controlled objects. Made of combination of integrated-circuit microchips and film circuitry. Temperature-sensing chips scanned at speeds approaching 10 kHz. Operating range minus 40 degrees C to 120 degrees C. Flexibility of array conforms to curved surfaces. Multiplexer eliminates numerous monitoring cables. Control of acquisition and recording of data effected by connecting array to microcomputers via suitable interface circuitry.

  5. System for Multiplexing Acoustic Emission (AE) Instrumentation

    NASA Technical Reports Server (NTRS)

    Prosser, William H. (Inventor); Perey, Daniel F. (Inventor); Gorman, Michael R. (Inventor); Scales, Edgar F. (Inventor)

    2003-01-01

    An acoustic monitoring device has at least two acoustic sensors with a triggering mechanism and a multiplexing circuit. After the occurrence of a triggering event at a sensor, the multiplexing circuit allows a recording component to record acoustic emissions at adjacent sensors. The acoustic monitoring device is attached to a solid medium to detect the occurrence of damage.

  6. A fully sealed plastic chip for multiplex PCR and its application in bacteria identification.

    PubMed

    Xu, Youchun; Yan, He; Zhang, Yan; Jiang, Kewei; Lu, Ying; Ren, Yonghong; Wang, Hui; Wang, Shan; Xing, Wanli

    2015-07-07

    Multiplex PCR is an effective tool for simultaneous multiple target detection but is limited by the intrinsic interference and competition among primer pairs when it is performed in one reaction tube. Dividing a multiplex PCR into many single PCRs is a simple strategy to overcome this issue. Here, we constructed a plastic, easy-to-use, fully sealed multiplex PCR chip based on reversible centrifugation for the simultaneous detection of 63 target DNA sequences. The structure of the chip is quite simple, which contains sine-shaped infusing channels and a number of reaction chambers connecting to one side of these channels. Primer pairs for multiplex PCR were sequentially preloaded in the different reaction chambers, and the chip was enclosed with PCR-compatible adhesive tape. For usage, the PCR master mix containing a DNA template is pipetted into the infusing channels and centrifuged into the reaction chambers, leaving the infusing channels filled with air to avoid cross-contamination of the different chambers. Then, the chip is sealed and placed on a flat thermal cycler for PCR. Finally, amplification products can be detected in situ using a fluorescence scanner or recovered by reverse centrifugation for further analyses. Therefore, our chip possesses two functions: 1) it can be used for multi-target detection based on end-point in situ fluorescence detection; and 2) it can work as a sample preparation unit for analyses that need multiplex PCR such as hybridization and target sequencing. The performance of this chip was carefully examined and further illustrated in the identification of 8 pathogenic bacterial genomic DNA samples and 13 drug-resistance genes. Due to simplicity of its structure and operation, accuracy and generality, high-throughput capacity, and versatile functions (i.e., for in situ detection and sample preparation), our multiplex PCR chip has great potential in clinical diagnostics and nucleic acid-based point-of-care testing.

  7. Assessing the diagnostic accuracy of PCR-based detection of Streptococcus pneumoniae from nasopharyngeal swabs collected for viral studies in Canadian adults hospitalised with community-acquired pneumonia: a Serious Outcomes Surveillance (SOS) Network of the Canadian Immunization Research (CIRN) study.

    PubMed

    Gillis, Hayley D; Lang, Amanda L S; ElSherif, May; Martin, Irene; Hatchette, Todd F; McNeil, Shelly A; LeBlanc, Jason J

    2017-06-08

    Detection and serotyping of Streptococcus pneumoniae are important to assess the impact of pneumococcal vaccines. This study describes the diagnostic accuracy of PCR-based detection of S. pneumoniae directly from nasopharyngeal (NP) swabs collected for respiratory virus studies. Active surveillance for community-acquired pneumonia (CAP) in hospitalised adults was performed from December 2010 to 2013. Detection of pneumococcal CAP (CAPSpn) was performed by urine antigen detection (UAD), identification of S. pneumoniae in sputum or blood cultures. S. pneumoniae was detected in NP swabs using lytA and cpsA real-time PCR, and serotyping was performed using conventional and real-time multiplex PCRs. For serotyping, the Quellung reaction, PCR-based serotyping or a serotype-specific UAD was used. NP swab results were compared against CAP cases where all pneumococcal tests were performed (n=434), or where at least one test was performed (n=1616). CAPSpn was identified in 22.1% (96/434) and 14.9% (240/1616), respectively. The sensitivity of NP swab PCR for the detection of S. pneumoniae was poor for CAPSpn (35.4% (34/96) and 34.17% (82/240)), but high specificity was observed (99.4% (336/338) and 97.89% (1347/1376)). Of the positive NP swabs, a serotype could be deduced by PCR in 88.2% (30/34) and 93.9% (77/82), respectively. While further optimisation may be needed to increase the sensitivity of PCR-based detection, its high specificity suggests there is a value for pneumococcal surveillance. With many laboratories archiving specimens for influenza virus surveillance, this specimen type could provide a non-culture-based method for pneumococcal surveillance. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  8. SQUID Multiplexers for Cryogenic Detector Arrays

    NASA Technical Reports Server (NTRS)

    Irwin, Kent; Beall, James; Deiker, Steve; Doriese, Randy; Duncan, William; Hilton, Gene; Moseley, S. Harvey; Reintsema, Carl; Stahle, Caroline; Ullom, Joel; hide

    2004-01-01

    SQUID multiplexers make it possible to build arrays of thousands of cryogenic detectors with a manageable number of readout channels. We are developing time-division SQUID multiplexers based on Nb trilayer SQUIDs to read arrays of superconducting transition-edge sensors. Our first-generation, 8-channel SQUID multiplexer was used in FIBRE, a one-dimensional TES array for submillimeter astronomy. Our second-generation 32-pixel multiplexer, based on an improved architecture, has been developed for instruments including Constellation-X, SCUBA-2, and solar x-ray astronomy missions. SCUBA-2, which is being developed for the James Clerk Maxwell Telescope, will have more than 10,000 pixels. We are now developing a third-generation architecture based on superconducting hot-electron switches. The use of SQUID multiplexers in instruments operating at above 2 K will also be discussed.

  9. SQUID Multiplexers for Cryogenic Detector Arrays

    NASA Technical Reports Server (NTRS)

    Irwin, Kent; Beall, James; Deiker, Steve; Doriese, Randy; Duncan, William; Hilton, Gene; Moseley, S. Harvey; Reintsema, Carl; Stahle, Caroline; Ullom, Joel; Vale, Leila

    2004-01-01

    SQUID multiplexers make it possible to build arrays of thousands of cryogenic detectors with a manageable number of readout channels. We are developing time-division SQUID multiplexers based on Nb trilayer SQUIDs to read arrays of superconducting transition-edge sensors. Our first-generation, 8-channel SQUID multiplexer was used in FIBRE, a one-dimensional TES array for submillimeter astronomy. Our second-generation 32-pixel multiplexer, based on an improved architecture, has been developed for instruments including Constellation-X, SCUBA-2, and solar x-ray astronomy missions. SCUBA-2, which is being developed for the James Clerk Maxwell Telescope, will have more than 10,000 pixels. We are now developing a third-generation architecture based on superconducting hot-electron switches. The use of SQUID multiplexers in instruments operating at above 2 K will also be discussed.

  10. Accelerated Genome Engineering through Multiplexing

    PubMed Central

    Zhao, Huimin

    2015-01-01

    Throughout the biological sciences, the past fifteen years have seen a push towards the analysis and engineering of biological systems at the organism level. Given the complexity of even the simplest organisms, though, to elicit a phenotype of interest often requires genotypic manipulation of several loci. By traditional means, sequential editing of genomic targets requires a significant investment of time and labor, as the desired editing event typically occurs at a very low frequency against an overwhelming unedited background. In recent years, the development of a suite of new techniques has greatly increased editing efficiency, opening up the possibility for multiple editing events to occur in parallel. Termed as multiplexed genome engineering, this approach to genome editing has greatly expanded the scope of possible genome manipulations in diverse hosts, ranging from bacteria to human cells. The enabling technologies for multiplexed genome engineering include oligonucleotide-based and nuclease-based methodologies, and their application has led to the great breadth of successful examples described in this review. While many technical challenges remain, there also exists a multiplicity of opportunities in this rapidly expanding field. PMID:26394307

  11. Information transport in multiplex networks

    NASA Astrophysics Data System (ADS)

    Pu, Cunlai; Li, Siyuan; Yang, Xianxia; Yang, Jian; Wang, Kai

    2016-04-01

    In this paper, we study information transport in multiplex networks comprised of two coupled subnetworks. The upper subnetwork, called the logical layer, employs the shortest paths protocol to determine the logical paths for packets transmission, while the lower subnetwork acts as the physical layer, in which packets are delivered by the biased random walk mechanism characterized with a parameter α. Through simulation, we obtain the optimal α corresponding to the maximum network lifetime and the maximum number of the arrival packets. Assortative coupling is better than random coupling and disassortative coupling, since it achieves better transmission performance. Generally, the more homogeneous the lower subnetwork is, the better the transmission performance, which is the opposite for the upper subnetwork. Finally, we propose an attack centrality for nodes based on the topological information of both subnetworks, and investigate the transmission performance under targeted attacks. Our work aids in understanding the spread and robustness issues of multiplex networks and provides some clues about the design of more efficient and robust routing architectures in communication systems.

  12. Implementation of a novel PCR based method for detecting malaria parasites from naturally infected mosquitoes in Papua New Guinea

    PubMed Central

    Hasan, Arif U; Suguri, Setsuo; Sattabongkot, Jetsumon; Fujimoto, Chigusa; Amakawa, Masao; Harada, Masakazu; Ohmae, Hiroshi

    2009-01-01

    Background Detection of Plasmodium species in mosquitoes is important for designing vector control studies. However, most of the PCR-based detection methods show some potential limitations. The objective of this study was to introduce an effective PCR-based method for detecting Plasmodium vivax and Plasmodium falciparum from the field-caught mosquitoes of Papua New Guinea. Methods A method has been developed to concurrently detect mitochondrial cytochrome b (Cyt b) of four human Plasmodium species using PCR (Cytb-PCR). To particularly discriminate P. falciparum from P. vivax, Plasmodium ovale and Plasmodium malariae, a polymerase chain reaction-repeated fragment length polymorphism (PCR-RFLP) has further been developed to use with this method. However, due to limited samples number of P. ovale and P. malariae; this study was mainly confined to P. vivax and P. falciparum. The efficiency of Cytb-PCR was evaluated by comparing it with two 'gold standards' enzyme linked immunosorbent assay specific for circumsporozoite protein (CS-ELISA) using artificially infected mosquitoes; and nested PCR specific for small subunit ribosomal RNA (SSUrRNA) using field caught mosquitoes collected from three areas (Kaboibus, Wingei, and Jawia) of the East Sepic Province of Papua New Guinea. Results A total of 90 mosquitoes were artificially infected with three strains of Plasmodium: P. vivax-210 (n = 30), P. vivax-247 (n = 30) and P. falciparum (n = 30). These infected mosquitoes along with another 32 unfed mosquitoes were first checked for the presence of Plasmodium infection by CS-ELISA, and later the same samples were compared with the Cytb-PCR. CS-ELISA for P. vivax-210, P. vivax-247 and P. falciparum detected positive infection in 30, 19 and 18 mosquitoes respectively; whereas Cytb-PCR detected 27, 16 and 16 infections, respectively. The comparison revealed a close agreement between the two assays (κ = 0.862, 0.842 and 0.894, respectively for Pv-210, Pv-247 and P. falciparum

  13. Reversible Computing

    DTIC Science & Technology

    1980-02-01

    will have been introduced. 9. Reversible celular autemata We shall assume the reader to have some familiarity with the concept of cel- lular...10003 Mr. Kin B. Thcmpson 1 copy Technical Director Information Systems Divisia.i Naval Research Laboratory (OP-91T) Technical Information Division

  14. Reverse mortgages.

    PubMed

    Farnesi, D

    1995-09-01

    Elders and their families are often caught in a financial bind when it comes to paying for much-needed home care services. Reverse mortgages may offer a solution to elderly home care clients who own their homes but have a limited income with which to maintain their independence.

  15. REVERSE OSMOSIS,

    DTIC Science & Technology

    acetate membranes. Mechanisms of the process and porous cellulose acetate membrane technology are briefly reviewed. Based on a general capillary...The reverse osmosis process is discussed with particular reference to systems involving aqueous solutions and Loeb-Sourirajan-type porous cellulose

  16. PCR-based specific techniques used for detecting the most important pathogens on strawberry: a systematic review.

    PubMed

    Mirmajlessi, Seyed Mahyar; Destefanis, Marialaura; Gottsberger, Richard Alexander; Mänd, Marika; Loit, Evelin

    2015-01-15

    Strawberry diseases are a major limiting factor that severely impact plant agronomic performance. Regarding limitations of traditional techniques for detection of pathogens, researchers have developed specific DNA-based tests as sensitive and specific techniques. The aim of this review is to provide an overview of polymerase chain reaction (PCR)-based methods used for detection or quantification of the most widespread strawberry pathogens, such as Fusarium oxysporum f.sp. fragariae, Phytophthora fragariae, Colletotrichum acutatum, Verticillium dahliae, Botrytis cinerea, Macrophomina phaseolina, and Xanthomonas fragariae. An updated and detailed list of published PCR protocols is presented and discussed, aimed at facilitating access to information that could be particularly useful for diagnostic laboratories in order to develop a rapid, cost-effective, and reliable monitoring technique. The study design was a systematic review of PCR-based techniques used for detection and quantification of strawberry pathogens. Using appropriate subject headings, AGRICOLA, AGRIS, BASE, Biological Abstracts, CAB Abstracts, Google Scholar, Scopus, Web of Knowledge, and SpringerLink databases were searched from their inception up to April 2014. Two assessors independently reviewed the titles, abstracts, and full articles of all identified citations. Selected articles were included if one of the mentioned strawberry pathogens was investigated based on PCR methods, and a summary of pre-analytical requirements for PCR was provided. A total of 259 titles and abstracts were reviewed, of which 22 full texts met all the inclusion criteria. Our systematic review identified ten different protocols for X. fragariae, eight for P. fragariae, four for B. cinerea, six for C. acutatum, three for V. dahlia, and only one for F. oxysporum. The accuracy and sensitivity of PCR diagnostic methods is the focus of most studies included in this review. However, a large proportion of errors in laboratories

  17. Analog bus driver and multiplexer

    NASA Technical Reports Server (NTRS)

    Pain, Bedabrata (Inventor); Hancock, Bruce (Inventor); Cunningham, Thomas J. (Inventor)

    2012-01-01

    For a source-follower signal chain, the ohmic drop in the selection switch causes unacceptable voltage offset, non-linearity, and reduced small signal gain. For an op amp signal chain, the required bias current and the output noise rises rapidly with increasing the array format due to a rapid increase in the effective capacitance caused by the Miller effect boosting up the contribution of the bus capacitance. A new switched source-follower signal chain circuit overcomes limitations of existing op-amp based or source follower based circuits used in column multiplexers and data readout. This will improve performance of CMOS imagers, and focal plane read-out integrated circuits for detectors of infrared or ultraviolet light.

  18. Cooperative epidemics on multiplex networks

    NASA Astrophysics Data System (ADS)

    Azimi-Tafreshi, N.

    2016-04-01

    The spread of one disease, in some cases, can stimulate the spreading of another infectious disease. Here, we treat analytically a symmetric coinfection model for spreading of two diseases on a two-layer multiplex network. We allow layer overlapping, but we assume that each layer is random and locally loopless. Infection with one of the diseases increases the probability of getting infected with the other. Using the generating function method, we calculate exactly the fraction of individuals infected with both diseases (so-called coinfected clusters) in the stationary state, as well as the epidemic spreading thresholds and the phase diagram of the model. With increasing cooperation, we observe a tricritical point and the type of transition changes from continuous to hybrid. Finally, we compare the coinfected clusters in the case of cooperating diseases with the so-called "viable" clusters in networks with dependencies.

  19. Multiplexed Primer Prediction for PCR

    SciTech Connect

    2007-07-23

    MPP predicts sets of multiplex-compatible primers for Polymerase Chain Reaction (PCR), finding a near minimal set of primers such that at least one amplicon will be generated from every target sequence in the input file. The code finds highly conserved oligos that are suitable as primers, according to user-specified desired primer characteristics such as length, melting temperature, and amplicon length. The primers are predicted not to form unwanted dimer or hairpin structures. The target sequences used as input can be diverse, since no multiple sequence alighment is required. The code is scalable, taking up to tens of thousands of sequences as input, and works, for example, to find a "universal primer set" for all viral genomes provided as a single input file. The code generates a periodic check-point file, thus in the event of premature execution termination, the application can be restarted from the last check-point file.

  20. Multiwavelength metasurfaces through spatial multiplexing

    PubMed Central

    Arbabi, Ehsan; Arbabi, Amir; Kamali, Seyedeh Mahsa; Horie, Yu; Faraon, Andrei

    2016-01-01

    Metasurfaces are two-dimensional arrangements of optical scatterers rationally arranged to control optical wavefronts. Despite the significant advances made in wavefront engineering through metasurfaces, most of these devices are designed for and operate at a single wavelength. Here we show that spatial multiplexing schemes can be applied to increase the number of operation wavelengths. We use a high contrast dielectric transmittarray platform with amorphous silicon nano-posts to demonstrate polarization insensitive metasurface lenses with a numerical aperture of 0.46, that focus light at 915 and 1550 nm to the same focal distance. We investigate two different methods, one based on large scale segmentation and one on meta-atom interleaving, and compare their performances. An important feature of this method is its simple generalization to adding more wavelengths or new functionalities to a device. Therefore, it provides a relatively straightforward method for achieving multi-functional and multiwavelength metasurface devices. PMID:27597568

  1. Simultaneous detection of four causal agents of tobacco bushy top disease by a multiplex one-step RT-PCR

    USDA-ARS?s Scientific Manuscript database

    Tobacco bushy top disease is a complex disease caused by mixed infection of Tobacco bushy top virus (TBTV), Tobacco vein distorting virus (TVDV), satellite RNA of TBTV (Sat-TBTV) and Tobacco vein distorting virus associate RNA (TVDVaRNA). A one-tube multiplex reverse transcription-PCR (RT-PCR) assay...

  2. Rapid identification of Acinetobacter baumannii, Acinetobacter nosocomialis and Acinetobacter pittii with a multiplex PCR assay.

    PubMed

    Chen, Te-Li; Lee, Yi-Tzu; Kuo, Shu-Chen; Yang, Su-Pen; Fung, Chang-Phone; Lee, Shou-Dong

    2014-09-01

    Acinetobacter baumannii, Acinetobacter nosocomialis and Acinetobacter pittii are clinically relevant members of the Acinetobacter calcoaceticus-A. baumannii (Acb) complex and important nosocomial pathogens. These three species are genetically closely related and phenotypically similar; however, they differ in their epidemiology, antibiotic resistance and pathogenicity. In this study, we investigated the use of a multiplex PCR-based assay designed to detect internal fragments of the 16S-23S rRNA intergenic region and the gyrB and recA genes. The assay was capable of differentiating A. baumannii, A. nosocomialis and A. pittii in a reliable manner. In 23 different reference strains and 89 clinical isolates of Acinetobacter species, the assay accurately identified clinically relevant Acb complex species except those 'between 1 and 3' or 'close to 13TU'. None of the non-Acb complex species was misidentified. In an analysis of 1034 positive blood cultures, the assay had a sensitivity of 92.4 % and specificity of 98.2 % for Acb complex identification. Our results show that a single multiplex PCR assay can reliably differentiate clinically relevant Acb complex species. Thus, this method may be used to better understand the clinical differences between infections caused by these species.

  3. Multiplex PCR for colony direct detection of Gram-positive histamine- and tyramine-producing bacteria.

    PubMed

    Coton, Emmanuel; Coton, Monika

    2005-12-01

    Formation of biogenic amines (BA) may occur in fermented foods and beverages due to the amino acid decarboxylase activities of Gram-positive bacteria. These compounds may cause food poisoning and therefore could imply food exportation problems. A set of consensual primers based on histidine decarboxylase gene (hdc) sequences of different bacteria was designed for the detection of histamine-producing Gram-positive bacteria. A multiplex PCR based on these hdc primers and recently designed primers targeting the tyrosine decarboxylase (tyrdc) gene was created. A third set of primers targeting the 16S rRNA gene of eubacteria was also used as an internal control. This multiplex PCR was performed on extracted DNA as well as directly on cell colonies. The results obtained show that this new molecular tool allowed for the detection of Gram-positive histamine- and/or tyramine-producing bacteria. The use of this molecular tool for early and rapid detection of Gram-positive BA-producing bacteria is of interest in evaluating the potential of cultured indigenous strains to produce biogenic amines in a fermented food product as well as to validate the innocuity of potential starter strains in the food industry.

  4. 3D multiplexed immunoplasmonics microscopy

    NASA Astrophysics Data System (ADS)

    Bergeron, Éric; Patskovsky, Sergiy; Rioux, David; Meunier, Michel

    2016-07-01

    Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K+ channel subunit KV1.1) on human cancer CD44+ EGFR+ KV1.1+ MDA-MB-231 cells and reference CD44- EGFR- KV1.1+ 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third, the developed

  5. A novel reversible carry-selected adder with low latency

    NASA Astrophysics Data System (ADS)

    Li, Ming-Cui; Zhou, Ri-Gui

    2016-07-01

    Reversible logic is getting more and more attention in quantum computing, optical computing, nanotechnology and low-power complementary metal oxide semiconductor designs since reversible circuits do not loose information during computation and have only small energy dissipation. In this paper, a novel carry-selected reversible adder is proposed primarily optimised for low latency. A 4-bit reversible full adder with two kinds of outputs, minimum delay and optimal quantum cost is presented as the building block for ?-bit reversible adder. Three new reversible gates NPG (new Peres gate), TEPG (triple extension of Peres gate) and RMUX21 (reversible 2-to-1 multiplexer) are proposed and utilised to design efficient adder units. The secondary carry propagation chain is carefully designed to reduce the time consumption. The novelty of the proposed design is the consideration of low latency. The comparative study shows that the proposed adder achieves the improvement from 61.46% to 95.29% in delay over the existing designs.

