Interferometric Reflectance Imaging Sensor (IRIS)—A Platform Technology for Multiplexed Diagnostics and Digital Detection

PubMed Central

Avci, Oguzhan; Lortlar Ünlü, Nese; Yalçın Özkumur, Ayça; Ünlü, M. Selim

2015-01-01

Over the last decade, the growing need in disease diagnostics has stimulated rapid development of new technologies with unprecedented capabilities. Recent emerging infectious diseases and epidemics have revealed the shortcomings of existing diagnostics tools, and the necessity for further improvements. Optical biosensors can lay the foundations for future generation diagnostics by providing means to detect biomarkers in a highly sensitive, specific, quantitative and multiplexed fashion. Here, we review an optical sensing technology, Interferometric Reflectance Imaging Sensor (IRIS), and the relevant features of this multifunctional platform for quantitative, label-free and dynamic detection. We discuss two distinct modalities for IRIS: (i) low-magnification (ensemble biomolecular mass measurements) and (ii) high-magnification (digital detection of individual nanoparticles) along with their applications, including label-free detection of multiplexed protein chips, measurement of single nucleotide polymorphism, quantification of transcription factor DNA binding, and high sensitivity digital sensing and characterization of nanoparticles and viruses. PMID:26205273

Multiplex Detection of Toxigenic Penicillium Species.

PubMed

Rodríguez, Alicia; Córdoba, Juan J; Rodríguez, Mar; Andrade, María J

2017-01-01

Multiplex PCR-based methods for simultaneous detection and quantification of different mycotoxin-producing Penicillia are useful tools to be used in food safety programs. These rapid and sensitive techniques allow taking corrective actions during food processing or storage for avoiding accumulation of mycotoxins in them. In this chapter, three multiplex PCR-based methods to detect at least patulin- and ochratoxin A-producing Penicillia are detailed. Two of them are different multiplex real-time PCR suitable for monitoring and quantifying toxigenic Penicillium using the nonspecific dye SYBR Green and specific hydrolysis probes (TaqMan). All of them successfully use the same target genes involved in the biosynthesis of such mycotoxins for designing primers and/or probes.

Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6

PubMed Central

Gootenberg, Jonathan S.; Abudayyeh, Omar O.; Kellner, Max J.; Joung, Julia; Collins, James J.; Zhang, Feng

2018-01-01

Rapid detection of nucleic acids is integral for clinical diagnostics and biotechnological applications. We recently developed a platform termed SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) that combines isothermal pre-amplification with Cas13 to detect single molecules of RNA or DNA. Through characterization of CRISPR enzymology and application development, we report here four advances integrated into SHERLOCKv2: 1) 4-channel single reaction multiplexing using orthogonal CRISPR enzymes; 2) quantitative measurement of input down to 2 aM; 3) 3.5-fold increase in signal sensitivity by combining Cas13 with Csm6, an auxilary CRISPR-associated enzyme; and 4) lateral flow read-out. SHERLOCKv2 can detect Dengue or Zika virus ssRNA as well as mutations in patient liquid biopsy samples via lateral flow, highlighting its potential as a multiplexable, portable, rapid, and quantitative detection platform of nucleic acids. PMID:29449508

A multiplex branched DNA assay for parallel quantitative gene expression profiling.

PubMed

Flagella, Michael; Bui, Son; Zheng, Zhi; Nguyen, Cung Tuong; Zhang, Aiguo; Pastor, Larry; Ma, Yunqing; Yang, Wen; Crawford, Kimberly L; McMaster, Gary K; Witney, Frank; Luo, Yuling

2006-05-01

We describe a novel method to quantitatively measure messenger RNA (mRNA) expression of multiple genes directly from crude cell lysates and tissue homogenates without the need for RNA purification or target amplification. The multiplex branched DNA (bDNA) assay adapts the bDNA technology to the Luminex fluorescent bead-based platform through the use of cooperative hybridization, which ensures an exceptionally high degree of assay specificity. Using in vitro transcribed RNA as reference standards, we demonstrated that the assay is highly specific, with cross-reactivity less than 0.2%. We also determined that the assay detection sensitivity is 25,000 RNA transcripts with intra- and interplate coefficients of variance of less than 10% and less than 15%, respectively. Using three 10-gene panels designed to measure proinflammatory and apoptosis responses, we demonstrated sensitive and specific multiplex gene expression profiling directly from cell lysates. The gene expression change data demonstrate a high correlation coefficient (R(2)=0.94) compared with measurements obtained using the single-plex bDNA assay. Thus, the multiplex bDNA assay provides a powerful means to quantify the gene expression profile of a defined set of target genes in large sample populations.

Multiplexed quantitation of protein expression and phosphorylation based on functionalized soluble nanopolymers

PubMed Central

Pan, Li; Iliuk, Anton; Yu, Shuai; Geahlen, Robert L.; Tao, W. Andy

2012-01-01

We report here for the first time the multiplexed quantitation of phosphorylation and protein expression based on a functionalized soluble nanopolymer. The soluble nanopolymer, pIMAGO, is functionalized with Ti (IV) ions for chelating phosphoproteins in high specificity, and with infrared fluorescent tags for direct, multiplexed assays. The nanopolymer allows for direct competition for epitopes on proteins of interest, thus facilitating simultaneous detection of phosphorylation by pIMAGO and total protein amount by protein antibody in the same well of microplates. The new strategy has a great potential to measure cell signaling events by clearly distinguishing actual phosphorylation signals from protein expression changes, thus providing a powerful tool to accurately profile cellular signal transduction in healthy and disease cells. We anticipate broad applications of this new strategy in monitoring cellular signaling pathways and discovering new signaling events. PMID:23088311

Optofluidic wavelength division multiplexing for single-virus detection

PubMed Central

Ozcelik, Damla; Parks, Joshua W.; Wall, Thomas A.; Stott, Matthew A.; Cai, Hong; Parks, Joseph W.; Hawkins, Aaron R.; Schmidt, Holger

2015-01-01

Optical waveguides simultaneously transport light at different colors, forming the basis of fiber-optic telecommunication networks that shuttle data in dozens of spectrally separated channels. Here, we reimagine this wavelength division multiplexing (WDM) paradigm in a novel context––the differentiated detection and identification of single influenza viruses on a chip. We use a single multimode interference (MMI) waveguide to create wavelength-dependent spot patterns across the entire visible spectrum and enable multiplexed single biomolecule detection on an optofluidic chip. Each target is identified by its time-dependent fluorescence signal without the need for spectral demultiplexing upon detection. We demonstrate detection of individual fluorescently labeled virus particles of three influenza A subtypes in two implementations: labeling of each virus using three different colors and two-color combinatorial labeling. By extending combinatorial multiplexing to three or more colors, MMI-based WDM provides the multiplexing power required for differentiated clinical tests and the growing field of personalized medicine. PMID:26438840

Microgels for multiplex and direct fluorescence detection

NASA Astrophysics Data System (ADS)

Causa, Filippo; Aliberti, Anna; Cusano, Angela M.; Battista, Edmondo; Netti, Paolo A.

2015-05-01

Blood borne oligonucleotides fragments contain useful clinical information whose detection and monitoring represent the new frontier in liquid biopsy as they can transform the current diagnosis procedure. For instance, recent studies have identified a new class of circulating biomarkers such as s miRNAs, and demonstrated that changes in their concentration are closely associated with the development of cancer and other pathologies. However, direct detection of miRNAs in body fluids is particularly challenging and demands high sensitivity -concentration range between atto to femtomolarspecificity, and multiplexing Here we report on engineered multifunctional microgels and innovative probe design for a direct and multiplex detection of relevant clinical miRNAs in fluorescence by single particle assay. Polyethyleneglycol-based microgels have a coreshell architecture with two spectrally encoded fluorescent dyes for multiplex analyses and are endowed with fluorescent probes for miRNA detection. Encoding and detection fluorescence signals are distinguishable by not overlapping emission spectra. Tuneable fluorescence probe conjugation and corresponding emission confinement on single microgel allows for enhanced target detection. Such suspension array has indeed high selectivity and sensitivity with a detection limit of 10-15 M and a dynamic range from 10-9 to 10-15 M. We believe that sensitivity in the fM concentration range, signal background minimization, multiplexed capability and direct measurement of such microgels will translate into diagnostic benefits opening up new roots toward liquid biopsy in the context of point-of-care testing through an easy and fast detection of sensitive diagnostic biomarkers directly in serum.

A multiplex PCR for detection of six viruses in ducks.

PubMed

Wang, Yongjuan; Zhu, Shanyuan; Hong, Weiming; Wang, Anping; Zuo, Weiyong

2017-10-01

In this study, six pairs of specific primers that can amplify DNA fragments of different sizes were designed and synthesized according to viral protein gene sequences published in GenBank. Then, a multiplex PCR method was established for rapid detection of duck hepatitis virus 1, duck plague virus, duck Tembusu virus, muscovy duck parvovirus, muscovy duck reovirus, and duck H9N2 avian influenza virus, and achieve simple and rapid detection of viral diseases in ducks. Single PCR was used to confirm primer specificity, and PCR conditions were optimized to construct a multiplex PCR system. Specificity and sensitivity assays were also developed. The multiplex PCR was used to detect duck embryos infected with mixed viruses and those with clinically suspected diseases to verify the feasibility of the multiplex PCR. Results show that the primers can specifically amplify target fragments, without any cross-amplification with other viruses. The multiplex PCR system can amplify six DNA fragments from the pooled viral genomes and specifically detect nucleic acids of the six duck susceptible viruses when the template amount is 10 2 copies/μl. In addition, the system can be used to detect viral nucleic acids in duck embryos infected with the six common viruses. The detection results for clinical samples are consistent with those detected by single PCR. Therefore, the established multiplex PCR method can perform specific, sensitive, and high-throughput detection of six duck-infecting viruses and can be applied to clinical identification and diagnosis of viral infection in ducks. Copyright © 2017. Published by Elsevier B.V.

Tuning a Parallel Segmented Flow Column and Enabling Multiplexed Detection.

PubMed

Pravadali-Cekic, Sercan; Kocic, Danijela; Hua, Stanley; Jones, Andrew; Dennis, Gary R; Shalliker, R Andrew

2015-12-15

Active flow technology (AFT) is new form of column technology that was designed to overcome flow heterogeneity to increase separation performance in terms of efficiency and sensitivity and to enable multiplexed detection. This form of AFT uses a parallel segmented flow (PSF) column. A PSF column outlet end-fitting consists of 2 or 4 ports, which can be multiplexed to connect up to 4 detectors. The PSF column not only allows a platform for multiplexed detection but also the combination of both destructive and non-destructive detectors, without additional dead volume tubing, simultaneously. The amount of flow through each port can also be adjusted through pressure management to suit the requirements of a specific detector(s). To achieve multiplexed detection using a PSF column there are a number of parameters which can be controlled to ensure optimal separation performance and quality of results; that is tube dimensions for each port, choice of port for each type of detector and flow adjustment. This protocol is intended to show how to use and tune a PSF column functioning in a multiplexed mode of detection.

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

Multiplex hydrolysis probe real-time PCR for simultaneous detection of hepatitis A virus and hepatitis E virus.

PubMed

Qiu, Feng; Cao, Jingyuan; Su, Qiudong; Yi, Yao; Bi, Shengli

2014-05-30

Detection of hepatitis viral infections has traditionally relied on the circulating antibody test using the enzyme-linked immunosorbent assay. However, multiplex real-time PCR has been increasingly used for a variety of viral nucleic acid detections and has proven to be superior to traditional methods. Hepatitis A virus (HAV) and hepatitis E virus (HEV) are the major causes of acute hepatitis worldwide; both HAV and HEV infection are a main public health problem. In the present study, a one-step multiplex reverse transcriptase quantitative polymerase chain reaction assay using hydrolysis probes was developed for simultaneously detecting HAV and HEV. This novel detection system proved specific to the target viruses, to be highly sensitive and to be applicable to clinical sera samples, making it useful for rapid, accurate and feasible identification of HAV and HEV.

Multiplex Hydrolysis Probe Real-Time PCR for Simultaneous Detection of Hepatitis A Virus and Hepatitis E Virus

PubMed Central

Qiu, Feng; Cao, Jingyuan; Su, Qiudong; Yi, Yao; Bi, Shengli

2014-01-01

Detection of hepatitis viral infections has traditionally relied on the circulating antibody test using the enzyme-linked immunosorbent assay. However, multiplex real-time PCR has been increasingly used for a variety of viral nucleic acid detections and has proven to be superior to traditional methods. Hepatitis A virus (HAV) and hepatitis E virus (HEV) are the major causes of acute hepatitis worldwide; both HAV and HEV infection are a main public health problem. In the present study, a one-step multiplex reverse transcriptase quantitative polymerase chain reaction assay using hydrolysis probes was developed for simultaneously detecting HAV and HEV. This novel detection system proved specific to the target viruses, to be highly sensitive and to be applicable to clinical sera samples, making it useful for rapid, accurate and feasible identification of HAV and HEV. PMID:24886818

One-step multiplex RT-qPCR detects three citrus viroids from different genera in a wide range of hosts.

PubMed

Osman, Fatima; Dang, Tyler; Bodaghi, Sohrab; Vidalakis, Georgios

2017-07-01

A one-step multiplex reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) based on species-specific minor groove binding (MGB) probes, was developed for the simultaneous detection, identification, and quantification of three citrus viroids belonging to different genera. Citrus exocortis viroid (Pospiviroid), Hop stunt viroid (Hostuviroid), and Citrus bark cracking viroid (Cocadviroid) cause a variety of maladies in agriculturally significant crops. Therefore, reliable assays for their detection are essential tools for various government and industry organizations implementing disease management programs. Singleplex qPCR primers and MGB probes were designed individually for the detection of the three targeted viroids, and subsequently combined in a one-step multiplex RT-qPCR reaction. A wide host range of woody plants, including citrus, grapevines, apricots, plums and herbaceous plants such as tomato, cucumber, eggplant and chrysanthemum different world regions were used to validate the assay. Single, double and triple viroid infections were identified in the tested samples. The developed multiplex RT-qPCR assay was compared with a previously reported SYBR Green I RT-qPCR for the universal detection of citrus viroids. Both assays accurately identified all citrus viroid infected samples. The multiplex assay complemented the SYBR Green I universal detection assay by differentiating among citrus viroid species in the positive samples. The developed multiplex RT-qPCR assay has the potential to simultaneously detect each targeted viroid and could be used in high throughput screenings for citrus viroids in field surveys, germplasm banks, nurseries and other viroid disease management programs. Copyright © 2017. Published by Elsevier B.V.

Simple, Sensitive and Accurate Multiplex Detection of Clinically Important Melanoma DNA Mutations in Circulating Tumour DNA with SERS Nanotags

PubMed Central

Wee, Eugene J.H.; Wang, Yuling; Tsao, Simon Chang-Hao; Trau, Matt

2016-01-01

Sensitive and accurate identification of specific DNA mutations can influence clinical decisions. However accurate diagnosis from limiting samples such as circulating tumour DNA (ctDNA) is challenging. Current approaches based on fluorescence such as quantitative PCR (qPCR) and more recently, droplet digital PCR (ddPCR) have limitations in multiplex detection, sensitivity and the need for expensive specialized equipment. Herein we describe an assay capitalizing on the multiplexing and sensitivity benefits of surface-enhanced Raman spectroscopy (SERS) with the simplicity of standard PCR to address the limitations of current approaches. This proof-of-concept method could reproducibly detect as few as 0.1% (10 copies, CV < 9%) of target sequences thus demonstrating the high sensitivity of the method. The method was then applied to specifically detect three important melanoma mutations in multiplex. Finally, the PCR/SERS assay was used to genotype cell lines and ctDNA from serum samples where results subsequently validated with ddPCR. With ddPCR-like sensitivity and accuracy yet at the convenience of standard PCR, we believe this multiplex PCR/SERS method could find wide applications in both diagnostics and research. PMID:27446486

Simple, Sensitive and Accurate Multiplex Detection of Clinically Important Melanoma DNA Mutations in Circulating Tumour DNA with SERS Nanotags.

PubMed

Wee, Eugene J H; Wang, Yuling; Tsao, Simon Chang-Hao; Trau, Matt

2016-01-01

Sensitive and accurate identification of specific DNA mutations can influence clinical decisions. However accurate diagnosis from limiting samples such as circulating tumour DNA (ctDNA) is challenging. Current approaches based on fluorescence such as quantitative PCR (qPCR) and more recently, droplet digital PCR (ddPCR) have limitations in multiplex detection, sensitivity and the need for expensive specialized equipment. Herein we describe an assay capitalizing on the multiplexing and sensitivity benefits of surface-enhanced Raman spectroscopy (SERS) with the simplicity of standard PCR to address the limitations of current approaches. This proof-of-concept method could reproducibly detect as few as 0.1% (10 copies, CV < 9%) of target sequences thus demonstrating the high sensitivity of the method. The method was then applied to specifically detect three important melanoma mutations in multiplex. Finally, the PCR/SERS assay was used to genotype cell lines and ctDNA from serum samples where results subsequently validated with ddPCR. With ddPCR-like sensitivity and accuracy yet at the convenience of standard PCR, we believe this multiplex PCR/SERS method could find wide applications in both diagnostics and research.

Application of a Multiplex Quantitative PCR to Assess Prevalence and Intensity Of Intestinal Parasite Infections in a Controlled Clinical Trial

PubMed Central

Llewellyn, Stacey; Inpankaew, Tawin; Nery, Susana Vaz; Gray, Darren J.; Verweij, Jaco J.; Clements, Archie C. A.; Gomes, Santina J.; Traub, Rebecca; McCarthy, James S.

2016-01-01

Background Accurate quantitative assessment of infection with soil transmitted helminths and protozoa is key to the interpretation of epidemiologic studies of these parasites, as well as for monitoring large scale treatment efficacy and effectiveness studies. As morbidity and transmission of helminth infections are directly related to both the prevalence and intensity of infection, there is particular need for improved techniques for assessment of infection intensity for both purposes. The current study aimed to evaluate two multiplex PCR assays to determine prevalence and intensity of intestinal parasite infections, and compare them to standard microscopy. Methodology/Principal Findings Faecal samples were collected from a total of 680 people, originating from rural communities in Timor-Leste (467 samples) and Cambodia (213 samples). DNA was extracted from stool samples and subject to two multiplex real-time PCR reactions the first targeting: Necator americanus, Ancylostoma spp., Ascaris spp., and Trichuris trichiura; and the second Entamoeba histolytica, Cryptosporidium spp., Giardia. duodenalis, and Strongyloides stercoralis. Samples were also subject to sodium nitrate flotation for identification and quantification of STH eggs, and zinc sulphate centrifugal flotation for detection of protozoan parasites. Higher parasite prevalence was detected by multiplex PCR (hookworms 2.9 times higher, Ascaris 1.2, Giardia 1.6, along with superior polyparasitism detection with this effect magnified as the number of parasites present increased (one: 40.2% vs. 38.1%, two: 30.9% vs. 12.9%, three: 7.6% vs. 0.4%, four: 0.4% vs. 0%). Although, all STH positive samples were low intensity infections by microscopy as defined by WHO guidelines the DNA-load detected by multiplex PCR suggested higher intensity infections. Conclusions/Significance Multiplex PCR, in addition to superior sensitivity, enabled more accurate determination of infection intensity for Ascaris, hookworms and

Improved assessment of accuracy and performance using a rotational paper-based device for multiplexed detection of heavy metals.

PubMed

Sun, Xiange; Li, Bowei; Qi, Anjin; Tian, Chongguo; Han, Jinglong; Shi, Yajun; Lin, Bingcheng; Chen, Lingxin

2018-02-01

In this work, a novel rotational microfluidic paper-based device was developed to improve the accuracy and performance of the multiplexed colorimetric detection by effectively avoiding the diffusion of colorimetric reagent on the detection zone. The integrated paper-based rotational valves were used to control the connection or disconnection between detection zones and fluid channels. Based on the manipulation of the rotational valves, this rotational paper-based device could prevent the random diffusion of colorimetric reagent and reduce the error of quantitative analysis considerably. The multiplexed colorimetric detection of heavy metals Ni(II), Cu(II) and Cr(VI) were implemented on the rotational device and the detection limits could be found to be 4.8, 1.6, and 0.18mg/L, respectively. The developed rotational device showed the great advantage in improving the detection accuracy and was expected to be a low-cost, portable analytical platform for the on-site detection. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Widefield quantitative multiplex surface enhanced Raman scattering imaging in vivo

NASA Astrophysics Data System (ADS)

McVeigh, Patrick Z.; Mallia, Rupananda J.; Veilleux, Israel; Wilson, Brian C.

2013-04-01

In recent years numerous studies have shown the potential advantages of molecular imaging in vitro and in vivo using contrast agents based on surface enhanced Raman scattering (SERS), however the low throughput of traditional point-scanned imaging methodologies have limited their use in biological imaging. In this work we demonstrate that direct widefield Raman imaging based on a tunable filter is capable of quantitative multiplex SERS imaging in vivo, and that this imaging is possible with acquisition times which are orders of magnitude lower than achievable with comparable point-scanned methodologies. The system, designed for small animal imaging, has a linear response from (0.01 to 100 pM), acquires typical in vivo images in <10 s, and with suitable SERS reporter molecules is capable of multiplex imaging without compensation for spectral overlap. To demonstrate the utility of widefield Raman imaging in biological applications, we show quantitative imaging of four simultaneous SERS reporter molecules in vivo with resulting probe quantification that is in excellent agreement with known quantities (R2>0.98).

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

PubMed

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

2013-12-01

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

Graphite nanocomposites sensor for multiplex detection of antioxidants in food.

PubMed

Ng, Khan Loon; Tan, Guan Huat; Khor, Sook Mei

2017-12-15

Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tert-butylhydroquinone (TBHQ) are synthetic antioxidants used in the food industry. Herein, we describe the development of a novel graphite nanocomposite-based electrochemical sensor for the multiplex detection and measurement of BHA, BHT, and TBHQ levels in complex food samples using a linear sweep voltammetry technique. Moreover, our newly established analytical method exhibited good sensitivity, limit of detection, limit of quantitation, and selectivity. The accuracy and reliability of analytical results were challenged by method validation and comparison with the results of the liquid chromatography method, where a linear correlation of more than 0.99 was achieved. The addition of sodium dodecyl sulfate as supporting additive further enhanced the LSV response (anodic peak current, I pa ) of BHA and BHT by 2- and 20-times, respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

PubMed

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

2011-01-01

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

Microfluidic platform for multiplexed detection in single cells and methods thereof

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

Wu, Meiye; Singh, Anup K.

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

Diagnosis of ocular toxoplasmosis by two polymerase chain reaction (PCR) examinations: qualitative multiplex and quantitative real-time.

PubMed

Sugita, Sunao; Ogawa, Manabu; Inoue, Shizu; Shimizu, Norio; Mochizuki, Manabu

2011-09-01

To establish a two-step polymerase chain reaction (PCR) diagnostic system for ocular toxoplasmosis. A total of 13 ocular fluid samples (11 aqueous humor and 2 vitreous fluid) were collected from 13 patients with clinically suspected ocular toxoplasmosis. Ten ocular samples from other uveitis patients and 20 samples from subjects without ocular inflammation were used as controls. Two polymerase chain reaction (PCR) methods, i.e., qualitative multiplex PCR and quantitative real-time PCR, were used to measure the toxoplasma genome (T. gondii B1 gene). Qualitative multiplex PCR detected T. gondii B1 gene in the ocular fluids of 11 out of 13 patients with clinically suspected ocular toxoplasmosis. In real-time PCR, we detected high copy numbers of T. gondii DNA (5.1 × 10(2)-2.1 × 10(6) copies/mL) in a total of 10 patients (10/13, 77%). Only ocular toxoplasmosis scar lesions were observed in the three real-time PCR-negative patients. PCR assay results for the samples from the two control groups were all negative. The two-step PCR examination to detect toxoplasma DNA is a useful tool for diagnosing ocular toxoplasmosis.

Indirect competitive assays on DVD for direct multiplex detection of drugs of abuse in oral fluids.

PubMed

Zhang, Lingling; Li, Xiaochun; Li, Yunchao; Shi, Xiaoli; Yu, Hua-Zhong

2015-02-03

On-site oral fluid testing for drugs of abuse has become prominent in order to take immediate administrative action in an enforcement process. Herein, we report a DVD technology-based indirect competitive immunoassay platform for the quantitative detection of drugs of abuse. A microfluidic approach was adapted to prepare multiplex immunoassays on a standard DVD-R, an unmodified multimode DVD/Blu-Ray drive to read signal, and a free disc-quality analysis software program to process the data. The DVD assay platform was successfully demonstrated for the simultaneous, quantitative detection of drug candidates (morphine and cocaine) in oral fluids with high selectivity. The detection limit achieved was as low as 1.0 ppb for morphine and 5.0 ppb for cocaine, comparable with that of standard mass spectrometry and ELISA methods.

Multiplex PCR detection of waterborne intestinal protozoa: microsporidia, Cyclospora, and Cryptosporidium.

PubMed

Lee, Seung-Hyun; Joung, Migyo; Yoon, Sejoung; Choi, Kyoungjin; Park, Woo-Yoon; Yu, Jae-Ran

2010-12-01

Recently, emerging waterborne protozoa, such as microsporidia, Cyclospora, and Cryptosporidium, have become a challenge to human health worldwide. Rapid, simple, and economical detection methods for these major waterborne protozoa in environmental and clinical samples are necessary to control infection and improve public health. In the present study, we developed a multiplex PCR test that is able to detect all these 3 major waterborne protozoa at the same time. Detection limits of the multiplex PCR method ranged from 10(1) to 10(2) oocysts or spores. The primers for microsporidia or Cryptosporidium used in this study can detect both Enterocytozoon bieneusi and Encephalitozoon intestinalis, or both Cryptosporidium hominis and Cryptosporidium parvum, respectively. Restriction enzyme digestion of PCR products with BsaBI or BsiEI makes it possible to distinguish the 2 species of microsporidia or Cryptosporidium, respectively. This simple, rapid, and cost-effective multiplex PCR method will be useful for detecting outbreaks or sporadic cases of waterborne protozoa infections.

Multiplexed detection of DNA sequences using a competitive displacement assay in a microfluidic SERRS-based device.

PubMed

Yazdi, Soroush H; Giles, Kristen L; White, Ian M

2013-11-05

We demonstrate sensitive and multiplexed detection of DNA sequences through a surface enhanced resonance Raman spectroscopy (SERRS)-based competitive displacement assay in an integrated microsystem. The use of the competitive displacement scheme, in which the target DNA sequence displaces a Raman-labeled reporter sequence that has lower affinity for the immobilized probe, enables detection of unlabeled target DNA sequences with a simple single-step procedure. In our implementation, the displacement reaction occurs in a microporous packed column of silica beads prefunctionalized with probe-reporter pairs. The use of a functionalized packed-bead column in a microfluidic channel provides two major advantages: (i) immobilization surface chemistry can be performed as a batch process instead of on a chip-by-chip basis, and (ii) the microporous network eliminates the diffusion limitations of a typical biological assay, which increases the sensitivity. Packed silica beads are also leveraged to improve the SERRS detection of the Raman-labeled reporter. Following displacement, the reporter adsorbs onto aggregated silver nanoparticles in a microfluidic mixer; the nanoparticle-reporter conjugates are then trapped and concentrated in the silica bead matrix, which leads to a significant increase in plasmonic nanoparticles and adsorbed Raman reporters within the detection volume as compared to an open microfluidic channel. The experimental results reported here demonstrate detection down to 100 pM of the target DNA sequence, and the experiments are shown to be specific, repeatable, and quantitative. Furthermore, we illustrate the advantage of using SERRS by demonstrating multiplexed detection. The sensitivity of the assay, combined with the advantages of multiplexed detection and single-step operation with unlabeled target sequences makes this method attractive for practical applications. Importantly, while we illustrate DNA sequence detection, the SERRS-based competitive

Quantitative, multiplexed workflow for deep analysis of human blood plasma and biomarker discovery by mass spectrometry.

PubMed

Keshishian, Hasmik; Burgess, Michael W; Specht, Harrison; Wallace, Luke; Clauser, Karl R; Gillette, Michael A; Carr, Steven A

2017-08-01

Proteomic characterization of blood plasma is of central importance to clinical proteomics and particularly to biomarker discovery studies. The vast dynamic range and high complexity of the plasma proteome have, however, proven to be serious challenges and have often led to unacceptable tradeoffs between depth of coverage and sample throughput. We present an optimized sample-processing pipeline for analysis of the human plasma proteome that provides greatly increased depth of detection, improved quantitative precision and much higher sample analysis throughput as compared with prior methods. The process includes abundant protein depletion, isobaric labeling at the peptide level for multiplexed relative quantification and ultra-high-performance liquid chromatography coupled to accurate-mass, high-resolution tandem mass spectrometry analysis of peptides fractionated off-line by basic pH reversed-phase (bRP) chromatography. The overall reproducibility of the process, including immunoaffinity depletion, is high, with a process replicate coefficient of variation (CV) of <12%. Using isobaric tags for relative and absolute quantitation (iTRAQ) 4-plex, >4,500 proteins are detected and quantified per patient sample on average, with two or more peptides per protein and starting from as little as 200 μl of plasma. The approach can be multiplexed up to 10-plex using tandem mass tags (TMT) reagents, further increasing throughput, albeit with some decrease in the number of proteins quantified. In addition, we provide a rapid protocol for analysis of nonfractionated depleted plasma samples analyzed in 10-plex. This provides ∼600 quantified proteins for each of the ten samples in ∼5 h of instrument time.

Challenges in designing a Taqman-based multiplex assay for the simultaneous detection of Herpes simplex virus types 1 and 2 and Varicella-zoster virus.

PubMed

Weidmann, Manfred; Armbruster, Katrin; Hufert, Frank T

2008-08-01

To optimise molecular detection of herpesviruses an internally controlled multiplex Taqman-PCR for the detection of Herpes simplex virus 1 (HSV1), Herpes simplex virus 2 (HSV2) and Varicella-zoster virus (VZV) was developed. The selection of the dye combination working on the ABI 7700 cycler for this multiplex PCR revealed crosstalk phenomena between several combinations of reference dyes and reporter dyes. A final dye combination with CY5 as reference dye and FAM/JOE/TXR as reporter dyes was selected. The influence of the concentration of the internal positive control (IPC) concentration on the quantitative results of HSV1, HSV2 and VZV positive patient samples was analysed. The results indicate that high IPC concentrations are detrimental for the sensitivity of the multiplex assay and that the presence of the IPC molecule narrows the dynamic range of the duplex PCRs between any of the virus PCRs and the IPC-PCR. The optimised multiplex assay detecting HSV1, HSV2 and VZV using 10(3) IPC molecules showed a performance and sensitivity comparable to that of the individual assays.

Multiplex PCR for rapid detection of genes encoding class A carbapenemases.

PubMed

Hong, Sang Sook; Kim, Kyeongmi; Huh, Ji Young; Jung, Bochan; Kang, Myung Seo; Hong, Seong Geun

2012-09-01

In recent years, there have been increasing reports of KPC-producing Klebsiella pneumoniae in Korea. The modified Hodge test can be used as a phenotypic screening test for class A carbapenamase (CAC)-producing clinical isolates; however, it does not distinguish between carbapenemase types. The confirmation of type of CAC is important to ensure optimal therapy and to prevent transmission. This study applied a novel multiplex PCR assay to detect and differentiate CAC genes in a single reaction. Four primer pairs were designed to amplify fragments encoding 4 CAC families (SME, IMI/NMC-A, KPC, and GES). The multiplex PCR detected all genes tested for 4 CAC families that could be differentiated by fragment size according to gene type. This multiplex PCR offers a simple and useful approach for detecting and distinguishing CAC genes in carbapenem-resistant strains that are metallo-β-lactamase nonproducers.

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.

Using multiple PCR and CE with chemiluminescence detection for simultaneous qualitative and quantitative analysis of genetically modified organism.

PubMed

Guo, Longhua; Qiu, Bin; Chi, Yuwu; Chen, Guonan

2008-09-01

In this paper, an ultrasensitive CE-CL detection system coupled with a novel double-on-column coaxial flow detection interface was developed for the detection of PCR products. A reliable procedure based on this system had been demonstrated for qualitative and quantitative analysis of genetically modified organism-the detection of Roundup Ready Soy (RRS) samples was presented as an example. The promoter, terminator, function and two reference genes of RRS were amplified with multiplex PCR simultaneously. After that, the multiplex PCR products were labeled with acridinium ester at the 5'-terminal through an amino modification and then analyzed by the proposed CE-CL system. Reproducibility of analysis times and peak heights for the CE-CL analysis were determined to be better than 0.91 and 3.07% (RSD, n=15), respectively, for three consecutive days. It was shown that this method could accurately and qualitatively detect RRS standards and the simulative samples. The evaluation in terms of quantitative analysis of RRS provided by this new method was confirmed by comparing our assay results with those of the standard real-time quantitative PCR (RT-QPCR) using SYBR Green I dyes. The results showed a good coherence between the two methods. This approach demonstrated the possibility for accurate qualitative and quantitative detection of GM plants in a single run.

A multiplexed system for quantitative comparisons of chromatin landscapes

PubMed Central

van Galen, Peter; Viny, Aaron D.; Ram, Oren; Ryan, Russell J.H.; Cotton, Matthew J.; Donohue, Laura; Sievers, Cem; Drier, Yotam; Liau, Brian B.; Gillespie, Shawn M.; Carroll, Kaitlin M.; Cross, Michael B.; Levine, Ross L.; Bernstein, Bradley E.

2015-01-01

Genome-wide profiling of histone modifications can provide systematic insight into the regulatory elements and programs engaged in a given cell type. However, conventional chromatin immunoprecipitation and sequencing (ChIP-seq) does not capture quantitative information on histone modification levels, requires large amounts of starting material, and involves tedious processing of each individual sample. Here we address these limitations with a technology that leverages DNA barcoding to profile chromatin quantitatively and in multiplexed format. We concurrently map relative levels of multiple histone modifications across multiple samples, each comprising as few as a thousand cells. We demonstrate the technology by monitoring dynamic changes following inhibition of P300, EZH2 or KDM5, by linking altered epigenetic landscapes to chromatin regulator mutations, and by mapping active and repressive marks in purified human hematopoietic stem cells. Hence, this technology enables quantitative studies of chromatin state dynamics across rare cell types, genotypes, environmental conditions and drug treatments. PMID:26687680

Highly multiplexed and quantitative cell-surface protein profiling using genetically barcoded antibodies.

PubMed

Pollock, Samuel B; Hu, Amy; Mou, Yun; Martinko, Alexander J; Julien, Olivier; Hornsby, Michael; Ploder, Lynda; Adams, Jarrett J; Geng, Huimin; Müschen, Markus; Sidhu, Sachdev S; Moffat, Jason; Wells, James A

2018-03-13

Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states. Copyright © 2018 the Author(s). Published by PNAS.

Aqueous two-phase systems enable multiplexing of homogeneous immunoassays

PubMed Central

Simon, Arlyne B.; Frampton, John P.; Huang, Nien-Tsu; Kurabayashi, Katsuo; Paczesny, Sophie; Takayama, Shuichi

2014-01-01

Quantitative measurement of protein biomarkers is critical for biomarker validation and early disease detection. Current multiplex immunoassays are time consuming costly and can suffer from low accuracy. For example, multiplex ELISAs require multiple, tedious, washing and blocking steps. Moreover, they suffer from nonspecific antibody cross-reactions, leading to high background and false-positive signals. Here, we show that co-localizing antibody-bead pairs in an aqueous two-phase system (ATPS) enables multiplexing of sensitive, no-wash, homogeneous assays, while preventing nonspecific antibody cross-reactions. Our cross-reaction-free, multiplex assay can simultaneously detect picomolar concentrations of four protein biomarkers ((C-X-C motif) ligand 10 (CXCL10), CXCL9, interleukin (IL)-8 and IL-6) in cell supernatants using a single assay well. The potential clinical utility of the assay is demonstrated by detecting diagnostic biomarkers (CXCL10 and CXCL9) in plasma from 88 patients at the onset of the clinical symptoms of chronic graft-versus-host disease (GVHD). PMID:25083509

Impact of Aerosol Dust on xMAP Multiplex Detection of Different Class Pathogens

PubMed Central

Kleymenov, Denis A.; Gushchin, Vladimir A.; Gintsburg, Alexander L.; Tkachuk, Artem P.

2017-01-01

Environmental or city-scale bioaerosol surveillance can provide additional value for biodefense and public health. Efficient bioaerosol monitoring should rely on multiplex systems capable of detecting a wide range of biologically hazardous components potentially present in air (bacteria, viruses, toxins and allergens). xMAP technology from LuminexTM allows multiplex bead-based detection of antigens or nucleic acids, but its use for simultaneous detection of different classes of pathogens (bacteria, virus, toxin) is questionable. Another problem is the detection of pathogens in complex matrices, e.g., in the presence of dust. In the this research, we developed the model xMAP multiplex test-system aiRDeTeX 1.0, which enables detection of influenza A virus, Adenovirus type 6 Salmonella typhimurium, and cholera toxin B subunit representing RNA virus, DNA virus, gram-negative bacteria and toxin respectively as model organisms of biologically hazardous components potentially present in or spreadable through the air. We have extensively studied the effect of matrix solution (PBS, distilled water), environmental dust and ultrasound treatment for monoplex and multiplex detection efficiency of individual targets. All targets were efficiently detectable in PBS and in the presence of dust. Ultrasound does not improve the detection except for bacterial LPS. PMID:29238328

Multiplex polymerase chain reaction-capillary gel electrophoresis: a promising tool for GMO screening--assay for simultaneous detection of five genetically modified cotton events and species.

PubMed

Nadal, Anna; Esteve, Teresa; Pla, Maria

2009-01-01

A multiplex polymerase chain reaction assay coupled to capillary gel electrophoresis for amplicon identification by size and color (multiplex PCR-CGE-SC) was developed for simultaneous detection of cotton species and 5 events of genetically modified (GM) cotton. Validated real-time-PCR reactions targeting Bollgard, Bollgard II, Roundup Ready, 3006-210-23, and 281-24-236 junction sequences, and the cotton reference gene acp1 were adapted to detect more than half of the European Union-approved individual or stacked GM cotton events in one reaction. The assay was fully specific (<1.7% of false classification rate), with limit of detection values of 0.1% for each event, which were also achieved with simulated mixtures at different relative percentages of targets. The assay was further combined with a second multiplex PCR-CGE-SC assay to allow simultaneous detection of 6 cotton and 5 maize targets (two endogenous genes and 9 GM events) in two multiplex PCRs and a single CGE, making the approach more economic. Besides allowing simultaneous detection of many targets with adequate specificity and sensitivity, the multiplex PCR-CGE-SC approach has high throughput and automation capabilities, while keeping a very simple protocol, e.g., amplification and labeling in one step. Thus, it is an easy and inexpensive tool for initial screening, to be complemented with quantitative assays if necessary.

Multiplex PCR for Rapid Detection of Genes Encoding Class A Carbapenemases

PubMed Central

Hong, Sang Sook; Kim, Kyeongmi; Huh, Ji Young; Jung, Bochan; Kang, Myung Seo

2012-01-01

In recent years, there have been increasing reports of KPC-producing Klebsiella pneumoniae in Korea. The modified Hodge test can be used as a phenotypic screening test for class A carbapenamase (CAC)-producing clinical isolates; however, it does not distinguish between carbapenemase types. The confirmation of type of CAC is important to ensure optimal therapy and to prevent transmission. This study applied a novel multiplex PCR assay to detect and differentiate CAC genes in a single reaction. Four primer pairs were designed to amplify fragments encoding 4 CAC families (SME, IMI/NMC-A, KPC, and GES). The multiplex PCR detected all genes tested for 4 CAC families that could be differentiated by fragment size according to gene type. This multiplex PCR offers a simple and useful approach for detecting and distinguishing CAC genes in carbapenem-resistant strains that are metallo-β-lactamase nonproducers. PMID:22950072

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

PubMed

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

2016-10-18

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

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.

Ultrasensitive Detection of Multiplexed Somatic Mutations Using MALDI-TOF Mass Spectrometry.

PubMed

Mosko, Michael J; Nakorchevsky, Aleksey A; Flores, Eunice; Metzler, Heath; Ehrich, Mathias; van den Boom, Dirk J; Sherwood, James L; Nygren, Anders O H

2016-01-01

Multiplex detection of low-frequency mutations is becoming a necessary diagnostic tool for clinical laboratories interested in noninvasive prognosis and prediction. Challenges include the detection of minor alleles among abundant wild-type alleles, the heterogeneous nature of tumors, and the limited amount of available tissue. A method that can reliably detect minor variants <1% in a multiplexed reaction using a platform amenable to a variety of throughputs would meet these requirements. We developed a novel approach, UltraSEEK, for high-throughput, multiplexed, ultrasensitive mutation detection and used it for detection of mutant sequence mixtures as low as 0.1% minor allele frequency. The process consisted of multiplex PCR, followed by mutation-specific, single-base extension using chain terminators labeled with a moiety for solid phase capture. The captured and enriched products were then identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. For verification, we successfully analyzed ultralow fractions of mutations in a set of characterized cell lines, and included a direct comparison to droplet digital PCR. Finally, we verified the specificity in a set of 122 paired tumor and circulating cell-free DNA samples from melanoma patients. Our results show that the UltraSEEK chemistry is a particularly powerful approach for the detection of somatic variants, with the potential to be an invaluable resource to investigators in saving time and material without compromising analytical sensitivity and accuracy. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Assembling substrate-less plasmonic metacrystals at the oil/water interface for multiplex ultratrace analyte detection.

PubMed

Lee, Yih Hong; Lee, Hiang Kwee; Ho, Jonathan Yong Chew; Yang, Yijie; Ling, Xing Yi

2016-08-15

Current substrate-less SERS platforms are limited to uncontrolled aggregation of plasmonic nanoparticles or quasi-crystalline arrays of spherical nanoparticles, with no study on how the lattice structures formed by nanoparticle self-assembly affect their detection capabilities. Here, we organize Ag octahedral building blocks into two large-area plasmonic metacrystals at the oil/water interface, and investigate their in situ SERS sensing capabilities. Amphiphilic octahedra assemble into a hexagonal close-packed metacrystal, while hydrophobic octahedra assemble into an open square metacrystal. The lower packing density square metacrystal gives rise to much stronger SERS enhancement than the denser packing hexagonal metacrystal, arising from the larger areas of plasmonic hotspots within the square metacrystal at the excitation wavelength. We further demonstrate the ability of the square metacrystal to achieve quantitative ultratrace detection of analytes from both the aqueous and organic phases. Detection limits are at the nano-molar levels, with analytical enhancement factors reaching 10(8). In addition, multiplex detection across both phases can be achieved in situ without any loss of signal quantitation.

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

PubMed Central

Wernike, Kerstin; Hoffmann, Bernd

2013-01-01

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

Simultaneous detection, typing and quantitation of oncogenic human papillomavirus by multiplex consensus real-time PCR.

PubMed

Jenkins, Andrew; Allum, Anne-Gry; Strand, Linda; Aakre, Randi Kersten

2013-02-01

A consensus multiplex real-time PCR test (PT13-RT) for the oncogenic human papillomavirus (HPV) types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 66 is described. The test targets the L1 gene. Analytical sensitivity is between 4 and 400 GU (genomic units) in the presence of 500 ng of human DNA, corresponding to 75,000 human cells. HPV types are grouped into multiplex groups of 3 or 4 resulting in the use of 4 wells per sample and permitting up to 24 samples per run (including controls) in a standard 96-well real-time PCR instrument. False negative results are avoided by (a) measuring sample DNA concentration to control that sufficient cellular material is present and (b) including HPV type 6 as a homologous internal control in order to detect PCR inhibition or competition from other (non-oncogenic) HPV types. Analysis time from refrigerator to report is 8 h, including 2.5 h hands-on time. Relative to the HC2 test, the sensitivity and specificity were respectively 98% and 83%, the lower specificity being attributable to the higher analytical sensitivity of PT13-RT. To assess type determination comparison was made with a reversed line-blot test. Type concordance was high (κ=0.79) with discrepancies occurring mostly in multiple-positive samples. Copyright © 2012 Elsevier B.V. All rights reserved.

A multiplexable, microfluidic platform for the rapid quantitation of a biomarker panel for early ovarian cancer detection at the point-of-care

PubMed Central

Shadfan, Basil H.; Simmons, Archana R.; Simmons, Glennon W.; Ho, Andy; Wong, Jorge; Lu, Karen H.; Bast, Robert C.; McDevitt, John T.

2015-01-01

Point-of-care (POC) diagnostic platforms have the potential to enable low-cost, large-scale screening. As no single biomarker is shed by all ovarian cancers, multiplexed biomarker panels promise improved sensitivity and specificity to address the unmet need for early detection of ovarian cancer. We have configured the programmable bio-nano-chip (p-BNC) - a multiplexable, microfluidic, modular platform - to quantify a novel multimarker panel comprising CA125, HE4, MMP-7 and CA72-4. The p-BNC is a bead-based immunoanalyzer system with a credit-card-sized footprint that integrates automated sample metering, bubble and debris removal, reagent storage and waste disposal, permitting POC analysis. Multiplexed p-BNC immunoassays demonstrated high specificity, low cross-reactivity, low limits of detection suitable for early detection, and a short analysis time of 43 minutes. Day-to-day variability, a critical factor for longitudinally monitoring biomarkers, ranged between 5.4–10.5%, well below the biological variation for all four markers. Biomarker concentrations for 31 late-stage sera correlated well (R2 = 0.71 to 0.93 for various biomarkers) with values obtained on the Luminex® platform. In a 31 patient cohort encompassing early- and late-stage ovarian cancers along with benign and healthy controls, the multiplexed p-BNC panel was able to distinguish cases from controls with 68.7% sensitivity at 80% specificity. Utility for longitudinal biomarker monitoring was demonstrated with pre-diagnostic sera from 2 cases and 4 controls. Taken together, the p-BNC shows strong promise as a diagnostic tool for large-scale screening that takes advantage of faster results and lower costs while leveraging possible improvement in sensitivity and specificity from biomarker panels. PMID:25388014

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

PubMed

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

2009-05-01

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

Development and validation of a multiplex quantitative polymerase chain reaction assay for the detection of Mollicutes impurities in human cells, cultured under good manufacturing practice conditions, and following European Pharmacopoeia requirements and the International Conference on Harmonization guidelines.

PubMed

Vanni, Irene; Ugolotti, Elisabetta; Raso, Alessandro; Di Marco, Eddi; Melioli, Giovanni; Biassoni, Roberto

2012-07-01

The clinical applications of in vitro manipulated cultured cells and their precursors are often made use of in therapeutic trials. However, tissue cultures can be easily contaminated by the ubiquitous Mollicutes micro-organisms, which can cause various and severe alterations in cellular function. Thus methods able to detect and trace Mollicutes impurities contaminating cell cultures are required before starting any attempt to grow cells under good manufacturing practice (GMP) conditions. We developed a multiplex quantitative polymerase chain reaction (qPCR) assay specific for the 16S-23S rRNA intergenic spacer regions, for the Tuf and P1 cytoadhesin genes, able to detect contaminant Mollicutes species in a single tube reaction. The system was validated by analyzing different cell lines and the positive samples were confirmed by 16S and P1 cytoadhesin gene dideoxy sequencing. Our multiplex qPCR detection system was able to reach a sensitivity, specificity and robustness comparable with the culture and the indicator cell culture method, as required by the European Pharmacopoeia guidelines. We have developed a multiplex qPCR method, validated following International Conference on Harmonization (ICH) guidelines, as a qualitative limit test for impurities, assessing the validation characteristics of limit of detection and specificity. It also follows the European Pharmacopoeia guidelines and Food and Drug Administration (FDA) requirements.

Multiplex polymerase chain reaction detection of black-pigmented bacteria in infections of endodontic origin.

PubMed

Seol, Jung-Hwan; Cho, Byung-Hoon; Chung, Chong-Pyoung; Bae, Kwang-Shik

2006-02-01

The purpose of this study was to detect the presence of Porphyromonas endodontalis, P. gingivalis, Prevotella intermedia, P. nigrescens, and P. tannerae from clinical samples using multiplex polymerase chain reactions (PCR). Two different multiplex PCR protocols were used (one for the two Porphyromonas species and the other for the three Prevotella species), each one using a primer pair specific for each target species. The results were compared to those of the conventional culture procedures. Microbial samples were taken aseptically from 40 infected root canals and abscesses from patients. Samples were cultured in an anaerobic condition for conventional identification using a Rapid ID 32 A kit. Multiplex PCR was processed using the DNA extracted from each sample. At least one of the five species of black-pigmented bacteria (BPB) were detected in 65% (26 of 40) of the samples using multiplex PCR, and in 15% (6 of 40) using the conventional culture procedures. Multiplex PCR was more rapid, sensitive, specific, and effective in detecting BPB than the conventional culture procedures.

Rapid detection method for Bacillus anthracis using a combination of multiplexed real-time PCR and pyrosequencing and its application for food biodefense.

PubMed

Janzen, Timothy W; Thomas, Matthew C; Goji, Noriko; Shields, Michael J; Hahn, Kristen R; Amoako, Kingsley K

2015-02-01

Bacillus anthracis, the causative agent of anthrax, has the capacity to form highly resilient spores as part of its life cycle. The potential for the dissemination of these spores using food as a vehicle is a huge public health concern and, hence, requires the development of a foodborne bioterrorism response approach. In this work, we address a critical gap in food biodefense by presenting a novel, combined, sequential method involving the use of real-time PCR and pyrosequencing for the rapid, specific detection of B. anthracis spores in three food matrices: milk, apple juice, and bottled water. The food samples were experimentally inoculated with 40 CFU ml(-1), and DNA was extracted from the spores and analyzed after immunomagnetic separation. Applying the combination of multiplex real-time PCR and pyrosequencing, we successfully detected the presence of targets on both of the virulence plasmids and the chromosome. The results showed that DNA amplicons generated from a five-target multiplexed real-time PCR detection using biotin-labeled primers can be used for single-plex pyrosequencing detection. The combined use of multiplexed real-time PCR and pyrosequencing is a novel, rapid detection method for B. anthracis from food and provides a tool for accurate, quantitative identification with potential biodefense applications.

Detection of common diarrhea-causing pathogens in Northern Taiwan by multiplex polymerase chain reaction.

PubMed

Huang, Shu-Huan; Lin, Yi-Fang; Tsai, Ming-Han; Yang, Shuan; Liao, Mei-Ling; Chao, Shao-Wen; Hwang, Cheng-Cheng

2018-06-01

Conventional methods for identifying gastroenteritis pathogens are time consuming, more likely to result in a false-negative, rely on personnel with diagnostic expertise, and are dependent on the specimen status. Alternatively, molecular diagnostic methods permit the rapid, simultaneous detection of multiple pathogens with high sensitivity and specificity. The present study compared conventional methods with the Luminex xTAG Gastrointestinal Pathogen Panel (xTAG GPP) for the diagnosis of infectious gastroenteritis in northern Taiwan. From July 2015 to April 2016, 217 clinical fecal samples were collected from patients with suspected infectious gastroenteritis. All specimens were tested using conventional diagnostic techniques following physicians' orders as well as with the xTAG GPP. The multiplex polymerase chain reaction (PCR) approach detected significantly more positive samples with bacterial, viral, and/or parasitic infections as compared to conventional analysis (55.8% vs 40.1%, respectively; P < .001). Moreover, multiplex PCR could detect Escherichia coli O157, enterotoxigenic E coli, Shiga-like toxin-producing E coli, Cryptosporidium, and Giardia, which were undetectable by conventional methods. Furthermore, 48 pathogens in 23 patients (10.6%) with coinfections were identified only using the multiplex PCR approach. Of which, 82.6% were from pediatric patients. Because the detection rates using multiplex PCR are higher than conventional methods, and some pediatric pathogens could only be detected by multiplex PCR, this approach may be useful in rapidly diagnosing diarrheal disease in children and facilitating treatment initiation. Further studies are necessary to determine if multiplex PCR improves patient outcomes and reduces costs.

Detection of common diarrhea-causing pathogens in Northern Taiwan by multiplex polymerase chain reaction

PubMed Central

Huang, Shu-Huan; Lin, Yi-Fang; Tsai, Ming-Han; Yang, Shuan; Liao, Mei-Ling; Chao, Shao-Wen; Hwang, Cheng-Cheng

2018-01-01

Abstract Conventional methods for identifying gastroenteritis pathogens are time consuming, more likely to result in a false-negative, rely on personnel with diagnostic expertise, and are dependent on the specimen status. Alternatively, molecular diagnostic methods permit the rapid, simultaneous detection of multiple pathogens with high sensitivity and specificity. The present study compared conventional methods with the Luminex xTAG Gastrointestinal Pathogen Panel (xTAG GPP) for the diagnosis of infectious gastroenteritis in northern Taiwan. From July 2015 to April 2016, 217 clinical fecal samples were collected from patients with suspected infectious gastroenteritis. All specimens were tested using conventional diagnostic techniques following physicians’ orders as well as with the xTAG GPP. The multiplex polymerase chain reaction (PCR) approach detected significantly more positive samples with bacterial, viral, and/or parasitic infections as compared to conventional analysis (55.8% vs 40.1%, respectively; P < .001). Moreover, multiplex PCR could detect Escherichia coli O157, enterotoxigenic E coli, Shiga-like toxin-producing E coli, Cryptosporidium, and Giardia, which were undetectable by conventional methods. Furthermore, 48 pathogens in 23 patients (10.6%) with coinfections were identified only using the multiplex PCR approach. Of which, 82.6% were from pediatric patients. Because the detection rates using multiplex PCR are higher than conventional methods, and some pediatric pathogens could only be detected by multiplex PCR, this approach may be useful in rapidly diagnosing diarrheal disease in children and facilitating treatment initiation. Further studies are necessary to determine if multiplex PCR improves patient outcomes and reduces costs. PMID:29879060

Multiplexed quantitative real-time PCR to detect 22q11.2 deletion in patients with congenital heart disease

PubMed Central

Mahnke, Donna K.; Larson, Joshua M.; Ghanta, Sujana; Feng, Ying; Simpson, Pippa M.; Broeckel, Ulrich; Duffy, Kelly; Tweddell, James S.; Grossman, William J.; Routes, John M.; Mitchell, Michael E.

2010-01-01

22q11.2 Deletion syndrome (22q11.2 DS) [DiGeorge syndrome type 1 (DGS1)] occurs in ∼1:3,000 live births; 75% of children with DGS1 have severe congenital heart disease requiring early intervention. The gold standard for detection of DGS1 is fluorescence in situ hybridization (FISH) with a probe at the TUPLE1 gene. However, FISH is costly and is typically ordered in conjunction with a karyotype analysis that takes several days. Therefore, FISH is underutilized and the diagnosis of 22q11.2 DS is frequently delayed, often resulting in profound clinical consequences. Our goal was to determine whether multiplexed, quantitative real-time PCR (MQPCR) could be used to detect the haploinsufficiency characteristic of 22q11.2 DS. A retrospective blinded study was performed on 382 subjects who had undergone congenital heart surgery. MQPCR was performed with a probe localized to the TBX1 gene on human chromosome 22, a gene typically deleted in 22q11.2 DS. Cycle threshold (Ct) was used to calculate the relative gene copy number (rGCN). Confirmation analysis was performed with the Affymetrix 6.0 Genome-Wide SNP Array. With MQPCR, 361 subjects were identified as nondeleted with an rGCN near 1.0 and 21 subjects were identified as deleted with an rGCN near 0.5, indicative of a hemizygous deletion. The sensitivity (21/21) and specificity (361/361) of MQPCR to detect 22q11.2 deletions was 100% at an rGCN value drawn at 0.7. One of 21 subjects with a prior clinical (not genetically confirmed) DGS1 diagnosis was found not to carry the deletion, while another subject, not previously identified as DGS1, was detected as deleted and subsequently confirmed via microarray. The MQPCR assay is a rapid, inexpensive, sensitive, and specific assay that can be used to screen for 22q11.2 deletion syndrome. The assay is readily adaptable to high throughput. PMID:20551144

Advances in multiplexed MRM-based protein biomarker quantitation toward clinical utility.

PubMed

Percy, Andrew J; Chambers, Andrew G; Yang, Juncong; Hardie, Darryl B; Borchers, Christoph H

2014-05-01

Accurate and rapid protein quantitation is essential for screening biomarkers for disease stratification and monitoring, and to validate the hundreds of putative markers in human biofluids, including blood plasma. An analytical method that utilizes stable isotope-labeled standard (SIS) peptides and selected/multiple reaction monitoring-mass spectrometry (SRM/MRM-MS) has emerged as a promising technique for determining protein concentrations. This targeted approach has analytical merit, but its true potential (in terms of sensitivity and multiplexing) has yet to be realized. Described herein is a method that extends the multiplexing ability of the MRM method to enable the quantitation 142 high-to-moderate abundance proteins (from 31mg/mL to 44ng/mL) in undepleted and non-enriched human plasma in a single run. The proteins have been reported to be associated to a wide variety of non-communicable diseases (NCDs), from cardiovascular disease (CVD) to diabetes. The concentrations of these proteins in human plasma are inferred from interference-free peptides functioning as molecular surrogates (2 peptides per protein, on average). A revised data analysis strategy, involving the linear regression equation of normal control plasma, has been instituted to enable the facile application to patient samples, as demonstrated in separate nutrigenomics and CVD studies. The exceptional robustness of the LC/MS platform and the quantitative method, as well as its high throughput, makes the assay suitable for application to patient samples for the verification of a condensed or complete protein panel. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge. © 2013.