  6. Multiple linked β and α globin genes in Atlantic cod: A PCR based strategy of genomic exploration.

    PubMed

    Halldórsdóttir, Katrín; Arnason, Einar

    2009-01-01

    Allozyme variation in Atlantic cod hemoglobins shows various signs of natural selection. We report a genomic exploration of globin genes in this non-model organism. Applying a PCR based strategy with a strict criterion of phylogenetically informative sites we estimate the number of linked β and α globin genes. We estimate PCR error rate by PCR of cloned DNA and recloning and by analysis of singleton variable sites among clones. Based on the error rate we exclude variable sites so that the remaining variation meets successively stricter criteria of doubleton and triplet variable site. Applying these criteria we find ten clusters of linked β/α globin genes in the genome of Atlantic cod. Six variable amino acid changes in both genes were found in linkage disequilibrium with silent nucleotide substitutions. A phylogenetic tree, based on our strictly phylogenetically informative sites among 57 clones from 19 individuals, is split into two major branches by an amino acid change in a β gene. This change is supported by extensive linkage disequilibrium between the amino acid change and numerous other phylogenetically informative silent nucleotide sites. The different gene sets in the genome may represent different loci encoding different globins and/or allelic variation at some loci.

  7. PCR-based study of the presence of Y-chromosome sequences in patients with Ullrich-Turner syndrome

    SciTech Connect

    Coto, E.; Menendez, M.J.; Lopez-Larrea, C.

    1995-07-03

    The presence of Y chromosome sequences in Ullrich-Turner syndrome (UTS) patients has been suggested in previous work. Karyotype analysis estimated at about 60% of patients with a 45, X constitution and molecular analysis (Southern blot analysis with several Y chromosome probes and PCR of specific sequences) identified the presence of Y chromosome material in about 40% of 45, X patients. We have developed a very sensitive, PCR-based method to detect Y specific sequences in DNA from UTS patients. This protocol permits the detection of a single cell carrying a Y sequence among 10{sup 5} Y-negative cells. We studied 18 UTS patients with 4 Y-specific sequences. In 11 patients we detected a positive amplification for at least one Y sequence. The existence of a simple and sensitive method for the detection of Y sequences has important implications for UTS patients, in view of the risk for some of the females carrying Y chromosome material of developing gonadoblastoma and virilization. Additionally, some of the UTS-associated phenotypes, such as renal anomalies, could be correlated with the presence of Y chromosome-specific sequences. 27 refs., 2 figs., 1 tab.

  8. Comparison of Three PCR-based Methods for Simplicity and Cost Effectiveness Identification of Cutaneous Leishmaniasis Due to Leishmania tropica.

    PubMed

    Mohammadi, Mohammad Ali; Bamorovat, Mehdi; Fasihi Harandi, Majid; Karimi, Tayyebeh; Sharifi, Iraj; Aflatoonian, Mohammad Reza

    2017-01-01

    To compare three molecular methods, PCR-RFLP for internal transcribed spacer, PCR sequencing and high resolution melting analysis shown reliable sensitivity and specificity for detecting Leishmania tropica as a model for cutaneous leishmaniasis (CL) as the perspective overview for scientific and economic approaches. This study was carried out between 2015 and 2016 in Leishmaniasis Research Center in Kerman University of Medical Sciences, Kerman, Iran. The positives smears (n=50) were obtained from patients referred from the health clinics in a major anthroponotic CL (ACL) focus, southeastern Iran. Only smear preparations with the same grade were selected according to the method described by the WHO for future PCR assays. All three molecular methods had capability to identify positive samples at species level with the same specificity and sensitivity. However, these techniques were different in simplicity, consuming time, and cost effectiveness. Although additional enzymatic process in PCR-RFLP provided good resolution to find Leishmania species but this would cause time and cost increases. HRM (high resolution melting) is a relatively new technique that allows direct characterization of PCR amplicons in a closed system with more simplicity, cost effectiveness and time-consuming compared with other PCR-based assays for epidemiological or clinical identification purposes.

  9. Comparison of Three PCR-based Methods for Simplicity and Cost Effectiveness Identification of Cutaneous Leishmaniasis Due to Leishmania tropica

    PubMed Central

    MOHAMMADI, Mohammad Ali; BAMOROVAT, Mehdi; FASIHI HARANDI, Majid; KARIMI, Tayyebeh; SHARIFI, Iraj; AFLATOONIAN, Mohammad Reza

    2017-01-01

    Background: To compare three molecular methods, PCR-RFLP for internal transcribed spacer, PCR sequencing and high resolution melting analysis shown reliable sensitivity and specificity for detecting Leishmania tropica as a model for cutaneous leishmaniasis (CL) as the perspective overview for scientific and economic approaches. Methods: This study was carried out between 2015 and 2016 in Leishmaniasis Research Center in Kerman University of Medical Sciences, Kerman, Iran. The positives smears (n=50) were obtained from patients referred from the health clinics in a major anthroponotic CL (ACL) focus, southeastern Iran. Only smear preparations with the same grade were selected according to the method described by the WHO for future PCR assays. Results: All three molecular methods had capability to identify positive samples at species level with the same specificity and sensitivity. However, these techniques were different in simplicity, consuming time, and cost effectiveness. Although additional enzymatic process in PCR-RFLP provided good resolution to find Leishmania species but this would cause time and cost increases. Conclusion: HRM (high resolution melting) is a relatively new technique that allows direct characterization of PCR amplicons in a closed system with more simplicity, cost effectiveness and time-consuming compared with other PCR-based assays for epidemiological or clinical identification purposes. PMID:28761481

  10. A PCR-Based Method to Construct Lentiviral Vector Expressing Double Tough Decoy for miRNA Inhibition

    PubMed Central

    Luo, Lan; Liu, Nian; Kang, Kang; Qu, Junle; Peng, Wenda; Gou, Deming

    2015-01-01

    DNA vector-encoded Tough Decoy (TuD) miRNA inhibitor is attracting increased attention due to its high efficiency in miRNA suppression. The current methods used to construct TuD vectors are based on synthesizing long oligonucleotides (~90 mer), which have been costly and problematic because of mutations during synthesis. In this study, we report a PCR-based method for the generation of double Tough Decoy (dTuD) vector in which only two sets of shorter oligonucleotides (< 60 mer) were used. Different approaches were employed to test the inhibitory potency of dTuDs. We demonstrated that dTuD is the most efficient method in miRNA inhibition in vitro and in vivo. Using this method, a mini dTuD library against 88 human miRNAs was constructed and used for a high-throughput screening (HTS) of AP-1 pathway-related miRNAs. Seven miRNAs (miR-18b-5p, -101-3p, -148b-3p, -130b-3p, -186-3p, -187-3p and -1324) were identified as candidates involved in AP-1 pathway regulation. This novel method allows for an accurate and cost-effective generation of dTuD miRNA inhibitor, providing a powerful tool for efficient miRNA suppression in vitro and in vivo. PMID:26624995

  11. An improved method of DNA isolation suitable for PCR-based detection of begomoviruses from jute and other mucilaginous plants.

    PubMed

    Ghosh, Raju; Paul, Sujay; Ghosh, Subrata Kumar; Roy, Anirban

    2009-07-01

    A relatively quick and inexpensive modified cetyl trimethylammonium bromide method for extraction of DNA from leaf materials containing large quantities of mucilage is described. The modification including use of more volume of extraction buffer and dissolving crude nucleic acid pellet in 1 M NaCl, reduced markedly the viscosity of the mucilage and thus in the final purification step yielded a larger quantity of mucilage-free DNA suitable for subsequent PCR-based detection of begomoviruses. The method was standardized with jute samples with yellow mosaic disease and validated with different other mucilaginous-hosts with low titre of begomoviruses. DNA isolated using this method showed consistency in yield and compatibility with PCR for detection of begomoviruses from different mucilaginous plant species. The method was compared for efficacy with other reported methods and it was found to be superior over the existing methods described for isolation of DNA from mucilaginous hosts. Thus the method described could be used on a wider scale for reliable and consistent detection of begomoviruses from mucilaginous hosts for characterization and variability study.

  12. Neutralization Assay for Zika and Dengue Viruses by Use of Real-Time-PCR-Based Endpoint Assessment.

    PubMed

    Wilson, Heather L; Tran, Thomas; Druce, Julian; Dupont-Rouzeyrol, Myrielle; Catton, Michael

    2017-10-01

    The global spread and infective complications of Zika virus (ZKV) and dengue virus (DENV) have made them flaviviruses of public health concern. Serological diagnosis can be challenging due to antibody cross-reactivity, particularly in secondary flavivirus infections or when there is a history of flavivirus vaccination. The virus neutralization assay is considered to be the most specific assay for measurement of anti-flavivirus antibodies. This study describes an assay where the neutralization endpoint is measured by real-time PCR, providing results within 72 h. It demonstrated 100% sensitivity (24/24 ZKV and 15/15 DENV) and 100% specificity (11/11 specimens) when testing well-characterized sera. In addition, the assay was able to determine the correct DENV serotype in 91.7% of cases. The high sensitivity and specificity of the real-time PCR neutralization assay makes it suitable to use as a confirmatory test for sera that are reactive in commercial IgM/IgG enzyme immunoassays. Results are objective and the PCR-based measurement of the neutralization endpoint lends itself to automation so that throughput may be increased in times of high demand. Copyright © 2017 American Society for Microbiology.

  13. A PCR based protocol for detecting indel mutations induced by TALENs and CRISPR/Cas9 in zebrafish.

    PubMed

    Yu, Chuan; Zhang, Yaguang; Yao, Shaohua; Wei, Yuquan

    2014-01-01

    Genome editing techniques such as the zinc-finger nucleases (ZFNs), transcription activator-like effecter nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system Cas9 can induce efficient DNA double strand breaks (DSBs) at the target genomic sequence and result in indel mutations by the error-prone non-homologous end joining (NHEJ) DNA repair system. Several methods including sequence specific endonuclease assay, T7E1 assay and high resolution melting curve assay (HRM) etc have been developed to detect the efficiency of the induced mutations. However, these assays have some limitations in that they either require specific sequences in the target sites or are unable to generate sequencing-ready mutant DNA fragments or unable to distinguish induced mutations from natural nucleotide polymorphism. Here, we developed a simple PCR-based protocol for detecting indel mutations induced by TALEN and Cas9 in zebrafish. We designed 2 pairs of primers for each target locus, with one putative amplicon extending beyond the putative indel site and the other overlapping it. With these primers, we performed a qPCR assay to efficiently detect the frequencies of newly induced mutations, which was accompanied with a T-vector-based colony analysis to generate single-copy mutant fragment clones for subsequent DNA sequencing. Thus, our work has provided a very simple, efficient and fast assay for detecting induced mutations, which we anticipate will be widely used in the area of genome editing.

  14. Use of Occult Blood Detection Cards for Real-Time PCR-Based Diagnosis of Schistosoma Mansoni Infection.

    PubMed

    Schunk, Mirjam; Kebede Mekonnen, Seleshi; Wondafrash, Beyene; Mengele, Carolin; Fleischmann, Erna; Herbinger, Karl-Heinz; Verweij, Jaco J; Geldmacher, Christof; Bretzel, Gisela; Löscher, Thomas; Zeynudin, Ahmed

    2015-01-01

    In Schistosoma mansoni infection, diagnosis and control after treatment mainly rely on parasitological stool investigations which are laborious and have limited sensitivity. PCR methods have shown equal or superior sensitivity but preservation and storage methods limit their use in the field. Therefore, the use of occult blood detection cards (fecal cards) for easy sampling and storage of fecal samples for further PCR testing was evaluated in a pilot study. Stool specimens were collected in a highly endemic area for S. mansoni in Ethiopia and submitted in an investigator-blinded fashion to microscopic examination by Kato-Katz thick smear as well as to real-time PCR using either fresh frozen stool samples or stool smears on fecal cards which have been stored at ambient temperature for up to ten months. Out of 55 stool samples, 35 were positive by microscopy, 33 and 32 were positive by PCR of frozen samples and of fecal card samples, respectively. When microscopy was used as diagnostic "gold standard", the sensitivity of PCR on fresh stool was 94.3% (95%-CI: 86.6; 100) and on fecal cards 91.4% (95%-CI: 82.2; 100). The use of fecal cards proved to be a simple and useful method for stool collection and prolonged storage prior to PCR based diagnosis of S. mansoni infection. This technique may be a valuable approach for large scale surveillance and post treatment assessments.

  15. DNA and RNA Extraction and Quantitative Real-Time PCR-Based Assays for Biogas Biocenoses in an Interlaboratory Comparison

    PubMed Central

    Lebuhn, Michael; Derenkó, Jaqueline; Rademacher, Antje; Helbig, Susanne; Munk, Bernhard; Pechtl, Alexander; Stolze, Yvonne; Prowe, Steffen; Schwarz, Wolfgang H.; Schlüter, Andreas; Liebl, Wolfgang; Klocke, Michael

    2016-01-01

    Five institutional partners participated in an interlaboratory comparison of nucleic acid extraction, RNA preservation and quantitative Real-Time PCR (qPCR) based assays for biogas biocenoses derived from different grass silage digesting laboratory and pilot scale fermenters. A kit format DNA extraction system based on physical and chemical lysis with excellent extraction efficiency yielded highly reproducible results among the partners and clearly outperformed a traditional CTAB/chloroform/isoamylalcohol based method. Analytical purpose, sample texture, consistency and upstream pretreatment steps determine the modifications that should be applied to achieve maximum efficiency in the trade-off between extract purity and nucleic acid recovery rate. RNA extraction was much more variable, and the destination of the extract determines the method to be used. RNA stabilization with quaternary ammonium salts was an as satisfactory approach as flash freezing in liquid N2. Due to co-eluted impurities, spectrophotometry proved to be of limited value for nucleic acid qualification and quantification in extracts obtained with the kit, and picoGreen® based quantification was more trustworthy. Absorbance at 230 nm can be extremely high in the presence of certain chaotropic guanidine salts, but guanidinium isothiocyanate does not affect (q)PCR. Absolute quantification by qPCR requires application of a reliable internal standard for which correct PCR efficiency and Y-intercept values are important and must be reported. PMID:28952569

  16. High frequency of false-positive signals in a real-time PCR-based "Plus/Minus" assay.

    PubMed

    Nowrouzian, Forough L; Adlerberth, Ingegerd; Wold, Agnes E

    2009-01-01

    Molecular biological methods using real-time polymerase chain reaction (RT-PCR) for detection of bacterial and viral genes in different environments have been developed into assays from different commercial sources. Applied Biosystems include and support two applications with their TaqMan instrument: the "Plus/Minus" and the "Allelic Discrimination" assays. These approaches are RT-PCR based, use short primers and fluorescent-labeled TaqMan probes and include three processes: a pre-read run, a PCR-amplification run, and a post-read run. In the "Plus/Minus" assay, samples and controls (distilled water) are loaded into the instrument, which calculates a positive or a negative outcome based on differences in signals between samples and the controls. When testing the "Plus/Minus" assay for detection of usp genes encoding a uropathogenic specific protein in Escherichia coli, an inordinately high proportion of false-positive signals was observed. This was shown to be due to a serious methodological deficiency. Our observations indicate that an adequate no-template control closely matching the target samples in all aspects, including amount of DNA, is required to establish a correct threshold in the pre-read run that forms the basis for further calculations in the post-read run of the "Plus/Minus" assay.

  17. Revealing constitutively expressed resistance genes in Agrostis species using PCR-based motif-directed RNA fingerprinting.

    PubMed

    Budak, Hikmet; Su, Senem; Ergen, Neslihan

    2006-12-01

    Agrostis species are mainly used in athletic fields and golf courses. Their integrity is maintained by fungicides, which makes the development of disease-resistance varieties a high priority. However, there is a lack of knowledge about resistance (R) genes and their use for genetic improvement in Agrostis species. The objective of this study was to identify and clone constitutively expressed cDNAs encoding R gene-like (RGL) sequences from three Agrostis species (colonial bentgrass (A. capillaris L.), creeping bentgrass (A. stolonifera L.) and velvet bentgrass (A. canina L.)) by PCR-based motif-directed RNA fingerprinting towards relatively conserved nucleotide binding site (NBS) domains. Sixty-one constitutively expressed cDNA sequences were identified and characterized. Sequence analysis of ESTs and probable translation products revealed that RGLs are highly conserved among these three Agrostis species. Fifteen of them were shown to share conserved motifs found in other plant disease resistance genes such as MLA13, Xa1, YR6, YR23 and RPP5. The molecular evolutionary forces, analysed using the Ka/Ks ratio, reflected purifying selection both on NBS and leucine-rich repeat (LRR) intervening regions of discovered RGL sequences in these species. This study presents, for the first time, isolation and characterization of constitutively expressed RGL sequences from Agrostis species revealing the presence of TNL (TIR-NBS-LRR) type R genes in monocot plants. The characterized RGLs will further enhance knowledge on the molecular evolution of the R gene family in grasses.

  18. Performance of human fecal anaerobe-associated PCR-based assays in a multi-laboratory method evaluation study

    USGS Publications Warehouse

    Layton, Blythe A.; Cao, Yiping; Ebentier, Darcy L.; Hanley, Kaitlyn; Ballesté, Elisenda; Brandão, João; Byappanahalli, Muruleedhara N.; Converse, Reagan; Farnleitner, Andreas H.; Gentry-Shields, Jennifer; Gourmelon, Michèle; Lee, Chang Soo; Lee, Jiyoung; Lozach, Solen; Madi, Tania; Meijer, Wim G.; Noble, Rachel; Peed, Lindsay; Reischer, Georg H.; Rodrigues, Raquel; Rose, Joan B.; Schriewer, Alexander; Sinigalliano, Chris; Srinivasan, Sangeetha; Stewart, Jill; ,; Laurie, C.; Wang, Dan; Whitman, Richard; Wuertz, Stefan; Jay, Jenny; Holden, Patricia A.; Boehm, Alexandria B.; Shanks, Orin; Griffith, John F.

    2013-01-01

    A number of PCR-based methods for detecting human fecal material in environmental waters have been developed over the past decade, but these methods have rarely received independent comparative testing in large multi-laboratory studies. Here, we evaluated ten of these methods (BacH, BacHum-UCD, Bacteroides thetaiotaomicron (BtH), BsteriF1, gyrB, HF183 endpoint, HF183 SYBR, HF183 Taqman®, HumM2, and Methanobrevibacter smithii nifH (Mnif)) using 64 blind samples prepared in one laboratory. The blind samples contained either one or two fecal sources from human, wastewater or non-human sources. The assay results were assessed for presence/absence of the human markers and also quantitatively while varying the following: 1) classification of samples that were detected but not quantifiable (DNQ) as positive or negative; 2) reference fecal sample concentration unit of measure (such as culturable indicator bacteria, wet mass, total DNA, etc); and 3) human fecal source type (stool, sewage or septage). Assay performance using presence/absence metrics was found to depend on the classification of DNQ samples. The assays that performed best quantitatively varied based on the fecal concentration unit of measure and laboratory protocol. All methods were consistently more sensitive to human stools compared to sewage or septage in both the presence/absence and quantitative analysis. Overall, HF183 Taqman® was found to be the most effective marker of human fecal contamination in this California-based study.

  19. PCR-based Methodologies Used to Detect and Differentiate the Burkholderia pseudomallei complex: B. pseudomallei, B. mallei, and B. thailandensis.

    PubMed

    Lowe, Woan; March, Jordon K; Bunnell, Annette J; O'Neill, Kim L; Robison, Richard A

    2014-01-01

    Methods for the rapid detection and differentiation of the Burkholderia pseudomallei complex comprising B. pseudomallei, B. mallei, and B. thailandensis, have been the topic of recent research due to the high degree of phenotypic and genotypic similarities of these species. B. pseudomallei and B. mallei are recognized by the CDC as tier 1 select agents. The high mortality rates of glanders and melioidosis, their potential use as bioweapons, and their low infectious dose, necessitate the need for rapid and accurate detection methods. Although B. thailandensis is generally avirulent in mammals, this species displays very similar phenotypic characteristics to that of B. pseudomallei. Optimal identification of these species remains problematic, due to the difficulty in developing a sensitive, selective, and accurate assay. The development of PCR technologies has revolutionized diagnostic testing and these detection methods have become popular due to their speed, sensitivity, and accuracy. The purpose of this review is to provide a comprehensive overview and evaluation of the advancements in PCR-based detection and differentiation methodologies for the B. pseudomallei complex, and examine their potential uses in diagnostic and environmental testing.

  20. A dual PCR-based sequencing approach for the identification and discrimination of Echinococcus and Taenia taxa.

    PubMed

    Boubaker, Ghalia; Marinova, Irina; Gori, Francesca; Hizem, Amani; Müller, Norbert; Casulli, Adriano; Jerez Puebla, Luis Enrique; Babba, Hamouda; Gottstein, Bruno; Spiliotis, Markus

    2016-08-01

    Reliable and rapid molecular tools for the genetic identification and differentiation of Echinococcus species and/or genotypes are crucial for studying spatial and temporal transmission dynamics. Here, we describe a novel dual PCR targeting regions in the small (rrnS) and large (rrnL) subunits of mitochondrial ribosomal RNA (rRNA) genes, which enables (i) the specific identification of species and genotypes of Echinococcus (rrnS + L-PCR) and/or (ii) the identification of a range of taeniid cestodes, including different species of Echinococcus, Taenia and some others (17 species of diphyllidean helminths). This dual PCR approach was highly sensitive, with an analytical detection limit of 1 pg for genomic DNA of Echinococcus. Using concatenated sequence data derived from the two gene markers (1225 bp), we identified five unique and geographically informative single nucleotide polymorphisms (SNPs) that allowed genotypes (G1 and G3) of Echinococcus granulosus sensu stricto to be distinguished, and 25 SNPs that allowed differentiation within Echinococcus canadensis (G6/7/8/10). In conclusion, we propose that this dual PCR-based sequencing approach can be used for molecular epidemiological studies of Echinococcus and other taeniid cestodes.