The validation and utility of a quantitative one-step multiplex RT real-time PCR targeting Rotavirus A and Norovirus

PubMed Central

Dung, Tran Thi Ngoc; Phat, Voong Vinh; Nga, Tran Vu Thieu; My, Phan Vu Tra; Duy, Pham Thanh; Campbell, James I.; Thuy, Cao Thu; Hoang, Nguyen Van Minh; Van Minh, Pham; Le Phuc, Hoang; Tuyet, Pham Thi Ngoc; Vinh, Ha; Kien, Duong Thi Hue; Huy, Huynh Le Anh; Vinh, Nguyen Thanh; Nga, Tran Thi Thu; Hau, Nguyen Thi Thu; Chinh, Nguyen Tran; Thuong, Tang Chi; Tuan, Ha Manh; Simmons, Cameron; Farrar, Jeremy J.; Baker, Stephen

2013-01-01

Rotavirus (RoV) and Norovirus (NoV) are the main causes of viral gastroenteritis. Currently, there is no validated multiplex real-time PCR that can detect and quantify RoV and NoV simultaneously. The aim of the study was to develop, validate, and internally control a multiplex one-step RT real-time PCR to detect and quantify RoV and NoV in stool samples. PCR sensitivity was assessed by comparing amplification against the current gold standard, enzyme immunoassay (EIA), on stool samples from 94 individuals with diarrhea and 94 individuals without diarrhea. PCR detected 10% more RoV positive samples than EIA in stools samples from patients with diarrhea. PCR detected 23% more NoV genogroup II positive samples from individuals with diarrhea and 9% more from individuals without diarrhea than EIA, respectively. Genotyping of the PCR positive/EIA negative samples suggested the higher rate of PCR positivity, in comparison to EIA, was due to increased sensitivity, rather than nonspecific hybridization. Quantitation demonstrated that the viral loads of RoV and NoV in the stools of diarrheal patients were an order of magnitude greater than in individuals without diarrhea. This internally controlled real-time PCR method is robust, exhibits a high degree of reproducibility, and may have a greater utility and sensitivity than commercial EIA kits. PMID:23046990

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

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

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

2000-05-05

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

Upconversion Nanoparticles-Encoded Hydrogel Microbeads-Based Multiplexed Protein Detection

NASA Astrophysics Data System (ADS)

Shikha, Swati; Zheng, Xiang; Zhang, Yong

2018-06-01

Fluorescently encoded microbeads are in demand for multiplexed applications in different fields. Compared to organic dye-based commercially available Luminex's xMAP technology, upconversion nanoparticles (UCNPs) are better alternatives due to their large anti-Stokes shift, photostability, nil background, and single wavelength excitation. Here, we developed a new multiplexed detection system using UCNPs for encoding poly(ethylene glycol) diacrylate (PEGDA) microbeads as well as for labeling reporter antibody. However, to prepare UCNPs-encoded microbeads, currently used swelling-based encapsulation leads to non-uniformity, which is undesirable for fluorescence-based multiplexing. Hence, we utilized droplet microfluidics to obtain encoded microbeads of uniform size, shape, and UCNPs distribution inside. Additionally, PEGDA microbeads lack functionality for probe antibodies conjugation on their surface. Methods to functionalize the surface of PEGDA microbeads (acrylic acid incorporation, polydopamine coating) reported thus far quench the fluorescence of UCNPs. Here, PEGDA microbeads surface was coated with silica followed by carboxyl modification without compromising the fluorescence intensity of UCNPs. In this study, droplet microfluidics-assisted UCNPs-encoded microbeads of uniform shape, size, and fluorescence were prepared. Multiple color codes were generated by mixing UCNPs emitting red and green colors at different ratios prior to encapsulation. UCNPs emitting blue color were used to label the reporter antibody. Probe antibodies were covalently immobilized on red UCNPs-encoded microbeads for specific capture of human serum albumin (HSA) as a model protein. The system was also demonstrated for multiplexed detection of both human C-reactive protein (hCRP) and HSA protein by immobilizing anti-hCRP antibodies on green UCNPs.

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.

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

PubMed Central

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

2014-01-01

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

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

PubMed

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

2012-11-14

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

Clinical Validation of Multiplex Real-Time PCR Assays for Detection of Bacterial Meningitis Pathogens

PubMed Central

Theodore, M. Jordan; Mair, Raydel; Trujillo-Lopez, Elizabeth; du Plessis, Mignon; Wolter, Nicole; Baughman, Andrew L.; Hatcher, Cynthia; Vuong, Jeni; Lott, Lisa; von Gottberg, Anne; Sacchi, Claudio; McDonald, J. Matthew; Messonnier, Nancy E.; Mayer, Leonard W.

2012-01-01

Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae are important causes of meningitis and other infections, and rapid, sensitive, and specific laboratory assays are critical for effective public health interventions. Singleplex real-time PCR assays have been developed to detect N. meningitidis ctrA, H. influenzae hpd, and S. pneumoniae lytA and serogroup-specific genes in the cap locus for N. meningitidis serogroups A, B, C, W135, X, and Y. However, the assay sensitivity for serogroups B, W135, and Y is low. We aimed to improve assay sensitivity and develop multiplex assays to reduce time and cost. New singleplex real-time PCR assays for serogroup B synD, W135 synG, and Y synF showed 100% specificity for detecting N. meningitidis species, with high sensitivity (serogroup B synD, 99% [75/76]; W135 synG, 97% [38/39]; and Y synF, 100% [66/66]). The lower limits of detection (LLD) were 9, 43, and 10 copies/reaction for serogroup B synD, W135 synG, and Y synF assays, respectively, a significant improvement compared to results for the previous singleplex assays. We developed three multiplex real-time PCR assays for detection of (i) N. meningitidis ctrA, H. influenzae hpd, and S. pneumoniae lytA (NHS assay); (ii) N. meningitidis serogroups A, W135, and X (AWX assay); and (iii) N. meningitidis serogroups B, C, and Y (BCY assay). Each multiplex assay was 100% specific for detecting its target organisms or serogroups, and the LLD was similar to that for the singleplex assay. Pairwise comparison of real-time PCR between multiplex and singleplex assays showed that cycle threshold values of the multiplex assay were similar to those for the singleplex assay. There were no substantial differences in sensitivity and specificity between these multiplex and singleplex real-time PCR assays. PMID:22170919

Clinical validation of multiplex real-time PCR assays for detection of bacterial meningitis pathogens.

PubMed

Wang, Xin; Theodore, M Jordan; Mair, Raydel; Trujillo-Lopez, Elizabeth; du Plessis, Mignon; Wolter, Nicole; Baughman, Andrew L; Hatcher, Cynthia; Vuong, Jeni; Lott, Lisa; von Gottberg, Anne; Sacchi, Claudio; McDonald, J Matthew; Messonnier, Nancy E; Mayer, Leonard W

2012-03-01

Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae are important causes of meningitis and other infections, and rapid, sensitive, and specific laboratory assays are critical for effective public health interventions. Singleplex real-time PCR assays have been developed to detect N. meningitidis ctrA, H. influenzae hpd, and S. pneumoniae lytA and serogroup-specific genes in the cap locus for N. meningitidis serogroups A, B, C, W135, X, and Y. However, the assay sensitivity for serogroups B, W135, and Y is low. We aimed to improve assay sensitivity and develop multiplex assays to reduce time and cost. New singleplex real-time PCR assays for serogroup B synD, W135 synG, and Y synF showed 100% specificity for detecting N. meningitidis species, with high sensitivity (serogroup B synD, 99% [75/76]; W135 synG, 97% [38/39]; and Y synF, 100% [66/66]). The lower limits of detection (LLD) were 9, 43, and 10 copies/reaction for serogroup B synD, W135 synG, and Y synF assays, respectively, a significant improvement compared to results for the previous singleplex assays. We developed three multiplex real-time PCR assays for detection of (i) N. meningitidis ctrA, H. influenzae hpd, and S. pneumoniae lytA (NHS assay); (ii) N. meningitidis serogroups A, W135, and X (AWX assay); and (iii) N. meningitidis serogroups B, C, and Y (BCY assay). Each multiplex assay was 100% specific for detecting its target organisms or serogroups, and the LLD was similar to that for the singleplex assay. Pairwise comparison of real-time PCR between multiplex and singleplex assays showed that cycle threshold values of the multiplex assay were similar to those for the singleplex assay. There were no substantial differences in sensitivity and specificity between these multiplex and singleplex real-time PCR assays.

Systematic Methodological Evaluation of a Multiplex Bead-Based Flow Cytometry Assay for Detection of Extracellular Vesicle Surface Signatures

PubMed Central

Wiklander, Oscar P. B.; Bostancioglu, R. Beklem; Welsh, Joshua A.; Zickler, Antje M.; Murke, Florian; Corso, Giulia; Felldin, Ulrika; Hagey, Daniel W.; Evertsson, Björn; Liang, Xiu-Ming; Gustafsson, Manuela O.; Mohammad, Dara K.; Wiek, Constanze; Hanenberg, Helmut; Bremer, Michel; Gupta, Dhanu; Björnstedt, Mikael; Giebel, Bernd; Nordin, Joel Z.; Jones, Jennifer C.; EL Andaloussi, Samir; Görgens, André

2018-01-01

Extracellular vesicles (EVs) can be harvested from cell culture supernatants and from all body fluids. EVs can be conceptually classified based on their size and biogenesis as exosomes and microvesicles. Nowadays, it is however commonly accepted in the field that there is a much higher degree of heterogeneity within these two subgroups than previously thought. For instance, the surface marker profile of EVs is likely dependent on the cell source, the cell’s activation status, and multiple other parameters. Within recent years, several new methods and assays to study EV heterogeneity in terms of surface markers have been described; most of them are being based on flow cytometry. Unfortunately, such methods generally require dedicated instrumentation, are time-consuming and demand extensive operator expertise for sample preparation, acquisition, and data analysis. In this study, we have systematically evaluated and explored the use of a multiplex bead-based flow cytometric assay which is compatible with most standard flow cytometers and facilitates a robust semi-quantitative detection of 37 different potential EV surface markers in one sample simultaneously. First, assay variability, sample stability over time, and dynamic range were assessed together with the limitations of this assay in terms of EV input quantity required for detection of differently abundant surface markers. Next, the potential effects of EV origin, sample preparation, and quality of the EV sample on the assay were evaluated. The findings indicate that this multiplex bead-based assay is generally suitable to detect, quantify, and compare EV surface signatures in various sample types, including unprocessed cell culture supernatants, cell culture-derived EVs isolated by different methods, and biological fluids. Furthermore, the use and limitations of this assay to assess heterogeneities in EV surface signatures was explored by combining different sets of detection antibodies in EV samples derived

Systematic Methodological Evaluation of a Multiplex Bead-Based Flow Cytometry Assay for Detection of Extracellular Vesicle Surface Signatures.

PubMed

Wiklander, Oscar P B; Bostancioglu, R Beklem; Welsh, Joshua A; Zickler, Antje M; Murke, Florian; Corso, Giulia; Felldin, Ulrika; Hagey, Daniel W; Evertsson, Björn; Liang, Xiu-Ming; Gustafsson, Manuela O; Mohammad, Dara K; Wiek, Constanze; Hanenberg, Helmut; Bremer, Michel; Gupta, Dhanu; Björnstedt, Mikael; Giebel, Bernd; Nordin, Joel Z; Jones, Jennifer C; El Andaloussi, Samir; Görgens, André

2018-01-01

Extracellular vesicles (EVs) can be harvested from cell culture supernatants and from all body fluids. EVs can be conceptually classified based on their size and biogenesis as exosomes and microvesicles. Nowadays, it is however commonly accepted in the field that there is a much higher degree of heterogeneity within these two subgroups than previously thought. For instance, the surface marker profile of EVs is likely dependent on the cell source, the cell's activation status, and multiple other parameters. Within recent years, several new methods and assays to study EV heterogeneity in terms of surface markers have been described; most of them are being based on flow cytometry. Unfortunately, such methods generally require dedicated instrumentation, are time-consuming and demand extensive operator expertise for sample preparation, acquisition, and data analysis. In this study, we have systematically evaluated and explored the use of a multiplex bead-based flow cytometric assay which is compatible with most standard flow cytometers and facilitates a robust semi-quantitative detection of 37 different potential EV surface markers in one sample simultaneously. First, assay variability, sample stability over time, and dynamic range were assessed together with the limitations of this assay in terms of EV input quantity required for detection of differently abundant surface markers. Next, the potential effects of EV origin, sample preparation, and quality of the EV sample on the assay were evaluated. The findings indicate that this multiplex bead-based assay is generally suitable to detect, quantify, and compare EV surface signatures in various sample types, including unprocessed cell culture supernatants, cell culture-derived EVs isolated by different methods, and biological fluids. Furthermore, the use and limitations of this assay to assess heterogeneities in EV surface signatures was explored by combining different sets of detection antibodies in EV samples derived

Rapid Differentiation and In Situ Detection of 16 Sourdough Lactobacillus Species by Multiplex PCR

PubMed Central

Settanni, Luca; van Sinderen, Douwe; Rossi, Jone; Corsetti, Aldo

2005-01-01

A two-step multiplex PCR-based method was designed for the rapid detection of 16 species of lactobacilli known to be commonly present in sourdough. The first step of multiplex PCR was developed with a mixture of group-specific primers, while the second step included three multiplex PCR assays with a mixture of species-specific primers. Primers were derived from sequences that specify the 16S rRNA, the 16S-23S rRNA intergenic spacer region, and part of the 23S rRNA gene. The primer pairs designed were shown to exclusively amplify the targeted rrn operon fragment of the corresponding species. Due to the reliability of simultaneously identifying Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum, a previously described multiplex PCR method employing recA gene-derived primers was included in the multiplex PCR system. The combination of a newly developed, quick bacterial DNA extraction method from sourdough and this multiplex PCR assay allows the rapid in situ detection of several sourdough-associated lactobacilli, including the recently described species Lactobacillus rossii, and thus represents a very useful alternative to culture-based methodologies. PMID:15933001

Multiplex quantitative PCR for detection of lower respiratory tract infection and meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis

PubMed Central

2010-01-01

Background Streptococcus pneumoniae and Haemophilus influenzae cause pneumonia and as Neisseria meningitidis they are important agents of meningitis. Although several PCR methods have been described for these bacteria the specificity is an underestimated problem. Here we present a quantitative multiplex real-time PCR (qmPCR) for detection of S. pneumoniae (9802 gene fragment), H. influenzae (omp P6 gene) and N. meningitidis (ctrA gene). The method was evaluated on bronchoalveolar lavage (BAL) samples from 156 adults with lower respiratory tract infection (LRTI) and 31 controls, and on 87 cerebrospinal fluid (CSF) samples from meningitis patients. Results The analytical sensitivity was not affected by using a combined mixture of reagents and a combined DNA standard (S. pneumoniae/H. influenzae/N. meningitidis) in single tubes. By blood- and BAL-culture and S. pneumoniae urinary antigen test, S. pneumoniae and H. influenzae were aetiological agents in 21 and 31 of the LTRI patients, respectively. These pathogens were identified by qmPCR in 52 and 72 of the cases, respectively, yielding sensitivities and specificities of 95% and 75% for S. pneumoniae, and 90% and 65% for H. influenzae, respectively. When using a cut-off of 105 genome copies/mL for clinical positivity the sensitivities and specificities were 90% and 80% for S. pneumoniae, and 81% and 85% for H. influenzae, respectively. Of 44 culture negative but qmPCR positive for H. influenzae, 41 were confirmed by fucK PCR as H. influenzae. Of the 103 patients who had taken antibiotics prior to sampling, S. pneumoniae and H. influenzae were identified by culture in 6% and 20% of the cases, respectively, and by the qmPCR in 36% and 53% of the cases, respectively. In 87 CSF samples S. pneumoniae and N. meningitidis were identified by culture and/or 16 S rRNA in 14 and 10 samples and by qmPCR in 14 and 10 samples, respectively, giving a sensitivity of 100% and a specificity of 100% for both bacteria. Conclusions The

Multiplex quantitative PCR for detection of lower respiratory tract infection and meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis.

PubMed

Abdeldaim, Guma M K; Strålin, Kristoffer; Korsgaard, Jens; Blomberg, Jonas; Welinder-Olsson, Christina; Herrmann, Björn

2010-12-03

Streptococcus pneumoniae and Haemophilus influenzae cause pneumonia and as Neisseria meningitidis they are important agents of meningitis. Although several PCR methods have been described for these bacteria the specificity is an underestimated problem. Here we present a quantitative multiplex real-time PCR (qmPCR) for detection of S. pneumoniae (9802 gene fragment), H. influenzae (omp P6 gene) and N. meningitidis (ctrA gene). The method was evaluated on bronchoalveolar lavage (BAL) samples from 156 adults with lower respiratory tract infection (LRTI) and 31 controls, and on 87 cerebrospinal fluid (CSF) samples from meningitis patients. The analytical sensitivity was not affected by using a combined mixture of reagents and a combined DNA standard (S. pneumoniae/H. influenzae/N. meningitidis) in single tubes. By blood- and BAL-culture and S. pneumoniae urinary antigen test, S. pneumoniae and H. influenzae were aetiological agents in 21 and 31 of the LTRI patients, respectively. These pathogens were identified by qmPCR in 52 and 72 of the cases, respectively, yielding sensitivities and specificities of 95% and 75% for S. pneumoniae, and 90% and 65% for H. influenzae, respectively. When using a cut-off of 10⁵ genome copies/mL for clinical positivity the sensitivities and specificities were 90% and 80% for S. pneumoniae, and 81% and 85% for H. influenzae, respectively. Of 44 culture negative but qmPCR positive for H. influenzae, 41 were confirmed by fucK PCR as H. influenzae. Of the 103 patients who had taken antibiotics prior to sampling, S. pneumoniae and H. influenzae were identified by culture in 6% and 20% of the cases, respectively, and by the qmPCR in 36% and 53% of the cases, respectively.In 87 CSF samples S. pneumoniae and N. meningitidis were identified by culture and/or 16 S rRNA in 14 and 10 samples and by qmPCR in 14 and 10 samples, respectively, giving a sensitivity of 100% and a specificity of 100% for both bacteria. The PCR provides increased

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

PubMed Central

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

2014-01-01

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

New primers for the detection Leishmania species by multiplex polymerase chain reaction.

PubMed

Conter, Carolina Cella; Lonardoni, Maria Valdrinez Campana; Aristides, Sandra Mara Alessi; Cardoso, Rosilene Fressatti; Silveira, Thaís Gomes Verzignassi

2018-02-01

Leishmaniasis is caused by protozoa of the Leishmania genus, which is divided into subgenus Viannia and Leishmania. In humans, the course of infection largely depends on the host-parasite relationship and primarily of the infective species. The objective of the present study was to design specific primers to the identification of Leishmania species using multiplex PCR. Four primers were designed, based on the GenBank sequences of the kDNA minicircle, amplifying 127 bp for subgenus Viannia, 100 bp for L. amazonensis, and 60 bp for Leishmania donovani complex and L. major. None of the primers amplified Trypanosoma cruzi or L. mexicana. The limit of detection of multiplex PCR was 2 × 10 -5 parasites for L. braziliensis, 2 x 10 -3 parasites for L. amazonensis, and 1.4 × 10 -3 parasites for L. infantum. The high sensitivity of multiplex PCR was confirmed by the detection of parasites in different biological samples, including lesion scrapings, spleen imprinting of a hamster, sandflies, and blood. The multiplex PCR that was developed herein presented good performance with regard to detecting and identifying the parasite in different biological samples and may thus be useful for diagnosis, decision making with regard to the proper therapeutic approach, and determining the geographic distribution of Leishmania species.

Multiplex real-time PCR assay for detection of pathogenic Vibrio parahaemolyticus strains.

PubMed

He, Peiyan; Chen, Zhongwen; Luo, Jianyong; Wang, Henghui; Yan, Yong; Chen, Lixia; Gao, Wenjie

2014-01-01

Foodborne disease caused by pathogenic Vibrio parahaemolyticus has become a serious public health problem in many countries. Rapid diagnosis and the identification of pathogenic V. parahaemolyticus are very important in the context of public health. In this study, an EvaGreen-based multiplex real-time PCR assay was established for the detection of pathogenic V. parahaemolyticus. This assay targeted three genetic markers of V. parahaemolyticus (species-specific gene toxR and virulence genes tdh and trh). The assay could unambiguously identify pathogenic V. parahaemolyticus with a minimum detection limit of 1.4 pg genomic DNA per reaction (concentration giving a positive multiplex real-time PCR result in 95% of samples). The specificity of the assay was evaluated using 72 strains of V. parahaemolyticus and other bacteria. A validation of the assay with clinical samples confirmed its sensitivity and specificity. Our data suggest the newly established multiplex real-time PCR assay is practical, cost-effective, specific, sensitive and capable of high-throughput detection of pathogenic V. parahaemolyticus. Copyright © 2014. Published by Elsevier Ltd.

Interferometric biosensing platform for multiplexed digital detection of viral pathogens and biomarkers

NASA Astrophysics Data System (ADS)

Daaboul, George

Label-free optical biosensors have been established as proven tools for monitoring specific biomolecular interactions. However, compact and robust embodiments of such instruments have yet to be introduced in order to provide sensitive, quantitative, and high-throughput biosensing for low-cost research and clinical applications. Here we present the interferometric reflectance-imaging sensor (IRIS). IRIS allows sensitive label free analysis using an inexpensive and durable multi-color LED illumination source on a silicon based surface. IRIS monitors biomolecular interaction through measurement of biomass addition to the sensor's surface. We demonstrate the capability of this system to dynamically monitor antigen---antibody interactions with a noise floor of 5.2 pg/mm 2 and DNA single mismatch detection under isothermal melting conditions in an array format. Ensemble detection of binding events using IRIS did not provide the sensitivity needed for detection of infectious disease and biomarkers at clinically relevant concentrations. Therefore, a new approach was adapted to the IRIS platform that allowed the detection and identification of individual nanoparticles on the sensor's surface. The new detection method was termed single-particle IRIS (SP-IRIS). We developed two detection modalities for SP-IRIS. The first modality is when the target is a nanoparticle such as a virus. We verified that SP-IRIS can accurately detect and size individual viral particles. Then we demonstrated that single nanoparticle counting and sizing methodology on SP-IRIS leads to a specific and sensitive virus sensor that can be multiplexed. Finally, we developed an assay for the detection of Ebola and Marburg. A detection limit of 3 x 103 PFU/ml was demonstrated for vesicular stomatitis virus (VSV) pseudotyped with Ebola or Marburg virus glycoprotein. We have demonstrated that virus detection can be done in human whole blood directly without the need for sample preparation. The second modality

Multiplexed screening assay for mRNA combining nuclease protection with luminescent array detection.

PubMed

Martel, Ralph R; Botros, Ihab W; Rounseville, Matthew P; Hinton, James P; Staples, Robin R; Morales, David A; Farmer, John B; Seligmann, Bruce E

2002-11-01

The principles and performance are described for the ArrayPlate mRNA assay, a multiplexed mRNA assay for high-throughput and high-content screening and drug development. THP-1 monocytes grown and subjected to compound treatments in 96-well plates were subjected to a multiplexed nuclease protection assay in situ. The nuclease protection assay destroyed all cell-derived mRNA, but left intact stoichiometric amounts of 16 target-specific oligonucleotide probes. Upon transfer of processed cell lysates to a microplate that contained a 16-element oligonucleotide array at the bottom of each well, the various probe species were separated by immobilization at predefined elements of the array. Quantitative detection of array-bound probes was by enzyme-mediated chemiluminescence. A high-resolution charge-coupled device imager was used for the simultaneous readout of all 1536 array elements in a 96-well plate. For the measurement of 16 genes in samples of 25000 cells, the average standard deviation from well to well within a plate was 8.6% of signal intensity and was 10.8% from plate to plate. Assay response was linear and reproducibility was constant for all detected genes in samples ranging from 1000 to 50000 cells. When THP-1 monocytes were differentiated with phorbol ester and subsequently activated with bacterial lipopolysaccharide that contained different concentrations of dexamethasone, dose-dependent effects of dexamethasone on the mRNA levels of several genes were observed.

Design of multiplex calibrant plasmids, their use in GMO detection and the limit of their applicability for quantitative purposes owing to competition effects.

PubMed

Debode, Frédéric; Marien, Aline; Janssen, Eric; Berben, Gilbert

2010-03-01

Five double-target multiplex plasmids to be used as calibrants for GMO quantification were constructed. They were composed of two modified targets associated in tandem in the same plasmid: (1) a part of the soybean lectin gene and (2) a part of the transgenic construction of the GTS40-3-2 event. Modifications were performed in such a way that each target could be amplified with the same primers as those for the original target from which they were derived but such that each was specifically detected with an appropriate probe. Sequence modifications were done to keep the parameters of the new target as similar as possible to those of its original sequence. The plasmids were designed to be used either in separate reactions or in multiplex reactions. Evidence is given that with each of the five different plasmids used in separate wells as a calibrant for a different copy number, a calibration curve can be built. When the targets were amplified together (in multiplex) and at different concentrations inside the same well, the calibration curves showed that there was a competition effect between the targets and this limits the range of copy numbers for calibration over a maximum of 2 orders of magnitude. Another possible application of multiplex plasmids is discussed.

Multiplex detection of pathogen biomarkers in human blood, serum, and saliva using silicon photonic microring resonators

NASA Astrophysics Data System (ADS)

Estrada, I. A.; Burlingame, R. W.; Wang, A. P.; Chawla, K.; Grove, T.; Wang, J.; Southern, S. O.; Iqbal, M.; Gunn, L. C.; Gleeson, M. A.

2015-05-01

Genalyte has developed a multiplex silicon photonic chip diagnostics platform (MaverickTM) for rapid detection of up to 32 biological analytes from a drop of sample in just 10 to 20 minutes. The chips are manufactured with waveguides adjacent to ring resonators, and probed with a continuously variable wavelength laser. A shift in the resonant wavelength as mass binds above the ring resonators is measured and is directly proportional to the amount of bound macromolecules. We present here the ability to multiplex the detection of hemorrhagic fever antigens in whole blood, serum, and saliva in a 16 minute assay. Our proof of concept testing of a multiplex antigencapture chip has the ability to detect Zaire Ebola (ZEBOV) recombinant soluble glycoprotein (rsGP), Marburg virus (MARV) Angola recombinant glycoprotein (rGP) and dengue nonstructural protein I (NS1). In parallel, detection of 2 malaria antigens has proven successful, but has yet to be incorporated into multiplex with the others. Each assay performs with sensitivity ranging from 1.6 ng/ml to 39 ng/ml depending on the antigen detected, and with minimal cross-reactivity.

Fiber-optic microsphere-based arrays for multiplexed biological warfare agent detection.

PubMed

Song, Linan; Ahn, Soohyoun; Walt, David R

2006-02-15

We report a multiplexed high-density DNA array capable of rapid, sensitive, and reliable identification of potential biological warfare agents. An optical fiber bundle containing 6000 individual 3.1-mum-diameter fibers was chemically etched to yield microwells and used as the substrate for the array. Eighteen different 50-mer single-stranded DNA probes were covalently attached to 3.1-mum microspheres. Probe sequences were designed for Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella melitensis, Clostridium botulinum, Vaccinia virus, and one biological warfare agent (BWA) simulant, Bacillus thuringiensis kurstaki. The microspheres were distributed into the microwells to form a randomized multiplexed high-density DNA array. A detection limit of 10 fM in a 50-microL sample volume was achieved within 30 min of hybridization for B. anthracis, Y. pestis, Vaccinia virus, and B. thuringiensis kurstaki. We used both specific responses of probes upon hybridization to complementary targets as well as response patterns of the multiplexed array to identify BWAs with high accuracy. We demonstrated the application of this multiplexed high-density DNA array for parallel identification of target BWAs in spiked sewage samples after PCR amplification. The array's miniaturized feature size, fabrication flexibility, reusability, and high reproducibility may enable this array platform to be integrated into a highly sensitive, specific, and reliable portable instrument for in situ BWA detection.

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

PubMed Central

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

2016-01-01

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

Real-time, multiplexed electrochemical DNA detection using an active complementary metal-oxide-semiconductor biosensor array with integrated sensor electronics.

PubMed

Levine, Peter M; Gong, Ping; Levicky, Rastislav; Shepard, Kenneth L

2009-03-15

Optical biosensing based on fluorescence detection has arguably become the standard technique for quantifying extents of hybridization between surface-immobilized probes and fluorophore-labeled analyte targets in DNA microarrays. However, electrochemical detection techniques are emerging which could eliminate the need for physically bulky optical instrumentation, enabling the design of portable devices for point-of-care applications. Unlike fluorescence detection, which can function well using a passive substrate (one without integrated electronics), multiplexed electrochemical detection requires an electronically active substrate to analyze each array site and benefits from the addition of integrated electronic instrumentation to further reduce platform size and eliminate the electromagnetic interference that can result from bringing non-amplified signals off chip. We report on an active electrochemical biosensor array, constructed with a standard complementary metal-oxide-semiconductor (CMOS) technology, to perform quantitative DNA hybridization detection on chip using targets conjugated with ferrocene redox labels. A 4 x 4 array of gold working electrodes and integrated potentiostat electronics, consisting of control amplifiers and current-input analog-to-digital converters, on a custom-designed 5 mm x 3 mm CMOS chip drive redox reactions using cyclic voltammetry, sense DNA binding, and transmit digital data off chip for analysis. We demonstrate multiplexed and specific detection of DNA targets as well as real-time monitoring of hybridization, a task that is difficult, if not impossible, with traditional fluorescence-based microarrays.

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

PubMed Central

Nolan, John P.; Mandy, Francis

2008-01-01

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

A multiplex microplatform for the detection of multiple DNA methylation events using gold-DNA affinity.

PubMed

Sina, Abu Ali Ibn; Foster, Matthew Thomas; Korbie, Darren; Carrascosa, Laura G; Shiddiky, Muhammad J A; Gao, Jing; Dey, Shuvashis; Trau, Matt

2017-10-07

We report a new multiplexed strategy for the electrochemical detection of regional DNA methylation across multiple regions. Using the sequence dependent affinity of bisulfite treated DNA towards gold surfaces, the method integrates the high sensitivity of a micro-fabricated multiplex device comprising a microarray of gold electrodes, with the powerful multiplexing capability of multiplex-PCR. The synergy of this combination enables the monitoring of the methylation changes across several genomic regions simultaneously from as low as 500 pg μl -1 of DNA with no sequencing requirement.

Improving membrane based multiplex immunoassays for semi-quantitative detection of multiple cytokines in a single sample

PubMed Central

2014-01-01

Background Inflammatory mediators can serve as biomarkers for the monitoring of the disease progression or prognosis in many conditions. In the present study we introduce an adaptation of a membrane-based technique in which the level of up to 40 cytokines and chemokines can be determined in both human and rodent blood in a semi-quantitative way. The planar assay was modified using the LI-COR (R) detection system (fluorescence based) rather than chemiluminescence and semi-quantitative outcomes were achieved by normalizing the outcomes using the automated exposure settings of the Odyssey readout device. The results were compared to the gold standard assay, namely ELISA. Results The improved planar assay allowed the detection of a considerably higher number of analytes (n = 30 and n = 5 for fluorescent and chemiluminescent detection, respectively). The improved planar method showed high sensitivity up to 17 pg/ml and a linear correlation of the normalized fluorescence intensity with the results from the ELISA (r = 0.91). Conclusions The results show that the membrane-based technique is a semi-quantitative assay that correlates satisfactorily to the gold standard when enhanced by the use of fluorescence and subsequent semi-quantitative analysis. This promising technique can be used to investigate inflammatory profiles in multiple conditions, particularly in studies with constraints in sample sizes and/or budget. PMID:25022797

Multiplexed capillary microfluidic immunoassay with smartphone data acquisition for parallel mycotoxin detection.

PubMed

Machado, Jessica M D; Soares, Ruben R G; Chu, Virginia; Conde, João P

2018-01-15

The field of microfluidics holds great promise for the development of simple and portable lab-on-a-chip systems. The use of capillarity as a means of fluidic manipulation in lab-on-a-chip systems can potentially reduce the complexity of the instrumentation and allow the development of user-friendly devices for point-of-need analyses. In this work, a PDMS microchannel-based, colorimetric, autonomous capillary chip provides a multiplexed and semi-quantitative immunodetection assay. Results are acquired using a standard smartphone camera and analyzed with a simple gray scale quantification procedure. The performance of this device was tested for the simultaneous detection of the mycotoxins ochratoxin A (OTA), aflatoxin B1 (AFB1) and deoxynivalenol (DON) which are strictly regulated food contaminants with severe detrimental effects on human and animal health. The multiplexed assay was performed approximately within 10min and the achieved sensitivities of<40, 0.1-0.2 and<10ng/mL for OTA, AFB1 and DON, respectively, fall within the majority of currently enforced regulatory and/or recommended limits. Furthermore, to assess the potential of the device to analyze real samples, the immunoassay was successfully validated for these 3 mycotoxins in a corn-based feed sample after a simple sample preparation procedure. Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed

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

2009-12-09

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

Rapid diagnosis of sepsis with TaqMan-Based multiplex real-time PCR.

PubMed

Liu, Chang-Feng; Shi, Xin-Ping; Chen, Yun; Jin, Ye; Zhang, Bing

2018-02-01

The survival rate of septic patients mainly depends on a rapid and reliable diagnosis. A rapid, broad range, specific and sensitive quantitative diagnostic test is the urgent need. Thus, we developed a TaqMan-Based Multiplex real-time PCR assays to identify bloodstream pathogens within a few hours. Primers and TaqMan probes were designed to be complementary to conserved regions in the 16S rDNA gene of different kinds of bacteria. To evaluate accurately, sensitively, and specifically, the known bacteria samples (Standard strains, whole blood samples) are determined by TaqMan-Based Multiplex real-time PCR. In addition, 30 blood samples taken from patients with clinical symptoms of sepsis were tested by TaqMan-Based Multiplex real-time PCR and blood culture. The mean frequency of positive for Multiplex real-time PCR was 96% at a concentration of 100 CFU/mL, and it was 100% at a concentration greater than 1000 CFU/mL. All the known blood samples and Standard strains were detected positively by TaqMan-Based Multiplex PCR, no PCR products were detected when DNAs from other bacterium were used in the multiplex assay. Among the 30 patients with clinical symptoms of sepsis, 18 patients were confirmed positive by Multiplex real-time PCR and seven patients were confirmed positive by blood culture. TaqMan-Based Multiplex real-time PCR assay with highly sensitivity, specificity and broad detection range, is a rapid and accurate method in the detection of bacterial pathogens of sepsis and should have a promising usage in the diagnosis of sepsis. © 2017 Wiley Periodicals, Inc.

Multiplex Detection of Aspergillus Species.

PubMed

Martínez-Culebras, Pedro; Selma, María Victoria; Aznar, Rosa

2017-01-01

Multiplex real-time polymerase chain reaction (PCR) provides a fast and accurate DNA-based tool for the simultaneous amplification of more than one target sequence in a single reaction. Here a duplex real-time PCR assay is described for the simultaneous detection of Aspergillus carbonarius and members of the Aspergillus niger aggregate, which are the main responsible species for ochratoxin A (OTA) contamination in grapes. This single tube reaction targets the beta-ketosynthase and the acyl transferase domains of the polyketide synthase of A. carbonarius and the A. niger aggregate, respectively.Besides, a rapid and efficient fungi DNA extraction procedure is described suitable to be applied in wine grapes. It includes a pulsifier equipment to remove conidia from grapes which prevents releasing of PCR inhibitors.

Centrifugal Microfluidic Platform for Rapid, Multiplexed Detection of TB and HIV Biomarkers in Whole Blood Samples

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

Litvinov, Julia; Moen, Scott T.; Berry, Gregory J.

Infection with Mycobacterium Tuberculosis represents a significant threat to people with immune disorders, such as HIV-positive individuals, and can result in significant health complications or death if not diagnosed and treated early. We present a centrifugal microfluidic platform for multiplexed detection of tuberculosis and HIV biomarkers in human whole blood with minimal sample preparation and a sample-to-answer time of 30 minutes. This multiplexed assay was developed for the detection of two M.tuberculosis secreted proteins, whose secretion represents an active and ongoing infection, as well as detection of HIV p24 protein and human anti-p24 antibodies. The limit of detection for thismore » multiplex assay is in the pg/mL range for both HIV and M.tuberculosis proteins, making this assay potentially useful in the clinical diagnosis of both HIV and Tuberculosis proteins indicative of active infection. Antigen detection for the HIV assay sensitivity was 89%, the specificity 85%. Serological detection had 100% sensitivity and specificity for the limited sample pool. The centrifugal microfluidic platform presented here offers the potential for a portable, fast and inexpensive multiplexed diagnostic device that can be used in resource-limited settings for diagnosis of TB and HIV.« less

Centrifugal Microfluidic Platform for Rapid, Multiplexed Detection of TB and HIV Biomarkers in Whole Blood Samples

DOE PAGES

Litvinov, Julia; Moen, Scott T.; Berry, Gregory J.; ...

2017-05-30

Infection with Mycobacterium Tuberculosis represents a significant threat to people with immune disorders, such as HIV-positive individuals, and can result in significant health complications or death if not diagnosed and treated early. We present a centrifugal microfluidic platform for multiplexed detection of tuberculosis and HIV biomarkers in human whole blood with minimal sample preparation and a sample-to-answer time of 30 minutes. This multiplexed assay was developed for the detection of two M.tuberculosis secreted proteins, whose secretion represents an active and ongoing infection, as well as detection of HIV p24 protein and human anti-p24 antibodies. The limit of detection for thismore » multiplex assay is in the pg/mL range for both HIV and M.tuberculosis proteins, making this assay potentially useful in the clinical diagnosis of both HIV and Tuberculosis proteins indicative of active infection. Antigen detection for the HIV assay sensitivity was 89%, the specificity 85%. Serological detection had 100% sensitivity and specificity for the limited sample pool. The centrifugal microfluidic platform presented here offers the potential for a portable, fast and inexpensive multiplexed diagnostic device that can be used in resource-limited settings for diagnosis of TB and HIV.« less

Toward photostable multiplex analyte detection on a single mode planar optical waveguide

NASA Astrophysics Data System (ADS)

Mukundan, Harshini; Xie, Hongzhi; Anderson, Aaron; Grace, W. Kevin; Martinez, Jennifer S.; Swanson, Basil

2009-02-01

We have developed a waveguide-based optical biosensor for the sensitive and specific detection of biomarkers associated with disease. Our technology combines the superior optical properties of single-mode planar waveguides, the robust nature of functionalized self-assembled monolayer sensing films and the specificity of fluorescence sandwich immunoassays to detect biomarkers in complex biological samples such as serum, urine and sputum. We have previously reported the adaptation of our technology to the detection of biomarkers associated with breast cancer and anthrax. However, these approaches primarily used phospholipid bilayers as the functional film and organic dyes (ex: AlexaFluors) as the fluorescence reporter. Organic dyes are easily photodegraded and are not amenable to multiplexing because of their narrow Stokes' shift. Here we have developed strategies for conjugation of the detector antibodies with quantum dots for use in a multiplex detection platform. We have previously evaluated dihydroxylipoic acid quantum dots for the detection of a breast cancer biomarker. In this manuscript, we investigate the detection of the Bacillus anthracis protective antigen using antibodies conjugated with polymer-coated quantum dots. Kinetics of binding on the waveguide-based biosensor is reported. We compare the sensitivity of quantum dot labeled antibodies to those labeled with AlexaFluor and demonstrate the photostability of the former in our assay platform. In addition, we compare sulfydryl labeling of the antibody in the hinge region to that of nonspecific amine labeling. This is but the first step in developing a multiplex assay for such biomarkers on our waveguide platform.

Prognostic Implications of Multiplex Detection of KRAS Mutations in Cell-Free DNA from Patients with Pancreatic Ductal Adenocarcinoma.

PubMed

Kim, Min Kyeong; Woo, Sang Myung; Park, Boram; Yoon, Kyong-Ah; Kim, Yun-Hee; Joo, Jungnam; Lee, Woo Jin; Han, Sung-Sik; Park, Sang-Jae; Kong, Sun-Young

2018-04-01

Cell-free DNA (cfDNA) is known to provide potential biomarkers for predicting clinical outcome, but its value in pancreatic ductal adenocarcinoma (PDAC) has not been fully evaluated. The aim of this study was to evaluate the clinical applicability of quantitative analysis of multiplex KRAS mutations in cell-free DNA from patients with PDAC. A total of 106 patients with PDAC were enrolled in this prospective study. The concentration and fraction of KRAS mutations were determined through multiplex detection of KRAS mutations in plasma samples by use of a droplet digital PCR kit (Bio-Rad). KRAS mutations were detected in 96.1% of tissue samples. Eighty patients (80.5%) harbored KRAS mutations in cfDNA, with a median KRAS mutation concentration of 0.165 copies/μL and a median fractional abundance of 0.415%. Multivariable analyses demonstrated that the KRAS mutation concentration [hazard ratio (HR), 2.08; 95% CI, 1.20-3.63] and KRAS fraction (HR, 1.73; 95% CI, 1.02-2.95) were significant factors for progression-free survival. KRAS mutation concentration (HR, 1.97; 95% CI, 1.05-3.67) also had prognostic implications for overall survival. Subgroup analyses showed that KRAS mutation concentration and fractional abundance significantly affected progression-free survival in resectable PDAC ( P = 0.016). Moreover, when combined with the cancer biomarker CA19-9, the KRAS mutation concentration in cfDNA showed additive benefits for the prediction of overall survival. This study demonstrates that multiplex detection of KRAS mutations in plasma cfDNA is clinically relevant, providing a potential candidate biomarker for prognosis of PDAC. © 2018 American Association for Clinical Chemistry.

Highly sensitive and quantitative detection of rare pathogens through agarose droplet microfluidic emulsion PCR at the single-cell level.

PubMed

Zhu, Zhi; Zhang, Wenhua; Leng, Xuefei; Zhang, Mingxia; Guan, Zhichao; Lu, Jiangquan; Yang, Chaoyong James

2012-10-21

Genetic alternations can serve as highly specific biomarkers to distinguish fatal bacteria or cancer cells from their normal counterparts. However, these mutations normally exist in very rare amount in the presence of a large excess of non-mutated analogs. Taking the notorious pathogen E. coli O157:H7 as the target analyte, we have developed an agarose droplet-based microfluidic ePCR method for highly sensitive, specific and quantitative detection of rare pathogens in the high background of normal bacteria. Massively parallel singleplex and multiplex PCR at the single-cell level in agarose droplets have been successfully established. Moreover, we challenged the system with rare pathogen detection and realized the sensitive and quantitative analysis of a single E. coli O157:H7 cell in the high background of 100,000 excess normal K12 cells. For the first time, we demonstrated rare pathogen detection through agarose droplet microfluidic ePCR. Such a multiplex single-cell agarose droplet amplification method enables ultra-high throughput and multi-parameter genetic analysis of large population of cells at the single-cell level to uncover the stochastic variations in biological systems.

Phage-protease-peptide: a novel trifecta enabling multiplex detection of viable bacterial pathogens.

PubMed

Alcaine, S D; Tilton, L; Serrano, M A C; Wang, M; Vachet, R W; Nugen, S R

2015-10-01

Bacteriophages represent rapid, readily targeted, and easily produced molecular probes for the detection of bacterial pathogens. Molecular biology techniques have allowed researchers to make significant advances in the bioengineering of bacteriophage to further improve speed and sensitivity of detection. Despite their host specificity, bacteriophages have not been meaningfully leveraged in multiplex detection of bacterial pathogens. We propose a proof-of-principal phage-based scheme to enable multiplex detection. Our scheme involves bioengineering bacteriophage to carry a gene for a specific protease, which is expressed during infection of the target cell. Upon lysis, the protease is released to cleave a reporter peptide, and the signal detected. Here we demonstrate the successful (i) modification of T7 bacteriophage to carry tobacco etch virus (TEV) protease; (ii) expression of TEV protease by Escherichia coli following infection by our modified T7, an average of 2000 units of protease per phage are produced during infection; and (iii) proof-of-principle detection of E. coli in 3 h after a primary enrichment via TEV protease activity using a fluorescent peptide and using a designed target peptide for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI-TOF MS) analysis. This proof-of-principle can be translated to other phage-protease-peptide combinations to enable multiplex bacterial detection and readily adopted on multiple platforms, like MALDI-TOF MS or fluorescent readers, commonly found in labs.

Surface-enhanced Raman scattering based nonfluorescent probe for multiplex DNA detection.

PubMed

Sun, Lan; Yu, Chenxu; Irudayaraj, Joseph

2007-06-01

To provide rapid and accurate detection of DNA markers in a straightforward, inexpensive, and multiplex format, an alternative surface-enhanced Raman scattering based probe was designed and fabricated to covalently attach both DNA probing sequence and nonfluorescent Raman tags to the surface of gold nanoparticles (DNA-AuP-RTag). The intensity of Raman signal of the probes could be controlled through the surface coverage of the nonfluorescent Raman tags (RTags). Detection sensitivity of these probes could be optimized by fine-tuning the amount of DNA molecules and RTags on the probes. Long-term stability of the DNA-AuP-RTag probes was found to be good (over 3 months). Excellent multiplexing capability of the DNA-AuP-RTag scheme was demonstrated by simultaneous identification of up to eight probes in a mixture. Detection of hybridization of single-stranded DNA to its complementary targets was successfully accomplished with a long-term goal to use nonfluorescent RTags in a Raman-based DNA microarray platform.

Rapid, portable, multiplexed detection of bacterial pathogens directly from clinical sample matrices

DOE PAGES

Phaneuf, Christopher R.; Mangadu, Betty Lou Bosano; Piccini, Matthew E.; ...

2016-09-23

Enteric and diarrheal diseases are a major cause of childhood illness and death in countries with developing economies. Each year, more than half of a million children under the age of five die from these diseases. We have developed a portable, microfluidic platform capable of simultaneous, multiplexed detection of several of the bacterial pathogens that cause these diseases. Furthermore, this platform can perform fast, sensitive immunoassays directly from relevant, complex clinical matrices such as stool without extensive sample cleanup or preparation. Using only 1 µL of sample per assay, we demonstrate simultaneous multiplexed detection of four bacterial pathogens implicated inmore » diarrheal and enteric diseases in less than 20 min.« less

Rapid, portable, multiplexed detection of bacterial pathogens directly from clinical sample matrices

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

Phaneuf, Christopher R.; Mangadu, Betty Lou Bosano; Piccini, Matthew E.

Enteric and diarrheal diseases are a major cause of childhood illness and death in countries with developing economies. Each year, more than half of a million children under the age of five die from these diseases. We have developed a portable, microfluidic platform capable of simultaneous, multiplexed detection of several of the bacterial pathogens that cause these diseases. Furthermore, this platform can perform fast, sensitive immunoassays directly from relevant, complex clinical matrices such as stool without extensive sample cleanup or preparation. Using only 1 µL of sample per assay, we demonstrate simultaneous multiplexed detection of four bacterial pathogens implicated inmore » diarrheal and enteric diseases in less than 20 min.« less

Multiplexing of ChIP-Seq Samples in an Optimized Experimental Condition Has Minimal Impact on Peak Detection.

PubMed

Kacmarczyk, Thadeous J; Bourque, Caitlin; Zhang, Xihui; Jiang, Yanwen; Houvras, Yariv; Alonso, Alicia; Betel, Doron

2015-01-01

Multiplexing samples in sequencing experiments is a common approach to maximize information yield while minimizing cost. In most cases the number of samples that are multiplexed is determined by financial consideration or experimental convenience, with limited understanding on the effects on the experimental results. Here we set to examine the impact of multiplexing ChIP-seq experiments on the ability to identify a specific epigenetic modification. We performed peak detection analyses to determine the effects of multiplexing. These include false discovery rates, size, position and statistical significance of peak detection, and changes in gene annotation. We found that, for histone marker H3K4me3, one can multiplex up to 8 samples (7 IP + 1 input) at ~21 million single-end reads each and still detect over 90% of all peaks found when using a full lane for sample (~181 million reads). Furthermore, there are no variations introduced by indexing or lane batch effects and importantly there is no significant reduction in the number of genes with neighboring H3K4me3 peaks. We conclude that, for a well characterized antibody and, therefore, model IP condition, multiplexing 8 samples per lane is sufficient to capture most of the biological signal.

Multiplexing of ChIP-Seq Samples in an Optimized Experimental Condition Has Minimal Impact on Peak Detection

PubMed Central

Kacmarczyk, Thadeous J.; Bourque, Caitlin; Zhang, Xihui; Jiang, Yanwen; Houvras, Yariv; Alonso, Alicia; Betel, Doron

2015-01-01

Multiplexing samples in sequencing experiments is a common approach to maximize information yield while minimizing cost. In most cases the number of samples that are multiplexed is determined by financial consideration or experimental convenience, with limited understanding on the effects on the experimental results. Here we set to examine the impact of multiplexing ChIP-seq experiments on the ability to identify a specific epigenetic modification. We performed peak detection analyses to determine the effects of multiplexing. These include false discovery rates, size, position and statistical significance of peak detection, and changes in gene annotation. We found that, for histone marker H3K4me3, one can multiplex up to 8 samples (7 IP + 1 input) at ~21 million single-end reads each and still detect over 90% of all peaks found when using a full lane for sample (~181 million reads). Furthermore, there are no variations introduced by indexing or lane batch effects and importantly there is no significant reduction in the number of genes with neighboring H3K4me3 peaks. We conclude that, for a well characterized antibody and, therefore, model IP condition, multiplexing 8 samples per lane is sufficient to capture most of the biological signal. PMID:26066343

Multiplexing detection of IgG against Plasmodium falciparum pregnancy-specific antigens

PubMed Central

Fonseca, Ana Maria; Quinto, Llorenç; Jiménez, Alfons; González, Raquel; Bardají, Azucena; Maculuve, Sonia; Dobaño, Carlota; Rupérez, Maria; Vala, Anifa; Aponte, John J.; Sevene, Esperanza; Macete, Eusebio; Menéndez, Clara

2017-01-01

Background Pregnant women exposed to Plasmodium falciparum generate antibodies against VAR2CSA, the parasite protein that mediates adhesion of infected erythrocytes to the placenta. There is a need of high-throughput tools to determine the fine specificity of these antibodies that can be used to identify immune correlates of protection and exposure. Here we aimed at developing a multiplex-immunoassay to detect antibodies against VAR2CSA antigens. Methods and findings We constructed two multiplex-bead arrays, one composed of 3 VAR2CSA recombinant-domains (DBL3X, DBL5Ɛ and DBL6Ɛ) and another composed of 46 new peptides covering VAR2CSA conserved and semi-conserved regions. IgG reactivity was similar in multiplexed and singleplexed determinations (Pearson correlation, protein array: R2 = 0.99 and peptide array: R2 = 0.87). IgG recognition of 25 out of 46 peptides and all recombinant-domains was higher in pregnant Mozambican women (n = 106) than in Mozambican men (n = 102) and Spanish individuals (n = 101; p<0.05). Agreement of IgG levels detected in cryopreserved plasma and in elutions from dried blood spots was good after exclusion of inappropriate filter papers. Under heterogeneous levels of exposure to malaria, similar seropositivity cutoffs were obtained using finite mixture models applied to antibodies measured on pregnant Mozambican women and average of antibodies measured on pregnant Spanish women never exposed to malaria. The application of the multiplex-bead array developed here, allowed the assessment of higher IgG levels and seroprevalences against VAR2CSA-derived antigens in women pregnant during 2003–2005 than during 2010–2012, in accordance with the levels of malaria transmission reported for these years in Mozambique. Conclusions The multiplex bead-based immunoassay to detect antibodies against selected 25 VAR2CSA new-peptides and recombinant-domains was successfully implemented. Analysis of field samples showed that responses were specific among

An integrated passive micromixer-magnetic separation-capillary electrophoresis microdevice for rapid and multiplex pathogen detection at the single-cell level.

PubMed

Jung, Jae Hwan; Kim, Gha-Young; Seo, Tae Seok

2011-10-21

Here we report an integrated microdevice consisting of an efficient passive mixer, a magnetic separation chamber, and a capillary electrophoretic microchannel in which DNA barcode assay, target pathogen separation, and barcode DNA capillary electrophoretic analysis were performed sequentially within 30 min for multiplex pathogen detection at the single-cell level. The intestine-shaped serpentine 3D micromixer provides a high mixing rate to generate magnetic particle-pathogenic bacteria-DNA barcode labelled AuNP complexes quantitatively. After magnetic separation and purification of those complexes, the barcode DNA strands were released and analyzed by the microfluidic capillary electrophoresis within 5 min. The size of the barcode DNA strand was controlled depending on the target bacteria (Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella typhimurium), and the different elution time of the barcode DNA peak in the electropherogram allows us to recognize the target pathogen with ease in the monoplex as well as in the multiplex analysis. In addition, the quantity of the DNA barcode strand (∼10(4)) per AuNP is enough to be observed in the laser-induced confocal fluorescence detector, thereby making single-cell analysis possible. This novel integrated microdevice enables us to perform rapid, sensitive, and multiplex pathogen detection with sample-in-answer-out capability to be applied for biosafety testing, environmental screening, and clinical trials.

Screening DNA chip and event-specific multiplex PCR detection methods for biotech crops.

PubMed

Lee, Seong-Hun

2014-11-01

There are about 80 biotech crop events that have been approved by safety assessment in Korea. They have been controlled by genetically modified organism (GMO) and living modified organism (LMO) labeling systems. The DNA-based detection method has been used as an efficient scientific management tool. Recently, the multiplex polymerase chain reaction (PCR) and DNA chip have been developed as simultaneous detection methods for several biotech crops' events. The event-specific multiplex PCR method was developed to detect five biotech maize events: MIR604, Event 3272, LY 038, MON 88017 and DAS-59122-7. The specificity was confirmed and the sensitivity was 0.5%. The screening DNA chip was developed from four endogenous genes of soybean, maize, cotton and canola respectively along with two regulatory elements and seven genes: P35S, tNOS, pat, bar, epsps1, epsps2, pmi, cry1Ac and cry3B. The specificity was confirmed and the sensitivity was 0.5% for four crops' 12 events: one soybean, six maize, three cotton and two canola events. The multiplex PCR and DNA chip can be available for screening, gene-specific and event-specific analysis of biotech crops as efficient detection methods by saving on workload and time. © 2014 Society of Chemical Industry. © 2014 Society of Chemical Industry.

Detection of pathogenic Vibrio spp. in shellfish by using multiplex PCR and DNA microarrays.

PubMed

Panicker, Gitika; Call, Douglas R; Krug, Melissa J; Bej, Asim K

2004-12-01

This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50 degrees C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 10(2) to 10(3) CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers.

Detection of Pathogenic Vibrio spp. in Shellfish by Using Multiplex PCR and DNA Microarrays

PubMed Central

Panicker, Gitika; Call, Douglas R.; Krug, Melissa J.; Bej, Asim K.

2004-01-01

This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50°C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 102 to 103 CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers. PMID:15574946

A multiplex calibrated real-time PCR assay for quantitation of DNA of EBV-1 and 2.

PubMed

Gatto, Francesca; Cassina, Giulia; Broccolo, Francesco; Morreale, Giuseppe; Lanino, Edoardo; Di Marco, Eddi; Vardas, Efthiya; Bernasconi, Daniela; Buttò, Stefano; Principi, Nicola; Esposito, Susanna; Scarlatti, Gabriella; Lusso, Paolo; Malnati, Mauro S

2011-12-01

Accurate and highly sensitive tests for the diagnosis of active Epstein-Barr virus (EBV) infection are essential for the clinical management of individuals infected with EBV. A calibrated quantitative real-time PCR assay for the measurement of EBV DNA of both EBV-1 and 2 subtypes was developed, combining the detection of the EBV DNA and a synthetic DNA calibrator in a multiplex PCR format. The assay displays a wide dynamic range and a high degree of accuracy even in the presence of 1μg of human genomic DNA. This assay measures with the same efficiency EBV DNA from strains prevalent in different geographic areas. The clinical sensitivity and specificity of the system were evaluated by testing 181 peripheral blood mononuclear cell (PBMCs) and plasma specimens obtained from 21 patients subjected to bone marrow transplantation, 70 HIV-seropositive subjects and 23 healthy controls. Patients affected by EBV-associated post-transplant lymphoprolipherative disorders had the highest frequency of EBV detection and the highest viral load. Persons infected with HIV had higher levels of EBV DNA load in PBMCs and a higher frequency of EBV plasma viremia compared to healthy controls. In conclusion, this new assay provides a reliable high-throughput method for the quantitation of EBV DNA in clinical samples. Copyright © 2011 Elsevier B.V. All rights reserved.

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

Multiplex biosensing with highly sensitive magnetic nanoparticle quantification method

NASA Astrophysics Data System (ADS)

Nikitin, M. P.; Orlov, A. V.; Znoyko, S. L.; Bragina, V. A.; Gorshkov, B. G.; Ksenevich, T. I.; Cherkasov, V. R.; Nikitin, P. I.

2018-08-01

Unique properties of magnetic nanoparticles (MNP) have provided many breakthrough solutions for life science. The immense potential of MNP as labels in advanced immunoassays stems from the fact that they, unlike optical labels, can be easily detected inside 3D opaque porous biosensing structures or in colored mediums, manipulated by an external magnetic field, exhibit high stability and negligible background signal in biological samples, etc. In this research, the magnetic nanolabels and an original technique of their quantification by non-linear magnetization have permitted development of novel methods of multiplex biosensing. Several types of highly sensitive multi-channel readers that offer an extremely wide linear dynamic range are developed to count MNP in different recognition zones for quantitative concentration measurements of various analytes. Four approaches to multiplex biosensing based on MNP have been demonstrated in one-run tests based on several 3D porous structures; flat and micropillar microfluidic sensor chips; multi-line lateral flow strips and modular architecture of the strips, which is the first 3D multiplexing method that goes beyond the traditional planar techniques. Detection of cardio- and cancer markers, small molecules and oligonucleotides were used in the experiments. The analytical characteristics of the developed multiplex methods are on the level of the modern time-consuming laboratory techniques. The developed multiplex biosensing platforms are promising for medical and veterinary diagnostics, food inspection, environmental and security monitoring, etc.

Development and Validation of a Multiplexed Protein Quantitation Assay for the Determination of Three Recombinant Proteins in Soybean Tissues by Liquid Chromatography with Tandem Mass Spectrometry.

PubMed

Hill, Ryan C; Oman, Trent J; Shan, Guomin; Schafer, Barry; Eble, Julie; Chen, Cynthia

2015-08-26

Currently, traditional immunochemistry technologies such as enzyme-linked immunosorbent assays (ELISA) are the predominant analytical tool used to measure levels of recombinant proteins expressed in genetically engineered (GE) plants. Recent advances in agricultural biotechnology have created a need to develop methods capable of selectively detecting and quantifying multiple proteins in complex matrices because of increasing numbers of transgenic proteins being coexpressed or "stacked" to achieve tolerance to multiple herbicides or to provide multiple modes of action for insect control. A multiplexing analytical method utilizing liquid chromatography with tandem mass spectrometry (LC-MS/MS) has been developed and validated to quantify three herbicide-tolerant proteins in soybean tissues: aryloxyalkanoate dioxygenase (AAD-12), 5-enol-pyruvylshikimate-3-phosphate synthase (2mEPSPS), and phosphinothricin acetyltransferase (PAT). Results from the validation showed high recovery and precision over multiple analysts and laboratories. Results from this method were comparable to those obtained with ELISA with respect to protein quantitation, and the described method was demonstrated to be suitable for multiplex quantitation of transgenic proteins in GE crops.

Detection of sexually transmitted infection and human papillomavirus in negative cytology by multiplex-PCR

PubMed Central

2010-01-01

Background The aim of this study was to determine the prevalence of human papillomavirus (HPV) and 15 species that cause sexually transmitted infections (STIs) in negative cytology. In addition, we compared the diagnostic performance of multiplex polymerase chain reaction (PCR) with widely available techniques used to detect HPV. Methods We recruited 235 women of reproductive age who had negative cytology findings in a liquid-based cervical smear. STIs were identified by multiplex PCR, and HPV genotypes by multiplex PCR, hybrid capture 2, and DNA microaray; discordant results were analyzed by direct sequencing. Results Approximately 96.6% of patients with negative cytology results were positive for pathogens that cause STIs. The pathogens most frequently detected were Gardnerella vaginalis, Ureaplasma urealyticum. The incidence of HPV in negative cytology was 23.3%. Low-risk HPV infection was significantly correlated with Chalmaydia trachomatis, and high-risk HPV infection was significantly correlated with Group β streptococcus. The analytical sensitivities of the multiplex PCR and DNA microarray were higher than 80%, and the analytical specificity was nearly 100% for all tests. Conclusions Multiplex PCR yielded results that most of patients with negative cytology were positive for pathogens that cause STIs, and were more similar to that of DNA microarray, than that of hybrid capture 2 in terms of analytical sensitivity and prediction value of HPV infection. PMID:20920170

Decision-feedback detection strategy for nonlinear frequency-division multiplexing

NASA Astrophysics Data System (ADS)

Civelli, Stella; Forestieri, Enrico; Secondini, Marco

2018-04-01

By exploiting a causality property of the nonlinear Fourier transform, a novel decision-feedback detection strategy for nonlinear frequency-division multiplexing (NFDM) systems is introduced. The performance of the proposed strategy is investigated both by simulations and by theoretical bounds and approximations, showing that it achieves a considerable performance improvement compared to previously adopted techniques in terms of Q-factor. The obtained improvement demonstrates that, by tailoring the detection strategy to the peculiar properties of the nonlinear Fourier transform, it is possible to boost the performance of NFDM systems and overcome current limitations imposed by the use of more conventional detection techniques suitable for the linear regime.