  1. Use of Occult Blood Detection Cards for Real-Time PCR-Based Diagnosis of Schistosoma Mansoni Infection

    PubMed Central

    Schunk, Mirjam; Kebede Mekonnen, Seleshi; Wondafrash, Beyene; Mengele, Carolin; Fleischmann, Erna; Herbinger, Karl-Heinz; Verweij, Jaco J.; Geldmacher, Christof; Bretzel, Gisela; Löscher, Thomas; Zeynudin, Ahmed

    2015-01-01

    Background In Schistosoma mansoni infection, diagnosis and control after treatment mainly rely on parasitological stool investigations which are laborious and have limited sensitivity. PCR methods have shown equal or superior sensitivity but preservation and storage methods limit their use in the field. Therefore, the use of occult blood detection cards (fecal cards) for easy sampling and storage of fecal samples for further PCR testing was evaluated in a pilot study. Methodology Stool specimens were collected in a highly endemic area for S. mansoni in Ethiopia and submitted in an investigator-blinded fashion to microscopic examination by Kato-Katz thick smear as well as to real-time PCR using either fresh frozen stool samples or stool smears on fecal cards which have been stored at ambient temperature for up to ten months. Principal Findings Out of 55 stool samples, 35 were positive by microscopy, 33 and 32 were positive by PCR of frozen samples and of fecal card samples, respectively. When microscopy was used as diagnostic “gold standard”, the sensitivity of PCR on fresh stool was 94.3% (95%-CI: 86.6; 100) and on fecal cards 91.4% (95%-CI: 82.2; 100). Conclusions The use of fecal cards proved to be a simple and useful method for stool collection and prolonged storage prior to PCR based diagnosis of S. mansoni infection. This technique may be a valuable approach for large scale surveillance and post treatment assessments PMID:26360049

  2. Specific PCR-based assays for the identification of Fasciola species: their development, evaluation and potential usefulness in prevalence surveys.

    PubMed

    Ai, L; Dong, S J; Zhang, W Y; Elsheikha, H M; Mahmmod, Y S; Lin, R Q; Yuan, Z G; Shi, Y L; Huang, W Y; Zhu, X Q

    2010-01-01

    Among the helminths infecting ruminants in China are three taxa belonging to the genus Fasciola: F. hepatica, F. gigantica and the so-called 'intermediate form' that appears to lie between these two species. Based on the sequences of the second internal-transcribed spacers (ITS-2) within the parasites' nuclear ribosomal DNA (rDNA), a pair of primers (DSJf/DSJ3) specific for F. hepatica and a pair (DSJf/DSJ4) specific for F. gigantica were designed and used to develop PCR-based assays. These assays allowed the identification and differentiation of F. hepatica, F. gigantica and the 'intermediate' Fasciola, with no amplicons produced from heterologous DNA samples. The results of sequencing confirmed the species-specific identity of the amplified products. The assays showed good sensitivity, giving positive results with as little as 0.11 ng of F. hepatica DNA and 0.35 ng of F. gigantica DNA. This meant that the DNA from a single Fasciola egg or a single infected snail was sufficient for identification of the Fasciola taxon. The developed PCR assays could provide useful tools for the detection, identification and epidemiological investigation of Fasciola infection in humans, other mammals and snails.

  3. 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.

  4. 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.

  5. 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.

  6. Vasectomy reversal.

    PubMed

    Belker, A M

    1987-02-01

    A vasovasostomy may be performed on an outpatient basis with local anesthesia, but also may be performed on an outpatient basis with epidural or general anesthesia. Local anesthesia is preferred by most of my patients, the majority of whom choose this technique. With proper preoperative and intraoperative sedation, patients sleep lightly through most of the procedure. Because of the length of time often required for bilateral microsurgical vasoepididymostomy, epidural or general anesthesia and overnight hospitalization are usually necessary. Factors influencing the preoperative choice for vasovasostomy or vasoepididymostomy in patients undergoing vasectomy reversal are considered. The preoperative planned choice of vasovasostomy or vasoepididymostomy for patients having vasectomy reversal described herein does not have the support of all urologists who regularly perform these procedures. My present approach has evolved as the data reported in Tables 1 and 2 have become available, but it may change as new information is evaluated. However, it offers a logical method for planning choices of anesthesia and inpatient or outpatient status for patients undergoing vasectomy reversal procedures.

  7. Recent developments in multiplexing techniques for immunohistochemistry

    PubMed Central

    Dixon, Angela R; Bathany, Cédric; Tsuei, Michael; White, Joshua; Barald, Kate F; Takayama, Shuichi

    2016-01-01

    Methods to detect immuno-labelled molecules at increasingly higher resolution, even when present at low levels, are revolutionizing immunohistochemistry (IHC). These technologies can be valuable for management and examination of rare patient tissue specimens, and for improved accuracy of early disease detection. The purpose of this mini-review is to highlight recent multiplexing methods that are candidates for more prevalent use in clinical research and potential translation to the clinic. Multiplex IHC methods, which permit identification of at least 3 and up to 30 discrete antigens, have been divided into whole section staining and spatially-patterned staining categories. Associated signal enhancement technologies that can enhance performance and throughput of multiplex IHC assays are also discussed. Each multiplex IHC technique, detailed herein, is associated with several advantages as well as tradeoffs that must be taken into consideration for proper evaluation and use of the methods. PMID:26289603

  8. 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.

  9. A multiplex real-time PCR assay for detection of Xanthomonas campestris from brassicas.

    PubMed

    Berg, T; Tesoriero, L; Hailstones, D L

    2006-06-01

    To develop a sensitive real-time PCR-based protocol for the detection of Xanthomonas campestris pathovars from Brassica seed. A 5' nuclease real-time PCR assay was developed to screen Brassica spp. seed for the presence of X. campestris pathovars that cause black rot. The assay amplifies a 78-bp segment of the X. campestris hrpF gene and a 100-bp segment of the Brassica spp. 18S-25S internal transcribed spacer region. The Brassica spp. target provides an internal control for the amplification process to prevent false negatives that may arise from inhibitors that are often present in extracts from plant material. Whilst the primers were compatible with SYBR Green I assays, the use of fluorescently labelled probes in a 5' nuclease assay afforded greatest sensitivity and specificity. Seed batches carrying one artificially infected seed among 10,000 were readily detected using the assay. The multiplex real-time PCR assay permitted the rapid detection of pathogenic strains of X. campestris from bacterial colonies, Brassica seed and plants. Strains of X. campestris pathogenic to brassicas were readily detected from seed via a multiplex 5' nuclease real-time PCR assay. The real-time assay offers an improvement in sensitivity and a reduced turn-around time over the conventional multiplex PCR. Real-time PCR can be used to rapidly screen Brassica spp. seed batches for the presence of X. campestris pathovars. This assay provides a means for growers and the seed industry to be aware of the black rot status of their planting material, so that they may more effectively employ disease control measures or seed disinfection.

  10. Shift multiplexing by planar waveguide referencing

    NASA Astrophysics Data System (ADS)

    Yi, Tao; Zhang, Jiasen; Yan, Lifen; Gong, Qihuang

    2005-09-01

    We present a new method with which to implement shift multiplexing by planar waveguide referencing. In this method, a planar waveguide is used to steer the reference beam, and we implement shift multiplexing by shifting the recording medium. A spatial selectivity as high as 1.1 μm is obtained. By using waveguide referencing we can make a compact and simple holographic system.

  11. Correlated edge overlaps in multiplex networks

    NASA Astrophysics Data System (ADS)

    Baxter, Gareth J.; Bianconi, Ginestra; da Costa, Rui A.; Dorogovtsev, Sergey N.; Mendes, José F. F.

    2016-07-01

    We develop the theory of sparse multiplex networks with partially overlapping links based on their local treelikeness. This theory enables us to find the giant mutually connected component in a two-layer multiplex network with arbitrary correlations between connections of different types. We find that correlations between the overlapping and nonoverlapping links markedly change the phase diagram of the system, leading to multiple hybrid phase transitions. For assortative correlations we observe recurrent hybrid phase transitions.

  12. Multiplexed RNA trafficking in oligodendrocytes and neurons.

    PubMed

    Carson, John H; Gao, Yuanzheng; Tatavarty, Vedakumar; Levin, Mikhail K; Korza, George; Francone, Victor P; Kosturko, Linda D; Maggipinto, Michael J; Barbarese, Elisa

    2008-08-01

    In oligodendrocytes and neurons genetic information is transmitted from the nucleus to dendrites in the form of RNA granules. Here we describe how transport of multiple different RNA molecules in individual granules is analogous to the process of multiplexing in telecommunications. In both cases multiple messages are combined into a composite signal for transmission on a single carrier. Multiplexing provides a mechanism to coordinate local expression of ensembles of genes in myelin in oligodendrocytes and at synapses in neurons.

  13. Primary oral Penicillium marneffei infection diagnosed by PCR-based molecular identification and transmission electron microscopic observation from formalin-fixed paraffin-embedded tissues.

    PubMed

    Hua, Xia; Zhang, Ruifeng; Yang, Hanjun; Lei, Song; Zhang, Yizhi; Ran, Yuping

    2012-11-07

    We report a case of primary oral Penicillium marneffei infection in a 39-year-old man without HIV infection. Although fungal culture was negative, the patient was finally confirmed to have P. marneffei infection by PCR-based molecular identification and transmission electron microscopic observation from formalin-fixed, paraffin-embedded tissues. The patient was cured with taking itraconazole for 3 months.

  14. Total bacterial load within Echinacea purpurea, determined using a new PCR-based quantification method, is correlated with LPS levels and In vitro macrophage activity

    USDA-ARS?s Scientific Manuscript database

    In the present study, total bacterial load was determined within E. purpurea samples and ranged from 6.4 × 106 to 3.3 × 108 bacteria/g of dry plant material. To estimate total bacterial load, we developed a PCR-based quantification method that circumvents the problems associated with nonviable/noncu...

  15. k-core percolation on multiplex networks

    NASA Astrophysics Data System (ADS)

    Azimi-Tafreshi, N.; Gómez-Gardeñes, J.; Dorogovtsev, S. N.

    2014-09-01

    We generalize the theory of k-core percolation on complex networks to k-core percolation on multiplex networks, where k ≡(k1,k2,...,kM). Multiplex networks can be defined as networks with vertices of one kind but M different types of edges, representing different types of interactions. For such networks, the k-core is defined as the largest subgraph in which each vertex has at least ki edges of each type, i =1,2,...,M. We derive self-consistency equations to obtain the birth points of the k-cores and their relative sizes for uncorrelated multiplex networks with an arbitrary degree distribution. To clarify our general results, we consider in detail multiplex networks with edges of two types and solve the equations in the particular case of Erdős-Rényi and scale-free multiplex networks. We find hybrid phase transitions at the emergence points of k-cores except the (1,1)-core for which the transition is continuous. We apply the k-core decomposition algorithm to air-transportation multiplex networks, composed of two layers, and obtain the size of (k1,k2)-cores.

  16. Real-time PCR-based assay for quantitative detection of Hematodinium sp. in the blue crab Callinectes sapidus.

    PubMed

    Nagle, L; Place, A R; Schott, E J; Jagus, R; Messick, G; Pitula, J S

    2009-03-09

    Hematodinium sp. is a parasitic dinoflagellate infecting the blue crab Callinectes sapidus and other crustaceans. PCR-based assays are currently being used to identify infections in crabs that would have been undetectable by traditional microscopic examination. We therefore sought to define the limits of quantitative PCR (qPCR) detection within the context of field collection protocols. We present a qPCR assay based on the Hematodinium sp. 18S rRNA gene that can detect 10 copies of the gene per reaction. Analysis of a cell dilution series vs. defined numbers of a cloned Hematodinium sp. 18S rRNA gene suggests a copy number of 10,000 per parasite and predicts a sensitivity of 0.001 cell equivalents. In practice, the assays are based on analysis of 1% of the DNA extracted from 200 microl of serum, yielding a theoretical detection limit of 5 cells ml(-1) hemolymph, assuming that 1 cell is present per sample. When applied to a limited field survey of blue crabs collected in Maryland coastal bays from May to August 2005, 24 of 128 crabs (18.8%) were identified as positive for Hematodinium sp. infection using qPCR. In comparison, only 6 of 128 crabs (4.7%) were identified as positive using traditional hemolymph microscopic examination. The qPCR method also detected the parasite in gill, muscle, heart and hepatopancreas tissues, with 17.2% of the crabs showing infection in at least one of these tissues. Importantly, it is now possible to enumerate parasites within defined quantities of crab tissue, which permits collection of more detailed information on the epizootiology of the pathogen.

  17. [Evaluation of COBAS TaqMan: a real-time PCR-based diagnostic kit for mycobacteria].

    PubMed

    Yonemaru, Makoto; Horiba, Masahide; Tada, Atsuhiko; Nagai, Takayuki

    2009-12-01

    The real-time PCR-based diagnostic kits, COBAS TaqMan MTB and COBAS TaqMan MAI (Roche Diagnostics, Tokyo, Japan), were developed to detect Mycobacterium tuberculosis (MTB) and M. avium (MAV)/M. intracellulare (MIN), respectively. The TaqMan kits simultaneously perform amplification and detection of mycobacterial DNA to reduce assay time. We evaluated the diagnostic accuracy of both TaqMan kits in 781 clinical specimens, and compared the results with those obtained from the AMPLICOR MTB and MAI kits. With smear-positive specimens, the TaqMan kits showed 100% concordance with AMPLICOR in MTB, MAV and MIN. With all specimens, the concordances of TaqMan with AMPLICOR were 99.1%, 99.0%, and 99.7% in MTB, MAV and MIN, respectively. Four specimens for MTB and one for MAV were AMPLICOR positive/TaqMan negative. Among them, two specimens were culture-positive for MTB and one for MAV. Three specimens for MTB, seven for MAV, and two for MIN were AMPLICOR negative/TaqMan positive. Among them, two specimens were culture-positive for MTB, seven for MAV, and one for MIN. In twelve out of 21 specimens in which AMPLICOR failed to activate PCR, TaqMan successfully determined the results which were in concordance with those of mycobacterial culture. Thus, our data suggest that the accuracy of TaqMan in detecting mycobacterial DNA is superior to that of AMPLICOR. We conclude that TaqMan, which is an easy and rapid DNA amplification test, is useful for detecting MTB, MAV and MIN.

  18. Evaluation and validation of reference genes for normalization of quantitative real-time PCR based gene expression studies in peanut.

    PubMed

    Reddy, Dumbala Srinivas; Bhatnagar-Mathur, Pooja; Cindhuri, Katamreddy Sri; Sharma, Kiran K

    2013-01-01

    The quantitative real-time PCR (qPCR) based techniques have become essential for gene expression studies and high-throughput molecular characterization of transgenic events. Normalizing to reference gene in relative quantification make results from qPCR more reliable when compared to absolute quantification, but requires robust reference genes. Since, ideal reference gene should be species specific, no single internal control gene is universal for use as a reference gene across various plant developmental stages and diverse growth conditions. Here, we present validation studies of multiple stably expressed reference genes in cultivated peanut with minimal variations in temporal and spatial expression when subjected to various biotic and abiotic stresses. Stability in the expression of eight candidate reference genes including ADH3, ACT11, ATPsyn, CYP2, ELF1B, G6PD, LEC and UBC1 was compared in diverse peanut plant samples. The samples were categorized into distinct experimental sets to check the suitability of candidate genes for accurate and reliable normalization of gene expression using qPCR. Stability in expression of the references genes in eight sets of samples was determined by geNorm and NormFinder methods. While three candidate reference genes including ADH3, G6PD and ELF1B were identified to be stably expressed across experiments, LEC was observed to be the least stable, and hence must be avoided for gene expression studies in peanut. Inclusion of the former two genes gave sufficiently reliable results; nonetheless, the addition of the third reference gene ELF1B may be potentially better in a diverse set of tissue samples of peanut.

  19. Development of a PCR-based strategy for CYP2D6 genotyping including gene multiplication of worldwide potential use.

    PubMed

    Dorado, Pedro; Cáceres, Macarena; Pozo-Guisado, Eulalia; Wong, Ma-Li; Licinio, Julio; Llerena, Adrián

    2005-10-01

    There is growing consensus on the potential use of pharmacogenetics in clinical practice, and hopes have been expressed for application to the improvement of global health. However, two major challenges may lead to widening the "biotechnological gap" between the developing and the industrial world; first the unaffordability of some current technologies for poorer countries, and second the necessity of analyzing all described alleles for every clinical case due to the inability to predict the ethnic group of a given patient. Because of its role in the metabolism of a number of drugs, cytochrome P450 2D6 (CYP2D6) is an excellent candidate for use in the optimization of drug therapy. CYP2D6 is a highly polymorphic gene locus with more than 50 variant alleles, and subjects can be classified as poor metabolizers (PM), extensive metabolizers (EM), or ultrarapid metabolizers (UM) of a given CYP2D6 substrate. Several strategies and methods for CYP2D6 genotyping exist. Some, however, are expensive and laborious. The aim of this study was to design a PCR-based genotyping methodology to allow rapid, straightforward, and inexpensive identification of 90%-95% of CYP2D6 PM or UM genotypes for routine clinical use, independent of the individual's ethnic group. CYP2D6 is amplified in initial extra long PCRs (XL-PCRs), which subsequently undergo fragment-length polymorphism analysis for the determination of carriers of CYP2D6 allelic variants. The same XL-PCRs are also used for the determination of CYP2D6 multiplication and 2D6*5 allele (abolished activity). The application of this new strategy for the detection of CYP2D6 mutated alleles and multiplications to routine clinical analysis will enable the PM and UM phenotypes to be predicted and identified at a reasonable cost in a large number of individuals at most locations.

  20. Development of a PCR-based strategy for CYP2D6 genotyping including gene multiplication of worldwide potential use.

    PubMed

    Dorado, Pedro; Cáceres, Macarena C; Pozo-Guisado, Eulalia; Wong, Ma-Li; Licinio, Julio; Llerena, Adrian

    2005-10-01

    There is growing consensus on the potential use of pharmacogenetics in clinical practice, and hopes have been expressed for application to the improvement of global health. However, two major challenges may lead to widening the "biotechnological gap" between the developing and the industrial world;first the unaffordability of some current technologies for poorer countries, and second the necessity of analyzing all described alleles for every clinical case due to the inability to predict the ethnic group of a given patient. Because of its role in the metabolism of a number of drugs, cytochrome P450 2D6 (CYP2D6) is an excellent candidate for use in the optimization of drug therapy. CYP2D6 is a highly polymorphic gene locus with more than 50 variant alleles, and subjects can be classified as poor metabolizers (PM), extensive metabolizers (EM), or ultrarapid metabolizers (UM) of a given CYP2D6 substrate. Several strategies and methods for CYP2D6 genotyping exist. Some, however, are expensive and laborious. The aim of this study was to design a PCR-based genotyping methodology to allow rapid, straightforward, and inexpensive identification of 90%-95% of CYP2D6 PM or UM genotypes for routine clinical use, independent of the individual's ethnic group. CYP2D6 is amplified in initial extra long PCRs (XL-PCRs), which subsequently undergo fragment-length polymorphism analysis for the determination of carriers of CYP2D6 allelic variants. The same XL-PCRs are also used for the determination of CYP2D6 multiplication and 2D6*5 allele (abolished activity). The application of this new strategy for the detection of CYP2D6 mutated alleles and multiplications to routine clinical analysis will enable the PM and UM phenotypes to be predicted and identified at a reasonable cost in a large number of individuals at most locations.

  1. Comparison of array comparative genomic hybridization and quantitative real-time PCR-based aneuploidy screening of blastocyst biopsies.

    PubMed

    Capalbo, Antonio; Treff, Nathan R; Cimadomo, Danilo; Tao, Xin; Upham, Kathleen; Ubaldi, Filippo Maria; Rienzi, Laura; Scott, Richard T

    2015-07-01

    Comprehensive chromosome screening (CCS) methods are being extensively used to select chromosomally normal embryos in human assisted reproduction. Some concerns related to the stage of analysis and which aneuploidy screening method to use still remain. In this study, the reliability of blastocyst-stage aneuploidy screening and the diagnostic performance of the two mostly used CCS methods (quantitative real-time PCR (qPCR) and array comparative genome hybridization (aCGH)) has been assessed. aCGH aneuploid blastocysts were rebiopsied, blinded, and evaluated by qPCR. Discordant cases were subsequently rebiopsied, blinded, and evaluated by single-nucleotide polymorphism (SNP) array-based CCS. Although 81.7% of embryos showed the same diagnosis when comparing aCGH and qPCR-based CCS, 18.3% (22/120) of embryos gave a discordant result for at least one chromosome. SNP array reanalysis showed that a discordance was reported in ten blastocysts for aCGH, mostly due to false positives, and in four cases for qPCR. The discordant aneuploidy call rate per chromosome was significantly higher for aCGH (5.7%) compared with qPCR (0.6%; P<0.01). To corroborate these findings, 39 embryos were simultaneously biopsied for aCGH and qPCR during blastocyst-stage aneuploidy screening cycles. 35 matched including all 21 euploid embryos. Blinded SNP analysis on rebiopsies of the four embryos matched qPCR. These findings demonstrate the high reliability of diagnosis performed at the blastocyst stage with the use of different CCS methods. However, the application of aCGH can be expected to result in a higher aneuploidy rate than other contemporary methods of CCS.