Multiplex real-time PCR for the detection and quantification of latent and persistent viral genomes in cellular or plasma blood fractions.

PubMed

Compston, Lara Isobel; Sarkobie, Francis; Li, Chengyao; Candotti, Daniel; Opare-Sem, Ohene; Allain, Jean-Pierre

2008-07-01

In common with latent viruses such as herpesviruses, parvovirus B19, HBV and GBV-C are contained successfully by the immune response and persist in the host. When immune control breaks down, reactivation of both latent and persistent viruses occurs. Two multiplex assays were developed (B19, HBV, HHV-8), (EBV, CMV, VZV) for blood screening, and tested on blood donor samples from Ghana to determine baseline prevalence of viraemia in immunocompetent persons. Single-virus real-time quantitative PCR (qPCR) assays were optimised for viral load determination of positive initial screening. The qPCR method utilised was absolute quantification with external standards. Multiplex and single-virus qPCR assays had similar sensitivity, except for the B19 assay in which sensitivity was 100-fold lower. Assays were optimised for reproducibility and repeatability, with R(2) of 0.9 being obtained for most assays. With the exception of B19 and CMV, assays had 100% detection limit ranging between 10(1) and 10(2) copies, IU or arbitrary units under single-virus and multiplex assay conditions. The prevalence of viraemia was 1.6% HBV (0.8% DNA+/HBsAg-, 0.8% DNA+/HBsAg+), 0.8% parvovirus B19, and 3.3% GBV-C viraemia in the plasma fraction. The prevalence of four herpesviruses was 1.0% HHV-8, 0.85% CMV, and 8.3% EBV, and no detectable VZV viraemia.

Surface-Enhanced Raman Scattering Based Nonfluorescent Probe for Multiplex DNA Detection

PubMed Central

Sun, Lan; Yu, Chenxu; Irudayaraj, Joseph

2008-01-01

To provide rapid and accurate detection of DNA markers in a straightforward, inexpensive and multiplex format, an alternative surface enhanced Raman scattering (SERS) based probe was designed and fabricated to covalently attach both DNA probing sequence and non-fluorescent Raman tags to the surface of gold nanoparticles (DNA-AuP-RTag). The intensity of Raman signal of the probes could be controlled through the surface coverage of the non-fluorescent Raman tags (RTags). Detection sensitivity of these probes could be optimized by fine-tuning the amount of DNA molecules and RTags on the probes. Long-term stability of the DNA-AuP-RTag probes was found to be good (over 3 months). Excellent multiplexing capability of the DNA-AuP-RTag scheme was demonstrated by simultaneous identification of up to eight probes in a mixture. Detection of hybridization of single-stranded DNA (ssDNA) to its complementary targets was successfully accomplished with a long-term goal to use non-fluorescent RTags in a Raman-based DNA microarray platform. PMID:17465531

A Novel Multiplex HRM Assay to Detect Clopidogrel Resistance.

PubMed

Zhang, Lichen; Ma, Xiaowei; You, Guoling; Zhang, Xiaoqing; Fu, Qihua

2017-11-22

Clopidogrel is an antiplatelet medicine used to prevent blood clots in patients who have had a heart attack, stroke, or other symptoms. Variability in the clinical response to clopidogrel treatment has been attributed to genetic factors. In particular, five SNPs of rs4244285, rs4986893, rs12248560, rs662 and rs1045642 have been associated with resistance to clopidogrel therapy in Chinese population. This work involves the development of a multiplex high-resolution melting (HRM) assay to genotype all five of these loci in 2 tubes. Amplicons corresponding to distinct SNPs in a common tube were designed with the aid of uMelt prediction software to have different melting temperatures Tm by addition of a GC-rich tail to the 5' end of the certain primers. Two kinds of commercial methods, Digital Fluorescence Molecular Hybridization (DFMH) and Sanger sequencing, were used as a control. Three hundred sixteen DFMH pretested samples from consecutive acute coronary syndrome patients were used for a blinded study of multiplex HRM. The sensitivity of HRM was 100% and the specificity was 99.93% reflecting detection of variants other than the known resistance SNPs. Multiplex HRM is an effective closed-tube, highly accurate, fast, and inexpensive method for genotyping the 5 clopidogrel resistance associated SNPs.

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

PubMed Central

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

2014-01-01

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

Single quantum dot analysis enables multiplexed point mutation detection by gap ligase chain reaction.

PubMed

Song, Yunke; Zhang, Yi; Wang, Tza-Huei

2013-04-08

Gene point mutations present important biomarkers for genetic diseases. However, existing point mutation detection methods suffer from low sensitivity, specificity, and a tedious assay processes. In this report, an assay technology is proposed which combines the outstanding specificity of gap ligase chain reaction (Gap-LCR), the high sensitivity of single-molecule coincidence detection, and the superior optical properties of quantum dots (QDs) for multiplexed detection of point mutations in genomic DNA. Mutant-specific ligation products are generated by Gap-LCR and subsequently captured by QDs to form DNA-QD nanocomplexes that are detected by single-molecule spectroscopy (SMS) through multi-color fluorescence burst coincidence analysis, allowing for multiplexed mutation detection in a separation-free format. The proposed assay is capable of detecting zeptomoles of KRAS codon 12 mutation variants with near 100% specificity. Its high sensitivity allows direct detection of KRAS mutation in crude genomic DNA without PCR pre-amplification. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Detection of enteroviruses and hepatitis a virus in water by consensus primer multiplex RT-PCR

PubMed Central

Li, Jun-Wen; Wang, Xin-Wei; Yuan, Chang-Qing; Zheng, Jin-Lai; Jin, Min; Song, Nong; Shi, Xiu-Quan; Chao, Fu-Huan

2002-01-01

AIM: To develop a rapid detection method of enteroviruses and Hepatitis A virus (HAV). METHODS: A one-step, single-tube consensus primers multiplex RT-PCR was developed to simultaneously detect Poliovirus, Coxsackie virus, Echovirus and HAV. A general upstream primer and a HAV primer and four different sets of primers (5 primers) specific for Poliovirus, Coxsacki evirus, Echovirus and HAV cDNA were mixed in the PCR mixture to reverse transcript and amplify the target DNA. Four distinct amplified DNA segments representing Poliovirus, Coxsackie virus, Echovirus and HAV were identified by gel electrophoresis as 589-, 671-, 1084-, and 1128 bp sequences, respectively. Semi-nested PCR was used to confirm the amplified products for each enterovirus and HAV. RESULTS: All four kinds of viral genome RNA were detected, and producing four bands which could be differentiated by the band size on the gel. To confirm the specificity of the multiplex PCR products, semi-nested PCR was performed. For all the four strains tested gave positive results. The detection sensitivity of multiplex PCR was similar to that of monoplex RT-PCR which was 24 PFU for Poliovrus, 21 PFU for Coxsackie virus, 60 PFU for Echovirus and 105 TCID50 for HAV. The minimum amount of enteric viral RNA detected by semi-nested PCR was equivalent to 2.4 PFU for Poliovrus, 2.1 PFU for Coxsackie virus, 6.0 PFU for Echovirus and 10.5 TCID50 for HAV. CONCLUSION: The consensus primers multiplex RT-PCR has more advantages over monoplex RT-PCR for enteric viruses detection, namely, the rapid turnaround time and cost effectiveness. PMID:12174381

A novel, multiplexed, probe-based quantitative PCR assay for the soybean root- and stem-rot pathogen, Phytophthora sojae, utilizes its transposable element

PubMed Central

Haudenshield, James S.; Song, Jeong Y.; Hartman, Glen L.

2017-01-01

Phytophthora root rot of soybean [Glycine max (L.) Merr.] is caused by the oomycete Phytophthora sojae (Kaufm. & Gerd.). P. sojae has a narrow host range, consisting primarily of soybean, and it is a serious pathogen worldwide. It exists in root and stem tissues as mycelium, wherein it can form oospores which subsequently germinate to release motile, infectious zoospores. Molecular assays detecting DNA of P. sojae are useful in disease diagnostics, and for determining the presence of the organism in host tissues, soils, and runoff or ponded water from potentially infested fields. Such assays as published have utilized ITS sequences from the nuclear ribosomal RNA genes in conventional PCR or dye-binding quantitative PCR (Q-PCR) but are not amenable to multiplexing, and some of these assays did not utilize control strategies for type I or type II errors. In this study, we describe primers and a bifunctional probe with specificity to a gypsy-like retroelement in the P. sojae genome to create a fluorogenic 5’-exonuclease linear hydrolysis assay, with a multiplexed internal control reaction detecting an exogenous target to validate negative calls, and with uracil-deglycosylase-mediated protection against carryover contamination. The assay specifically detected 13 different P. sojae isolates, and excluded 17 other Phytophthora species along with 20 non-Phytophthora fungal and oomycete species pathogenic on soybean. A diagnostic limit of detection of 34 fg total P. sojae DNA was observed in serial dilutions, equivalent to 0.3 genome, and a practical detection sensitivity of four zoospores per sample was achieved, despite losses during DNA extraction. PMID:28441441

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

PubMed

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

2018-04-01

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

Single-shot quantitative phase microscopy with color-multiplexed differential phase contrast (cDPC)

PubMed Central

2017-01-01

We present a new technique for quantitative phase and amplitude microscopy from a single color image with coded illumination. Our system consists of a commercial brightfield microscope with one hardware modification—an inexpensive 3D printed condenser insert. The method, color-multiplexed Differential Phase Contrast (cDPC), is a single-shot variant of Differential Phase Contrast (DPC), which recovers the phase of a sample from images with asymmetric illumination. We employ partially coherent illumination to achieve resolution corresponding to 2× the objective NA. Quantitative phase can then be used to synthesize DIC and phase contrast images or extract shape and density. We demonstrate amplitude and phase recovery at camera-limited frame rates (50 fps) for various in vitro cell samples and c. elegans in a micro-fluidic channel. PMID:28152023

Single-shot quantitative phase microscopy with color-multiplexed differential phase contrast (cDPC).

PubMed

Phillips, Zachary F; Chen, Michael; Waller, Laura

2017-01-01

We present a new technique for quantitative phase and amplitude microscopy from a single color image with coded illumination. Our system consists of a commercial brightfield microscope with one hardware modification-an inexpensive 3D printed condenser insert. The method, color-multiplexed Differential Phase Contrast (cDPC), is a single-shot variant of Differential Phase Contrast (DPC), which recovers the phase of a sample from images with asymmetric illumination. We employ partially coherent illumination to achieve resolution corresponding to 2× the objective NA. Quantitative phase can then be used to synthesize DIC and phase contrast images or extract shape and density. We demonstrate amplitude and phase recovery at camera-limited frame rates (50 fps) for various in vitro cell samples and c. elegans in a micro-fluidic channel.

Comparison of Conventional PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Arcobacter Species

PubMed Central

Wang, Xiaoyu; Seo, Dong Joo; Lee, Min Hwa

2014-01-01

This study aimed to develop a loop-mediated isothermal amplification (LAMP) method for the rapid detection of Arcobacter species. Specific primers targeting the 23S ribosomal RNA gene were used to detect Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii. The specificity of the LAMP primer set was assessed using DNA samples from a panel of Arcobacter and Campylobacter species, and the sensitivity was determined using serial dilutions of Arcobacter species cultures. LAMP showed a 10- to 1,000-fold-higher sensitivity than multiplex PCR, with a detection limit of 2 to 20 CFU per reaction in vitro. Whereas multiplex PCR showed cross-reactivity with Campylobacter species, the LAMP method developed in this study was more sensitive and reliable than conventional PCR or multiplex PCR for the detection of Arcobacter species. PMID:24478488

Direct PCR - A rapid method for multiplexed detection of different serotypes of Salmonella in enriched pork meat samples.

PubMed

Chin, Wai Hoe; Sun, Yi; Høgberg, Jonas; Quyen, Than Linh; Engelsmann, Pia; Wolff, Anders; Bang, Dang Duong

2017-04-01

Salmonellosis, an infectious disease caused by Salmonella spp., is one of the most common foodborne diseases. Isolation and identification of Salmonella by conventional bacterial culture method is time consuming. In response to the demand for rapid on line or at site detection of pathogens, in this study, we developed a multiplex Direct PCR method for rapid detection of different Salmonella serotypes directly from pork meat samples without any DNA purification steps. An inhibitor-resistant Phusion Pfu DNA polymerase was used to overcome PCR inhibition. Four pairs of primers including a pair of newly designed primers targeting Salmonella spp. at subtype level were incorporated in the multiplex Direct PCR. To maximize the efficiency of the Direct PCR, the ratio between sample and dilution buffer was optimized. The sensitivity and specificity of the multiplex Direct PCR were tested using naturally contaminated pork meat samples for detecting and subtyping of Salmonella spp. Conventional bacterial culture methods were used as reference to evaluate the performance of the multiplex Direct PCR. Relative accuracy, sensitivity and specificity of 98.8%; 97.6% and 100%, respectively, were achieved by the method. Application of the multiplex Direct PCR to detect Salmonella in pork meat at slaughter reduces the time of detection from 5 to 6 days by conventional bacterial culture and serotyping methods to 14 h (including 12 h enrichment time). Furthermore, the method poses a possibility of miniaturization and integration into a point-of-need Lab-on-a-chip system for rapid online pathogen detection. Copyright © 2016 Elsevier Ltd. All rights reserved.

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.

Development and application of a quantitative multiplexed small GTPase activity assay using targeted proteomics.

PubMed

Zhang, Cheng-Cheng; Li, Ru; Jiang, Honghui; Lin, Shujun; Rogalski, Jason C; Liu, Kate; Kast, Juergen

2015-02-06

Small GTPases are a family of key signaling molecules that are ubiquitously expressed in various types of cells. Their activity is often analyzed by western blot, which is limited by its multiplexing capability, the quality of isoform-specific antibodies, and the accuracy of quantification. To overcome these issues, a quantitative multiplexed small GTPase activity assay has been developed. Using four different binding domains, this assay allows the binding of up to 12 active small GTPase isoforms simultaneously in a single experiment. To accurately quantify the closely related small GTPase isoforms, a targeted proteomic approach, i.e., selected/multiple reaction monitoring, was developed, and its functionality and reproducibility were validated. This assay was successfully applied to human platelets and revealed time-resolved coactivation of multiple small GTPase isoforms in response to agonists and differential activation of these isoforms in response to inhibitor treatment. This widely applicable approach can be used for signaling pathway studies and inhibitor screening in many cellular systems.

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.

Development of multiplex polymerase chain reaction for detection of Ehrlichia canis, Babesia spp and Hepatozoon canis in canine blood.

PubMed

Kledmanee, Kan; Suwanpakdee, Sarin; Krajangwong, Sakranmanee; Chatsiriwech, Jarin; Suksai, Parut; Suwannachat, Pongpun; Sariya, Ladawan; Buddhirongawatr, Ruangrat; Charoonrut, Phingphol; Chaichoun, Kridsada

2009-01-01

A multiplex polymerase chain reaction (PCR) has been developed for simultaneous detection of canine blood parasites, Ehrlichia canis, Babesia spp and Hepatozoon canis, from blood samples in a single reaction. The multiplex PCR primers were specific to E. canis VirB9, Babesia spp 16S rRNA and H. canis 16S rRNA genes. Specificity of the amplicons was confirmed by DNA sequencing. The assay was evaluated using normal canine and infected blood samples, which were detected by microscopic examination. This multiplex PCR offers scope for simultaneous detection of three important canine blood parasites and should be valuable in monitoring parasite infections in dogs and ticks.

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.

Aqueous two-phase system patterning of detection antibody solutions for cross-reaction-free multiplex ELISA

NASA Astrophysics Data System (ADS)

Frampton, John P.; White, Joshua B.; Simon, Arlyne B.; Tsuei, Michael; Paczesny, Sophie; Takayama, Shuichi

2014-05-01

Accurate disease diagnosis, patient stratification and biomarker validation require the analysis of multiple biomarkers. This paper describes cross-reactivity-free multiplexing of enzyme-linked immunosorbent assays (ELISAs) using aqueous two-phase systems (ATPSs) to confine detection antibodies at specific locations in fully aqueous environments. Antibody cross-reactions are eliminated because the detection antibody solutions are co-localized only to corresponding surface-immobilized capture antibody spots. This multiplexing technique is validated using plasma samples from allogeneic bone marrow recipients. Patients with acute graft versus host disease (GVHD), a common and serious condition associated with allogeneic bone marrow transplantation, display higher mean concentrations for four multiplexed biomarkers (HGF, elafin, ST2 and TNFR1) relative to healthy donors and transplant patients without GVHD. The antibody co-localization capability of this technology is particularly useful when using inherently cross-reactive reagents such as polyclonal antibodies, although monoclonal antibody cross-reactivity can also be reduced. Because ATPS-ELISA adapts readily available antibody reagents, plate materials and detection instruments, it should be easily transferable into other research and clinical settings.

Simultaneous mutation and copy number variation (CNV) detection by multiplex PCR-based GS-FLX sequencing.

PubMed

Goossens, Dirk; Moens, Lotte N; Nelis, Eva; Lenaerts, An-Sofie; Glassee, Wim; Kalbe, Andreas; Frey, Bruno; Kopal, Guido; De Jonghe, Peter; De Rijk, Peter; Del-Favero, Jurgen

2009-03-01

We evaluated multiplex PCR amplification as a front-end for high-throughput sequencing, to widen the applicability of massive parallel sequencers for the detailed analysis of complex genomes. Using multiplex PCR reactions, we sequenced the complete coding regions of seven genes implicated in peripheral neuropathies in 40 individuals on a GS-FLX genome sequencer (Roche). The resulting dataset showed highly specific and uniform amplification. Comparison of the GS-FLX sequencing data with the dataset generated by Sanger sequencing confirmed the detection of all variants present and proved the sensitivity of the method for mutation detection. In addition, we showed that we could exploit the multiplexed PCR amplicons to determine individual copy number variation (CNV), increasing the spectrum of detected variations to both genetic and genomic variants. We conclude that our straightforward procedure substantially expands the applicability of the massive parallel sequencers for sequencing projects of a moderate number of amplicons (50-500) with typical applications in resequencing exons in positional or functional candidate regions and molecular genetic diagnostics. 2008 Wiley-Liss, Inc.

Aqueous two-phase system patterning of detection antibody solutions for cross-reaction-free multiplex ELISA

PubMed Central

Frampton, John P.; White, Joshua B.; Simon, Arlyne B.; Tsuei, Michael; Paczesny, Sophie; Takayama, Shuichi

2014-01-01

Accurate disease diagnosis, patient stratification and biomarker validation require the analysis of multiple biomarkers. This paper describes cross-reactivity-free multiplexing of enzyme-linked immunosorbent assays (ELISAs) using aqueous two-phase systems (ATPSs) to confine detection antibodies at specific locations in fully aqueous environments. Antibody cross-reactions are eliminated because the detection antibody solutions are co-localized only to corresponding surface-immobilized capture antibody spots. This multiplexing technique is validated using plasma samples from allogeneic bone marrow recipients. Patients with acute graft versus host disease (GVHD), a common and serious condition associated with allogeneic bone marrow transplantation, display higher mean concentrations for four multiplexed biomarkers (HGF, elafin, ST2 and TNFR1) relative to healthy donors and transplant patients without GVHD. The antibody co-localization capability of this technology is particularly useful when using inherently cross-reactive reagents such as polyclonal antibodies, although monoclonal antibody cross-reactivity can also be reduced. Because ATPS-ELISA adapts readily available antibody reagents, plate materials and detection instruments, it should be easily transferable into other research and clinical settings. PMID:24786974

Nucleic acid sequence detection using multiplexed oligonucleotide PCR

DOEpatents

Nolan, John P [Santa Fe, NM; White, P Scott [Los Alamos, NM

2006-12-26

Methods for rapidly detecting single or multiple sequence alleles in a sample nucleic acid are described. Provided are all of the oligonucleotide pairs capable of annealing specifically to a target allele and discriminating among possible sequences thereof, and ligating to each other to form an oligonucleotide complex when a particular sequence feature is present (or, alternatively, absent) in the sample nucleic acid. The design of each oligonucleotide pair permits the subsequent high-level PCR amplification of a specific amplicon when the oligonucleotide complex is formed, but not when the oligonucleotide complex is not formed. The presence or absence of the specific amplicon is used to detect the allele. Detection of the specific amplicon may be achieved using a variety of methods well known in the art, including without limitation, oligonucleotide capture onto DNA chips or microarrays, oligonucleotide capture onto beads or microspheres, electrophoresis, and mass spectrometry. Various labels and address-capture tags may be employed in the amplicon detection step of multiplexed assays, as further described herein.

Nano metal-organic framework (NMOF)-based strategies for multiplexed microRNA detection in solution and living cancer cells

NASA Astrophysics Data System (ADS)

Wu, Yafeng; Han, Jianyu; Xue, Peng; Xu, Rong; Kang, Yuejun

2015-01-01

MiRNAs are an emerging type of biomarker for diagnostics and prognostics. A reliable sensing strategy that can monitor miRNA expression in living cancer cells would be critical in view of its extensive advantages for fundamental research related to miRNA-associated bioprocesses and biomedical applications. Conventional miRNA sensing methods include northern blot, microarrays and real-time quantitative PCR. However, none of them is able to monitor miRNA levels expressed in living cancer cells in a real-time fashion. Some fluorescennt biosensors developed recently from carbon nanomaterials, such as single-walled carbon nanotubes (SWNTs), graphene oxide (GO), and carbon nanoparticles, have been successfully used for assaying miRNA in vitro; however the preparation processes are often expensive, complicated and time-consuming, which have motivated the research on other substitute and novel materials. Herein we present a novel sensing strategy based on peptide nucleic acid (PNA) probes labeled with fluorophores and conjugated with an NMOF vehicle to monitor multiplexed miRNAs in living cancer cells. The NMOF works as a fluorescence quencher of the labelled PNA that is firmly bound with the metal center. In the presence of a target miRNA, PNA is hybridized and released from the NMOF leading to the recovery of fluorescence. This miRNA sensor not only enables the quantitative and highly specific detection of multiplexed miRNAs in living cancer cells, but it also allows the precise and in situ monitoring of the spatiotemporal changes of miRNA expression.MiRNAs are an emerging type of biomarker for diagnostics and prognostics. A reliable sensing strategy that can monitor miRNA expression in living cancer cells would be critical in view of its extensive advantages for fundamental research related to miRNA-associated bioprocesses and biomedical applications. Conventional miRNA sensing methods include northern blot, microarrays and real-time quantitative PCR. However, none of

The increasing application of multiplex nucleic acid detection tests to the diagnosis of syndromic infections.

PubMed

Gray, J; Coupland, L J

2014-01-01

On 14 January 2013, the US Food and Drug Administration (FDA) announced permission for a multiplex nucleic acid test, the xTAG® Gastrointestinal Pathogen Panel (GPP) (Luminex Corporation, USA), which simultaneously detects 11 common viral, bacterial and parasitic causes of infectious gastroenteritis, to be marketed in the USA. This announcement reflects the current move towards the development and commercialization of detection technologies based on nucleic acid amplification techniques for diagnosis of syndromic infections. We discuss the limitations and advantages of nucleic acid amplification techniques and the recent advances in Conformité Européene - in-vitro diagnostic (CE-IVD)-approved multiplex real-time PCR kits for the simultaneous detection of multiple targets within the clinical diagnostics market.

Simultaneous Detection of Four Foodborne Viruses in Food Samples Using a One-Step Multiplex Reverse Transcription PCR.

PubMed

Lee, Shin-Young; Kim, Mi-Ju; Kim, Hyun-Joong; Jeong, KwangCheol Casey; Kim, Hae-Yeong

2018-02-28

A one-step multiplex reverse transcription PCR (RT-PCR) method comprising six primer sets (for the detection of norovirus GI and GII, hepatitis A virus, rotavirus, and astrovirus) was developed to simultaneously detect four kinds of pathogenic viruses. The size of the PCR products for norovirus GI and GII, hepatitis A virus (VP3/VP1 and P2A regions), rotavirus, and astrovirus were 330, 164, 244, 198, 629, and 449 bp, respectively. The RT-PCR with the six primer sets showed specificity for the pathogenic viruses. The detection limit of the developed multiplex RT-PCR, as evaluated using serially diluted viral RNAs, was comparable to that of one-step single RT-PCR. Moreover, this multiplex RT-PCR was evaluated using food samples such as water, oysters, lettuce, and vegetable product. These food samples were artificially spiked with the four kinds of viruses in diverse combinations, and the spiked viruses in all food samples were detected successfully.

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

PubMed

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

2016-05-28

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

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

PubMed Central

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

2016-01-01

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

Simultaneous detection of Plasmodium vivax and Plasmodium falciparum gametocytes in clinical isolates by multiplex-nested RT-PCR

PubMed Central

2012-01-01

Background Gametocyte carriage is essential for malaria transmission and endemicity of disease; thereby it is a target for malaria control strategies. Malaria-infected individuals may harbour gametocytes below the microscopic detection threshold that can be detected by reverse transcription polymerase chain reaction (RT-PCR) targeting gametocyte-specific mRNA. To date, RT-PCR has mainly been applied to the diagnosis of Plasmodium falciparum gametocytes but very limited for that of Plasmodium vivax. Methods A multiplex-nested RT-PCR targeting Pfs25 and Pvs25 mRNA specific to mature gametocytes of P. falciparum and P. vivax, respectively, was developed. The assay was evaluated using blood samples collected in rainy and dry seasons from febrile patients,in a malaria-endemic area in Thailand. Malaria diagnosis was performed by Giemsa-stained blood smears and 18S rRNA PCR. Results The multiplex-nested RT-PCR detected Pfs25 mRNA in 75 of 86 (87.2%) P. falciparum-infected individuals and Pvs25 mRNA in 82 of 90 (91.1%) P. vivax malaria patients diagnosed by 18S rRNA PCR. Gametocytes were detected in 38 (eight P. falciparum and 30 P. vivax) of 157 microscopy positive samples, implying that a large number of patients harbour sub-microscopic gametocytaemia. No seasonal differences in gametocyte carriage were observed for both malaria species diagnosed by multiplex-nested RT-PCR. With single-nested RT-PCR targeting Pfs25 or Pvs25 mRNA as standard, the multiplex-nested RT-PCR offered sensitivities of 97.4% and 98.9% and specificities of 100% and 98.8% for diagnosing mature gametocytes of P. falciparum and P. vivax, respectively. The minimum detection limit of the multiplex-nested PCR was 10 copies of templates. Conclusions The multiplex-nested RT-PCR developed herein is useful for simultaneous assessment of both P. falciparum and P. vivax gametocyte carriage that is prevalent and generally sympatric in several malaria-endemic areas outside Africa. PMID:22682065

Simultaneous detection of Plasmodium vivax and Plasmodium falciparum gametocytes in clinical isolates by multiplex-nested RT-PCR.

PubMed

Kuamsab, Napaporn; Putaporntip, Chaturong; Pattanawong, Urassaya; Jongwutiwes, Somchai

2012-06-10

Gametocyte carriage is essential for malaria transmission and endemicity of disease; thereby it is a target for malaria control strategies. Malaria-infected individuals may harbour gametocytes below the microscopic detection threshold that can be detected by reverse transcription polymerase chain reaction (RT-PCR) targeting gametocyte-specific mRNA. To date, RT-PCR has mainly been applied to the diagnosis of Plasmodium falciparum gametocytes but very limited for that of Plasmodium vivax. A multiplex-nested RT-PCR targeting Pfs25 and Pvs25 mRNA specific to mature gametocytes of P. falciparum and P. vivax, respectively, was developed. The assay was evaluated using blood samples collected in rainy and dry seasons from febrile patients,in a malaria-endemic area in Thailand. Malaria diagnosis was performed by Giemsa-stained blood smears and 18S rRNA PCR. The multiplex-nested RT-PCR detected Pfs25 mRNA in 75 of 86 (87.2%) P. falciparum-infected individuals and Pvs25 mRNA in 82 of 90 (91.1%) P. vivax malaria patients diagnosed by 18S rRNA PCR. Gametocytes were detected in 38 (eight P. falciparum and 30 P. vivax) of 157 microscopy positive samples, implying that a large number of patients harbour sub-microscopic gametocytaemia. No seasonal differences in gametocyte carriage were observed for both malaria species diagnosed by multiplex-nested RT-PCR. With single-nested RT-PCR targeting Pfs25 or Pvs25 mRNA as standard, the multiplex-nested RT-PCR offered sensitivities of 97.4% and 98.9% and specificities of 100% and 98.8% for diagnosing mature gametocytes of P. falciparum and P. vivax, respectively. The minimum detection limit of the multiplex-nested PCR was 10 copies of templates. The multiplex-nested RT-PCR developed herein is useful for simultaneous assessment of both P. falciparum and P. vivax gametocyte carriage that is prevalent and generally sympatric in several malaria-endemic areas outside Africa.