  2. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences

    PubMed Central

    Ågren, Joakim; Hamidjaja, Raditijo A; Hansen, Trine; Ruuls, Robin; Thierry, Simon; Vigre, Håkan; Janse, Ingmar; Sundström, Anders; Segerman, Bo; Koene, Miriam; Löfström, Charlotta; Van Rotterdam, Bart; Derzelle, Sylviane

    2013-01-01

    Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. For the detection of chromosomal DNA, different genes have been used, such as BA813, rpoB, gyrA, plcR, S-layer, and prophage-lambda. Following a review of the literature, an in silico analysis of all signature sequences reported for identification of B. anthracis was conducted. Published primer and probe sequences were compared for specificity against 134 available Bacillus spp. genomes. Although many of the chromosomal targets evaluated are claimed to be specific to B. anthracis, cross-reactions with closely related B. cereus and B. thuringiensis strains were often observed. Of the 35 investigated PCR assays, only 4 were 100% specific for the B. anthracis chromosome. An interlaboratory ring trial among five European laboratories was then performed to evaluate six assays, including the WHO recommended procedures, using a collection of 90 Bacillus strains. Three assays performed adequately, yielding no false positive or negative results. All three assays target chromosomal markers located within the lambdaBa03 prophage region (PL3, BA5345, and BA5357). Detection limit was further assessed for one of these highly specific assays. PMID:24005110

  3. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences.

    PubMed

    Ågren, Joakim; Hamidjaja, Raditijo A; Hansen, Trine; Ruuls, Robin; Thierry, Simon; Vigre, Håkan; Janse, Ingmar; Sundström, Anders; Segerman, Bo; Koene, Miriam; Löfström, Charlotta; Van Rotterdam, Bart; Derzelle, Sylviane

    2013-11-15

    Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. For the detection of chromosomal DNA, different genes have been used, such as BA813, rpoB, gyrA, plcR, S-layer, and prophage-lambda. Following a review of the literature, an in silico analysis of all signature sequences reported for identification of B. anthracis was conducted. Published primer and probe sequences were compared for specificity against 134 available Bacillus spp. genomes. Although many of the chromosomal targets evaluated are claimed to be specific to B. anthracis, cross-reactions with closely related B. cereus and B. thuringiensis strains were often observed. Of the 35 investigated PCR assays, only 4 were 100% specific for the B. anthracis chromosome. An interlaboratory ring trial among five European laboratories was then performed to evaluate six assays, including the WHO recommended procedures, using a collection of 90 Bacillus strains. Three assays performed adequately, yielding no false positive or negative results. All three assays target chromosomal markers located within the lambdaBa03 prophage region (PL3, BA5345, and BA5357). Detection limit was further assessed for one of these highly specific assays.

  4. Evaluation of a Novel PCR-Based Assay for Detection and Identification of Chlamydia trachomatis Serovars in Cervical Specimens▿

    PubMed Central

    Quint, Koen; Porras, Carolina; Safaeian, Mahboobeh; González, Paula; Hildesheim, Allan; Quint, Wim; van Doorn, Leen-Jan; Silva, Sandra; Melchers, Willem; Schiffman, Mark; Rodríguez, Ana Cecilia; Wacholder, Sholom; Freer, Enrique; Cortes, Bernal; Herrero, Rolando

    2007-01-01

    The aims of this study were to compare a novel PCR-based Chlamydia trachomatis detection and genotyping (Ct-DT) assay with the FDA-approved, commercially available C. trachomatis detection Hybrid Capture 2 (HC2) assay and to investigate the C. trachomatis serovar distribution among young women in a rural Costa Rican study population. A total of 5,828 sexually active women participating in a community-based trial in Costa Rica were tested for C. trachomatis by HC2. A sample of 1,229 specimens consisting of 100% HC2 C. trachomatis-positive specimens (n = 827) and a random sample of 8% HC2 C. trachomatis-negative specimens (n = 402) were tested with the Ct-DT assay. Agreement between the two assays was determined by the unweighted kappa statistic. Discrepant specimens were tested with a second commercially available test (COBAS TaqMan). The Ct-DT-positive specimens were further analyzed with the Ct-DT genotyping step to investigate the distribution of 14 different C. trachomatis serovars (A, B/Ba, C, D/Da, E, F, G/Ga, H, I/Ia, J, K, L1, L2/L2a, and L3). After accounting for the sampling fraction selected for Ct-DT testing, crude agreement with the HC2 assay was 98% and the kappa was 0.92 (95% confidence interval [CI], 0.89 to 0.97). The 33 discordant samples that were further analyzed with the COBAS TaqMan test showed better agreement with the Ct-DT assay (31/33, P < 0.001). Among the 806 Ct-DT-positive samples, serovar E was the most common serovar (31%), followed by serovars F and D (both 21%) and serovar I (15%). In conclusion, the novel Ct-DT assay permits reliable detection and identification of C. trachomatis serovars. PMID:17959760

  5. PCR-based diagnosis of surra-targeting VSG gene: experimental studies in small laboratory rodents and buffalo.

    PubMed

    Sengupta, P P; Balumahendiran, M; Suryanaryana, V V S; Raghavendra, A G; Shome, B R; Gajendragad, M R; Prabhudas, K

    2010-07-15

    Trypanosoma evansi, the causative organism of 'surra' expresses its variable surface glycoprotein (VSG) at early, middle and late stages of infection in animals. The variable antigenic nature of VSG caused by switching its expression type favours evasion from the host immune response and leads to chronic and persistent infection. Developing a polymerase chain reaction (PCR)-based diagnostic tool targeting the VSG gene is expected to be highly specific and sensitive for diagnosis of surra. Hence, in the present study, we have designed EXP3F/4R primer pair and amplified the 1.4 kb of VSG gene of T. evansi and studied the phylogenetic relationship by in silico analysis. The PCR method was standardised using another set of primer, DITRYF/R, and 400 bp was amplified from blood and tissue samples of experimentally infected animals. Applying the PCR method, we were able to detect as low as 0.15 trypanosomeml(-1). Considering the number of parasite-to-DNA concentration, the PCR method has a sensitivity of 0.015 pg ml(-1). The PCR could detect the presence of the parasite as early as 24h post-infection (p.i.) and 72 h p.i., respectively, in experimentally infected rats and buffalo. No amplification was observed with DNA of Babesia bigemina and Theileria annulata, indicating the primers are specific for T. evansi. The PCR method could detect the dog, lion and leopard isolates of T. evansi. Similarly, amplifying the DNA from the experimentally infected tissues was also found to be sensitive. Thus, the findings of this study favour the application of PCR over the parasitological methods for the detection of the early and/or chronic stage of surra in domestic and wild animals.

  6. Evaluation and Validation of Reference Genes for Normalization of Quantitative Real-Time PCR Based Gene Expression Studies in Peanut

    PubMed Central

    Cindhuri, Katamreddy Sri; Sharma, Kiran K.

    2013-01-01

    The quantitative real-time PCR (qPCR) based techniques have become essential for gene expression studies and high-throughput molecular characterization of transgenic events. Normalizing to reference gene in relative quantification make results from qPCR more reliable when compared to absolute quantification, but requires robust reference genes. Since, ideal reference gene should be species specific, no single internal control gene is universal for use as a reference gene across various plant developmental stages and diverse growth conditions. Here, we present validation studies of multiple stably expressed reference genes in cultivated peanut with minimal variations in temporal and spatial expression when subjected to various biotic and abiotic stresses. Stability in the expression of eight candidate reference genes including ADH3, ACT11, ATPsyn, CYP2, ELF1B, G6PD, LEC and UBC1 was compared in diverse peanut plant samples. The samples were categorized into distinct experimental sets to check the suitability of candidate genes for accurate and reliable normalization of gene expression using qPCR. Stability in expression of the references genes in eight sets of samples was determined by geNorm and NormFinder methods. While three candidate reference genes including ADH3, G6PD and ELF1B were identified to be stably expressed across experiments, LEC was observed to be the least stable, and hence must be avoided for gene expression studies in peanut. Inclusion of the former two genes gave sufficiently reliable results; nonetheless, the addition of the third reference gene ELF1B may be potentially better in a diverse set of tissue samples of peanut. PMID:24167633

  7. PCR-based screening and lineage identification of Trypanosoma cruzi directly from faecal samples of triatomine bugs from northwestern Argentina

    PubMed Central

    MARCET, P. L.; DUFFY, T.; CARDINAL, M. V.; BURGOS, J. M.; LAURICELLA, M. A.; LEVIN, M. J.; KITRON, U.; GÜRTLER, R. E.; SCHIJMAN, A. G.

    2007-01-01

    SUMMARY This study applied improved DNA extraction and polymerase chain reaction strategies for screening and identification of Trypanosoma cruzi lineages directly from faeces of triatomines collected in a well-defined rural area in northwestern Argentina. Amplification of the variable regions of the kinetoplastid minicircle genome (kDNA-PCR) was performed in faecal lysates from 33 microscope (MO)-positive and 93 MO-negative Triatoma infestans, 2 MO-positive and 38 MO-negative Triatoma guasayana and 2 MO-positive and 73 MO-negative Triatoma garciabesi. kDNA-PCR detected T. cruzi in 91% MO-positive and 7.5% MO-negative T. infestans, which were confirmed by amplification of the minicircle conserved region. In contrast, kDNA-PCR was negative in all faecal samples from the other triatomine species. A panel of PCR-based genomic markers (intergenic region of spliced-leader DNA, 24Sα and 18S rRNA genes and A10 sequence) was implemented to identify the parasite lineages directly in DNA lysates from faeces and culture isolates from 28 infected specimens. Two were found to be infected with TCI, 24 with TCIIe, 1 with TCIId and 1 revealed a mixed TCI+TCII infection in the faecal sample whose corresponding culture only showed TCII, providing evidence of the advantages of direct typing of biological samples. This study provides an upgrade in the current diagnosis and lineage identification of T. cruzi in field-collected triatomines and shows T. cruzi II strains as predominant in the region. PMID:16393354

  8. Codominant PCR-based markers and candidate genes for powdery mildew resistance in melon (Cucumis melo L.).

    PubMed

    Yuste-Lisbona, Fernando J; Capel, Carmen; Gómez-Guillamón, María L; Capel, Juan; López-Sesé, Ana I; Lozano, Rafael

    2011-03-01

    Powdery mildew caused by Podosphaera xanthii is a major disease in melon crops, and races 1, 2, and 5 of this fungus are those that occur most frequently in southern Europe. The genotype TGR-1551 bears a dominant gene that provides resistance to these three races of P. xanthii. By combining bulked segregant analysis and amplified fragment length polymorphisms (AFLP), we identified eight markers linked to this dominant gene. Cloning and sequencing of the selected AFLP fragments allowed the development of six codominant PCR-based markers which mapped on the linkage group (LG) V. Sequence analysis of these markers led to the identification of two resistance-like genes, MRGH5 and MRGH63, belonging to the nucleotide binding site (NBS)-leucine-rich repeat (LRR) gene family. Quantitative trait loci (QTL) analysis detected two QTLs, Pm-R1-2 and Pm-R5, the former significantly associated with the resistance to races 1 and 2 (LOD score of 26.5 and 33.3; 53.6 and 61.9% of phenotypic variation, respectively), and the latter with resistance to race 5 (LOD score of 36.8; 65.5% of phenotypic variation), which have been found to be colocalized with the MRGH5 and MRGH63 genes, respectively. The results suggest that the cluster of NBS-LRR genes identified in LG V harbours candidate genes for resistance to races 1, 2, and 5 of P. xanthii. The evaluation of other resistant germplasm showed that the codominant markers here reported are also linked to the Pm-w resistance gene carried by the accession 'WMR-29' proving their usefulness as genotyping tools in melon breeding programmes.

  9. Evaluation of PCR Based Assays for the Improvement of Proportion Estimation of Bacterial and Viral Pathogens in Diarrheal Surveillance

    PubMed Central

    Guan, Hongxia; Zhang, Jingyun; Xiao, Yong; Sha, Dan; Ling, Xia; Kan, Biao

    2016-01-01

    Diarrhea can be caused by a variety of bacterial, viral and parasitic organisms. Laboratory diagnosis is essential in the pathogen-specific burden assessment. In the pathogen spectrum monitoring in the diarrheal surveillance, culture methods are commonly used for the bacterial pathogens' detection whereas nucleic acid based amplification, the non-cultural methods are used for the viral pathogens. Different methodology may cause the inaccurate pathogen spectrum for the bacterial pathogens because of their different culture abilities with the different media, and for the comparison of bacterial vs. viral pathogens. The application of nucleic acid-based methods in the detection of viral and bacterial pathogens will likely increase the number of confirmed positive diagnoses, and will be comparable since all pathogens will be detected based on the same nucleic acid extracts from the same sample. In this study, bacterial pathogens, including diarrheagenic Escherichia coli (DEC), Salmonella spp., Shigella spp., Vibrio parahaemolyticus and V. cholerae, were detected in 334 diarrheal samples by PCR-based methods using nucleic acid extracted from stool samples and associated enrichment cultures. A protocol was established to facilitate the consistent identification of bacterial pathogens in diarrheal patients. Five common enteric viruses were also detected by RT-PCR, including rotavirus, sapovirus, norovirus (I and II), human astrovirus, and enteric adenovirus. Higher positive rates were found for the bacterial pathogens, showing the lower proportion estimation if only using culture methods. This application will improve the quality of bacterial diarrheagenic pathogen survey, providing more accurate information pertaining to the pathogen spectrum associated with finding of food safety problems and disease burden evaluation. PMID:27065958

  10. Aspergillus Collagen-Like Genes (acl): Identification, Sequence Polymorphism, and Assessment for PCR-Based Pathogen Detection

    PubMed Central

    Tuntevski, Kiril; Durney, Brandon C.; Snyder, Anna K.; LaSala, P. Rocco; Nayak, Ajay P.; Green, Brett J.; Beezhold, Donald H.; Rio, Rita V. M.; Holland, Lisa A.

    2013-01-01

    The genus Aspergillus is a burden to public health due to its ubiquitous presence in the environment, its production of allergens, and wide demographic susceptibility among cystic fibrosis, asthmatic, and immunosuppressed patients. Current methods of detection of Aspergillus colonization and infection rely on lengthy morphological characterization or nonstandardized serological assays that are restricted to identifying a fungal etiology. Collagen-like genes have been shown to exhibit species-specific conservation across the noncollagenous regions as well as strain-specific polymorphism in the collagen-like regions. Here we assess the conserved region of the Aspergillus collagen-like (acl) genes and explore the application of PCR amplicon size-based discrimination among the five most common etiologic species of the Aspergillus genus, including Aspergillus fumigatus, A. flavus, A. nidulans, A. niger, and A. terreus. Genetic polymorphism and phylogenetic analysis of the aclF1 gene were additionally examined among the available strains. Furthermore, the applicability of the PCR-based assay to identification of these five species in cultures derived from sputum and bronchoalveolar fluid from 19 clinical samples was explored. Application of capillary electrophoresis on nanogels was additionally demonstrated to improve the discrimination between Aspergillus species. Overall, this study demonstrated that Aspergillus acl genes could be used as PCR targets to discriminate between clinically relevant Aspergillus species. Future studies aim to utilize the detection of Aspergillus acl genes in PCR and microfluidic applications to determine the sensitivity and specificity for the identification of Aspergillus colonization and invasive aspergillosis in immunocompromised subjects. PMID:24123732

  11. PCR-Based Method To Differentiate the Subspecies of the Mycobacterium tuberculosis Complex on the Basis of Genomic Deletions

    PubMed Central

    Huard, Richard C.; de Oliveira Lazzarini, Luiz Claudio; Butler, W. Ray; van Soolingen, Dick; Ho, John L.

    2003-01-01

    The classical Mycobacterium tuberculosis complex (MtbC) subspecies include Mycobacterium tuberculosis, Mycobacterium africanum (subtypes I and II), Mycobacterium bovis (along with the attenuated M. bovis bacillus Calmette-Guérin [BCG]), and Mycobacterium microti; increasingly recognized MtbC groupings include Mycobacterium bovis subsp. caprae and “Mycobacterium tuberculosis subsp. canettii.” Previous investigations have documented each MtbC subspecies as a source of animal and/or human tuberculosis. However, study of these organisms is hindered by the lack of a single protocol that quickly and easily differentiates all of the MtbC groupings. Towards this end we have developed a rapid, simple, and reliable PCR-based MtbC typing method that makes use of MtbC chromosomal region-of-difference deletion loci. Here, seven primer pairs (which amplify within the loci 16S rRNA, Rv0577, IS1561′, Rv1510, Rv1970, Rv3877/8, and Rv3120) were run in separate but simultaneous reactions. Each primer pair either specifically amplified a DNA fragment of a unique size or failed, depending upon the source mycobacterial DNA. The pattern of amplification products from all of the reactions, visualized by agarose gel electrophoresis, allowed immediate identification either as MtbC composed of M. tuberculosis (or M. africanum subtype II), M. africanum subtype I, M. bovis, M. bovis BCG, M. caprae, M. microti, or “M. canettii” or as a Mycobacterium other than MtbC (MOTT). This MtbC PCR typing panel provides an advanced approach to determine the subspecies of MtbC isolates and to differentiate them from clinically important MOTT species. It has proven beneficial in the management of Mycobacterium collections and may be applied for practical clinical and epidemiological use. PMID:12682155

  12. A method of multiplex PCR for detection of field released Beauveria bassiana, a fungal entomopathogen applied for pest management in jute (Corchorus olitorius).

    PubMed

    Biswas, Chinmay; Dey, Piyali; Gotyal, B S; Satpathy, Subrata

    2015-04-01

    The fungal entomopathogen Beauveria bassiana is a promising biocontrol agent for many pests. Some B. bassiana strains have been found effective against jute pests. To monitor the survival of field released B. bassiana a rapid and efficient detection technique is essential. Conventional methods such as plating method or direct culture method which are based on cultivation on selective media followed by microscopy are time consuming and not so sensitive. PCR based methods are rapid, sensitive and reliable. A single primer PCR may fail to amplify some of the strains. However, multiplex PCR increases the possibility of detection as it uses multiple primers. Therefore, in the present investigation a multiplex PCR protocol was developed by multiplexing three primers SCA 14, SCA 15 and SCB 9 to detect field released B. bassiana strains from soil as well as foliage of jute field. Using our multiplex PCR protocol all the five B. bassiana strains could be detected from soil and three strains viz., ITCC 6063, ITCC 4563 and ITCC 4796 could be detected even from the crop foliage after 45 days of spray.

  13. Multiplexing Prisms for Field Expansion.

    PubMed

    Peli, Eli; Jung, Jae-Hyun

    2017-08-01

    Prisms used for field expansion are limited by the optical scotoma at a prism apex (apical scotoma). For a patient with two functioning eyes, fitting prisms unilaterally allows the other eye to compensate for the apical scotoma. A monocular patient's field loss cannot be expanded with a conventional or Fresnel prism because of the apical scotoma. A newly invented optical device, the multiplexing prism (MxP), was developed to overcome the apical scotoma limitation in monocular field expansion. A Fresnel-prism-like device with alternating prism and flat elements superimposes shifted and see-through views, thus creating the (monocular) visual confusion required for field expansion and eliminating the apical scotoma. Several implementations are demonstrated and preliminarily evaluated for different monocular conditions with visual field loss. The field expansion of the MxP is compared with the effect of conventional prisms using calculated and measured perimetry. Field expansion without apical scotomas is shown to be effective for monocular patients with hemianopia or constricted peripheral field. The MxPs are shown to increase the nasal field for a patient with only one eye and for patients with bitemporal hemianopia. The MxPs placed at the far temporal field are shown to expand the normal visual field. The ability to control the contrast ratio between the two images is verified. A novel optical device is demonstrated to have the potential for field expansion technology in a variety of conditions. The devices may be inexpensive and can be constructed in a cosmetically acceptable format.

  14. Integrated three channel laser and optical multiplexer for narrowband wavelength division multiplexing

    NASA Astrophysics Data System (ADS)

    Ragdale, C. M.; Reid, T. J.; Reid, D. C. J.; Carter, A. C.

    1994-05-01

    The fabrication and characterization of a monolithically integrated three channel narrowband wavelength multiplexer and DBR laser are reported. The multiplexers include Bragg gratings with an extinction ratio of greater than 20 dB anda bandwidth of approximately 1 nm to give channel spacings of less than 10 nm.

  15. A PCR-Based Assay by Sequence-Characterized DNA Markers for the Identification and Detection of Aphanomyces euteiches.

    PubMed

    Vandemark, G J; Kraft, J M; Larsen, R C; Gritsenko, M A; Boge, W L

    2000-10-01

    ABSTRACT Polymerase chain reaction (PCR) products were identified and amplified from isolates of Aphanomyces euteiches and A. cochlioides. The products were cloned and sequenced, and the data were used to design pairs of extended PCR primers to amplify sequence-characterized DNA markers. The primer pair OPC7-FS-30 and OPC7-RS-25 amplified a single 1,332-bp product from all isolates of A. euteiches that were not amplified from any other isolates tested. A single 718-bp product was selectively amplified only from isolates of A. cochlioides with the primer pair OPB10-FS-25 and OPB10-RS-25. A. euteiches was detected in roots of several varieties of field-grown peas collected from a root rot trial site. PCR also detected A. euteiches in the organic fraction of field soil samples. Both pairs of extended primers were used in a multiplex reaction to unambiguously discriminate between A. euteiches and A. cochlioides. Both pairs of primers were used in two-step PCR reactions in which annealing and extension was done in a single step at 72 degrees C. This reduced the time required for amplification of the diagnostic PCR product and its resolution by electrophoresis to less than 3 h.

  16. Interlaboratory transfer of a PCR multiplex method for simultaneous detection of four genetically modified maize lines: Bt11, MON810, T25, and GA21.

    PubMed

    Hernández, Marta; Rodríguez-Lázaro, David; Zhang, David; Esteve, Teresa; Pla, Maria; Prat, Salomé

    2005-05-04

    The number of cultured hectares and commercialized genetically modified organisms (GMOs) has increased exponentially in the past 9 years. Governments in many countries have established a policy of labeling all food and feed containing or produced by GMOs. Consequently, versatile, laboratory-transferable GMO detection methods are in increasing demand. Here, we describe a qualitative PCR-based multiplex method for simultaneous detection and identification of four genetically modified maize lines: Bt11, MON810, T25, and GA21. The described system is based on the use of five primers directed to specific sequences in these insertion events. Primers were used in a single optimized multiplex PCR reaction, and sequences of the amplified fragments are reported. The assay allows amplification of the MON810 event from the 35S promoter to the hsp intron yielding a 468 bp amplicon. Amplification of the Bt11 and T25 events from the 35S promoter to the PAT gene yielded two different amplicons of 280 and 177 bp, respectively, whereas amplification of the 5' flanking region of the GA21 gave rise to an amplicon of 72 bp. These fragments are clearly distinguishable in agarose gels and have been reproduced successfully in a different laboratory. Hence, the proposed method comprises a rapid, simple, reliable, and sensitive (down to 0.05%) PCR-based assay, suitable for detection of these four GM maize lines in a single reaction.

  17. A comparative evaluation of PCR- based methods for species- specific determination of African animal trypanosomes in Ugandan cattle

    PubMed Central

    2013-01-01

    Background In recent years, PCR has been become widely applied for the detection of trypanosomes overcoming many of the constraints of parasitological and serological techniques, being highly sensitive and specific for trypanosome detection. Individual species-specific multi-copy trypanosome DNA sequences can be targeted to identify parasites. Highly conserved ribosomal RNA (rRNA) genes are also useful for comparisons between closely related species. The internal transcribed spacer regions (ITS) in particular are relatively small, show variability among related species and are flanked by highly conserved segments to which PCR primers can be designed. Individual variations in inter-species length makes the ITS region a useful marker for identification of multiple trypanosome species within a sample. Methods Six hundred blood samples from cattle collected in Uganda on FTA cards were screened using individual species-specific primers for Trypanosoma congolense, Trypanosoma brucei and Trypanosoma vivax and compared to a modified (using eluate extracted using chelex) ITS-PCR reaction. Results The comparative analysis showed that the species-specific primer sets showed poor agreement with the ITS primer set. Using species-specific PCR for Trypanozoon, a prevalence of 10.5% was observed as compared to 0.2% using ITS PCR (Kappa = 0.03). For Trypanosoma congolense, the species-specific PCR reaction indicated a prevalence of 0% compared to 2.2% using ITS PCR (Kappa = 0). For T. vivax, species-specific PCR detected prevalence of 5.7% compared to 2.8% for ITS PCR (Kappa = 0.29). Conclusions When selecting PCR based tools to apply to epidemiological surveys for generation of prevalence data for animal trypanosomiasis, it is recommended that species-specific primers are used, being the most sensitive diagnostic tool for screening samples to identify members of Trypanozoon (T. b. brucei s.l). While ITS primers are useful for studying the prevalence of trypanosomes

  18. Antimicrobial resistance of Enterococcus species from meat and fermented meat products isolated by a PCR-based rapid screening method.

    PubMed

    Jahan, Musarrat; Krause, Denis O; Holley, Richard A

    2013-05-15

    Enterococci are predominantly found in the gastrointestinal tract of humans and animals, but species commonly resident on vegetation are known. Their presence in large numbers in foods may indicate a lapse in sanitation and their ability to serve as a genetic reservoir of transferable antibiotic resistance is of concern. Conventional culture methods for identification of enterococci are slow and sometimes give false results because of the biochemical diversity of the organisms in this genus. This work reports the development of a PCR-based assay to detect enterococci at the genus level by targeting a 16S rRNA sequence. Published 16S rRNA sequences were aligned and used to design genus specific primers (EntF and EntR). The primers were able to amplify a 678 bp target region from Enterococcus faecalis ATCC 7080 and 20 other strains of enterococci from 11 different species, but there was no amplification by 32 species from closely related genera (Pediococcus, Lactobacillus, Streptococcus and Listeria) or species of Escherichia coli and Salmonella. The PCR positive samples were plated, screened by a colony patch technique and their identities were confirmed by API 20 Strep panels and sequencing. When dry fermented sausage and ham as well as fresh meat batter for dry cured sausage manufacture were tested for enterococci by the method, 29 Enterococcus strains (15 E. faecalis, 13 E. faecium, and one E. gallinarum) were identified. When susceptibility of these enterococci to 12 antibiotics was tested, the highest incidence of resistance was to clindamycin (89.6%), followed by tetracycline hydrochloride (65.5%), tylosin (62%), erythromycin (45%), streptomycin and neomycin (17%), chloramphenicol (10.3%), penicillin (10.3%), ciprofloxacin (10.3%) and gentamicin (3.4%). None was resistant to the clinically important drugs vancomycin or ampicillin. Most strains (27/29) were resistant to more than one antibiotic while 17 of 29 strains were resistant to three to 8 antibiotics

  19. Super-multiplex vibrational imaging

    NASA Astrophysics Data System (ADS)

    Wei, Lu; Chen, Zhixing; Shi, Lixue; Long, Rong; Anzalone, Andrew V.; Zhang, Luyuan; Hu, Fanghao; Yuste, Rafael; Cornish, Virginia W.; Min, Wei

    2017-04-01

    potential of this 24-colour (super-multiplex) optical imaging approach for elucidating intricate interactions in complex biological systems.

  20. Multiplexing of volume holographic wavelet correlation processor

    NASA Astrophysics Data System (ADS)

    Feng, Wenyi; Yan, Yingbai; Jin, Guofan; Wu, Minxian; He, Qingsheng

    2000-03-01

    Volume holographic associative memory in a photorefractive crystal provides an inherent mechanism to develop a multi-channel correlation identification system with high parallelism. Wavelet transform is introduced to improve discrimination of the system. We first investigate parameters of the system for parallelism enhancement, and then study multiplexing of the system on input objects and wavelet filters. A general volume holographic wavelet correlation processor has a single input-object channel and a single wavelet-filtering channel. In other words, it can only process one input object with one wavelet filter at a same time. Based on the fact that a volume holographic correlator is not a shift-invariant system, multiplexing of input objects is proposed to improve parallelism of the processor. As a result, several input objects can be recognized simultaneously. Multiplexing of wavelet filters with different wavelet parameters is also achieved by a Dammann grating. Wavelet correlation outputs with different filters are synthesized to improve recognition accuracy of the processor. Corresponding experimental results in human face recognition are given. The combination of the input object multiplexing and the wavelet filter multiplexing is also described.

  1. Development of multiplex PCR for simultaneous detection and differentiation of six DNA and RNA viruses from clinical samples of sheep and goats.

    PubMed

    He, Ya-Peng; Zhang, Qi; Fu, Ming-Zhe; Xu, Xin-Gang

    2017-05-01

    Multiplex reverse transcription-polymerase chain reaction (RT-PCR) and PCR protocols were developed and subsequently evaluated for its effectiveness in detecting simultaneously single and mixed infections in sheep and goats. Specific primers for three DNA viruses and three RNA viruses, including foot and mouth disease virus (FMDV), Bluetongue virus (BTV), peste des petits ruminants virus (PPRV), sheeppox virus (SPPV), goatpox virus (GTPV) and orf virus (ORFV) were used for testing procedure. A single nucleic acid extraction protocol was adopted for the simultaneous extraction of both RNA and DNA viruses. The multiplex PCR consisted with two-step procedure which included reverse transcription of RNA virus and multiplex PCR of viral cDNA and DNA. The multiplex PCR assay was shown to be sensitive because it could detect at least 100pg of viral genomic DNA or RNA from a mixture of six viruses in a reaction. The assay was also highly specific in detecting one or more of the same viruses in various combinations in specimens. Thirty seven clinical samples collected from sheep and goats were detected among forty three samples tested by both uniplex and multiplex PCR, showing highly identification. As results of the sensitivity and specificity, the multiplex PCR is a useful approach for clinical diagnosis of mixed infections of DNA and RNA viruses in sheep and goats with a reaction. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Nested multiplex RT-PCR for detection and differentiation of West Nile virus and eastern equine encephalomyelitis virus in brain tissues.

    PubMed

    Johnson, Donna J; Ostlund, Eileen N; Schmitt, Beverly J

    2003-09-01

    A traditional nested reverse transcription-polymerase chain reaction (RT-PCR) assay specific for eastern equine encephalomyelitis (EEE) virus was designed to multiplex with a previously described West Nile (WN) virus nested RT-PCR assay. Differentiation of EEE and WN was based on base pair size of the amplified product. One hundred fifty-seven mammalian and avian brain tissues were tested by EEE/WN nested multiplex RT-PCR, EEE nested RT-PCR, and WN nested RT-PCR, and results were compared with other diagnostic test results from the same animals. Serological and virus isolation testing confirmed the results of the multiplex PCR assay. When compared with cell culture virus isolation, the multiplex assay was shown to be more sensitive in detecting the presence of EEE or WN virus in brain tissues. The multiplex assay was shown to be sensitive and specific for North American EEE and WN and provided a rapid means of identifying both viruses in brain tissues. No apparent sacrifice in sensitivity was observed in the multiplex procedure compared with the individual EEE and WN nested RT-PCR assays. Data collected from an additional 485 multiplex RT-PCR tests conducted during the summer and fall of 2002 further support the validity of the procedure.

  3. PCR-based verification of positive rapid diagnostic tests for intestinal protozoa infections with variable test band intensity.

    PubMed

    Becker, Sören L; Müller, Ivan; Mertens, Pascal; Herrmann, Mathias; Zondie, Leyli; Beyleveld, Lindsey; Gerber, Markus; du Randt, Rosa; Pühse, Uwe; Walter, Cheryl; Utzinger, Jürg

    2017-10-01

    Stool-based rapid diagnostic tests (RDTs) for pathogenic intestinal protozoa (e.g. Cryptosporidium spp. and Giardia intestinalis) allow for prompt diagnosis and treatment in resource-constrained settings. Such RDTs can improve individual patient management and facilitate population-based screening programmes in areas without microbiological laboratories for confirmatory testing. However, RDTs are difficult to interpret in case of 'trace' results with faint test band intensities and little is known about whether such ambiguous results might indicate 'true' infections. In a longitudinal study conducted in poor neighbourhoods of Port Elizabeth, South Africa, a total of 1428 stool samples from two cohorts of schoolchildren were examined on the spot for Cryptosporidium spp. and G. intestinalis using an RDT (Crypto/Giardia DuoStrip; Coris BioConcept). Overall, 121 samples were positive for G. intestinalis and the RDT suggested presence of cryptosporidiosis in 22 samples. After a storage period of 9-10 months in cohort 1 and 2-3 months in cohort 2, samples were subjected to multiplex PCR (BD Max™ Enteric Parasite Panel, Becton Dickinson). Ninety-three percent (112/121) of RDT-positive samples for G. intestinalis were confirmed by PCR, with a correlation between RDT test band intensity and quantitative pathogen load present in the sample. For Cryptosporidium spp., all positive RDTs had faintly visible lines and these were negative on PCR. The performance of the BD Max™ PCR was nearly identical in both cohorts, despite the prolonged storage at disrupted cold chain conditions in cohort 1. The Crypto/Giardia DuoStrip warrants further validation in communities with a high incidence of diarrhoea. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Primers to block the amplification of symbiotic apostome ciliate 18S rRNA gene in a PCR-based copepod diet study

    NASA Astrophysics Data System (ADS)

    Yi, Xiaoyan; Zhang, Huan; Liu, Guangxing

    2014-05-01

    Pelagic copepods play an important role in the marine food web. However, a full understanding of the ecological status of this zooplankton group depends on the careful study of their natural diets. In previous PCR-based copepod diet studies, we found many apostome ciliates that live symbiotically under the exoskeleton of the copepods, and their sequences were often over-represented in the 18S rRNA gene (18S rDNA) libraries. As a first step to address this issue, we designed three apostome ciliate 18S rDNA blocking primers, and tested their blocking efficiency against apostome ciliate 18s rDNA under various PCR conditions. Using a semi-quantitative PCR method, we optimized the conditions to efficiently amplify the 18S rDNA of the prey while simultaneously excluding the symbiotic apostome ciliates. This technique will facilitate PCR-based diet studies of copepods and other zooplankton in their natural environments.

  5. A qualitative look at multiplex gene expression of single cells using capillary electrophoresis.

    PubMed

    Zabzdyr, Jennifer L; Lillard, Sheri J

    2005-01-01

    We demonstrate the first use of capillary electrophoresis with laser-induced fluorescence (CE-LIF) for the qualitative analysis of single-cell multiplex products of the reverse transcriptase-polymerase chain reaction (RT-PCR). The expression of both estrogen receptor alpha (ERalpha) and beta-actin in individual MCF-7 cells was monitored using a one-pot reaction. Reverse transcription and a single round of touch-down PCR, performed in a multiplex format, were used to generate fragment sizes of 318 bp and 838 bp, for ERalpha and beta-actin, respectively. A replaceable hydroxypropylmethylcellulose sieving matrix was used to effect a size-based separation of ethidium bromide-bound DNA. As titration of RT-PCR reaction components did not appreciably influence multiplex product generation, the use of additives, including bovine serum albumin (BSA) and herring sperm DNA, was explored. The addition of BSA to the RT-PCR mixture only resulted in efficient amplification of beta-actin, whereas the DNA carrier allowed co-amplification of both ERalpha and beta-actin. Furthermore, the sensitivity of our CE-LIF method eliminated the need for a second round of nested PCR, typically required when RT-PCR products are analyzed using gel electrophoresis.

  6. Superconducting Digital Multiplexers for Sensor Arrays

    NASA Technical Reports Server (NTRS)

    Kadin, Alan M.; Brock, Darren K.; Gupta, Deepnarayan

    2004-01-01

    Arrays of cryogenic microbolometers and other cryogenic detectors are being developed for infrared imaging. If the signal from each sensor is amplified, multiplexed, and digitized using superconducting electronics, then this data can be efficiently read out to ambient temperature with a minimum of noise and thermal load. HYPRES is developing an integrated system based on SQUID amplifiers, a high-resolution analog-to-digital converter (ADC) based on RSFQ (rapid single flux quantum) logic, and a clocked RSFQ multiplexer. The ADC and SQUIDs have already been demonstrated for other projects, so this paper will focus on new results of a digital multiplexer. Several test circuits have been fabricated using Nb Josephson technology and are about to be tested at T = 4.2 K, with a more complete prototype in preparation.

  7. 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.

  8. ARMS-PCR for detection of BRAF V600E hotspot mutation in comparison with Real-Time PCR-based techniques.

    PubMed

    Machnicki, Marcin M; Glodkowska-Mrowka, Eliza; Lewandowski, Tomasz; Ploski, Rafał; Wlodarski, Pawel; Stoklosa, Tomasz

    2013-01-01

    BRAF mutation testing is one of the best examples how modern genetic testing may help to effectively use targeted therapies in cancer patients. Since many different genetic techniques are employed to assess BRAF mutation status with no available comparison of their sensitivity and usefulness for different types of samples, we decided to evaluate our own PCR-based assay employing the amplification refractory mutation system (ARMS-PCR) to detect the most common hotspot mutation c. T1799A (p. V600E) by comparing it with two qPCR based assays: a commercially available test with hybridizing probes (TIB MOLBIOL) and high resolution melting (HRM). Positive results were verified with Sanger sequencing. DNA from two cancer cell lines with known mutation status and from tissue samples from melanoma and gastric cancer was used. ARMS-PCR was the most sensitive method with the level of detection of the mutant allele at 2%. Similar sensitivity was observed for the qPCR-based commercial test employing hybridizing probes; however, this test cannot exclude negative results from poor or low quality samples. Another qPCR-based method, HRM, had lower sensitivity with the detection level of approximately 20%. An additional drawback of HRM methodology was the inability to distinguish between wild type and mutant homozygotes in a straightforward assay, probably due to the character of this particular mutation (T\\>A). Sanger sequencing had the sensitivity of the detection of mutant allele similar to HRM, approx. 20%. In conclusion, simple ARMS-PCR may be considered the method of choice for rapid, cost-effective screening for BRAF p. V600E mutation.

  9. Automated Methods for Multiplexed Pathogen Detection

    SciTech Connect

    Straub, Tim M.; Dockendorff, Brian P.; Quinonez-Diaz, Maria D.; Valdez, Catherine O.; Shutthanandan, Janani I.; Tarasevich, Barbara J.; Grate, Jay W.; Bruckner-Lea, Cindy 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

  10. 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

  11. Spectral analysis of multiplex Raman probe signatures.

    PubMed

    Lutz, Barry R; Dentinger, Claire E; Nguyen, Lienchi N; Sun, Lei; Zhang, Jingwu; Allen, April N; Chan, Selena; Knudsen, Beatrice S

    2008-11-25

    Raman nanoparticle probes are an emerging new class of optical labels for interrogation of physiological and pathological processes in bioassays, cells, and tissues. Although their unique emission signatures are ideal for multiplexing, the full potential of these probes has not been realized because conventional analysis methods are inadequate. We report a novel spectral fitting method that exploits the entire spectral signature to quantitatively extract individual probe signals from multiplex spectra. We evaluate the method in a series of multiplex assays using unconjugated and antibody-conjugated composite organic-inorganic nanoparticles (COINs). Results show sensitive multiplex detection of small signals (<2% of total signal) and similar detection limits in corresponding 4-plex and singlet plate binding assays. In a triplex assay on formalin-fixed human prostate tissue, two antibody-conjugated COINs and a conventional fluorophore are used to image expression of prostate-specific antigen, cytokeratin-18, and DNA. The spectral analysis method effectively removes tissue autofluorescence and other unknown background, allowing accurate and reproducible imaging (area under ROC curve 0.89 +/- 0.03) at subcellular spatial resolution. In all assay systems, the error attributable to spectral analysis constitutes multiplex spectra with overlapping peaks, (2) robust spot-by-spot removal of unknown background, (3) the opportunity to quantitatively assess the analysis error, (4) elimination of operator bias, and (5) simple automation appropriate for high-throughput analysis. The simple implementation and universal applicability of this approach significantly expands the potential of Raman probes for quantitative in vivo and ex vivo multiplex analysis.

  12. Advanced combinational microfluidic multiplexer for fuel cell reactors

    NASA Astrophysics Data System (ADS)

    Lee, D. W.; Doh, I.; Kim, Y.; Cho, Y.-H.

    2013-12-01

    An advanced combinational microfluidic multiplexer capable to address multiple fluidic channels for fuel cell reactors is proposed. Using only 4 control lines and two different levels of control pressures, the proposed multiplexer addresses up to 19 fluidic channels, at least two times larger than the previous microfluidic multiplexers. The present multiplexer providing high control efficiency and simple structure for channel addressing would be used in the application areas of the integrated microfluidic systems such as fuel cell reactors and dynamic pressure generators.

  13. Architecture of an all optical de-multiplexer for spatially multiplexed channels

    NASA Astrophysics Data System (ADS)

    Murshid, Syed H.; Finch, Michael F.; Lovell, Gregory L.

    2013-05-01

    Multiple channels of light can propagate through a multimode fiber without interfering with each other and can be independently detected at the output end of the fiber using spatial domain multiplexing (SDM). Each channel forms a separate concentric ring at the output. The typical single pin-diode structure cannot simultaneously detect and demultiplex the multiple channel propagation supported by the SDM architecture. An array of concentric circular pindiodes can be used to simultaneously detect and de-multiplex the SDM signals; however, an all optical solution is generally preferable. This paper presents simple architecture for an all optical SDM de-multiplexer.

  14. Multimode fiber optic wavelength division multiplexing

    NASA Technical Reports Server (NTRS)

    Spencer, J. L.

    1982-01-01

    Optical wavelength division multiplexing (WDM) systems, with signals transmitted on different wavelengths through a single optical fiber, can have increased bandwidth and fault isolation properties over single wavelength optical systems. Two WDM system designs that might be used with multimode fibers are considered and a general description of the components which could be used to implement the system are given. The components described are sources, multiplexers, demultiplexers, and detectors. Emphasis is given to the demultiplexer technique which is the major developmental component in the WDM system.

  15. Line graphs for a multiplex network.

    PubMed

    Criado, Regino; Flores, Julio; García Del Amo, Alejandro; Romance, Miguel; Barrena, Eva; Mesa, Juan A

    2016-06-01

    It is well known that line graphs offer a good summary of the graphs properties, which make them easier to analyze and highlight the desired properties. We extend the concept of line graph to multiplex networks in order to analyze multi-plexed and multi-layered networked systems. As these structures are very rich, different approaches to this notion are required to capture a variety of situations. Some relationships between these approaches are established. Finally, by means of some simulations, the potential utility of this concept is illustrated.

  16. Cooperative spreading processes in multiplex networks

    NASA Astrophysics Data System (ADS)

    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.

  17. A direct quantitative PCR-based measurement of herpes simplex virus susceptibility to antiviral drugs and neutralizing antibodies.

    PubMed

    Virók, Dezső P; Eszik, Ildikó; Mosolygó, Tímea; Önder, Kamil; Endrész, Valéria; Burián, Katalin

    2017-04-01

    the method, we applied the direct qPCR for antiviral inhibitory concentration 50 (IC50) measurements of known and novel antiviral compounds. The measured IC50 of acyclovir was ∼0.28μg/ml, similar to the previously published IC50 value. The IC50 of novel antiviral candidates was between 1.6-3.1μg/ml. The direct qPCR-based neutralization titres of HSV positive sera were 1:32-1:64, identical to the neutralization titres determined using a traditional neutralization assay. The negative sera did not inhibit the HSV-2 replication in either of the tests. Our direct qPCR method for the HSV-2 growth determination of antiviral IC50 and neutralization titre is less time-consuming, less subjective and a more accurate alternative to the traditional plaque titration and growth reduction assays.

  18. Simultaneous detection of Cymbidium mosaic virus and Odontoglossum ringspot virus in orchids using multiplex RT-PCR.

    PubMed

    Kim, Su Min; Choi, Sun Hee

    2015-12-01

    A system for simultaneous detection of two orchid-infecting viruses was developed and applied to several orchid species. The detection system involved multiplex reverse transcription-polymerase chain reaction (RT-PCR) and could simultaneously identify Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) from the orchid species studied. Multiplex RT-PCR was conducted using two virus-specific primer pairs and an internal control pair of primers to amplify the CymMV and ORSV coat protein regions, and orchid 18S rDNA, respectively. For optimization of multiplex RT-PCR conditions, serial dilutions of total RNA and cDNA were performed and the detection limit of the system was evaluated. The optimized multiplex detection system for CymMV and ORSV was applied to various orchid species, including several cultivars of Doritaenopsis, Cymbidium, Dendrobium, and Phalaenopsis to test the efficacy of this method. Our results indicate that the multiplex RT-PCR detection system will be a rapid, simple, and precise diagnosis tool in a range of orchid species.