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

Multiplex Nucleic Acid Sequence-Based Amplification for Simultaneous Detection of Several Enteric Viruses in Model Ready-To-Eat Foods†

PubMed Central

Jean, Julie; D'Souza, Doris H.; Jaykus, Lee-Ann

2004-01-01

Human enteric viruses are currently recognized as one of the most important causes of food-borne disease. Implication of enteric viruses in food-borne outbreaks can be difficult to confirm due to the inadequacy of the detection methods available. In this study, a nucleic acid sequence-based amplification (NASBA) method was developed in a multiplex format for the specific, simultaneous, and rapid detection of epidemiologically relevant human enteric viruses. Three previously reported primer sets were used in a single reaction for the amplification of RNA target fragments of 474, 371, and 165 nucleotides for the detection of hepatitis A virus and genogroup I and genogroup II noroviruses, respectively. Amplicons were detected by agarose gel electrophoresis and confirmed by electrochemiluminescence and Northern hybridization. Endpoint detection sensitivity for the multiplex NASBA assay was approximately 10−1 reverse transcription-PCR-detectable units (or PFU, as appropriate) per reaction. When representative ready-to-eat foods (deli sliced turkey and lettuce) were inoculated with various concentrations of each virus and processed for virus detection with the multiplex NASBA method, all three human enteric viruses were simultaneously detected at initial inoculum levels of 100 to 102 reverse transcription-PCR-detectable units (or PFU)/9 cm2 in both food commodities. The multiplex NASBA system provides rapid and simultaneous detection of clinically relevant food-borne viruses in a single reaction tube and may be a promising alternative to reverse transcription-PCR for the detection of viral contamination of foods. PMID:15528524

Multiplex nucleic acid sequence-based amplification for simultaneous detection of several enteric viruses in model ready-to-eat foods.

PubMed

Jean, Julie; D'Souza, Doris H; Jaykus, Lee-Ann

2004-11-01

Human enteric viruses are currently recognized as one of the most important causes of food-borne disease. Implication of enteric viruses in food-borne outbreaks can be difficult to confirm due to the inadequacy of the detection methods available. In this study, a nucleic acid sequence-based amplification (NASBA) method was developed in a multiplex format for the specific, simultaneous, and rapid detection of epidemiologically relevant human enteric viruses. Three previously reported primer sets were used in a single reaction for the amplification of RNA target fragments of 474, 371, and 165 nucleotides for the detection of hepatitis A virus and genogroup I and genogroup II noroviruses, respectively. Amplicons were detected by agarose gel electrophoresis and confirmed by electrochemiluminescence and Northern hybridization. Endpoint detection sensitivity for the multiplex NASBA assay was approximately 10(-1) reverse transcription-PCR-detectable units (or PFU, as appropriate) per reaction. When representative ready-to-eat foods (deli sliced turkey and lettuce) were inoculated with various concentrations of each virus and processed for virus detection with the multiplex NASBA method, all three human enteric viruses were simultaneously detected at initial inoculum levels of 10(0) to 10(2) reverse transcription-PCR-detectable units (or PFU)/9 cm2 in both food commodities. The multiplex NASBA system provides rapid and simultaneous detection of clinically relevant food-borne viruses in a single reaction tube and may be a promising alternative to reverse transcription-PCR for the detection of viral contamination of foods.

A multiplex nested PCR for the detection and identification of Candida species in blood samples of critically ill paediatric patients

PubMed Central

2014-01-01

Background Nosocomial candidaemia is associated with high mortality rates in critically ill paediatric patients; thus, the early detection and identification of the infectious agent is crucial for successful medical intervention. The PCR-based techniques have significantly increased the detection of Candida species in bloodstream infections. In this study, a multiplex nested PCR approach was developed for candidaemia detection in neonatal and paediatric intensive care patients. Methods DNA samples from the blood of 54 neonates and children hospitalised in intensive care units with suspected candidaemia were evaluated by multiplex nested PCR with specific primers designed to identify seven Candida species, and the results were compared with those obtained from blood cultures. Results The multiplex nested PCR had a detection limit of four Candida genomes/mL of blood for all Candida species. Blood cultures were positive in 14.8% of patients, whereas the multiplex nested PCR was positive in 24.0% of patients, including all culture-positive patients. The results obtained with the molecular technique were available within 24 hours, and the assay was able to identify Candida species with 100% of concordance with blood cultures. Additionally, the multiplex nested PCR detected dual candidaemia in three patients. Conclusions Our proposed PCR method may represent an effective tool for the detection and identification of Candida species in the context of candidaemia diagnosis in children, showing highly sensitive detection and the ability to identify the major species involved in this infection. PMID:25047415

A multiplex nested PCR for the detection and identification of Candida species in blood samples of critically ill paediatric patients.

PubMed

Taira, Cleison Ledesma; Okay, Thelma Suely; Delgado, Artur Figueiredo; Ceccon, Maria Esther Jurfest Rivero; de Almeida, Margarete Teresa Gottardo; Del Negro, Gilda Maria Barbaro

2014-07-21

Nosocomial candidaemia is associated with high mortality rates in critically ill paediatric patients; thus, the early detection and identification of the infectious agent is crucial for successful medical intervention. The PCR-based techniques have significantly increased the detection of Candida species in bloodstream infections. In this study, a multiplex nested PCR approach was developed for candidaemia detection in neonatal and paediatric intensive care patients. DNA samples from the blood of 54 neonates and children hospitalised in intensive care units with suspected candidaemia were evaluated by multiplex nested PCR with specific primers designed to identify seven Candida species, and the results were compared with those obtained from blood cultures. The multiplex nested PCR had a detection limit of four Candida genomes/mL of blood for all Candida species. Blood cultures were positive in 14.8% of patients, whereas the multiplex nested PCR was positive in 24.0% of patients, including all culture-positive patients. The results obtained with the molecular technique were available within 24 hours, and the assay was able to identify Candida species with 100% of concordance with blood cultures. Additionally, the multiplex nested PCR detected dual candidaemia in three patients. Our proposed PCR method may represent an effective tool for the detection and identification of Candida species in the context of candidaemia diagnosis in children, showing highly sensitive detection and the ability to identify the major species involved in this infection.

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.

Polarization-multiplexing ghost imaging

NASA Astrophysics Data System (ADS)

Dongfeng, Shi; Jiamin, Zhang; Jian, Huang; Yingjian, Wang; Kee, Yuan; Kaifa, Cao; Chenbo, Xie; Dong, Liu; Wenyue, Zhu

2018-03-01

A novel technique for polarization-multiplexing ghost imaging is proposed to simultaneously obtain multiple polarimetric information by a single detector. Here, polarization-division multiplexing speckles are employed for object illumination. The light reflected from the objects is detected by a single-pixel detector. An iterative reconstruction method is used to restore the fused image containing the different polarimetric information by using the weighted sum of the multiplexed speckles based on the correlation coefficients obtained from the detected intensities. Next, clear images of the different polarimetric information are recovered by demultiplexing the fused image. The results clearly demonstrate that the proposed method is effective.

Multiplex Quantitative PCR Assays for the Detection and Quantification of the Six Major Non-O157 Escherichia coli Serogroups in Cattle Feces.

PubMed

Shridhar, P B; Noll, L W; Shi, X; An, B; Cernicchiaro, N; Renter, D G; Nagaraja, T G; Bai, J

2016-01-01

Shiga toxin-producing Escherichia coli (STEC) serogroups O26, O45, O103, O111, O121, and O145, called non-O157 STEC, are important foodborne pathogens. Cattle, a major reservoir, harbor the organisms in the hindgut and shed them in the feces. Although limited data exist on fecal shedding, concentrations of non-O157 STEC in feces have not been reported. The objectives of our study were (i) to develop and validate two multiplex quantitative PCR (mqPCR) assays, targeting O-antigen genes of O26, O103, and O111 (mqPCR-1) and O45, O121, and O145 (mqPCR-2); (ii) to utilize the two assays, together with a previously developed four-plex qPCR assay (mqPCR-3) targeting the O157 antigen and three virulence genes (stx1, stx2, and eae), to quantify seven serogroups and three virulence genes in cattle feces; and (iii) to compare the three mqPCR assays to a 10-plex conventional PCR (cPCR) targeting seven serogroups and three virulence genes and culture methods to detect seven E. coli serogroups in cattle feces. The two mqPCR assays (1 and 2) were shown to be specific to the target genes, and the detection limits were 4 and 2 log CFU/g of pure culture-spiked fecal samples, before and after enrichment, respectively. A total of 576 fecal samples collected from a feedlot were enriched in E. coli broth and were subjected to quantification (before enrichment) and detection (after enrichment). Of the 576 fecal samples subjected, before enrichment, to three mqPCR assays for quantification, 175 (30.4%) were quantifiable (≥4 log CFU/g) for at least one of the seven serogroups, with O157 being the most common serogroup. The three mqPCR assays detected higher proportions of postenriched fecal samples (P > 0.01) as positive for one or more serogroups compared with cPCR and culture methods. This is the first study to assess the applicability of qPCR assays to detect and quantify six non-O157 serogroups in cattle feces and to generate data on fecal concentration of the six serogroups.

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

PubMed

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

2017-11-01

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

A Real-Time Clinical Endoscopic System for Intraluminal, Multiplexed Imaging of Surface-Enhanced Raman Scattering Nanoparticles

PubMed Central

Garai, Ellis; Loewke, Nathan O.; Rogalla, Stephan; Mandella, Michael J.; Felt, Stephen A.; Friedland, Shai; Liu, Jonathan T. C.; Gambhir, Sanjiv S.; Contag, Christopher H.

2015-01-01

The detection of biomarker-targeting surface-enhanced Raman scattering (SERS) nanoparticles (NPs) in the human gastrointestinal tract has the potential to improve early cancer detection; however, a clinically relevant device with rapid Raman-imaging capability has not been described. Here we report the design and in vivo demonstration of a miniature, non-contact, opto-electro-mechanical Raman device as an accessory to clinical endoscopes that can provide multiplexed molecular data via a panel of SERS NPs. This device enables rapid circumferential scanning of topologically complex luminal surfaces of hollow organs (e.g., colon and esophagus) and produces quantitative images of the relative concentrations of SERS NPs that are present. Human and swine studies have demonstrated the speed and simplicity of this technique. This approach also offers unparalleled multiplexing capabilities by simultaneously detecting the unique spectral fingerprints of multiple SERS NPs. Therefore, this new screening strategy has the potential to improve diagnosis and to guide therapy by enabling sensitive quantitative molecular detection of small and otherwise hard-to-detect lesions in the context of white-light endoscopy. PMID:25923788

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.

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

PubMed

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

2018-04-18

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

Polarization-interleave-multiplexed discrete multi-tone modulation with direct detection utilizing MIMO equalization.

PubMed

Zhou, Xian; Zhong, Kangping; Gao, Yuliang; Sui, Qi; Dong, Zhenghua; Yuan, Jinhui; Wang, Liang; Long, Keping; Lau, Alan Pak Tao; Lu, Chao

2015-04-06

Discrete multi-tone (DMT) modulation is an attractive modulation format for short-reach applications to achieve the best use of available channel bandwidth and signal noise ratio (SNR). In order to realize polarization-multiplexed DMT modulation with direct detection, we derive an analytical transmission model for dual polarizations with intensity modulation and direct diction (IM-DD) in this paper. Based on the model, we propose a novel polarization-interleave-multiplexed DMT modulation with direct diction (PIM-DMT-DD) transmission system, where the polarization de-multiplexing can be achieved by using a simple multiple-input-multiple-output (MIMO) equalizer and the transmission performance is optimized over two distinct received polarization states to eliminate the singularity issue of MIMO demultiplexing algorithms. The feasibility and effectiveness of the proposed PIM-DMT-DD system are investigated via theoretical analyses and simulation studies.

Multiplexed MRM-based quantitation of candidate cancer biomarker proteins in undepleted and non-enriched human plasma.

PubMed

Percy, Andrew J; Chambers, Andrew G; Yang, Juncong; Borchers, Christoph H

2013-07-01

An emerging approach for multiplexed targeted proteomics involves bottom-up LC-MRM-MS, with stable isotope-labeled internal standard peptides, to accurately quantitate panels of putative disease biomarkers in biofluids. In this paper, we used this approach to quantitate 27 candidate cancer-biomarker proteins in human plasma that had not been treated by immunoaffinity depletion or enrichment techniques. These proteins have been reported as biomarkers for a variety of human cancers, from laryngeal to ovarian, with breast cancer having the highest correlation. We implemented measures to minimize the analytical variability, improve the quantitative accuracy, and increase the feasibility and applicability of this MRM-based method. We have demonstrated excellent retention time reproducibility (median interday CV: 0.08%) and signal stability (median interday CV: 4.5% for the analytical platform and 6.1% for the bottom-up workflow) for the 27 biomarker proteins (represented by 57 interference-free peptides). The linear dynamic range for the MRM assays spanned four orders-of-magnitude, with 25 assays covering a 10(3) -10(4) range in protein concentration. The lowest abundance quantifiable protein in our biomarker panel was insulin-like growth factor 1 (calculated concentration: 127 ng/mL). Overall, the analytical performance of this assay demonstrates high robustness and sensitivity, and provides the necessary throughput and multiplexing capabilities required to verify and validate cancer-associated protein biomarker panels in human plasma, prior to clinical use. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparison of culture, single and multiplex real-time PCR for detection of Sabin poliovirus shedding in recently vaccinated Indian children.

PubMed

Giri, Sidhartha; Rajan, Anand K; Kumar, Nirmal; Dhanapal, Pavithra; Venkatesan, Jayalakshmi; Iturriza-Gomara, Miren; Taniuchi, Mami; John, Jacob; Abraham, Asha Mary; Kang, Gagandeep

2017-08-01

Although, culture is considered the gold standard for poliovirus detection from stool samples, real-time PCR has emerged as a faster and more sensitive alternative. Detection of poliovirus from the stool of recently vaccinated children by culture, single and multiplex real-time PCR was compared. Of the 80 samples tested, 55 (68.75%) were positive by culture compared to 61 (76.25%) and 60 (75%) samples by the single and one step multiplex real-time PCR assays respectively. Real-time PCR (singleplex and multiplex) is more sensitive than culture for poliovirus detection in stool, although the difference was not statistically significant. © 2017 Wiley Periodicals, Inc.

Development of an In-House Multiplex Nested RT-PCR Method for Detecting Acute HIV-1 Infection in High Risk Populations.

PubMed

Liu, Zhiying; Li, Wei; Xu, Meng; Sheng, Bo; Yang, Zixuan; Jiao, Yanmei; Zhang, Tong; Mou, Danlei; Chen, Dexi; Wu, Hao

2015-01-01

The detection of acute HIV infection (AHI) among high risk populations can help reduce secondary transmission of HIV. The nucleic acid testing (NAT) can shorten the test window period by up to 7-12 days. In this study, we describe an in-house NAT based on the multiplex nested RT-PCR method to detect the HIV RNA. We also evaluated it in a high risk cohort in Beijing. Four primer pairs were designed and evaluated for the detection of different HIV-1 subtypes in group M. Multiplex RT-PCR and nested PCR were performed. The sensitivity, specialty, primers compatibility among HIV subtypes were evaluated simultaneously. In an MSM cohort in Beijing during a 3-year period, a total of 11,808 blood samples that were negative by ELISA or indeterminate by Western blot were analyzed by this multiplex nested RT-PCR with pooling strategy. The multiplex nested RT-PCR was successfully applied for the detection of at least six HIV-1 subtypes. The sensitivity was 40 copies/ml and the specificity was 100%. A total of 29 people were tested HIV-1 positive with acute infection in a MSM cohort of Beijing during a 3 years period. This multiplex nested RT-PCR provides a useful tool for the rapid detection of acute HIV-1 infection. When used in combination with the 3(rd) generation ELISA, it can improve the detection rate of HIV infection, especially in the source limited regions.

Research on aircraft trailing vortex detection based on laser's multiplex information echo

NASA Astrophysics Data System (ADS)

Zhao, Nan-xiang; Wu, Yong-hua; Hu, Yi-hua; Lei, Wu-hu

2010-10-01

Airfoil trailing vortex is an important reason for the crash, and vortex detection is the basic premise for the civil aeronautics boards to make the flight measures and protect civil aviation's security. So a new method of aircraft trailing vortex detection based on laser's multiplex information echo has been proposed in this paper. According to the classical aerodynamics theories, the formation mechanism of the trailing vortex from the airfoil wingtip has been analyzed, and the vortex model of Boeing 737 in the taking-off phase has also been established on the FLUENT software platform. Combining with the unique morphological structure characteristics of trailing vortex, we have discussed the vortex's possible impact on the frequency, amplitude and phase information of laser echo, and expounded the principle of detecting vortex based on fusing this information variation of laser echo. In order to prove the feasibility of this detecting technique, the field experiment of detecting the vortex of civil Boeing 737 by laser has been carried on. The experimental result has shown that the aircraft vortex could be found really in the laser scanning area, and its diffusion characteristic has been very similar to the previous simulation result. Therefore, this vortex detection means based on laser's multiplex information echo was proved to be practicable relatively in this paper. It will provide the detection and identification of aircraft's trailing vortex a new way, and have massive research value and extensive application prospect as well.

Detection of pathogenic bacteria in shellfish using multiplex PCR followed by CovaLink NH microwell plate sandwich hybridization.

PubMed

Lee, Chi-Ying; Panicker, Gitika; Bej, Asim K

2003-05-01

Outbreak of diseases associated with consumption of raw shellfish especially oysters is a major concern to the seafood industry and public health agencies. A multiplex PCR amplification of targeted gene segments followed by DNA-DNA sandwich hybridization was optimized to detect the etiologic agents. First, a multiplex PCR amplification of hns, spvB, vvh, ctx and tl was developed enabling simultaneous detection of total Salmonella enterica serotype Typhimurium, Vibrio vulnificus, Vibrio cholerae and Vibrio parahaemolyticus from both pure cultures and seeded oysters. Amplicons were then subjected to a colorimetric CovaLink NH microwell plate sandwich hybridization using phosphorylated and biotinlylated oligonucleotide probes, the nucleotide sequences of which were located internal to the amplified DNA. The results from the hybridization with the multiplexed PCR amplified DNA exhibited a high signal/noise ratio ranging between 14.1 and 43.2 measured at 405 nm wavelength. The sensitivity of detection for each pathogen was 10(2) cells/g of oyster tissue homogenate. The results from this study showed that the combination of the multiplex PCR with a colorimetric microwell plate sandwich hybridization assay permits a specific, sensitive, and reproducible system for the detection of the microbial pathogens in shellfish, thereby improving the microbiological safety of shellfish to consumers.

Rapid and reliable detection and identification of GM events using multiplex PCR coupled with oligonucleotide microarray.

PubMed

Xu, Xiaodan; Li, Yingcong; Zhao, Heng; Wen, Si-yuan; Wang, Sheng-qi; Huang, Jian; Huang, Kun-lun; Luo, Yun-bo

2005-05-18

To devise a rapid and reliable method for the detection and identification of genetically modified (GM) events, we developed a multiplex polymerase chain reaction (PCR) coupled with a DNA microarray system simultaneously aiming at many targets in a single reaction. The system included probes for screening gene, species reference gene, specific gene, construct-specific gene, event-specific gene, and internal and negative control genes. 18S rRNA was combined with species reference genes as internal controls to assess the efficiency of all reactions and to eliminate false negatives. Two sets of the multiplex PCR system were used to amplify four and five targets, respectively. Eight different structure genes could be detected and identified simultaneously for Roundup Ready soybean in a single microarray. The microarray specificity was validated by its ability to discriminate two GM maizes Bt176 and Bt11. The advantages of this method are its high specificity and greatly reduced false-positives and -negatives. The multiplex PCR coupled with microarray technology presented here is a rapid and reliable tool for the simultaneous detection of GM organism ingredients.

Broadband quantitative phase microscopy with extended field of view using off-axis interferometric multiplexing.

PubMed

Girshovitz, Pinhas; Frenklach, Irena; Shaked, Natan T

2015-11-01

We propose a new portable imaging configuration that can double the field of view (FOV) of existing off-axis interferometric imaging setups, including broadband off-axis interferometers. This configuration is attached at the output port of the off-axis interferometer and optically creates a multiplexed interferogram on the digital camera, which is composed of two off-axis interferograms with straight fringes at orthogonal directions. Each of these interferograms contains a different FOV of the imaged sample. Due to the separation of these two FOVs in the spatial-frequency domain, they can be fully reconstructed separately, while obtaining two complex wavefronts from the sample at once. Since the optically multiplexed off-axis interferogram is recorded by the camera in a single exposure, fast dynamics can be recorded with a doubled imaging area. We used this technique for quantitative phase microscopy of biological samples with extended FOV. We demonstrate attaching the proposed module to a diffractive phase microscopy interferometer, illuminated by a broadband light source. The biological samples used for the experimental demonstrations include microscopic diatom shells, cancer cells, and flowing blood cells.

Graphene nano-ink biosensor arrays on a microfluidic paper for multiplexed detection of metabolites.

PubMed

Labroo, Pratima; Cui, Yue

2014-02-27

The development of a miniaturized and low-cost platform for the highly sensitive, selective and rapid detection of multiplexed metabolites is of great interest for healthcare, pharmaceuticals, food science, and environmental monitoring. Graphene is a delicate single-layer, two-dimensional network of carbon atoms with extraordinary electrical sensing capability. Microfluidic paper with printing technique is a low cost matrix. Here, we demonstrated the development of graphene-ink based biosensor arrays on a microfluidic paper for the multiplexed detection of different metabolites, such as glucose, lactate, xanthine and cholesterol. Our results show that the graphene biosensor arrays can detect multiple metabolites on a microfluidic paper sensitively, rapidly and simultaneously. The device exhibits a fast measuring time of less than 2 min, a low detection limit of 0.3 μM, and a dynamic detection range of 0.3-15 μM. The process is simple and inexpensive to operate and requires a low consumption of sample volume. We anticipate that these results could open exciting opportunities for a variety of applications. Copyright © 2014 Elsevier B.V. All rights reserved.

A Microfluidic Platform for High-Throughput Multiplexed Protein Quantitation

PubMed Central

Volpetti, Francesca; Garcia-Cordero, Jose; Maerkl, Sebastian J.

2015-01-01

We present a high-throughput microfluidic platform capable of quantitating up to 384 biomarkers in 4 distinct samples by immunoassay. The microfluidic device contains 384 unit cells, which can be individually programmed with pairs of capture and detection antibody. Samples are quantitated in each unit cell by four independent MITOMI detection areas, allowing four samples to be analyzed in parallel for a total of 1,536 assays per device. We show that the device can be pre-assembled and stored for weeks at elevated temperature and we performed proof-of-concept experiments simultaneously quantitating IL-6, IL-1β, TNF-α, PSA, and GFP. Finally, we show that the platform can be used to identify functional antibody combinations by screening 64 antibody combinations requiring up to 384 unique assays per device. PMID:25680117

Colorimetric Aptasensor Using Unmodified Gold Nanoparticles for Homogeneous Multiplex Detection

PubMed Central

Niu, Shucao; Lv, Zhenzhen; Liu, Jinchuan; Bai, Wenhui; Yang, Shuming; Chen, Ailiang

2014-01-01

Colorimetric aptasensors using unmodified gold nanoparticles (AuNPs) have attracted much attention because of their low cost, simplicity, and practicality, and they have been developed for various targets in the past several years. However, previous research has focused on developing single-target assays. Here, we report the development of a homogeneous multiplex aptasensor by using more than one class of aptamers to stabilize AuNPs. Using sulfadimethoxine (SDM), kanamycin (KAN) and adenosine (ADE) as example targets, a KAN aptamer (750 nM), an SDM aptamer (250 nM) and an ADE aptamer (500 nM) were mixed at a 1∶1∶1 volume ratio and adsorbed directly onto the surface of unmodified AuNPs by electrostatic interaction. Upon the addition of any of the three targets, the conformation of the corresponding aptamer changed from a random coil structure to a rigid folded structure, which could not adsorb and stabilize AuNPs. The AuNPs aggregated in a specific reaction buffer (20 mM Tris-HCl containing 20 mM NaCl and 5 mM KCl), which led to a color change from red to purple/blue. These results demonstrate that the multiplex colorimetric aptasensor detected three targets simultaneously while maintaining the same sensitivity as a single-target aptasensor for each individual target. The multiplex aptasensor could be extended to other aptamers for various molecular detection events. Due to its simple design, easy operation, fast response, cost effectiveness and lack of need for sophisticated instrumentation, the proposed strategy provides a powerful tool to examine large numbers of samples to screen for a small number of potentially positive samples containing more than one analyte, which can be further validated using sophisticated instruments. PMID:25279730

Multiplex fluorescent PCR for noninvasive prenatal detection of fetal-derived paternally inherited diseases using circulatory fetal DNA in maternal plasma.

PubMed

Tang, Dong-ling; Li, Yan; Zhou, Xin; Li, Xia; Zheng, Fang

2009-05-01

To develop a fluorescent polymerase chain reaction (PCR) assay for the detection of circulating fetal DNA in maternal plasma and use the established multiplex in noninvasive prenatal genetic diagnosis and its further applications in forensic casework. The DNA template was extracted from 47 pregnant women and the whole blood samples from the stated biological fathers were used to detect genotype. Using multiplex fluorescent PCR at 16 different polymorphic short tandem repeat (STR) loci, maternal DNA extracted from plasma samples at early pregnancy, medium pregnancy and late pregnancy were used to detect genotype. Their husbands' DNA was also used for fetal genotype ascertainment. Multiplex fluorescent PCR with 16 polymorphic short tandem repeats revealed the presence of fetal DNA in all cases. Every pregnant women/husband pair was informative in at least 3 of 16 loci. The chances of detecting paternally inherited fetal alleles ranged from 66.67 to 94.12%. They are 66.67% in early pregnancy, 85.71% in medium pregnancy and 94.12% in late pregnancy. The accuracy of Multiplex PCR assay to detect fetal DNA was 100%. Circulating fetal DNA analysis can be used as a possible alternative tool in routine laboratory prenatal diagnosis in the near future; this highly polymorphic STR multiplex has greatly improved the chances of detecting paternally inherited fetal alleles compared with other fetal DNA detection systems that use fetus-derived Y sequences to detect only male fetal DNA in maternal plasma. Our proposed technique can be applied to both female and male fetuses, which provides a sensitive, accurate and efficient method for noninvasive prenatal genetic diagnosis and forensic casework.

The plasma interleukin-6 response to acute psychosocial stress in humans is detected by a magnetic multiplex assay: comparison to high-sensitivity ELISA.

PubMed

Quinn, Andrea M; Williams, Allison R; Sivilli, Teresa I; Raison, Charles L; Pace, Thaddeus W W

2018-03-13

Circulating concentrations of interleukin (IL)-6, an inflammatory biomarker widely assessed in humans to study the inflammatory response to acute psychological stress, have for decades been quantified using enzyme-linked immunosorbent assay (ELISA). However, biobehavioral researchers are increasingly using cytokine multiplex assays instead of ELISA to measure IL-6 and other cytokines. Despite this trend, multiplex assays have not been directly compared to ELISA for their ability to detect subtle stress-induced changes of IL-6. Here, we tested the prediction that a high-sensitivity multiplex assay (human Magnetic Luminex Performance Assay, R&D Systems, Minneapolis, MN) would detect changes in IL-6 as a result of acute stress challenge in a manner comparable to high-sensitivity ELISA. Blood was collected from 12 healthy adults immediately before and then 90 and 210 min after the start of the Trier Social Stress Test (TSST), an acute laboratory psychosocial stress challenge. In addition to quantifying IL-6 concentrations in plasma with both multiplex and ELISA, we also assessed concentrations of tumor necrosis factor-alpha, IL-8, IL-10, IL-5, and IL-2 with multiplex. The multiplex detected IL-6 in all samples. Concentrations strongly correlated with values determined by ELISA across all samples (r = 0.941, p < .001) as well as among samples collected at individual TSST time points. IL-6 responses to the TSST (i.e. area under the curve) captured by multiplex and ELISA were also strongly correlated (r s   = 0.937, p < .001). While other cytokines were detected by multiplex, none changed as a result of TSST challenge at time points examined. These results suggest high-sensitivity magnetic multiplex assay is able to detect changes in plasma concentrations of IL-6 as a result of acute stress in humans.

Development of multiplex PCR assay for simultaneous detection of Salmonella genus, Salmonella subspecies I, Salm. Enteritidis, Salm. Heidelberg and Salm. Typhimurium.

PubMed

Park, S H; Ricke, S C

2015-01-01

The aim of this research was to develop multiplex PCR assay that could simultaneously detect Salmonella genus, Salmonella subsp. I, Salm. Enteritidis, Heidelberg and Typhimurium because these Salmonella serovars are the most common isolates associated with poultry products. Five primers were utilized to establish multiplex PCR and applied to Salmonella isolates from chickens and farm environments. These isolates were identified as Salmonella subsp. I and 16 of 66 isolates were classified as Salm. Enteritidis, while Heidelberg or Typhimurium was not detected. We also spiked three Salmonella strains on chicken breast meat to evaluate the specificity and sensitivity of multiplex PCR as well as qPCR to optimize quantification of Salmonella in these samples. The optimized multiplex PCR and qPCR could detect approx. 2·2 CFU of Salmonella per gram after 18 h enrichment. The multiplex PCR and qPCR would provide rapid and consistent results. Also, these techniques would be useful for the detection and quantification of Salmonella in contaminated poultry, foods and environmental samples. The strategy for the rapid detection of Salmonella serovars in poultry is needed to further reduce the incidence of salmonellosis in humans. The optimized multiplex PCR will be useful to detect prevalent Salmonella serovars in poultry products. © 2014 The Society for Applied Microbiology.

Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR.