  19. Rapid simultaneous detection of enterovirus and parechovirus RNAs in clinical samples by one-step real-time reverse transcription-PCR assay.

    PubMed

    Bennett, Susan; Harvala, Heli; Witteveldt, Jeroen; McWilliam Leitch, E Carol; McLeish, Nigel; Templeton, Kate; Gunson, Rory; Carman, William F; Simmonds, Peter

    2011-07-01

    Enteroviruses (EVs) are recognized as the major etiological agent in meningitis in children and young adults. The use of molecular techniques, such as PCR, has substantially improved the sensitivity of enterovirus detection compared to that of virus culture methods. PCR-based methods also can detect a much wider range of EV variants, including those within species A, as well as human parechoviruses (HPeVs) that often grow poorly in vitro and which previously have been underdiagnosed by traditional methods. To exploit these developments, we developed a real-time one-step reverse transcription-PCR (RT-PCR) for the rapid and sensitive detection of EV and HPeV in clinical specimens. Two commercially available RT-PCR kits were used (method I, Platinum one-step kit; method II, Express qPCR one-step kit) with primers and probes targeting the EV and HPeV 5'-untranslated regions (5'UTR). Amplification dynamics (threshold cycle [C(T)]values and efficiencies) of absolutely quantified full-length RNA transcripts representative of EV species A to D and HPeV were similar, demonstrating the effectiveness of both assays across the range of currently described human EV and HPeV variants. Probit analysis of multiple endpoint replicates demonstrated comparable sensitivities of the assays for EV and HPeV (method I, approximately 10 copies per reaction for both targets; method II, 20 copies per reaction). C(T) values were highly reproducible on repeat testing of positive controls within assays and between assay runs. Considering the sample turnaround time of less than 3 h, the multiplexed one-step RT-PCR method provides rapid diagnostic testing for EV and HPeV in cases of suspected central nervous system infections in a clinically relevant time frame.

  20. Rapid Simultaneous Detection of Enterovirus and Parechovirus RNAs in Clinical Samples by One-Step Real-Time Reverse Transcription-PCR Assay ▿

    PubMed Central

    Bennett, Susan; Harvala, Heli; Witteveldt, Jeroen; McWilliam Leitch, E. Carol; McLeish, Nigel; Templeton, Kate; Gunson, Rory; Carman, William F.; Simmonds, Peter

    2011-01-01

    Enteroviruses (EVs) are recognized as the major etiological agent in meningitis in children and young adults. The use of molecular techniques, such as PCR, has substantially improved the sensitivity of enterovirus detection compared to that of virus culture methods. PCR-based methods also can detect a much wider range of EV variants, including those within species A, as well as human parechoviruses (HPeVs) that often grow poorly in vitro and which previously have been underdiagnosed by traditional methods. To exploit these developments, we developed a real-time one-step reverse transcription-PCR (RT-PCR) for the rapid and sensitive detection of EV and HPeV in clinical specimens. Two commercially available RT-PCR kits were used (method I, Platinum one-step kit; method II, Express qPCR one-step kit) with primers and probes targeting the EV and HPeV 5′-untranslated regions (5′UTR). Amplification dynamics (threshold cycle [CT]values and efficiencies) of absolutely quantified full-length RNA transcripts representative of EV species A to D and HPeV were similar, demonstrating the effectiveness of both assays across the range of currently described human EV and HPeV variants. Probit analysis of multiple endpoint replicates demonstrated comparable sensitivities of the assays for EV and HPeV (method I, approximately 10 copies per reaction for both targets; method II, 20 copies per reaction). CT values were highly reproducible on repeat testing of positive controls within assays and between assay runs. Considering the sample turnaround time of less than 3 h, the multiplexed one-step RT-PCR method provides rapid diagnostic testing for EV and HPeV in cases of suspected central nervous system infections in a clinically relevant time frame. PMID:21593263

  1. Delay grid multiplexing: simple time-based multiplexing and readout method for silicon photomultipliers.

    PubMed

    Won, Jun Yeon; Ko, Guen Bae; Lee, Jae Sung

    2016-10-07

    In this paper, we propose a fully time-based multiplexing and readout method that uses the principle of the global positioning system. Time-based multiplexing allows simplifying the multiplexing circuits where the only innate traces that connect the signal pins of the silicon photomultiplier (SiPM) channels to the readout channels are used as the multiplexing circuit. Every SiPM channel is connected to the delay grid that consists of the traces on a printed circuit board, and the inherent transit times from each SiPM channel to the readout channels encode the position information uniquely. Thus, the position of each SiPM can be identified using the time difference of arrival (TDOA) measurements. The proposed multiplexing can also allow simplification of the readout circuit using the time-to-digital converter (TDC) implemented in a field-programmable gate array (FPGA), where the time-over-threshold (ToT) is used to extract the energy information after multiplexing. In order to verify the proposed multiplexing method, we built a positron emission tomography (PET) detector that consisted of an array of 4  ×  4 LGSO crystals, each with a dimension of 3  ×  3  ×  20 mm(3), and one- to-one coupled SiPM channels. We first employed the waveform sampler as an initial study, and then replaced the waveform sampler with an FPGA-TDC to further simplify the readout circuits. The 16 crystals were clearly resolved using only the time information obtained from the four readout channels. The coincidence resolving times (CRTs) were 382 and 406 ps FWHM when using the waveform sampler and the FPGA-TDC, respectively. The proposed simple multiplexing and readout methods can be useful for time-of-flight (TOF) PET scanners.

  2. Delay grid multiplexing: simple time-based multiplexing and readout method for silicon photomultipliers

    NASA Astrophysics Data System (ADS)

    Won, Jun Yeon; Ko, Guen Bae; Lee, Jae Sung

    2016-10-01

    In this paper, we propose a fully time-based multiplexing and readout method that uses the principle of the global positioning system. Time-based multiplexing allows simplifying the multiplexing circuits where the only innate traces that connect the signal pins of the silicon photomultiplier (SiPM) channels to the readout channels are used as the multiplexing circuit. Every SiPM channel is connected to the delay grid that consists of the traces on a printed circuit board, and the inherent transit times from each SiPM channel to the readout channels encode the position information uniquely. Thus, the position of each SiPM can be identified using the time difference of arrival (TDOA) measurements. The proposed multiplexing can also allow simplification of the readout circuit using the time-to-digital converter (TDC) implemented in a field-programmable gate array (FPGA), where the time-over-threshold (ToT) is used to extract the energy information after multiplexing. In order to verify the proposed multiplexing method, we built a positron emission tomography (PET) detector that consisted of an array of 4  ×  4 LGSO crystals, each with a dimension of 3  ×  3  ×  20 mm3, and one- to-one coupled SiPM channels. We first employed the waveform sampler as an initial study, and then replaced the waveform sampler with an FPGA-TDC to further simplify the readout circuits. The 16 crystals were clearly resolved using only the time information obtained from the four readout channels. The coincidence resolving times (CRTs) were 382 and 406 ps FWHM when using the waveform sampler and the FPGA-TDC, respectively. The proposed simple multiplexing and readout methods can be useful for time-of-flight (TOF) PET scanners.

  3. Moving through a multiplex holographic scene

    NASA Astrophysics Data System (ADS)

    Mrongovius, Martina

    2013-02-01

    This paper explores how movement can be used as a compositional element in installations of multiplex holograms. My holographic images are created from montages of hand-held video and photo-sequences. These spatially dynamic compositions are visually complex but anchored to landmarks and hints of the capturing process - such as the appearance of the photographer's shadow - to establish a sense of connection to the holographic scene. Moving around in front of the hologram, the viewer animates the holographic scene. A perception of motion then results from the viewer's bodily awareness of physical motion and the visual reading of dynamics within the scene or movement of perspective through a virtual suggestion of space. By linking and transforming the physical motion of the viewer with the visual animation, the viewer's bodily awareness - including proprioception, balance and orientation - play into the holographic composition. How multiplex holography can be a tool for exploring coupled, cross-referenced and transformed perceptions of movement is demonstrated with a number of holographic image installations. Through this process I expanded my creative composition practice to consider how dynamic and spatial scenes can be conveyed through the fragmented view of a multiplex hologram. This body of work was developed through an installation art practice and was the basis of my recently completed doctoral thesis: 'The Emergent Holographic Scene — compositions of movement and affect using multiplex holographic images'.

  4. A Wavelength Multiplexed Bidirectional Fiber Ring Network

    DTIC Science & Technology

    2007-11-02

    commercially available from many vendors. Depending on their application, they can be rather complex or quite simple in their functionality . A simple ADM...FBG implementation of circulators, it allows for bidirectional signal ths. The BADM in Figure 10 functions as multiplexer is also used in Figure 10...signature) _________________________ (date) Captain Robert Voigt , United States Navy Electrical Engineering Department Chair

  5. Microcomputer Multiplexes Alphanumeric Labels on CRT's

    NASA Technical Reports Server (NTRS)

    Cooper, T.

    1983-01-01

    External, low-power alphanumeric label generator eliminates costly video circuitry. Microprocessor-based system for multiplexing alphanumeric and analog data stores both program and data. Uses inexpensive circuits, consumes minimal current, is programmable by user, adapts to many CRT monitors. System generates 5-by-7 dot-matrix characters. System speed is adaquate for medical monitoring purposes.

  6. Fiber optics wavelength division multiplexing(components)

    NASA Technical Reports Server (NTRS)

    Hendricks, Herbert D.

    1985-01-01

    The long term objectives are to develop optical multiplexers/demultiplexers, different wavelength and modulation stable semiconductor lasers and high data rate transceivers, as well as to test and evaluate fiber optic networks applicable to the Space Station. Progress in each of the above areas is briefly discussed.

  7. Few Mode Multicore Photonic Lantern Multiplexer

    DTIC Science & Technology

    2016-01-01

    LP11a modes. To characterize the fabricated 21 mode multiplexer we used a super- luminescent diode centered at 1550 nm. Near field mode profiles...performed by coupling a super- luminescent diode centered at 1550nm. The experimental observations confirmed low insertion losses of less than 0.4 dB

  8. Determinants of public cooperation in multiplex networks

    NASA Astrophysics Data System (ADS)

    Battiston, Federico; Perc, Matjaž; Latora, Vito

    2017-07-01

    Synergies between evolutionary game theory and statistical physics have significantly improved our understanding of public cooperation in structured populations. Multiplex networks, in particular, provide the theoretical framework within network science that allows us to mathematically describe the rich structure of interactions characterizing human societies. While research has shown that multiplex networks may enhance the resilience of cooperation, the interplay between the overlap in the structure of the layers and the control parameters of the corresponding games has not yet been investigated. With this aim, we consider here the public goods game on a multiplex network, and we unveil the role of the number of layers and the overlap of links, as well as the impact of different synergy factors in different layers, on the onset of cooperation. We show that enhanced public cooperation emerges only when a significant edge overlap is combined with at least one layer being able to sustain some cooperation by means of a sufficiently high synergy factor. In the absence of either of these conditions, the evolution of cooperation in multiplex networks is determined by the bounds of traditional network reciprocity with no enhanced resilience. These results caution against overly optimistic predictions that the presence of multiple social domains may in itself promote cooperation, and they help us better understand the complexity behind prosocial behavior in layered social systems.

  9. Microwave multiplex readout for superconducting sensors

    NASA Astrophysics Data System (ADS)

    Ferri, E.; Becker, D.; Bennett, D.; Faverzani, M.; Fowler, J.; Gard, J.; Giachero, A.; Hays-Wehle, J.; Hilton, G.; Maino, M.; Mates, J.; Puiu, A.; Nucciotti, A.; Reintsema, C.; Schmidt, D.; Swetz, D.; Ullom, J.; Vale, L.

    2016-07-01

    The absolute neutrino mass scale is still an outstanding challenge in both particle physics and cosmology. The calorimetric measurement of the energy released in a nuclear beta decay is a powerful tool to determine the effective electron-neutrino mass. In the last years, the progress on low temperature detector technologies has allowed to design large scale experiments aiming at pushing down the sensitivity on the neutrino mass below 1 eV. Even with outstanding performances in both energy ( eV on keV) and time resolution ( 1 μs) on the single channel, a large number of detectors working in parallel is required to reach a sub-eV sensitivity. Microwave frequency domain readout is the best available technique to readout large array of low temperature detectors, such as Transition Edge Sensors (TESs) or Microwave Kinetic Inductance Detectors (MKIDs). In this way a multiplex factor of the order of thousands can be reached, limited only by the bandwidth of the available commercial fast digitizers. This microwave multiplexing system will be used to readout the HOLMES detectors, an array of 1000 microcalorimeters based on TES sensors in which the 163Ho will be implanted. HOLMES is a new experiment for measuring the electron neutrino mass by means of the electron capture (EC) decay of 163Ho. We present here the microwave frequency multiplex which will be used in the HOLMES experiment and the microwave frequency multiplex used to readout the MKID detectors developed in Milan as well.

  10. Immunity of multiplex networks via acquaintance vaccination

    NASA Astrophysics Data System (ADS)

    Wang, Zhen; Zhao, Da-Wei; Wang, Lin; Sun, Gui-Quan; Jin, Zhen

    2015-11-01

    How to find the effective approach of immunizing a population is one open question in the research of complex systems. Up to now, there have been a great number of works focusing on the efficiency of various immunization strategies. However, the majority of these existing achievements are limited to isolated networks, how immunization affects disease spreading in multiplex networks seems to need further exploration. In this letter, we explore the impact of the acquaintance immunization in multiplex networks, where two kinds of immunization strategies, multiplex node-based acquaintance immunization and layer node-based acquaintance immunization, are proposed. With the generating function method, our theoretical framework is able to accurately calculate the critical immunization threshold which is one of the most important indexes to predict the epidemic regime. Moreover, we further uncover that, with the increment of degree correlation between network layers, the immunization threshold declines for multiplex node-based acquaintance immunization, but slowly increases for layer node-based acquaintance immunization.

  11. Microcomputer Multiplexes Alphanumeric Labels on CRT's

    NASA Technical Reports Server (NTRS)

    Cooper, T.

    1983-01-01

    External, low-power alphanumeric label generator eliminates costly video circuitry. Microprocessor-based system for multiplexing alphanumeric and analog data stores both program and data. Uses inexpensive circuits, consumes minimal current, is programmable by user, adapts to many CRT monitors. System generates 5-by-7 dot-matrix characters. System speed is adaquate for medical monitoring purposes.

  12. High-speed multiplexing of keyboard data inputs

    NASA Technical Reports Server (NTRS)

    Anderson, T. O. (Inventor)

    1981-01-01

    A high speed multiplexing system is described in which keyboard entered data is sequentially and automatically sampled by the multiplexing system for input to a computer. A sequencer is provided which sequentially and automatically controls the multiplexer to sample each keyboard input in accordance with a predetermined sampling sequence. Whenever keyboard entered data appears on input lines to the multiplexer, the system inputs the keyboard data to the computer during a brief time interval in which the multiplexer remains at the particular keyboard address or port. Thus, a high speed sampling circuit is provided whereby the only operator action required is data entry through a keyboard. Priority or interrupt systems are not required.

  13. Error-Prone PCR-Based Mutagenesis Strategy for Rapidly Generating High-Yield Influenza Vaccine Candidates

    PubMed Central

    Ye, Jianqiang; Wen, Feng; Xu, Yifei; Zhao, Nan; Long, Liping; Sun, Hailiang; Yang, Jialiang; Cooley, Jim; Pharr, G. Todd; Webby, Richard; Wan, Xiu-Feng

    2015-01-01

    Vaccination is the primary strategy for the prevention and control of influenza outbreaks. However, the manufacture of influenza vaccine requires a high-yield seed strain, and the conventional methods for generating such strains are time consuming. In this study, we developed a novel method to rapidly generate high-yield candidate vaccine strains by integrating error-prone PCR, site-directed mutagenesis strategies, and reverse genetics. We used this method to generate seed strains for the influenza A(H1N1)pdm09 virus and produced six high-yield candidate strains. We used a mouse model to assess the efficacy of two of the six candidate strains as a vaccine seed virus: both strains provided complete protection in mice against lethal challenge, thus validating our method. Results confirmed that the efficacy of these candidate vaccine seed strains was not affected by the yield-optimization procedure. PMID:25899178

  14. The physiologic state of Escherichia coli O157:H7 does not affect its detection in two commercial real-time PCR-based tests

    USDA-ARS?s Scientific Manuscript database

    Multiplex real-time PCR detection of Escherichia coli O157:H7 is an efficient molecular tool with high sensitivity and specificity for meat safety and quality assurance in the beef industry. The Biocontrol GDS and the DuPont Qualicon BAX®-RT rapid detection systems are two commercial tests based on...

  15. Antibody-Based Protein Multiplex Platforms: Technical and Operational Challenges

    PubMed Central

    Ellington, Allison A.; Kullo, Iftikhar J.; Bailey, Kent R.; Klee, George G.

    2010-01-01

    BACKGROUND The measurement of multiple protein biomarkers may refine risk stratification in clinical settings. This concept has stimulated development of multiplexed immunoassay platforms that provide multiple, parallel protein measurements on the same specimen. CONTENT We provide an overview of antibody-based multiplexed immunoassay platforms and discuss technical and operational challenges. Multiplexed immunoassays use traditional immunoassay principles in which high-affinity capture ligands are immobilized in parallel arrays in either planar format or on microspheres in suspension. Development of multiplexed immunoassays requires rigorous validation of assay configuration and analytical performance to minimize assay imprecision and inaccuracy. Challenges associated with multiplex configuration include selection and immobilization of capture ligands, calibration, interference between antibodies and proteins and assay diluents, and compatibility of assay limits of quantification. We discuss potential solutions to these challenges. Criteria for assessing analytical multiplex assay performance include the range of linearity, analytical specificity, recovery, and comparison to a quality reference method. Quality control materials are not well developed for multiplexed protein immunoassays, and algorithms for interpreting multiplex quality control data are needed. SUMMARY Technical and operational challenges have hindered implementation of multiplexed assays in clinical settings. Formal procedures that guide multiplex assay configuration, analytical validation, and quality control are needed before broad application of multiplexed arrays can occur in the in vitro diagnostic market. PMID:19959625

  16. A novel IPTV program multiplex access system to EPON

    NASA Astrophysics Data System (ADS)

    Xu, Xian; Liu, Deming; He, Wei; Lu, Xi

    2007-11-01

    With the rapid development of high speed networks, such as Ethernet Passive Optical Network (EPON), traffic patterns in access networks have evolved from traditional text-oriented service to the mixed text-, voice- and video- based services, leading to so called "Triple Play". For supporting IPTV service in EPON access network infrastructure, in this article we propose a novel IPTV program multiplex access system to EPON, which enables multiple IPTV program source servers to seamlessly access to IPTV service access port of optical line terminal (OLT) in EPON. There are two multiplex schemes, namely static multiplex scheme and dynamic multiplex scheme, in implementing the program multiplexing. Static multiplex scheme is to multiplex all the IPTV programs and forward them to the OLT, regardless of the need of end-users. While dynamic multiplex scheme can dynamically multiplex and forward IPTV programs according to what the end-users actually demand and those watched by no end-user would not be multiplexed. By comparing these two schemes, a reduced traffic of EPON can be achieved by using dynamic multiplex scheme, especially when most end-users are watching the same few IPTV programs. Both schemes are implemented in our system, with their hardware and software designs described.

  17. A novel, Q-PCR based approach to measuring endogenous retroviral clearance by capture protein A chromatography.

    PubMed

    Zhang, Min; Lute, Scott; Norling, Lenore; Hong, Connie; Safta, Aurelia; O'Connor, Deborah; Bernstein, Lisa J; Wang, Hua; Blank, Greg; Brorson, Kurt; Chen, Qi

    2009-04-01

    Quantification of virus removal by the purification process during production is required for clinical use of biopharmaceuticals. The current validation approach for virus removal by chromatography steps typically involves time-consuming spiking experiments with expensive model viruses at bench scale. Here we propose a novel, alternative approach that can be applied in at least one instance: evaluating retroviral clearance by protein A chromatography. Our strategy uses a quantitative PCR (Q-PCR) assay that quantifies the endogenous type C retrovirus-like particle genomes directly in production Chinese Hamster Ovary (CHO) cell culture harvests and protein A pools. This eliminates the need to perform spiking with model viruses, and measures the real virus from the process. Using this new approach, clearance values were obtained that was comparable to those from the old model-virus spike/removal approach. We tested the concept of design space for CHO retrovirus removal using samples from a protein A characterization study, where a wide range of chromatographic operating conditions were challenged, including load density, flow rate, wash, pooling, temperature, and resin life cycles. Little impact of these variables on CHO retrovirus clearance was found, arguing for implementation of the design space approach for viral clearance to support operational ranges and manufacturing excursions. The viral clearance results from Q-PCR were confirmed by an orthogonal quantitative product-enhanced reverse transcriptase (Q-PERT) assay that quantifies CHO retrovirus by their reverse transcriptase (RT) enzyme activity. Overall, our results demonstrate that protein A chromatography is a robust retrovirus removal step and CHO retrovirus removal can be directly measured at large scale using Q-PCR assays.

  18. A Multiplexed Diagnostic Platform for Point-of-Care Pathogen Detection

    SciTech Connect

    Regan, J F; Letant, S E; Adams, K L; Mahnke, R C; Nguyen, N T; Dzenitis, J M; Hindson, B J; Hadley, D R; Makarewicz, T J; Henderer, B D; Breneman, J W; Tammero, L F; Ortiz, J I; Derlet, R W; Cohen, S; Colston, W W; McBride, M T; Birch, J M

    2008-02-04

    We developed an automated point-of-care diagnostic instrument that is capable of analyzing nasal swab samples for the presence of respiratory diseases. This robust instrument, called FluIDx, performs autonomous multiplexed RT-PCR reactions that are analyzed by microsphere xMAP technology. We evaluated the performance of FluIDx, in comparison rapid tests specific for influenza and respiratory syncytial virus, in a clinical study performed at the UC Davis Medical Center. The clinical study included samples positive for RSV (n = 71), influenza A (n = 16), influenza B (n = 4), adenovirus (n = 5), parainfluenza virus (n = 2), and 44 negative samples, according to a composite reference method. FluIDx and the rapid tests detected 85.9% and 62.0% of the RSV positive samples, respectively. Similar sensitivities were recorded for the influenza B samples; whereas the influenza A samples were poorly detected, likely due to the utilization of an influenza A signature that did not accurately match currently circulating influenza A strains. Data for all pathogens were compiled and indicate that FluIDx is more sensitive than the rapid tests, detecting 74.2% (95% C.I. of 64.7-81.9%) of the positive samples in comparison to 53.6% (95% C.I. of 43.7-63.2%) for the rapid tests. The higher sensitivity of FluIDx was partially offset by a lower specificity, 77.3% versus 100.0%. Overall, these data suggest automated flow-through PCR-based instruments that perform multiplexed assays can successfully screen clinical samples for infectious diseases.

  19. Development of a Multiplex PCR Assay for Rapid Molecular Serotyping of Haemophilus parasuis.

    PubMed

    Howell, Kate J; Peters, Sarah E; Wang, Jinhong; Hernandez-Garcia, Juan; Weinert, Lucy A; Luan, Shi-Lu; Chaudhuri, Roy R; Angen, Øystein; Aragon, Virginia; Williamson, Susanna M; Parkhill, Julian; Langford, Paul R; Rycroft, Andrew N; Wren, Brendan W; Maskell, Duncan J; Tucker, Alexander W

    2015-12-01

    Haemophilus parasuis causes Glässer's disease and pneumonia in pigs. Indirect hemagglutination (IHA) is typically used to serotype this bacterium, distinguishing 15 serovars with some nontypeable isolates. The capsule loci of the 15 reference strains have been annotated, and significant genetic variation was identified between serovars, with the exception of serovars 5 and 12. A capsule locus and in silico serovar were identified for all but two nontypeable isolates in our collection of >200 isolates. Here, we describe the development of a multiplex PCR, based on variation within the capsule loci of the 15 serovars of H. parasuis, for rapid molecular serotyping. The multiplex PCR (mPCR) distinguished between all previously described serovars except 5 and 12, which were detected by the same pair of primers. The detection limit of the mPCR was 4.29 × 10(5) ng/μl bacterial genomic DNA, and high specificity was indicated by the absence of reactivity against closely related commensal Pasteurellaceae and other bacterial pathogens of pigs. A subset of 150 isolates from a previously sequenced H. parasuis collection was used to validate the mPCR with 100% accuracy compared to the in silico results. In addition, the two in silico-nontypeable isolates were typeable using the mPCR. A further 84 isolates were analyzed by mPCR and compared to the IHA serotyping results with 90% concordance (excluding those that were nontypeable by IHA). The mPCR was faster, more sensitive, and more specific than IHA, enabling the differentiation of 14 of the 15 serovars of H. parasuis.

  20. New multiplex PCR method for the simultaneous diagnosis of the three known species of equine tapeworm.

    PubMed

    Bohórquez, G Alejandro; Luzón, Mónica; Martín-Hernández, Raquel; Meana, Aránzazu

    2015-01-15

    Although several techniques exist for the detection of equine tapeworms in serum and feces, the differential diagnosis of tapeworm infection is usually based on postmortem findings and the morphological identification of eggs in feces. In this study, a multiplex polymerase chain reaction (PCR)-based method for the simultaneuos detection of Anoplocephala magna, Anoplocephala perfoliata and Anoplocephaloides mamillana has been developed and validated. The method simultaneously amplifies hypervariable SSUrRNA gene regions in the three tapeworm species in a single reaction using three pairs of primers, which exclusively amplify the internal transcribed spacer 2 (ITS-2) in each target gene. The method was tested on three types of sample: (a) 1/10, 1/100, 1/500, 1/1000, 1/2000 and 1/5000 dilutions of 70 ng of genomic DNA of the three tapeworm species, (b) DNA extracted from negative aliquots of sediments of negative control fecal samples spiked with 500, 200, 100, 50 and 10 eggs (only for A. magna and A. perfoliata; no A. mamillana eggs available) and (c) DNA extracted from 80, 50, 40, 30, 10 and 1 egg per 2 μl of PCR reaction mix (only for A. magna and A. perfoliata; no A. mamillana eggs available). No amplification was observed against the DNA of Gasterophilus intestinalis, Parascaris equorum and Strongylus vulgaris. The multiplex PCR method emerged as specific for the three tapeworms and was able to identify as few as 50 eggs per fecal sample and as little as 0.7 ng of control genomic DNA obtained from the three species. The method proposed is able to differentiate infections caused by the two most frequent species A. magna or A. perfoliata when the eggs are present in feces and is also able to detect mixed infections by the three cestode species. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Development of a Multiplex PCR Assay for Rapid Molecular Serotyping of Haemophilus parasuis

    PubMed Central

    Peters, Sarah E.; Wang, Jinhong; Hernandez-Garcia, Juan; Weinert, Lucy A.; Luan, Shi-Lu; Chaudhuri, Roy R.; Angen, Øystein; Aragon, Virginia; Williamson, Susanna M.; Langford, Paul R.; Rycroft, Andrew N.; Wren, Brendan W.; Maskell, Duncan J.; Tucker, Alexander W.

    2015-01-01

    Haemophilus parasuis causes Glässer's disease and pneumonia in pigs. Indirect hemagglutination (IHA) is typically used to serotype this bacterium, distinguishing 15 serovars with some nontypeable isolates. The capsule loci of the 15 reference strains have been annotated, and significant genetic variation was identified between serovars, with the exception of serovars 5 and 12. A capsule locus and in silico serovar were identified for all but two nontypeable isolates in our collection of >200 isolates. Here, we describe the development of a multiplex PCR, based on variation within the capsule loci of the 15 serovars of H. parasuis, for rapid molecular serotyping. The multiplex PCR (mPCR) distinguished between all previously described serovars except 5 and 12, which were detected by the same pair of primers. The detection limit of the mPCR was 4.29 × 105 ng/μl bacterial genomic DNA, and high specificity was indicated by the absence of reactivity against closely related commensal Pasteurellaceae and other bacterial pathogens of pigs. A subset of 150 isolates from a previously sequenced H. parasuis collection was used to validate the mPCR with 100% accuracy compared to the in silico results. In addition, the two in silico-nontypeable isolates were typeable using the mPCR. A further 84 isolates were analyzed by mPCR and compared to the IHA serotyping results with 90% concordance (excluding those that were nontypeable by IHA). The mPCR was faster, more sensitive, and more specific than IHA, enabling the differentiation of 14 of the 15 serovars of H. parasuis. PMID:26424843

  2. Simultaneous and multiplexed detection of exosome microRNAs using molecular beacons.

    PubMed

    Lee, Ji Hye; Kim, Jeong Ah; Jeong, Seunga; Rhee, Won Jong

    2016-12-15

    Simultaneous and multiplexed detection of microRNAs (miRNAs) in a whole exosome is developed, which can be utilized as a PCR-free efficient diagnosis method for various diseases. Exosomes are small extracellular vesicles that contain biomarker miRNAs from parental cells. Because they circulate throughout bodily fluids, exosomal biomarkers offer great advantages for diagnosis in many aspects. In general, PCR-based methods can be used for exosomal miRNA detection but they are laborious, expensive, and time-consuming, which make them unsuitable for high-throughput diagnosis of diseases. Previously, we reported that single miRNA in the exosomes can be detected specifically using an oligonucleotide probe or molecular beacon. Herein, we demonstrate for the first time that multiple miRNAs can be detected simultaneously in exosomes using miRNA-targeting molecular beacons. Exosomes from a breast cancer cell line, MCF-7, were used for the production of exosomes because MCF-7 has a high level of miR-21, miR-375, and miR-27a as target miRNAs. Molecular beacons successfully hybridized with multiple miRNAs in the cancer cell-derived exosomes even in the presence of high human serum concentration. In addition, it is noteworthy that the choice of fluorophores for multiplexing biomarkers in an exosome is crucial because of its small size. The proposed method described in this article is beneficial to high-throughput analysis for disease diagnosis, prognosis, and response to treatment because it is a time-, labor-, and cost-saving technique.

  3. 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.

  4. IDH mutation detection in formalin-fixed paraffin-embedded gliomas using multiplex PCR and single-base extension.

    PubMed

    Perizzolo, Marco; Winkfein, Bob; Hui, Susan; Krulicki, Wally; Chan, Jennifer A; Demetrick, Douglas J

    2012-09-01

    Isocitrate dehydrogenase (IDH) genes are mutated in a significant portion of gliomas, myeloid leukemias and chondroid neoplasms. In gliomas, IDH mutations are prognostic, as those tumors with the mutation are associated with a proneural subclass and have longer survival compared with those without the mutation. We developed a simple, PCR-based SNaPshot® assay (Life Technologies, Carlsbad, CA, USA) to detect IDH1/2 mutations. This protocol combines a single, multiplexed PCR reaction using gene specific primers followed by a single, multiplexed SNaPshot reaction and detection by capillary electrophoresis. In a blinded study of 32 paraffin-embedded glioma specimens previously screened for IDH mutations by a PCR/direct sequencing method, concordance of our IDH SNaPshot test with sequencing was 100%. We performed the assay on an additional 57 specimens submitted for diagnostic IDH mutation evaluation. Data analysis was much faster and easier to perform than analysis of the sequencing data, and results could be obtained in 1 day from DNA extraction to analysis. Furthermore, we could readily identify a mixture of 5% mutant allele vs. 95% wild-type allele in our SNaPshot assay, in comparison to approximately 20% mutant allele in our PCR-sequencing assay. Our assay represents a fast, sensitive, straightforward method of reliably detecting common mutations of IDH genes in glial neoplasms, or other tumors.

  5. High throughput multiplex assay for species identification of Papua New Guinea malaria vectors: members of the Anopheles punctulatus (Diptera: Culicidae) species group.

    PubMed

    Henry-Halldin, Cara N; Reimer, Lisa; Thomsen, Edward; Koimbu, Gussy; Zimmerman, Allison; Keven, John B; Dagoro, Henry; Hetzel, Manuel W; Mueller, Ivo; Siba, Peter; Zimmerman, Peter A

    2011-01-01

    Malaria and filariasis are transmitted in the Southwest Pacific region by Anopheles punctulatus sibling species including An. punctulatus, An. koliensis, the An. farauti complex 1-8 (includes An. hinesorum [An. farauti 2], An. torresiensis [An. farauti 3]). Distinguishing these species from each other requires molecular diagnostic methods. We developed a multiplex polymerase chain reaction (PCR)-based assay specific for known species-specific nucleotide differences in the internal transcribed spacer 2 region and identified the five species most frequently implicated in transmitting disease (An. punctulatus, An. koliensis, An. farauti 1, An. hinesorum, and An. farauti 4). A set of 340 individual mosquitoes obtained from seven Papua New Guinea provinces representing a variety of habitats were analyzed by using this multiplex assay. Concordance between molecular and morphological diagnosis was 56.4% for An. punctulatus, 85.3% for An. koliensis, and 88.9% for An. farauti. Among 158 mosquitoes morphologically designated as An. farauti, 33 were re-classified by PCR as An. punctulatus, 4 as An. koliensis, 26 as An. farauti 1, 49 as An. hinesorum, and 46 as An. farauti 4. Misclassification results from variable coloration of the proboscis and overlap of An. punctulatus, An. koliensis, the An. farauti 4. This multiplex technology enables further mosquito strain identification and simultaneous detection of microbial pathogens.

  6. Multiplex PCR (polymerase chain reaction) assay for detection of E. coli O157:H7, Salmonella sp., Vibrio cholerae and Vibrio parahaemolyticus in spiked shrimps (Penaeus monodon).

    PubMed

    Fakruddin, M D; Sultana, Mahmuda; Ahmed, Monzur Morshed; Chowdhury, Abhijit; Choudhury, Naiyyum

    2013-03-15

    The coastal aquaculture mainly shrimps constitute major export sector in Bangladesh and is increasingly shaped by international trade conditions and by national responses to those stringent quality and safety standards. PCR based validated methods for detection of major bacterial pathogens in shrimp might be very useful tool for ensuring quality and safety standards of exportable shrimps. The objective of this study was to evaluate overall performance (sensitivity and specificity) of the multiplex PCR assay for detection of Vibrio cholerae, Vibrio parahaemolyticus, Salmonella sp. and Escherichia coli O157:H7 from spiked shrimp samples. The targeted genes were ompW for V. cholerae, tdh for V. parahaemolyticus, sefA for Salmonella spp. and hlyEHEC for E. coli O157:H7. The genomic DNA was extracted by using standard method and amplified accordingly. Sensitivity of the assay was tested by inoculating the shrimp homogenate with viable cells of laboratory references strains (target pathogens). The genes were amplified individually both from culture homogenate and spiked samples. Twenty different uniplex and multiplex PCR assay were performed; the results showed that the sensitivity and specificity of multiplex PCR are comparable to that of the results of uniplex PCR for the samples. DNA extracted from shrimp samples spiked with non-target pathogen (Bacillus cereus, Shigella flexneri and Staphylococcus aureus) yielded negative results.

  7. Development of a 24-locus multiplex system to incorporate the core loci in the Combined DNA Index System (CODIS) and the European Standard Set (ESS).

    PubMed

    Guo, Fei; Shen, Hongying; Tian, Huaizhou; Jin, Ping; Jiang, Xianhua

    2014-01-01

    The 24-locus multiplex system allows co-amplification and fluorescent detection of 24 loci (23 STR loci and Amelogenin), including STR loci in the Combined DNA Index System (CODIS) and the ESS (European Standard Set) as well as five additional loci (D2S1338, D6S1043, D19S433, Penta D and Penta E) commonly used in commercial kits. It facilitates data sharing and minimizes adventitious matches within national or between international DNA databases. Additionally, the system can amplify directly from blood and buccal samples spotted on filter paper and swabs and reduce the cycling time to less than one hour and a half. Primers, internal size standard, allelic ladders and matrix standard set were designed and created in-house with a design strategy to work in this multiplex. Developmental validation experiments followed the Scientific Working Group on DNA Analysis Methods (SWGDAM) and the Chinese National Standard (GA/T815-2009) guidelines. The system was evaluated by species specificity, sensitivity, stability, precision and accuracy, case-type samples, population, mixture and PCR-based studies. The results demonstrate that the 24-locus multiplex system is a robust and reliable identification assay as required for forensic DNA typing and databasing.

  8. A Multiplexed NMR-Reporter Approach to Measure Cellular Kinase and Phosphatase Activities in Real-Time.

    PubMed

    Thongwichian, Rossukon; Kosten, Jonas; Benary, Uwe; Rose, Honor May; Stuiver, Marchel; Theillet, Francois-Xavier; Dose, Alexander; Koch, Birgit; Yokoyama, Hideki; Schwarzer, Dirk; Wolf, Jana; Selenko, Philipp

    2015-05-27

    Cell signaling is governed by dynamic changes in kinase and phosphatase activities, which are difficult to assess with discontinuous readout methods. Here, we introduce an NMR-based reporter approach to directly identify active kinases and phosphatases in complex physiological environments such as cell lysates and to measure their individual activities in a semicontinuous fashion. Multiplexed NMR profiling of reporter phosphorylation states provides unique advantages for kinase inhibitor studies and reveals reversible modulations of cellular enzyme activities under different metabolic conditions.

  9. A BAC pooling strategy combined with PCR-based screenings in a large, highly repetitive genome enables integration of the maize genetic and physical maps

    PubMed Central

    Yim, Young-Sun; Moak, Patricia; Sanchez-Villeda, Hector; Musket, Theresa A; Close, Pamela; Klein, Patricia E; Mullet, John E; McMullen, Michael D; Fang, Zheiwei; Schaeffer, Mary L; Gardiner, Jack M; Coe, Edward H; Davis, Georgia L

    2007-01-01

    Background Molecular markers serve three important functions in physical map assembly. First, they provide anchor points to genetic maps facilitating functional genomic studies. Second, they reduce the overlap required for BAC contig assembly from 80 to 50 percent. Finally, they validate assemblies based solely on BAC fingerprints. We employed a six-dimensional BAC pooling strategy in combination with a high-throughput PCR-based screening method to anchor the maize genetic and physical maps. Results A total of 110,592 maize BAC clones (~ 6x haploid genome equivalents) were pooled into six different matrices, each containing 48 pools of BAC DNA. The quality of the BAC DNA pools and their utility for identifying BACs containing target genomic sequences was tested using 254 PCR-based STS markers. Five types of PCR-based STS markers were screened to assess potential uses for the BAC pools. An average of 4.68 BAC clones were identified per marker analyzed. These results were integrated with BAC fingerprint data generated by the Arizona Genomics Institute (AGI) and the Arizona Genomics Computational Laboratory (AGCoL) to assemble the BAC contigs using the FingerPrinted Contigs (FPC) software and contribute to the construction and anchoring of the physical map. A total of 234 markers (92.5%) anchored BAC contigs to their genetic map positions. The results can be viewed on the integrated map of maize [1,2]. Conclusion This BAC pooling strategy is a rapid, cost effective method for genome assembly and anchoring. The requirement for six replicate positive amplifications makes this a robust method for use in large genomes with high amounts of repetitive DNA such as maize. This strategy can be used to physically map duplicate loci, provide order information for loci in a small genetic interval or with no genetic recombination, and loci with conflicting hybridization-based information. PMID:17291341

  10. Detection of the KPC Gene in Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii during a PCR-Based Nosocomial Surveillance Study in Puerto Rico▿

    PubMed Central

    Robledo, Iraida E.; Aquino, Edna E.; Vázquez, Guillermo J.

    2011-01-01

    A 6-month, PCR-based, island-wide hospital surveillance study of beta-lactam resistance in Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii was conducted in Puerto Rico. Of 10,507 isolates, 1,239 (12%) unique, multi-beta-lactam-resistant isolates from all geographical regions were identified. The KPC gene was detected in 61 E. coli, 333 K. pneumoniae, 99 P. aeruginosa, and 41 A. baumannii isolates, indicating the widespread dissemination of the KPC gene in clinically significant nosocomial isolates. PMID:21444702

  11. Optofluidic devices for biomolecule sensing and multiplexing

    NASA Astrophysics Data System (ADS)

    Ozcelik, Damla

    Optofluidics which integrates photonics and microfluidics, has led to highly compact, sensitive and adaptable biomedical sensors. Optofluidic biosensors based on liquid-core anti-resonant reflecting optical waveguides (LC-ARROWs), have proven to be a highly sensitive, portable, and reconfigurable platform for fluorescence spectroscopy and detection of single biomolecules such as proteins, nucleic acids, and virus particles. However, continued improvements in sensitivity remain a major goal as we approach the ultimate limit of detecting individual bio-particles labeled by single or few fluorophores. Additionally, the ability to simultaneously detect and identify multiple biological particles or biomarkers is one of the key requirements for molecular diagnostic tests. The compactness and adaptability of these platforms can further be advanced by introducing tunability, integrating off-chip components, designing reconfigurable and customizable devices, which makes these platforms very good candidates for many different applications. The goal of this thesis was to introduce new elements in these LC-ARROW optofluidics platforms that provide major enhancements in their functionality, making them more sensitive, compact, customizable and multiplexed. First, a novel integrated tunable spectral filter that achieves effective elimination of background noise on the ARROW platform was demonstrated. A unique dual liquid-core design enabled the independent multi-wavelength tuning of the spectral filter by adjusting the refractive index and chemical properties of the liquid. In order to enhance the detection sensitivity of the platform, Y-splitter waveguides were integrated to create multiple excitation spots for each target molecule. A powerful signal processing algorithm was used to analyze the data to improve the signal-to-noise ratio (SNR) of the collected data. Next, the design, optimization and characterization of the Y-splitter waveguides are presented; and single

  12. Validation of endogenous reference genes in Buglossoides arvensis for normalizing RT-qPCR-based gene expression data.

    PubMed

    Gadkar, Vijay J; Filion, Martin

    2015-01-01

    Selection of a stably expressed reference gene (RG) is an important step for generating reliable and reproducible quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) gene expression data. We, in this study, have sought to validate RGs for Buglossoides arvensis, a high nutraceutical value plant whose refined seed oil is entering the market under the commercial trade name Ahiflower™. This weed plant has received attention for its natural ability to significantly accumulate the poly-unsaturated fatty acid (PUFA) stearidonic acid (SDA, C18:4n-3) in its seeds, which is uncommon for most plant species. Ten candidate RGs (β-Act, 18S rRNA, EF-1a, α-Tub, UBQ, α-actin, CAC, PP2a, RUBISCO, GAPDH) were isolated from B. arvensis and TaqMan™ compliant primers/probes were designed for RT-qPCR analysis. Abundance of these gene transcripts was analyzed across different tissues and growth regimes. Two of the most widely used algorithms, geNorm and NormFinder, showed variation in expression levels of these RGs. However, combinatorial analysis of the results clearly identified CAC and α-actin as the most stable and unstable RG candidates, respectively. This study has for the first time identified and validated RGs in the non-model system B. arvensis, a weed plant projected to become an important yet sustainable source of dietary omega-3 PUFA.

  13. Virtual-image generation in 360-degree viewable image-plane disk-type multiplex holography.

    PubMed

    Cheng, Yih-Shyang; Chen, Zheng-Feng; Chen, Chih-Hung

    2013-04-22

    By shifting the rotational axis of the recording film and recording the individual image-plane holograms in reversed sequence with the real-image holographic system [Opt. Express 18, 14012 (2010)], the disk-type multiplex hologram can be made to generate virtual image for walk-around viewing if the recording reference source point is maintained on the symmetry axis of hologram disk. Theoretical formulation and numerical simulation show the characteristics of the reconstructed image. Experimental results are also shown for qualitative comparison.