PubMed

Zhong, Qun; Bhattacharya, Smiti; Kotsopoulos, Steven; Olson, Jeff; Taly, Valérie; Griffiths, Andrew D; Link, Darren R; Larson, Jonathan W

2011-07-07

Quantitative polymerase chain reactions (qPCR) based on real-time PCR constitute a powerful and sensitive method for the analysis of nucleic acids. However, in qPCR, the ability to multiplex targets using differently colored fluorescent probes is typically limited to 4-fold by the spectral overlap of the fluorophores. Furthermore, multiplexing qPCR assays requires expensive instrumentation and most often lengthy assay development cycles. Digital PCR (dPCR), which is based on the amplification of single target DNA molecules in many separate reactions, is an attractive alternative to qPCR. Here we report a novel and easy method for multiplexing dPCR in picolitre droplets within emulsions-generated and read out in microfluidic devices-that takes advantage of both the very high numbers of reactions possible within emulsions (>10(6)) as well as the high likelihood that the amplification of only a single target DNA molecule will initiate within each droplet. By varying the concentration of different fluorogenic probes of the same color, it is possible to identify the different probes on the basis of fluorescence intensity. Adding multiple colors increases the number of possible reactions geometrically, rather than linearly as with qPCR. Accurate and precise copy numbers of up to sixteen per cell were measured using a model system. A 5-plex assay for spinal muscular atrophy was demonstrated with just two fluorophores to simultaneously measure the copy number of two genes (SMN1 and SMN2) and to genotype a single nucleotide polymorphism (c.815A>G, SMN1). Results of a pilot study with SMA patients are presented. This journal is © The Royal Society of Chemistry 2011

Wavelength-multiplexing surface plasmon holographic microscopy.

PubMed

Zhang, Jiwei; Dai, Siqing; Zhong, Jinzhan; Xi, Teli; Ma, Chaojie; Li, Ying; Di, Jianglei; Zhao, Jianlin

2018-05-14

Surface plasmon holographic microscopy (SPHM), which combines surface plasmon microscopy with digital holographic microscopy, can be applied for amplitude- and phase-contrast surface plasmon resonance (SPR) imaging. In this paper, we propose an improved SPHM with the wavelength multiplexing technique based on two laser sources and a common-path hologram recording configuration. Through recording and reconstructing the SPR images at two wavelengths simultaneously employing the improved SPHM, tiny variation of dielectric refractive index in near field is quantitatively monitored with an extended measurement range while maintaining the high sensitivity. Moreover, imaging onion tissues is performed to demonstrate that the detection sensitivities of two wavelengths can compensate for each other in SPR imaging. The proposed wavelength-multiplexing SPHM presents simple structure, high temporal stability and inherent capability of phase curvature compensation, as well as shows great potentials for further applications in monitoring diverse dynamic processes related with refractive index variations and imaging biological tissues with low-contrast refractive index distributions in the near field.

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

PubMed Central

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

2018-01-01

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

A Multiplex RT-PCR Assay for S. aureus, L. monocytogenes, and Salmonella spp. Detection in Raw Milk with Pre-enrichment.

PubMed

Ding, Tian; Suo, Yuanjie; Zhang, Zhaohuan; Liu, Donghong; Ye, Xingqian; Chen, Shiguo; Zhao, Yong

2017-01-01

This study firstly developed a multiplex real-time PCR (RT-PCR) technique combined with a pre-enrichment step to simultaneously detect Staphylococcus aureus ( S. aureus ), Listeria monocytogenes ( L. monocytogenes ) and Salmonella spp. in raw milk and the dairy farm environment (feces, soil, feed, water) in one reaction. Brain heart infusion (BHI) broth was selected for the enrichment step to increase the density of the target bacteria by using an incubation of 4 h before multiplex RT-PCR. The results showed that the detection limit of the multiplex real-time assay was approximately 10 2 CFU/mL for pure cultures and artificially contaminated milk without enrichment, while 12, 14, and 10 CFU/25 mL, respectively, for S. aureus, L. monocytogenes , and Salmonella spp. after pre-enrichment. The newly developed multiplex RT-PCR assay was applied to 46 dairy farm environmental samples and raw milk samples covering a wide variety of sample types. The results demonstrated that the multiplex RT-PCR assay coupled with the BHI enrichment broth was suitable for the simultaneous screening of S. aureus, L. monocytogenes , and Salmonella spp. in the pasture environment and in raw milk. The multiplex RT-PCR assay clearly and successfully shortened the total detection time and reduced labor compared to conventional culture-based methods for testing natural samples.

A Multiplex RT-PCR Assay for S. aureus, L. monocytogenes, and Salmonella spp. Detection in Raw Milk with Pre-enrichment

PubMed Central

Ding, Tian; Suo, Yuanjie; Zhang, Zhaohuan; Liu, Donghong; Ye, Xingqian; Chen, Shiguo; Zhao, Yong

2017-01-01

This study firstly developed a multiplex real-time PCR (RT-PCR) technique combined with a pre-enrichment step to simultaneously detect Staphylococcus aureus (S. aureus), Listeria monocytogenes (L. monocytogenes) and Salmonella spp. in raw milk and the dairy farm environment (feces, soil, feed, water) in one reaction. Brain heart infusion (BHI) broth was selected for the enrichment step to increase the density of the target bacteria by using an incubation of 4 h before multiplex RT-PCR. The results showed that the detection limit of the multiplex real-time assay was approximately 102 CFU/mL for pure cultures and artificially contaminated milk without enrichment, while 12, 14, and 10 CFU/25 mL, respectively, for S. aureus, L. monocytogenes, and Salmonella spp. after pre-enrichment. The newly developed multiplex RT-PCR assay was applied to 46 dairy farm environmental samples and raw milk samples covering a wide variety of sample types. The results demonstrated that the multiplex RT-PCR assay coupled with the BHI enrichment broth was suitable for the simultaneous screening of S. aureus, L. monocytogenes, and Salmonella spp. in the pasture environment and in raw milk. The multiplex RT-PCR assay clearly and successfully shortened the total detection time and reduced labor compared to conventional culture-based methods for testing natural samples. PMID:28620364

Detection and Typing of Human Papilloma Viruses by Nested Multiplex Polymerase Chain Reaction Assay in Cervical Cancer

PubMed Central

Jalal Kiani, Seyed; Shatizadeh Malekshahi, Somayeh; Yousefi Ghalejoogh, Zohreh; Ghavvami, Nastaran; Shafiei Jandaghi, Nazanin Zahra; Shahsiah, Reza; Jahanzad, Isa; Yavarian, Jila

2015-01-01

Background: Cervical cancer is the leading cause of death from cancer in under-developed countries. Human papilloma virus (HPV) 16 and 18 are the most prevalent types associated with carcinogenesis in the cervix. Conventional Polymerase Chain Reaction (PCR), type-specific and consensus primer-based PCR followed by sequencing, Restriction Fragment Length Polymorphism (RFLP) or hybridization by specific probes are common methods for HPV detection and typing. In addition, some researchers have developed a multiplex PCR for simultaneous detection and typing of different HPVs. Objectives: The aim of the present study was to investigate the prevalence of HPV infection and its types in cervical Squamous Cell Carcinoma (SCC) using the Nested Multiplex PCR (NMPCR) assay. Patients and Methods: Sixty-six samples with histologically confirmed SCC were evaluated. Total DNA was isolated by phenol–chloroform extraction and ethanol precipitation. Nested multiplex PCR was performed with first-round PCR by GP-E6/E7 consensus primers for amplification of the genomic DNA of all known mucosal HPV genotypes and second-round PCR by type-specific multiplex PCR primer cocktails. Results: Human papilloma virus infection was detected in 78.8% of samples, with the highest prevalence of HPV 16 (60.6%) while concurrent infections with two types was detected in 10.6%. Conclusions: The NMPCR assay is more convenient and easy for analysis of results, which is important for fast diagnosis and patient management, in a type-specific manner. PMID:26865940

Detection and quantitation of HPV in genital and oral tissues and fluids by real time PCR

PubMed Central

2010-01-01

Background Human papillomaviruses (HPVs) remain a serious world health problem due to their association with anogenital/oral cancers and warts. While over 100 HPV types have been identified, a subset is associated with malignancy. HPV16 and 18 are the most prevalent oncogenic types, while HPV6 and 11 are most commonly responsible for anogenital warts. While other quantitative PCR (qPCR) assays detect oncogenic HPV, there is no single tube assay distinguishing the most frequent oncogenic types and the most common types found in warts. Results A Sybr Green-based qPCR assay was developed utilizing degenerate primers to the highly conserved HPV E1 theoretically detecting any HPV type. A single tube multiplex qPCR assay was also developed using type-specific primer pairs and TaqMan probes that allowed for detection and quantitation of HPV6,11,16,18. Each HPV type was detected over a range from 2 × 101 to 2 × 106copies/reaction providing a reliable method of quantitating type-specific HPV in 140 anogenital/cutaneous/oral benign and malignant specimens. 35 oncogenic and low risk alpha genus HPV types were detected. Concordance was detected in previously typed specimens. Comparisons to the gold standard detected an overall sensitivity of 89% (95% CI: 77% - 96%) and specificity of 90% (95%CI: 52% - 98%). Conclusion There was good agreement between the ability of the qPCR assays described here to identify HPV types in malignancies previously typed using standard methods. These novel qPCR assays will allow rapid detection and quantitation of HPVs to assess their role in viral pathogenesis. PMID:20723234

Quantitative Tracking of Combinatorially Engineered Populations with Multiplexed Binary Assemblies.

PubMed

Zeitoun, Ramsey I; Pines, Gur; Grau, Willliam C; Gill, Ryan T

2017-04-21

Advances in synthetic biology and genomics have enabled full-scale genome engineering efforts on laboratory time scales. However, the absence of sufficient approaches for mapping engineered genomes at system-wide scales onto performance has limited the adoption of more sophisticated algorithms for engineering complex biological systems. Here we report on the development and application of a robust approach to quantitatively map combinatorially engineered populations at scales up to several dozen target sites. This approach works by assembling genome engineered sites with cell-specific barcodes into a format compatible with high-throughput sequencing technologies. This approach, called barcoded-TRACE (bTRACE) was applied to assess E. coli populations engineered by recursive multiplex recombineering across both 6-target sites and 31-target sites. The 31-target library was then tracked throughout growth selections in the presence and absence of isopentenol (a potential next-generation biofuel). We also use the resolution of bTRACE to compare the influence of technical and biological noise on genome engineering efforts.

Molecular beacon probes-base multiplex NASBA Real-time for detection of HIV-1 and HCV.

PubMed

Mohammadi-Yeganeh, S; Paryan, M; Mirab Samiee, S; Kia, V; Rezvan, H

2012-06-01

Developed in 1991, nucleic acid sequence-based amplification (NASBA) has been introduced as a rapid molecular diagnostic technique, where it has been shown to give quicker results than PCR, and it can also be more sensitive. This paper describes the development of a molecular beacon-based multiplex NASBA assay for simultaneous detection of HIV-1 and HCV in plasma samples. A well-conserved region in the HIV-1 pol gene and 5'-NCR of HCV genome were used for primers and molecular beacon design. The performance features of HCV/HIV-1 multiplex NASBA assay including analytical sensitivity and specificity, clinical sensitivity and clinical specificity were evaluated. The analysis of scalar concentrations of the samples indicated that the limit of quantification of the assay was <1000 copies/ml for HIV-1 and <500 copies/ml for HCV with 95% confidence interval. Multiplex NASBA assay showed a 98% sensitivity and 100% specificity. The analytical specificity study with BLAST software demonstrated that the primers do not attach to any other sequences except for that of HIV-1 or HCV. The primers and molecular beacon probes detected all HCV genotypes and all major variants of HIV-1. This method may represent a relatively inexpensive isothermal method for detection of HIV-1/HCV co-infection in monitoring of patients.

Detection of selected intestinal helminths and protozoa at Hospital Universiti Sains Malaysia using multiplex real-time PCR.

PubMed

Basuni, M; Mohamed, Z; Ahmad, M; Zakaria, N Z; Noordin, R

2012-09-01

Intestinal parasites are the causative agents of a number of important human infections in developing countries. The objective of this study was to determine the prevalence of selected helminths and protozoan infections among patients admitted with gastrointestinal disorders at Hospital Universiti Sains Malaysia, Kelantan, Malaysia using multiplex real-time PCR. In addition microscopic examination was also performed following direct smear, zinc sulphate concentration and Kato-Katz thick smear techniques; and the presence of protozoan parasites was confirmed using trichrome and acid-fast stains. Of the 225 faecal samples analysed, 26.2% were positive for intestinal parasites by the multiplex real-time PCR, while 5.3% were positive by microscopy. As compared to microscopy, the multiplex real-time PCR detected 5.8 and 4.5 times more positives for the selected helminth and protozoan infections respectively. Among the selected helminths detected in this study, hookworm was the most prevalent by real-time PCR, while Ascaris lumbricoides was detected the most by microscopy. Meanwhile, among the selected protozoa detected in this study, Entamoeba histolytica was the most prevalent by real-time PCR, however microscopy detected equal number of cases with E. histolytica and Giardia lamblia. This study showed that real-time PCR can be used to obtain a more accurate prevalence data on intestinal helminths and protozoa.

Multiplex PCR for the detection and differentiation of Vibrio parahaemolyticus strains using the groEL, tdh and trh genes.

PubMed

Hossain, Muhammad Tofazzal; Kim, Young-Ok; Kong, In-Soo

2013-01-01

Vibrio parahaemolyticus is a significant cause of human gastrointestinal disorders worldwide, transmitted primarily by ingestion of raw or undercooked contaminated seafood. In this study, a multiplex PCR assay for the detection and differentiation of V. parahaemolyticus strains was developed using primer sets for a species-specific marker, groEL, and two virulence markers, tdh and trh. Multiplex PCR conditions were standardised, and extracted genomic DNA of 70 V. parahaemolyticus strains was used for identification. The sensitivity and efficacy of this method were validated using artificially inoculated shellfish and seawater. The expected sizes of amplicons were 510 bp, 382 bp, and 171 bp for groEL, tdh and trh, respectively. PCR products were sufficiently different in size, and the detection limits of the multiplex PCR for groEL, tdh and trh were each 200 pg DNA. Specific detection and differentiation of virulent from non-virulent strains in shellfish homogenates and seawater was also possible after artificial inoculation with various V. parahaemolyticus strains. This newly developed multiplex PCR is a rapid assay for detection and differentiation of pathogenic V. parahaemolyticus strains, and could be used to prevent disease outbreaks and protect public health by helping the seafood industry maintain a safe shellfish supply. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

PubMed

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

2015-06-01

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

Large-Scale Interlaboratory Study to Develop, Analytically Validate and Apply Highly Multiplexed, Quantitative Peptide Assays to Measure Cancer-Relevant Proteins in Plasma*

PubMed Central

Abbatiello, Susan E.; Schilling, Birgit; Mani, D. R.; Zimmerman, Lisa J.; Hall, Steven C.; MacLean, Brendan; Albertolle, Matthew; Allen, Simon; Burgess, Michael; Cusack, Michael P.; Gosh, Mousumi; Hedrick, Victoria; Held, Jason M.; Inerowicz, H. Dorota; Jackson, Angela; Keshishian, Hasmik; Kinsinger, Christopher R.; Lyssand, John; Makowski, Lee; Mesri, Mehdi; Rodriguez, Henry; Rudnick, Paul; Sadowski, Pawel; Sedransk, Nell; Shaddox, Kent; Skates, Stephen J.; Kuhn, Eric; Smith, Derek; Whiteaker, Jeffery R.; Whitwell, Corbin; Zhang, Shucha; Borchers, Christoph H.; Fisher, Susan J.; Gibson, Bradford W.; Liebler, Daniel C.; MacCoss, Michael J.; Neubert, Thomas A.; Paulovich, Amanda G.; Regnier, Fred E.; Tempst, Paul; Carr, Steven A.

2015-01-01

There is an increasing need in biology and clinical medicine to robustly and reliably measure tens to hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility, and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here, we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and seven control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data, we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to subnanogram/ml sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and interlaboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy-isotope-labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an interlaboratory clinical study of patient samples. Our study further establishes that LC

Multiplexed Quantitation of Endogenous Proteins in Dried Blood Spots by Multiple Reaction Monitoring - Mass Spectrometry

PubMed Central

Chambers, Andrew G.; Percy, Andrew J.; Yang, Juncong; Camenzind, Alexander G.; Borchers, Christoph H.

2013-01-01

Dried blood spot (DBS) sampling, coupled with multiple reaction monitoring mass spectrometry (MRM-MS), is a well-established approach for quantifying a wide range of small molecule biomarkers and drugs. This sampling procedure is simpler and less-invasive than those required for traditional plasma or serum samples enabling collection by minimally trained personnel. Many analytes are stable in the DBS format without refrigeration, which reduces the cost and logistical challenges of sample collection in remote locations. These advantages make DBS sample collection desirable for advancing personalized medicine through population-wide biomarker screening. Here we expand this technology by demonstrating the first multiplexed method for the quantitation of endogenous proteins in DBS samples. A panel of 60 abundant proteins in human blood was targeted by monitoring proteotypic tryptic peptides and their stable isotope-labeled analogs by MRM. Linear calibration curves were obtained for 40 of the 65 peptide targets demonstrating multiple proteins can be quantitatively extracted from DBS collection cards. The method was also highly reproducible with a coefficient of variation of <15% for all 40 peptides. Overall, this assay quantified 37 proteins spanning a range of more than four orders of magnitude in concentration within a single 25 min LC/MRM-MS analysis. The protein abundances of the 33 proteins quantified in matching DBS and whole blood samples showed an excellent correlation, with a slope of 0.96 and an R2 value of 0.97. Furthermore, the measured concentrations for 80% of the proteins were stable for at least 10 days when stored at −20 °C, 4 °C and 37 °C. This work represents an important first step in evaluating the integration of DBS sampling with highly-multiplexed MRM for quantitation of endogenous proteins. PMID:23221968

Development of a multiplex PCR assay for detection and discrimination of Theileria annulata and Theileria sergenti in cattle.

PubMed

Junlong, Liu; Li, Youquan; Liu, Aihong; Guan, Guiquan; Xie, Junren; Yin, Hong; Luo, Jianxun

2015-07-01

Aim to construct a simple and efficient diagnostic assay for Theileria annulata and Theileria sergenti, a multiplex polymerase chain reaction (PCR) method was developed in this study. Following the alignment of the related sequences, two primer sets were designed specific targeting on T. annulata cytochrome b (COB) gene and T. sergenti internal transcribed spacer (ITS) sequences. It was found that the designed primers could react in one PCR system and generating amplifications of 818 and 393 base pair for T. sergenti and T. annulata, respectively. The standard genomic DNA of both species Theileria was serial tenfold diluted for testing the sensitivity, while specificity test confirmed both primer sets have no cross-reaction with other Theileria and Babesia species. In addition, 378 field samples were used for evaluation of the utility of the multiplex PCR assay for detection of the pathogens infection. The detection results were compared with the other two published PCR methods which targeting on T. annulata COB gene and T. sergenti major piroplasm surface protein (MPSP) gene, respectively. The developed multiplex PCR assay has similar efficient detection with COB and MPSP PCR, which indicates this multiplex PCR may be a valuable assay for the epidemiological studies for T. annulata and T. sergenti.

A Multiplexed Diagnostic Platform for Point-of-Care Pathogen Detection

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

Regan, J F; Letant, S E; Adams, K L

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,more » 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.« less

Multiplex real-time PCR for detection, identification and quantification of 'Candidatus Liberibacter solanacearum' in potato plants with zebra chip.

PubMed

Li, Wenbin; Abad, Jorge A; French-Monar, Ronald D; Rascoe, John; Wen, Aimin; Gudmestad, Neil C; Secor, Gary A; Lee, Ing-Ming; Duan, Yongping; Levy, Laurene

2009-07-01

The new Liberibacter species, 'Candidatus Liberibacter solanacearum' (Lso) recently associated with potato/tomato psyllid-transmitted diseases in tomato and capsicum in New Zealand, was found to be consistently associated with a newly emerging potato zebra chip (ZC) disease in Texas and other southwestern states in the USA. A species-specific primer LsoF was developed for both quantitative real-time PCR (qPCR) and conventional PCR (cPCR) to detect and quantify Lso in infected samples. In multiplex qPCR, a plant cytochrome oxidase (COX)-based probe-primer set was used as a positive internal control for host plants, which could be used to reliably access the DNA extraction quality and to normalize qPCR data for accurate quantification of the bacterial populations in environment samples. Neither the qPCR nor the cPCR using the primer and/or probe sets with LsoF reacted with other Liberibacter species infecting citrus or other potato pathogens. The low detection limit of the multiplex qPCR was about 20 copies of the target 16S rDNA templates per reaction for field samples. Lso was readily detected and quantified in various tissues of ZC-affected potato plants collected from fields in Texas. A thorough but uneven colonization of Lso was revealed in various tissues of potato plants. The highest Lso populations were about 3x10(8) genomes/g tissue in the root, which were 3-order higher than those in the above-ground tissues of potato plants. The Lso bacterial populations were normally distributed across the ZC-affected potato plants collected from fields in Texas, with 60% of ZC-affected potato plants harboring an average Lso population from 10(5) to 10(6) genomes/g tissue, 4% of plants hosting above 10(7) Lso genomes/g tissue, and 8% of plants holding below 10(3) Lso genomes/g tissue. The rapid, sensitive, specific and reliable multiplex qPCR showed its potential to become a powerful tool for early detection and quantification of the new Liberibacter species associated

High-Throughput Multiplexed Quantitation of Protein Aggregation and Cytotoxicity in a Huntington’s Disease Model

PubMed Central

Titus, Steven A; Southall, Noel; Marugan, Juan; Austin, Christopher P; Zheng, Wei

2012-01-01

A hallmark of Huntington’s disease is the presence of a large polyglutamine expansion in the first exon of the Huntingtin protein and the propensity of protein aggregation by the mutant proteins. Aberrant protein aggregation also occurs in other polyglutamine expansion disorders, as well as in other neurodegenerative diseases including Parkinson’s, Alzheimer’s, and prion diseases. However, the pathophysiological role of these aggregates in the cell death that characterizes the diseases remains unclear. Identification of small molecule probes that modulate protein aggregation and cytotoxicity caused by aggregated proteins may greatly facilitate the studies on pathogenesis of these diseases and potentially lead to development of new therapies. Based on a detergent insoluble property of the Huntingtin protein aggregates, we have developed a homogenous assay to rapidly quantitate the levels of protein aggregates in a cellular model of Huntington’s disease. The protein aggregation assay has also been multiplexed with a protease release assay for the measurement of cytotoxicity resulting from aggregated proteins in the same cells. Through a testing screen of a compound library, we have demonstrated that this multiplexed cytotoxicity and protein aggregation assay has ability to identify active compounds that prevent cell death and/or modulate protein aggregation in cells of the Huntington’s disease model. Therefore, this multiplexed screening approach is also useful for development of high-throughput screening assays for other neurodegenerative diseases involving protein aggregation. PMID:23346268

Comparison between Saliva and Nasopharyngeal Swab Specimens for Detection of Respiratory Viruses by Multiplex Reverse Transcription-PCR

PubMed Central

Kim, Young-gon; Kim, Min Young; Park, Kwisung; Cho, Chi Hyun; Yoon, Soo Young; Nam, Myung Hyun; Lee, Chang Kyu; Cho, Yun-Jung; Lim, Chae Seung

2016-01-01

ABSTRACT Nasopharyngeal swabs (NPSs) are being widely used as specimens for multiplex real-time reverse transcription (RT)-PCR for respiratory virus detection. However, it remains unclear whether NPS specimens are optimal for all viruses targeted by multiplex RT-PCR. In addition, the procedure to obtain NPS specimens causes coughing in most patients, which possibly increases the risk of nosocomial spread of viruses. In this study, paired NPS and saliva specimens were collected from 236 adult male patients with suspected acute respiratory illnesses. Specimens were tested for 16 respiratory viruses by multiplex real-time RT-PCR. Among the specimens collected from the 236 patients, at least 1 respiratory virus was detected in 183 NPS specimens (77.5%) and 180 saliva specimens (76.3%). The rates of detection of respiratory viruses were comparable for NPS and saliva specimens (P = 0.766). Nine virus species and 349 viruses were isolated, 256 from NPS specimens and 273 from saliva specimens (P = 0.1574). Adenovirus was detected more frequently in saliva samples (P < 0.0001), whereas influenza virus type A and human rhinovirus were detected more frequently in NPS specimens (P = 0.0001 and P = 0.0289, respectively). The possibility of false-positive adenovirus detection from saliva samples was excluded by direct sequencing. In conclusion, neither of the sampling methods was consistently more sensitive than the other. We suggest that these cost-effective methods for detecting respiratory viruses in mixed NPS-saliva specimens might be valuable for future studies. PMID:27807150

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. Copyright © 2016 Elsevier B.V. All rights reserved.

Dual-excitation upconverting nanoparticle and quantum dot aptasensor for multiplexed food pathogen detection.

PubMed

Kurt, Hasan; Yüce, Meral; Hussain, Babar; Budak, Hikmet

2016-07-15

In this report, a dual-excitation sensing method was developed using aptamer-functionalized quantum dots and upconverting nanoparticles, exhibiting Stokes and anti-Stokes type excitation profiles, respectively. Conjugation of the aptamer-functionalized luminescent nanoparticles with the magnetic beads, comprising short DNA sequences that were partially complementary to the aptamer sequences, enabled facile separation of the analyte-free conjugates for fluorescent measurement. UV-Visible spectroscopy, Circular Dichroism spectroscopy, Dynamic Light Scattering and Polyacrylamide Gel Electrophoresis techniques were used to characterize the aptamer probes developed. The target-specific luminescent conjugates were applied for multiplex detection of model food pathogens, Salmonella typhimurium, and Staphylococcus aureus, in which the fluorescent emission spectra were obtained under UV excitation at 325nm for quantum dots and NIR excitation at 980nm for upconverting nanoparticles, respectively. The dual-excitation strategy was aimed to minimize cross-talk between the luminescent signals for multiplexed detection, and yielded limit of detection values of 16 and 28cfumL(-1) for Staphylococcus aureus, and Salmonella typhimurium, respectively. By employing a greater number of quantum dots and upconverting nanoparticles with non-overlapping fluorescent emissions, the proposed methodology might be exploited further to detect several analytes, simultaneously. Copyright © 2016 Elsevier B.V. All rights reserved.

Analysis of sensitivity and rapid hybridization of a multiplexed Microbial Detection Microarray

DOE PAGES

Thissen, James B.; McLoughlin, Kevin; Gardner, Shea; ...

2014-06-01

Microarrays have proven to be useful in rapid detection of many viruses and bacteria. Pathogen detection microarrays have been used to diagnose viral and bacterial infections in clinical samples and to evaluate the safety of biological drug materials. A multiplexed version of the Lawrence Livermore Microbial Detection Array (LLMDA) was developed and evaluated with minimum detectable concentrations for pure unamplified DNA viruses, along with mixtures of viral and bacterial DNA subjected to different whole genome amplification protocols. In addition the performance of the array was tested when hybridization time was reduced from 17 h to 1 h. The LLMDA wasmore » able to detect unamplified vaccinia virus DNA at a concentration of 14 fM, or 100,000 genome copies in 12 μL of sample. With amplification, positive identification was made with only 100 genome copies of input material. When tested against human stool samples from patients with acute gastroenteritis, the microarray detected common gastroenteritis viral and bacterial infections such as rotavirus and E. coli. Accurate detection was found but with a 4-fold drop in sensitivity for a 1 h compared to a 17 h hybridization. The array detected 2 ng (equivalent concentration of 15.6 fM) of labeled DNA from a virus with 1 h hybridization without any amplification, and was able to identify the components of a mixture of viruses and bacteria at species and in some cases strain level resolution. Sensitivity improved by three orders of magnitude with random whole genome amplification prior to hybridization; for instance, the array detected a DNA virus with only 20 fg or 100 genome copies as input. This multiplexed microarray is an efficient tool to analyze clinical and environmental samples for the presence of multiple viral and bacterial pathogens rapidly.« less

Analysis of sensitivity and rapid hybridization of a multiplexed Microbial Detection Microarray

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

Thissen, James B.; McLoughlin, Kevin; Gardner, Shea

Microarrays have proven to be useful in rapid detection of many viruses and bacteria. Pathogen detection microarrays have been used to diagnose viral and bacterial infections in clinical samples and to evaluate the safety of biological drug materials. A multiplexed version of the Lawrence Livermore Microbial Detection Array (LLMDA) was developed and evaluated with minimum detectable concentrations for pure unamplified DNA viruses, along with mixtures of viral and bacterial DNA subjected to different whole genome amplification protocols. In addition the performance of the array was tested when hybridization time was reduced from 17 h to 1 h. The LLMDA wasmore » able to detect unamplified vaccinia virus DNA at a concentration of 14 fM, or 100,000 genome copies in 12 μL of sample. With amplification, positive identification was made with only 100 genome copies of input material. When tested against human stool samples from patients with acute gastroenteritis, the microarray detected common gastroenteritis viral and bacterial infections such as rotavirus and E. coli. Accurate detection was found but with a 4-fold drop in sensitivity for a 1 h compared to a 17 h hybridization. The array detected 2 ng (equivalent concentration of 15.6 fM) of labeled DNA from a virus with 1 h hybridization without any amplification, and was able to identify the components of a mixture of viruses and bacteria at species and in some cases strain level resolution. Sensitivity improved by three orders of magnitude with random whole genome amplification prior to hybridization; for instance, the array detected a DNA virus with only 20 fg or 100 genome copies as input. This multiplexed microarray is an efficient tool to analyze clinical and environmental samples for the presence of multiple viral and bacterial pathogens rapidly.« less

Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis.

PubMed

Stack, Edward C; Wang, Chichung; Roman, Kristin A; Hoyt, Clifford C

2014-11-01

Tissue sections offer the opportunity to understand a patient's condition, to make better prognostic evaluations and to select optimum treatments, as evidenced by the place pathology holds today in clinical practice. Yet, there is a wealth of information locked up in a tissue section that is only partially accessed, due mainly to the limitations of tools and methods. Often tissues are assessed primarily based on visual analysis of one or two proteins, or 2-3 DNA or RNA molecules. Even while analysis is still based on visual perception, image analysis is starting to address the variability of human perception. This is in contrast to measuring characteristics that are substantially out of reach of human perception, such as parameters revealed through co-expression, spatial relationships, heterogeneity, and low abundance molecules. What is not routinely accessed is the information revealed through simultaneous detection of multiple markers, the spatial relationships among cells and tissue in disease, and the heterogeneity now understood to be critical to developing effective therapeutic strategies. Our purpose here is to review and assess methods for multiplexed, quantitative, image analysis based approaches, using new multicolor immunohistochemistry methods, automated multispectral slide imaging, and advanced trainable pattern recognition software. A key aspect of our approach is presenting imagery in a workflow that engages the pathologist to utilize the strengths of human perception and judgment, while significantly expanding the range of metrics collectable from tissue sections and also provide a level of consistency and precision needed to support the complexities of personalized medicine. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Development of a multiplex lateral flow strip test for foot-and-mouth disease virus detection using monoclonal antibodies.