  14. A population genetic database of cat breeds developed in coordination with a domestic cat STR multiplex.

    PubMed

    Menotti-Raymond, Marilyn; David, Victor A; Weir, Bruce S; O'Brien, Stephen J

    2012-05-01

    A simple tandem repeat (STR) PCR-based typing system developed for the genetic individualization of domestic cat samples has been used to generate a population genetic database of domestic cat breeds. A panel of 10 tetranucleotide STR loci and a gender-identifying sequence tagged site (STS) were co-amplified in genomic DNA of 1043 individuals representing 38 cat breeds. The STR panel exhibits relatively high heterozygosity in cat breeds, with an average 10-locus heterozygosity of 0.71, which represents an average of 38 breed-specific heterozygosities for the 10-member panel. When the entire set of breed individuals was analyzed as a single population, a heterozygosity of 0.87 was observed. Heterozygosities obtained for the 10 loci range from 0.72 to 0.96. The power for genetic individualization of domestic cat samples of the multiplex is high, with a probability of match (p(m)) of 6.2E-14, using a conservative θ = 0.05.

  15. A Population Genetic Database of Cat Breeds Developed in Coordination with a Domestic Cat STR Multiplex*

    PubMed Central

    Menotti-Raymond, Marilyn; David, Victor A.; Weir, Bruce S.; O’Brien, Stephen J.

    2015-01-01

    A simple tandem repeat (STR) PCR-based typing system developed for the genetic individualization of domestic cat samples has been used to generate a population genetic database of domestic cat breeds. A panel of 10 tetranucleotide STR loci and a gender-identifying sequence tagged site (STS) were co-amplified in genomic DNA of 1043 individuals representing 38 cat breeds. The STR panel exhibits relatively high heterozygosity in cat breeds, with an average 10-locus heterozygosity of 0.71, which represents an average of 38 breed-specific heterozygosities for the 10-member panel. When the entire set of breed individuals was analyzed as a single population, a heterozygosity of 0.87 was observed. Heterozygosities obtained for the 10 loci range from 0.72 to 0.96. The power for genetic individualization of domestic cat samples of the multiplex is high, with a probability of match (pm) of 6.2E-14, using a conservative θ = 0.05. PMID:22268511

  16. On the Application of Time-Reversed Space-Time Block Code to Aeronautical Telemetry

    DTIC Science & Technology

    2014-06-01

    Keying (SOQPSK), bit error rate (BER), Orthogonal Frequency Division Multiplexing ( OFDM ), Generalized time-reversed space-time block codes (GTR-STBC) 16...Alamouti code [4]) is optimum [2]. Although OFDM is generally applied on a per subcarrier basis in frequency selective fading, it is not a viable

  17. 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.

  18. Observability of Boolean multiplex control networks

    PubMed Central

    Wu, Yuhu; Xu, Jingxue; Sun, Xi-Ming; Wang, Wei

    2017-01-01

    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. PMID:28452370

  19. Multispectral computational ghost imaging with multiplexed illumination

    NASA Astrophysics Data System (ADS)

    Huang, Jian; Shi, Dongfeng

    2017-07-01

    Computational ghost imaging has attracted wide attention from researchers in many fields over the last two decades. Multispectral imaging as one application of computational ghost imaging possesses spatial and spectral resolving abilities, and is very useful for surveying scenes and extracting detailed information. Existing multispectral imagers mostly utilize narrow band filters or dispersive optical devices to separate light of different wavelengths, and then use multiple bucket detectors or an array detector to record them separately. Here, we propose a novel multispectral ghost imaging method that uses one single bucket detector with multiplexed illumination to produce a colored image. The multiplexed illumination patterns are produced by three binary encoded matrices (corresponding to the red, green and blue colored information, respectively) and random patterns. The results of the simulation and experiment have verified that our method can be effective in recovering the colored object. Multispectral images are produced simultaneously by one single-pixel detector, which significantly reduces the amount of data acquisition.

  20. Cycles and clustering in multiplex networks

    NASA Astrophysics Data System (ADS)

    Baxter, Gareth J.; Cellai, Davide; Dorogovtsev, Sergey N.; Mendes, José F. F.

    2016-12-01

    In multiplex networks, cycles cannot be characterized only by their length, as edges may occur in different layers in different combinations. We define a classification of cycles by the number of edges in each layer and the number of switches between layers. We calculate the expected number of cycles of each type in the configuration model of a large sparse multiplex network. Our method accounts for the full degree distribution including correlations between degrees in different layers. In particular, we obtain the numbers of cycles of length 3 of all possible types. Using these, we give a complete set of clustering coefficients and their expected values. We show that correlations between the degrees of a vertex in different layers strongly affect the number of cycles of a given type, and the number of switches between layers. Both increase with assortative correlations and are strongly decreased by disassortative correlations. The effect of correlations on clustering coefficients is equally pronounced.

  1. Integrated mode converter for mode division multiplexing

    NASA Astrophysics Data System (ADS)

    Perez-Galacho, Diego; Alonso-Ramos, Carlos Alberto; Marris-Morini, Delphine; Vakarin, Vladyslav; Le Roux, Xavier; Ortega-Moñux, Alejandro; Wangüemert-Perez, Juan Gonzalo; Vivien, Laurent

    2016-05-01

    The ever growing demands of bandwidth in optical communication systems are making traditional Wavelength Division Multiplexing (WDM) based systems to reach its limit. In order to cope with future bandwidth demand is necessary to use new levels of orthogonality, such as the waveguide mode or the polarization state. Mode Division Multiplexing (MDM) has recently attracted attention as a possible solution to increase aggregate bandwidth. In this work we discuss the proposition a of mode converter that can cover the whole C-Band of optical communications. The Mode Converter is based on two Multimode Interference (MMI) couplers and a phase shifter. Insertion loss (IL) below 0.2 dB and Extinction ratio (ER) higher than 20 dB in a broad bandwidth range of 1.5 μm to 1.6 μm have been estimated. The total length of the device is less than 30 μm.

  2. Tunable lifetime multiplexing using luminescent nanocrystals

    NASA Astrophysics Data System (ADS)

    Lu, Yiqing; Zhao, Jiangbo; Zhang, Run; Liu, Yujia; Liu, Deming; Goldys, Ewa M.; Yang, Xusan; Xi, Peng; Sunna, Anwar; Lu, Jie; Shi, Yu; Leif, Robert C.; Huo, Yujing; Shen, Jian; Piper, James A.; Robinson, J. Paul; Jin, Dayong

    2014-01-01

    Optical multiplexing plays an important role in applications such as optical data storage, document security, molecular probes and bead assays for personalized medicine. Conventional fluorescent colour coding is limited by spectral overlap and background interference, restricting the number of distinguishable identities. Here, we show that tunable luminescent lifetimes τ in the microsecond region can be exploited to code individual upconversion nanocrystals. In a single colour band, one can generate more than ten nanocrystal populations with distinct lifetimes ranging from 25.6 µs to 662.4 µs and decode their well-separated lifetime identities, which are independent of both colour and intensity. Such `τ-dots' potentially suit multichannel bioimaging, high-throughput cytometry quantification, high-density data storage, as well as security codes to combat counterfeiting. This demonstration extends the optical multiplexing capability by adding the temporal dimension of luminescent signals, opening new opportunities in the life sciences, medicine and data security.

  3. Spin and wavelength multiplexed nonlinear metasurface holography

    NASA Astrophysics Data System (ADS)

    Ye, Weimin; Zeuner, Franziska; Li, Xin; Reineke, Bernhard; He, Shan; Qiu, Cheng-Wei; Liu, Juan; Wang, Yongtian; Zhang, Shuang; Zentgraf, Thomas

    2016-06-01

    Metasurfaces, as the ultrathin version of metamaterials, have caught growing attention due to their superior capability in controlling the phase, amplitude and polarization states of light. Among various types of metasurfaces, geometric metasurface that encodes a geometric or Pancharatnam-Berry phase into the orientation angle of the constituent meta-atoms has shown great potential in controlling light in both linear and nonlinear optical regimes. The robust and dispersionless nature of the geometric phase simplifies the wave manipulation tremendously. Benefitting from the continuous phase control, metasurface holography has exhibited advantages over conventional depth controlled holography with discretized phase levels. Here we report on spin and wavelength multiplexed nonlinear metasurface holography, which allows construction of multiple target holographic images carried independently by the fundamental and harmonic generation waves of different spins. The nonlinear holograms provide independent, nondispersive and crosstalk-free post-selective channels for holographic multiplexing and multidimensional optical data storages, anti-counterfeiting, and optical encryption.

  4. Optimal estimator for tomographic fluorescence lifetime multiplexing

    PubMed Central

    Hou, Steven S.; Bacskai, Brian J.; Kumar, Anand T. N.

    2016-01-01

    We use the model resolution matrix to analytically derive an optimal Bayesian estimator for multiparameter inverse problems that simultaneously minimizes inter-parameter cross talk and the total reconstruction error. Application of this estimator to time-domain diffuse fluorescence imaging shows that the optimal estimator for lifetime multiplexing is identical to a previously developed asymptotic time-domain (ATD) approach, except for the inclusion of a diagonal regularization term containing decay amplitude uncertainties. We show that, while the optimal estimator and ATD provide zero cross talk, the optimal estimator provides lower reconstruction error, while ATD results in superior relative quantitation. The framework presented here is generally applicable to other multiplexing problems where the simultaneous and accurate relative quantitation of multiple parameters is of interest. PMID:27192234

  5. An integrated microspectrometer for localised multiplexing measurements.

    PubMed

    Hu, Zhixiong; Glidle, Andrew; Ironside, Charles; Cooper, Jonathan M; Yin, Huabing

    2015-01-07

    We describe the development of an integrated lensed Arrayed Waveguide Grating (AWG) microspectrometer for localized multiplexing fluorescence measurements. The device, which has a footprint that is only 1 mm wide and 1 cm long, is capable of spectroscopic measurements on chip. Multiple fluorescence signals were measured simultaneously based upon simple intensity readouts from a CCD camera. We also demonstrate the integration of the AWG spectrometer with a microfluidic platform using a lensing function to confine the beam shape for focused illumination. This capability enhances signal collection, gives better spatial resolution, and provides a route for the analysis of small volume samples (e.g. cells) in flow. To show these capabilities we developed a novel "bead-AWG" platform with which we demonstrate localized multiplexed fluorescence detection either simultaneously or successively. Such an integrated system provides the basis for a portable system capable of optical detection of multi-wavelength fluorescence from a single defined location.

  6. 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.

  7. Spin and wavelength multiplexed nonlinear metasurface holography

    PubMed Central

    Ye, Weimin; Zeuner, Franziska; Li, Xin; Reineke, Bernhard; He, Shan; Qiu, Cheng-Wei; Liu, Juan; Wang, Yongtian; Zhang, Shuang; Zentgraf, Thomas

    2016-01-01

    Metasurfaces, as the ultrathin version of metamaterials, have caught growing attention due to their superior capability in controlling the phase, amplitude and polarization states of light. Among various types of metasurfaces, geometric metasurface that encodes a geometric or Pancharatnam–Berry phase into the orientation angle of the constituent meta-atoms has shown great potential in controlling light in both linear and nonlinear optical regimes. The robust and dispersionless nature of the geometric phase simplifies the wave manipulation tremendously. Benefitting from the continuous phase control, metasurface holography has exhibited advantages over conventional depth controlled holography with discretized phase levels. Here we report on spin and wavelength multiplexed nonlinear metasurface holography, which allows construction of multiple target holographic images carried independently by the fundamental and harmonic generation waves of different spins. The nonlinear holograms provide independent, nondispersive and crosstalk-free post-selective channels for holographic multiplexing and multidimensional optical data storages, anti-counterfeiting, and optical encryption. PMID:27306147

  8. Encrypted optical storage with angular multiplexing.

    PubMed

    Matoba, O; Javidi, B

    1999-12-11

    We present the first, to our knowledge, demonstration of an encrypted optical storage based on double-random phase encoding by using angular multiplexing in a photorefractive material. Original two-dimensional data are encrypted by use of two random phase codes located in the input and the Fourier planes and are then stored holographically in a LiNbO(3):Fe crystal. The retrieval of the original data can be achieved with a phase-conjugated readout scheme. We demonstrate the encryption and the decryption of multiple frames of two-dimensional digital data by using angular multiplexing. We also evaluate numerically the influence of the bandwidth of the optical system on the decrypted digital data. The bit error rate as a function of the optical system bandwidth is presented.

  9. Hidden geometric correlations in real multiplex networks

    NASA Astrophysics Data System (ADS)

    Kleineberg, Kaj-Kolja; Boguñá, Marián; Ángeles Serrano, M.; Papadopoulos, Fragkiskos

    2016-11-01

    Real networks often form interacting parts of larger and more complex systems. Examples can be found in different domains, ranging from the Internet to structural and functional brain networks. Here, we show that these multiplex systems are not random combinations of single network layers. Instead, they are organized in specific ways dictated by hidden geometric correlations between the layers. We find that these correlations are significant in different real multiplexes, and form a key framework for answering many important questions. Specifically, we show that these geometric correlations facilitate the definition and detection of multidimensional communities, which are sets of nodes that are simultaneously similar in multiple layers. They also enable accurate trans-layer link prediction, meaning that connections in one layer can be predicted by observing the hidden geometric space of another layer. And they allow efficient targeted navigation in the multilayer system using only local knowledge, outperforming navigation in the single layers only if the geometric correlations are sufficiently strong.

  10. Multiplexing Short Primers for Viral Family PCR

    SciTech Connect

    Gardner, S N; Hiddessen, A L; Hara, C A; Williams, P L; Wagner, M; Colston, B W

    2008-06-26

    We describe a Multiplex Primer Prediction (MPP) algorithm to build multiplex compatible primer sets for large, diverse, and unalignable sets of target sequences. The MPP algorithm is scalable to larger target sets than other available software, and it does not require a multiple sequence alignment. We applied it to questions in viral detection, and demonstrated that there are no universally conserved priming sequences among viruses and that it could require an unfeasibly large number of primers ({approx}3700 18-mers or {approx}2000 10-mers) to generate amplicons from all sequenced viruses. We then designed primer sets separately for each viral family, and for several diverse species such as foot-and-mouth disease virus, hemagglutinin and neuraminidase segments of influenza A virus, Norwalk virus, and HIV-1.

  11. Spectrally multiplexed chromatic confocal multipoint sensing.

    PubMed

    Hillenbrand, Matthias; Lorenz, Lucia; Kleindienst, Roman; Grewe, Adrian; Sinzinger, Stefan

    2013-11-15

    We present a concept for chromatic confocal distance sensing that employs two levels of spectral multiplexing for the parallelized evaluation of multiple lateral measurement points; at the first level, the chromatic confocal principle is used to encode distance information within the spectral distribution of the sensor signal. For lateral multiplexing, the total spectral bandwidth of the sensor is split into bands. Each band is assigned to a different lateral measurement point by a segmented diffractive element. Based on this concept, we experimentally demonstrate a chromatic confocal three-point sensor that is suitable for harsh production environments, since it works with a single-point spectrometer and does not require scanning functionality. The experimental system has a working distance of more than 50 mm, a measurement range of 9 mm, and an axial resolution of 50 μm.

  12. Sequential Multiplex Analyte Capturing for Phosphoprotein Profiling*

    PubMed Central

    Poetz, Oliver; Henzler, Tanja; Hartmann, Michael; Kazmaier, Cornelia; Templin, Markus F.; Herget, Thomas; Joos, Thomas O.

    2010-01-01

    Microarray-based sandwich immunoassays can simultaneously detect dozens of proteins. However, their use in quantifying large numbers of proteins is hampered by cross-reactivity and incompatibilities caused by the immunoassays themselves. Sequential multiplex analyte capturing addresses these problems by repeatedly probing the same sample with different sets of antibody-coated, magnetic suspension bead arrays. As a miniaturized immunoassay format, suspension bead array-based assays fulfill the criteria of the ambient analyte theory, and our experiments reveal that the analyte concentrations are not significantly changed. The value of sequential multiplex analyte capturing was demonstrated by probing tumor cell line lysates for the abundance of seven different receptor tyrosine kinases and their degree of phosphorylation and by measuring the complex phosphorylation pattern of the epidermal growth factor receptor in the same sample from the same cavity. PMID:20682761

  13. Fiber composite slices for multiplexed immunoassays

    PubMed Central

    Kim, Jiyun; Bae, Sangwook; Song, Seowoo; Chung, Keumsim; Kwon, Sunghoon

    2015-01-01

    Fabrication methods for the development of multiplexed immunoassay platforms primarily depend on the individual functionalization of reaction chambers to achieve a heterogeneous reacting substrate composition, which increases the overall manufacturing time and cost. Here, we describe a new type of low-cost fabrication method for a scalable immunoassay platform based on cotton threads. The manufacturing process involves the fabrication of functionalized fibers and the arrangement of these fibers into a bundle; this bundle is then sectioned to make microarray-like particles with a predefined surface architecture. With these sections, composed of heterogeneous thread fragments with different types of antibodies, we demonstrated quantitative and 7-plex immunoassays. We expect that this methodology will prove to be a versatile, low-cost, and highly scalable method for the fabrication of multiplexed bioassay platforms. PMID:26339310

  14. Fiber optic multiplex optical transmission system

    NASA Technical Reports Server (NTRS)

    Bell, C. H. (Inventor)

    1977-01-01

    A multiplex optical transmission system which minimizes external interference while simultaneously receiving and transmitting video, digital data, and audio signals is described. Signals are received into subgroup mixers for blocking into respective frequency ranges. The outputs of these mixers are in turn fed to a master mixer which produces a composite electrical signal. An optical transmitter connected to the master mixer converts the composite signal into an optical signal and transmits it over a fiber optic cable to an optical receiver which receives the signal and converts it back to a composite electrical signal. A de-multiplexer is coupled to the output of the receiver for separating the composite signal back into composite video, digital data, and audio signals. A programmable optic patch board is interposed in the fiber optic cables for selectively connecting the optical signals to various receivers and transmitters.

  15. Six mode selective fiber optic spatial multiplexer.

    PubMed

    Velazquez-Benitez, A M; Alvarado, J C; Lopez-Galmiche, G; Antonio-Lopez, J E; Hernández-Cordero, J; Sanchez-Mondragon, J; Sillard, P; Okonkwo, C M; Amezcua-Correa, R

    2015-04-15

    Low-loss all-fiber photonic lantern (PL) mode multiplexers (MUXs) capable of selectively exciting the first six fiber modes of a multimode fiber (LP01, LP11a, LP11b, LP21a, LP21b, and LP02) are demonstrated. Fabrication of the spatial mode multiplexers was successfully achieved employing a combination of either six step or six graded index fibers of four different core sizes. Insertion losses of 0.2-0.3 dB and mode purities above 9 dB are achieved. Moreover, it is demonstrated that the use of graded index fibers in a PL eases the length requirements of the adiabatic tapered transition and could enable scaling to large numbers.

  16. Demand and Congestion in Multiplex Transportation Networks.

    PubMed

    Chodrow, Philip S; Al-Awwad, Zeyad; Jiang, Shan; González, Marta C

    Urban transportation systems are multimodal, sociotechnical systems; however, while their multimodal aspect has received extensive attention in recent literature on multiplex networks, their sociotechnical aspect has been largely neglected. We present the first study of an urban transportation system using multiplex network analysis and validated Origin-Destination travel demand, with Riyadh's planned metro as a case study. We develop methods for analyzing the impact of additional transportation layers on existing dynamics, and show that demand structure plays key quantitative and qualitative roles. There exist fundamental geometrical limits to the metro's impact on traffic dynamics, and the bulk of environmental accrue at metro speeds only slightly faster than those planned. We develop a simple model for informing the use of additional, "feeder" layers to maximize reductions in global congestion. Our techniques are computationally practical, easily extensible to arbitrary transportation layers with complex transfer logic, and implementable in open-source software.

  17. Multiplexed Energy Coupler for Rotating Equipment

    NASA Technical Reports Server (NTRS)

    Zhao, Xiaoliang

    2011-01-01

    A multiplexing antenna assembly can efficiently couple AC signal/energy into, or out of, rotating equipment. The unit only passes AC energy while blocking DC energy. Concentric tubes that are sliced into multiple pieces are assembled together so that, when a piece from an outer tube aligns well with an inner tube piece, efficient energy coupling is achieved through a capacitive scheme. With N outer pieces and M inner pieces, an effective N x M combination can be achieved in a multiplexed manner. The energy coupler is non-contact, which is useful if isolation from rotating and stationary parts is required. Additionally, the innovation can operate in high temperatures. Applications include rotating structure sensing, non-contact energy transmission, etc.

  18. Spin and wavelength multiplexed nonlinear metasurface holography.

    PubMed

    Ye, Weimin; Zeuner, Franziska; Li, Xin; Reineke, Bernhard; He, Shan; Qiu, Cheng-Wei; Liu, Juan; Wang, Yongtian; Zhang, Shuang; Zentgraf, Thomas

    2016-06-16

    Metasurfaces, as the ultrathin version of metamaterials, have caught growing attention due to their superior capability in controlling the phase, amplitude and polarization states of light. Among various types of metasurfaces, geometric metasurface that encodes a geometric or Pancharatnam-Berry phase into the orientation angle of the constituent meta-atoms has shown great potential in controlling light in both linear and nonlinear optical regimes. The robust and dispersionless nature of the geometric phase simplifies the wave manipulation tremendously. Benefitting from the continuous phase control, metasurface holography has exhibited advantages over conventional depth controlled holography with discretized phase levels. Here we report on spin and wavelength multiplexed nonlinear metasurface holography, which allows construction of multiple target holographic images carried independently by the fundamental and harmonic generation waves of different spins. The nonlinear holograms provide independent, nondispersive and crosstalk-free post-selective channels for holographic multiplexing and multidimensional optical data storages, anti-counterfeiting, and optical encryption.

  19. 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.

  20. Demand and Congestion in Multiplex Transportation Networks

    PubMed Central

    al-Awwad, Zeyad; Jiang, Shan; González, Marta C.

    2016-01-01

    Urban transportation systems are multimodal, sociotechnical systems; however, while their multimodal aspect has received extensive attention in recent literature on multiplex networks, their sociotechnical aspect has been largely neglected. We present the first study of an urban transportation system using multiplex network analysis and validated Origin-Destination travel d