PubMed

Yang, Ming; Caterer, Nigel R; Xu, Wanhong; Goolia, Melissa

2015-09-01

Foot-and-mouth disease (FMD) is one of the world's most highly contagious animal diseases with tremendous economic consequences. A rapid and specific test for FMD diagnosis at the site of a suspected outbreak is crucial for the implementation of control measures. This project developed a multiplex lateral flow immunochromatographic strip test (multiplex-LFI) for the rapid detection and serotyping of FMD viruses. The monoclonal antibodies (mAbs) against serotypes O, A, and Asia 1 were used as capture mAbs. The mAbs were conjugated with fluorescein, rhodamine or biotin for serotype O, A and Asia 1, respectively. The detection mAbs which consisted of a serotype-independent mAb in combination with one serotype A-specific mAb and one Asia 1-specific mAb, were each colloidal gold-conjugated. The strips used in this study contained one control line and three test lines, which corresponded to one of the three serotypes, O, A or Asia 1. The newly developed multiplex-LFI strip test specifically identified serotype O (n=46), A (n=45) and Asia 1 (n=17) in all tested field isolates. The sensitivity of this strip test was comparable to the double antibody sandwich ELISA for serotypes O and A, but lower than the ELISA for serotype Asia 1. The multiplex-LFI strip test identified all tissue suspensions from animals that were experimentally inoculated with serotypes O, A or Asia 1. FMD viruses were detected in 38% and 50% of the swab samples from the lesion areas of experimentally inoculated sheep for serotypes O and A, respectively. The capability of the multiplex-LFI strip tests to produce rapid results with high specificity for FMD viruses of multiple serotypes makes this test a valuable tool to detect FMD viruses at outbreak sites. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

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

Development and validation of a multiplex conventional PCR assay for simultaneous detection and grouping of porcine bocaviruses.

PubMed

Zheng, Xiaowen; Liu, Gaopeng; Opriessnig, Tanja; Wang, Zining; Yang, Zongqi; Jiang, Yonghou

2016-10-01

Porcine bocavirus (PBoV), a newly described porcine parvovirus, has received attention because it can be commonly identified in clinically affected pigs including pigs with post-weaning multisystemic wasting syndrome (PWMS) and pigs with diarrhea. In recent years, novel PBoVs have been identified and were classified into three genogroups, but the ability to detect and classify these novel PBoVs is not comprehensive to date. In this study, a multiplex conventional PCR assay for simultaneous detection and grouping of PBoVs was developed by screening combinations of mixed primer pairs followed by optimization of the PCR conditions. This method exclusively amplifies targeted fragments of 531bp from the VP1 gene of PBoV G1, 291bp from the NP1 gene of PBoV G2, and 384bp from the NP1/VP1 gene of PBoV G3. The assay has a detection limit of 1.0×10(3)copies/μL for PBoV G1 4.5×10(3) for PBoV G2 and 3.8×10(3) for PBoV G3 based on testing mixed purified plasmid constructs containing the specific viral target fragments. The performance of the multiplex PCR assay was comparable to that of the single PCRs which used the same primer pairs. Using the newly established multiplex PCR assay, 227 field samples including faeces, serum and tissue samples from pigs were investigated. All three PBoV genogroups were detected in the clinical samples with a detection rate of 1.3%, 2.6% and 12.3%, respectively for PBoV G1, G2 and G3. Additionally, coinfections with two or more PBoV were detected in 1.7% of the samples investigated. These results indicate the multiplex PCR assay is specific, sensitive and rapid, and can be used for the detection and differentiation of single and multiple infections of the three PBoV genogroups in pigs. Copyright © 2016 Elsevier B.V. All rights reserved.

A volumetric meter chip for point-of-care quantitative detection of bovine catalase for food safety control.

PubMed

Cui, Xingye; Hu, Jie; Choi, Jane Ru; Huang, Yalin; Wang, Xuemin; Lu, Tian Jian; Xu, Feng

2016-09-07

A volumetric meter chip was developed for quantitative point-of-care (POC) analysis of bovine catalase, a bioindicator of bovine mastitis, in milk samples. The meter chip displays multiplexed quantitative results by presenting the distance of ink bar advancement that is detectable by the naked eye. The meter chip comprises a poly(methyl methacrylate) (PMMA) layer, a double-sided adhesive (DSA) layer and a glass slide layer fabricated by the laser-etching method, which is typically simple, rapid (∼3 min per chip), and cost effective (∼$0.2 per chip). Specially designed "U shape" reaction cells are covered by an adhesive tape that serves as an on-off switch, enabling the simple operation of the assay. As a proof of concept, we employed the developed meter chip for the quantification of bovine catalase in raw milk samples to detect catalase concentrations as low as 20 μg/mL. The meter chip has great potential to detect various target analytes for a wide range of POC applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Detection of clinically relevant copy number alterations in oral cancer progression using multiplexed droplet digital PCR.

PubMed

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

2017-09-19

Copy number alterations (CNAs), a common genomic event during carcinogenesis, are known to affect a large fraction of the genome. Common recurrent gains or losses of specific chromosomal regions occur at frequencies that they may be considered distinctive features of tumoral cells. Here we introduce a novel multiplexed droplet digital PCR (ddPCR) assay capable of detecting recurrent CNAs that drive tumorigenesis of oral squamous cell carcinoma. Applied to DNA extracted from oral cell lines and clinical samples of various disease stages, we found good agreement between CNAs detected by our ddPCR assay with those previously reported using comparative genomic hybridization or single nucleotide polymorphism arrays. Furthermore, we demonstrate that the ability to target specific locations of the genome permits detection of clinically relevant oncogenic events such as small, submicroscopic homozygous deletions. Additional capabilities of the multiplexed ddPCR assay include the ability to infer ploidy level, quantify the change in copy number of target loci with high-level gains, and simultaneously assess the status and viral load for high-risk human papillomavirus types 16 and 18. This novel multiplexed ddPCR assay therefore may have clinical value in differentiating between benign oral lesions from those that are at risk of progressing to oral cancer.

Development of a multiplex microsphere immunoassay for the quantitation of salivary antibody responses to selected waterborne pathogens

EPA Science Inventory

Saliva has an important advantage over serum as a medium for antibody detection due to non-invasive sampling, which is critical for community-based epidemiological surveys. The development of a Luminex multiplex immunoassay for measurement of salivary IgG and IgA responses to pot...

Multiplex detection of microRNAs by combining molecular beacon probes with T7 exonuclease-assisted cyclic amplification reaction.

PubMed

Liu, Yacui; Zhang, Jiangyan; Tian, Jingxiao; Fan, Xiaofei; Geng, Hao; Cheng, Yongqiang

2017-01-01

A simple, highly sensitive, and specific assay was developed for the homogeneous and multiplex detection of microRNAs (miRNAs) by combining molecular beacon (MB) probes and T7 exonuclease-assisted cyclic amplification. An MB probe with five base pairs in the stem region without special modification can effectively prevent the digestion by T7 exonuclease. Only in the presence of target miRNA is the MB probe hybridized with the target miRNA, and then digested by T7 exonuclease in the 5' to 3' direction. At the same time, the target miRNA is released and subsequently initiates the nuclease-assisted cyclic digestion process, generating enhanced fluorescence signal significantly. The results show that the combination of T7 exonuclease-assisted cyclic amplification reaction and MB probe possesses higher sensitivity for miRNA detection. Moreover, multiplex detection of miRNAs was successfully achieved by designing two MB probes labeled with FAM and Cy3, respectively. As a result, the method opens a new pathway for the sensitive and multiplex detection of miRNAs as well as clinical diagnosis. Graphical Abstract A simple, highly sensitive, and specific assay was developed for the detection of microRNAs by combining molecular beacon probes with T7 exonuclease-assisted cyclic amplification reaction.

A SIMPLE AND EFFECTIVE MULTIPLEX PCR TECHNIQUE FOR DETECTING HUMAN PATHOGENIC TAENIA EGGS IN HOUSEFLIES.

PubMed

Pornruseetriratn, Siritavee; Maipanich, Wanna; Sa-nguankiat, Surapol; Pubampen, Somchit; Poodeepiyasawat, Akkarin; Thaenkham, Urusa

2017-01-01

Taenia solium, T. saginata, and T. asiatica are cestode pathogens causing taeniasis in humans. Houseflies can transfer Taenia eggs to food. However, houseflies are thought to carry only small numbers of Taenia eggs, sometimes fewer than 10. Although several PCR-based methods have been developed to detect Taenia DNA, these require more than 10 eggs for adequate detection. We developed a multiplex PCR method with high specificity for the discrimination among the eggs of the three Taenia species, T. solium, T. saginata, and T. asiatica, using 18S ribosomal DNA (rDNA) as a genetic marker. This technique was found to be highly sensitive, capable of identifying the Taenia species from only one egg. This multiplex PCR technique using 18S rDNA specific primers should be suitable to diagnose Taenia eggs.

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.

Photocleavage-based affinity purification of biomarkers from serum: Application to multiplex allergy testing.

PubMed

Wan, Zhi; Ostendorff, Heather P; Liu, Ziying; Schneider, Lynda C; Rothschild, Kenneth J; Lim, Mark J

2018-01-01

Multiplex serological immunoassays, such as implemented on microarray or microsphere-based platforms, provide greater information content and higher throughput, while lowering the cost and blood volume required. These features are particularly attractive in pediatric food allergy testing to facilitate high throughput multi-allergen analysis from finger- or heel-stick collected blood. However, the miniaturization and microfluidics necessary for creating multiplex assays make them highly susceptible to the "matrix effect" caused by interference from non-target agents in serum and other biofluids. Such interference can result in lower sensitivity, specificity, reproducibility and quantitative accuracy. These problems have in large part prevented wide-spread implementation of multiplex immunoassays in clinical laboratories. We report the development of a novel method to eliminate the matrix effect by utilizing photocleavable capture antibodies to purify and concentrate blood-based biomarkers (a process termed PC-PURE) prior to detection in a multiplex immunoassay. To evaluate this approach, it was applied to blood-based allergy testing. Patient total IgE was purified and enriched using PC-PURE followed by multiplex microsphere-based detection of allergen-specific IgEs (termed the AllerBead assay). AllerBead was formatted to detect the eight most common pediatric food allergens: milk, soy, wheat, egg, peanuts, tree nuts, fin fish and shellfish, which account for >90% of all pediatric food allergies. 205 serum samples obtained from Boston Children's Hospital were evaluated. When PC-PURE was employed with AllerBead, excellent agreement was obtained with the standard, non-multiplex, ImmunoCAP® assay (average sensitivity above published negative predictive cutoffs = 96% and average Pearson r = 0.90; average specificity = 97%). In contrast, poor ImmunoCAP®-correlation was observed when PC-PURE was not utilized (average sensitivity above published negative predictive

Photocleavage-based affinity purification of biomarkers from serum: Application to multiplex allergy testing

PubMed Central

Wan, Zhi; Ostendorff, Heather P.; Liu, Ziying; Schneider, Lynda C.; Rothschild, Kenneth J.

2018-01-01

Multiplex serological immunoassays, such as implemented on microarray or microsphere-based platforms, provide greater information content and higher throughput, while lowering the cost and blood volume required. These features are particularly attractive in pediatric food allergy testing to facilitate high throughput multi-allergen analysis from finger- or heel-stick collected blood. However, the miniaturization and microfluidics necessary for creating multiplex assays make them highly susceptible to the “matrix effect” caused by interference from non-target agents in serum and other biofluids. Such interference can result in lower sensitivity, specificity, reproducibility and quantitative accuracy. These problems have in large part prevented wide-spread implementation of multiplex immunoassays in clinical laboratories. We report the development of a novel method to eliminate the matrix effect by utilizing photocleavable capture antibodies to purify and concentrate blood-based biomarkers (a process termed PC-PURE) prior to detection in a multiplex immunoassay. To evaluate this approach, it was applied to blood-based allergy testing. Patient total IgE was purified and enriched using PC-PURE followed by multiplex microsphere-based detection of allergen-specific IgEs (termed the AllerBead assay). AllerBead was formatted to detect the eight most common pediatric food allergens: milk, soy, wheat, egg, peanuts, tree nuts, fin fish and shellfish, which account for >90% of all pediatric food allergies. 205 serum samples obtained from Boston Children’s Hospital were evaluated. When PC-PURE was employed with AllerBead, excellent agreement was obtained with the standard, non-multiplex, ImmunoCAP® assay (average sensitivity above published negative predictive cutoffs = 96% and average Pearson r = 0.90; average specificity = 97%). In contrast, poor ImmunoCAP®-correlation was observed when PC-PURE was not utilized (average sensitivity above published negative predictive

Multiplex detection of pancreatic cancer biomarkers using a SERS-based immunoassay

NASA Astrophysics Data System (ADS)

Banaei, Nariman; Foley, Anne; Houghton, Jean Marie; Sun, Yubing; Kim, Byung

2017-11-01

Early diagnosis of pancreatic cancer (PC) is critical to reduce the mortality rate of this disease. Current biological analysis approaches cannot robustly detect several low abundance PC biomarkers in sera, limiting the clinical application of these biomarkers. Enzyme linked immunosorbent assay and radioimmunoassay are two common platforms for detection of biomarkers; however, they suffer from some limitation. This study demonstrates a novel system for multiplex detection of pancreatic biomarkers CA19-9, MMP7 and MUC4 in sera samples with high sensitivity using surface enhanced Raman spectroscopy. Measuring the levels of these biomarkers in PC patients, pancreatitis patients, and healthy individuals reveals the unique expression pattern of these markers in PC patients, suggesting the great potential of using this approach for early diagnostics of PCs.

Simultaneous Detection of 13 Key Bacterial Respiratory Pathogens by Combination of Multiplex PCR and Capillary Electrophoresis.

PubMed

Jiang, Lu Xi; Ren, Hong Yu; Zhou, Hai Jian; Zhao, Si Hong; Hou, Bo Yan; Yan, Jian Ping; Qin, Tian; Chen, Yu

2017-08-01

Lower respiratory tract infections continue to pose a significant threat to human health. It is important to accurately and rapidly detect respiratory bacteria. To compensate for the limits of current respiratory bacteria detection methods, we developed a combination of multiplex polymerase chain reaction (PCR) and capillary electrophoresis (MPCE) assay to detect thirteen bacterial pathogens responsible for lower respiratory tract infections, including Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Mycoplasma pneumoniae, Legionella spp., Bordetella pertussis, Mycobacterium tuberculosis complex, Corynebacterium diphtheriae, and Streptococcus pyogenes. Three multiplex PCR reactions were built, and the products were analyzed by capillary electrophoresis using the high-throughput DNA analyzer. The specificity of the MPCE assay was examined and the detection limit was evaluated using DNA samples from each bacterial strain and the simulative samples of each strain. This assay was further evaluated using 152 clinical specimens and compared with real-time PCR reactions. For this assay, three nested-multiplex-PCRs were used to detect these clinical specimens. The detection limits of the MPCE assay for the 13 pathogens were very low and ranged from 10-7 to 10-2 ng/μL. Furthermore, analysis of the 152 clinical specimens yielded a specificity ranging from 96.5%-100.0%, and a sensitivity of 100.0% for the 13 pathogens. This study revealed that the MPCE assay is a rapid, reliable, and high-throughput method with high specificity and sensitivity. This assay has great potential in the molecular epidemiological survey of respiratory pathogens. Copyright © 2017 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Functionalization of optical nanotip arrays with an electrochemical microcantilever for multiplexed DNA detection.

PubMed

Descamps, Emeline; Duroure, Nathalie; Deiss, Frédérique; Leichlé, Thierry; Adam, Catherine; Mailley, Pascal; Aït-Ikhlef, Ali; Livache, Thierry; Nicu, Liviu; Sojic, Neso

2013-08-07

Optical nanotip arrays fabricated on etched fiber bundles were functionalized with DNA spots. Such unconventional substrates (3D and non-planar) are difficult to pattern with standard microfabrication techniques but, using an electrochemical cantilever, up to 400 spots were electrodeposited on the nanostructured optical surface in 5 min. This approach allows each spot to be addressed individually and multiplexed fluorescence detection is demonstrated. Finally, remote fluorescence detection was performed by imaging through the optical fiber bundle itself after hybridisation with the complementary sequence.

[Detection of large deletions in X linked Alport syndrome using competitive multiplex fluorescence polymerase chain reaction].

PubMed

Wang, F; Zhang, Y Q; Ding, J; Yu, L X

2017-10-18

To evaluate the ability of multiplex competitive fluorescence polymerase chain reaction in detection of large deletion and duplication genotypes of X-linked Alport syndrome. Clinical diagnosis of X-linked Alport syndrome was based on either abnormal staining of type IV collagen α5 chain in the epidermal basement membrane alone or with abnormal staining of type IV collagen α5 chain in the glomerular basement membrane and Bowman's capsule/ultrastructural changes in the glomerular basement membrane typical of Alport syndrome. A total of 20 unrelated Chinese patients (13 males and 7 females) clinically diagnosed as X-linked Alport syndrome were included in the study. Their genotypes were unknown. Control subjects included a male patient with other renal disease and two patients who had large deletions in COL4A5 gene detected by multiplex ligation-dependent probe amplification. Genomic DNA was isolated from peripheral blood leukocytes in all the participants. Multiplex competitive fluorescence polymerase chain reaction was used to coamplify 53 exons of COL4A5 gene and four reference genes in a single reaction. When a deletion removed exon 1 of COL4A5 gene was identified, the same method was used to coamplify the first 4 exons of COL4A5 and COL4A6 genes, a promoter shared by COL4A5 and COL4A6 genes, and three reference genes in a single reaction. Any copy number loss suggested by this method was verified by electrophoresis of corresponding polymerase chain reaction amplified products or DNA sequencing to exclude possible DNA variations in the primer regions. Genotypes of two positive controls identified by multiplex competitive fluorescence polymerase chain reaction were consistent with those detected by multiplex ligation-dependent probe amplification. Deletions were identified in 6 of the 20 patients, including two large deletions removing the 5' part of both COL4A5 and COL4A6 genes with the breakpoint located in the second intron of COL4A6, two large deletions

Sensitive and quantitative measurement of gene expression directly from a small amount of whole blood.

PubMed

Zheng, Zhi; Luo, Yuling; McMaster, Gary K

2006-07-01

Accurate and precise quantification of mRNA in whole blood is made difficult by gene expression changes during blood processing, and by variations and biases introduced by sample preparations. We sought to develop a quantitative whole-blood mRNA assay that eliminates blood purification, RNA isolation, reverse transcription, and target amplification while providing high-quality data in an easy assay format. We performed single- and multiplex gene expression analysis with multiple hybridization probes to capture mRNA directly from blood lysate and used branched DNA to amplify the signal. The 96-well plate singleplex assay uses chemiluminescence detection, and the multiplex assay combines Luminex-encoded beads with fluorescent detection. The single- and multiplex assays could quantitatively measure as few as 6000 and 24,000 mRNA target molecules (0.01 and 0.04 amoles), respectively, in up to 25 microL of whole blood. Both formats had CVs < 10% and dynamic ranges of 3-4 logs. Assay sensitivities allowed quantitative measurement of gene expression in the minority of cells in whole blood. The signals from whole-blood lysate correlated well with signals from purified RNA of the same sample, and absolute mRNA quantification results from the assay were similar to those obtained by quantitative reverse transcription-PCR. Both single- and multiplex assay formats were compatible with common anticoagulants and PAXgene-treated samples; however, PAXgene preparations induced expression of known antiapoptotic genes in whole blood. Both the singleplex and the multiplex branched DNA assays can quantitatively measure mRNA expression directly from small volumes of whole blood. The assay offers an alternative to current technologies that depend on RNA isolation and is amenable to high-throughput gene expression analysis of whole blood.

Detection of four important Eimeria species by multiplex PCR in a single assay.

PubMed

You, Myung-Jo

2014-06-01

The oocysts of some of the recognized species of chicken coccidiosis are difficult to distinguish morphologically. Diagnostic laboratories are increasingly utilizing DNA-based technologies for the specific identification of Eimeria species. This study reports a multiplex polymerase chain reaction (PCR) assay based on internal transcribed spacer-1 (ITS-1) for the simultaneous diagnosis of the Eimeria tenella, Eimeria acervulina, Eimeria maxima, and Eimeria necatrix species, which infect domestic fowl. Primer pairs specific to each species were designed in order to generate a ladder of amplification products ranging from 20 to 25 bp, and a common optimum annealing temperature for these species was determined to be 52.5 °C. Sensitivity tests were performed for each species, showing a detection threshold of 1-5 pg. All the species were amplified homogeneously, and a homogenous band ladder was observed, indicating that the assay permitted the simultaneous detection of all the species in a single-tube reaction. In the phylogenic study, there was a clear species clustering, which was irrespective of geographical location, for all the ITS-1 sequences used. This multiplex PCR assay represents a rapid and potential cost-effective diagnostic method for the detection of some key Eimeria species that infect domestic fowl. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Quantitative multiplex quantum dot in-situ hybridisation based gene expression profiling in tissue microarrays identifies prognostic genes in acute myeloid leukaemia

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

Tholouli, Eleni; MacDermott, Sarah; Hoyland, Judith

2012-08-24

Highlights: Black-Right-Pointing-Pointer Development of a quantitative high throughput in situ expression profiling method. Black-Right-Pointing-Pointer Application to a tissue microarray of 242 AML bone marrow samples. Black-Right-Pointing-Pointer Identification of HOXA4, HOXA9, Meis1 and DNMT3A as prognostic markers in AML. -- Abstract: Measurement and validation of microarray gene signatures in routine clinical samples is problematic and a rate limiting step in translational research. In order to facilitate measurement of microarray identified gene signatures in routine clinical tissue a novel method combining quantum dot based oligonucleotide in situ hybridisation (QD-ISH) and post-hybridisation spectral image analysis was used for multiplex in-situ transcript detection inmore » archival bone marrow trephine samples from patients with acute myeloid leukaemia (AML). Tissue-microarrays were prepared into which white cell pellets were spiked as a standard. Tissue microarrays were made using routinely processed bone marrow trephines from 242 patients with AML. QD-ISH was performed for six candidate prognostic genes using triplex QD-ISH for DNMT1, DNMT3A, DNMT3B, and for HOXA4, HOXA9, Meis1. Scrambled oligonucleotides were used to correct for background staining followed by normalisation of expression against the expression values for the white cell pellet standard. Survival analysis demonstrated that low expression of HOXA4 was associated with poorer overall survival (p = 0.009), whilst high expression of HOXA9 (p < 0.0001), Meis1 (p = 0.005) and DNMT3A (p = 0.04) were associated with early treatment failure. These results demonstrate application of a standardised, quantitative multiplex QD-ISH method for identification of prognostic markers in formalin-fixed paraffin-embedded clinical samples, facilitating measurement of gene expression signatures in routine clinical samples.« less

Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria, Cylindrospermopsis and Microcystis

PubMed Central

Te, Shu Harn; Chen, Enid Yingru

2015-01-01

The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques—qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples. PMID:26025892

Synovial fluid multiplex PCR is superior to culture for detection of low-virulent pathogens causing periprosthetic joint infection.

PubMed

Morgenstern, Christian; Cabric, Sabrina; Perka, Carsten; Trampuz, Andrej; Renz, Nora

2018-02-01

Analysis of joint aspirate is the standard preoperative investigation for diagnosis of periprosthetic joint infection (PJI). We compared the diagnostic performance of culture and multiplex polymerase chain reaction (PCR) of synovial fluid for diagnosis of PJI. Patients in whom aspiration of the prosthetic hip or knee joint was performed before revision arthroplasty were prospectively included. The performance of synovial fluid culture and multiplex PCR was compared by McNemar's chi-squared test. A total of 142 patients were included, 82 with knee and 60 with hip prosthesis. PJI was diagnosed in 77 patients (54%) and aseptic failure in 65 patients (46%). The sensitivity of synovial fluid culture and PCR was 52% and 60%, respectively, showing concordant results in 116 patients (82%). In patients with PJI, PCR missed 6 high-virulent pathogens (S. aureus, streptococci, E. faecalis, E. coli) which grew in synovial fluid culture, whereas synovial fluid culture missed 12 pathogens detected by multiplex PCR, predominantly low-virulent pathogens (Cutibacterium acnes and coagulase-negative staphylococci). In patients with aseptic failure, PCR detected 6 low-virulent organisms (predominantly C. acnes). While the overall performance of synovial fluid PCR was comparable to culture, PCR was superior for detection of low-virulent bacteria such as Cutibacterium spp. and coagulase-negative staphylococci. In addition, synovial fluid culture required several days for growth, whereas multiplex PCR provided results within 5hours in an automated manner. Copyright © 2017 Elsevier Inc. All rights reserved.

Qualis-SIS: automated standard curve generation and quality assessment for multiplexed targeted quantitative proteomic experiments with labeled standards.

PubMed

Mohammed, Yassene; Percy, Andrew J; Chambers, Andrew G; Borchers, Christoph H

2015-02-06

Multiplexed targeted quantitative proteomics typically utilizes multiple reaction monitoring and allows the optimized quantification of a large number of proteins. One challenge, however, is the large amount of data that needs to be reviewed, analyzed, and interpreted. Different vendors provide software for their instruments, which determine the recorded responses of the heavy and endogenous peptides and perform the response-curve integration. Bringing multiplexed data together and generating standard curves is often an off-line step accomplished, for example, with spreadsheet software. This can be laborious, as it requires determining the concentration levels that meet the required accuracy and precision criteria in an iterative process. We present here a computer program, Qualis-SIS, that generates standard curves from multiplexed MRM experiments and determines analyte concentrations in biological samples. Multiple level-removal algorithms and acceptance criteria for concentration levels are implemented. When used to apply the standard curve to new samples, the software flags each measurement according to its quality. From the user's perspective, the data processing is instantaneous due to the reactivity paradigm used, and the user can download the results of the stepwise calculations for further processing, if necessary. This allows for more consistent data analysis and can dramatically accelerate the downstream data analysis.

Metal-doped inorganic nanoparticles for multiplex detection of biomarkers by a sandwich-type ICP-MS immunoassay.

PubMed

Ko, Jung Aa; Lim, H B

2016-09-28

Metal-doped inorganic nanoparticles were synthesized for the multiplex detection of biomarkers by a sandwich-type inductively coupled plasma mass spectrometry (ICP-MS) immunoassay. The synthesized Cs-doped multicore magnetic nanoparticles (MMNPs) were used not only for magnetic extraction of targets but also for ratiometric measurement in ICP-MS. In addition, three different metal/dye-doped silica nanoparticles (SNPs) were synthesized as probes for multiplex detection: Y/RhBITC (rhodamine B isothiocyanate)-doped SNPs for CRP (cardiovascular disease), Cd/RhBITC-doped SNPs for AFP (tumor), and Au/5(6)-XRITC (X-rhodamine-5-(and-6)-isothiocyanate)-doped SNPs for NSE (heart disease). For quantification, the doped metals of SNPs were measured by ICP-MS and then the signal ratio to Cs of MMNPs was plotted with respect to the concentration of targets by a ratiometry. Limits of detection (LOD) of 0.35 ng/mL to 77 ng mL(-1) and recoveries of 83%-125% were obtained for serum samples spiked with the biomarkers. Since no sample treatment was necessary prior to the extraction, the proposed method provided short analysis time and convenience for the multiplex determination of biomarkers, which will be valuable for clinical application. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of multiplex PCR for the detection of total coliform bacteria for Escherichia coli and Clostridium perfringens in drinking water.

PubMed

Tantawiwat, Suwalee; Tansuphasiri, Unchalee; Wongwit, Waranya; Wongchotigul, Varee; Kitayaporn, Dwip

2005-01-01

Multiplex PCR amplification of lacZ, uidA and plc genes was developed for the simultaneous detection of total coliform bacteria for Escherichia coli and Clostridium perfringens, in drinking water. Detection by agarose gel electrophoresis yielded a band of 876 bp for the lacZ gene of all coliform bacteria; a band of 147 bp for the uidA gene and a band of 876 bp for the lacZ gene of all strains of E. coli; a band of 280 bp for the p/c gene for all strains of C. perfringens; and a negative result for all three genes when tested with other bacteria. The detection limit was 100 pg for E. coli and C. perfringens, and 1 ng for coliform bacteria when measured with purified DNA. This assay was applied to the detection of these bacteria in spiked water samples. Spiked water samples with 0-1,000 CFU/ml of coliform bacteria and/or E. coli and/or C. perfringens were detected by this multiplex PCR after a pre-enrichment step to increase the sensitivity and to ensure that the detection was based on the presence of cultivable bacteria. The result of bacterial detection from the multiplex PCR was comparable with that of a standard plate count on selective medium (p=0.62). When using standard plate counts as a gold standard, the sensitivity for this test was 99.1% (95% CI 95.33, 99.98) and the specificity was 90.9 % (95% CI 75.67, 98.08). Multiplex PCR amplification with a pre-enrichment step was shown to be an effective, sensitive and rapid method for the simultaneous detection of these three microbiological parameters in drinking water.

Multiplexing 200 spatial modes with a single hologram

NASA Astrophysics Data System (ADS)

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

2017-11-01

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

A multiplex competitive ELISA for the detection and characterization of gluten in fermented-hydrolyzed foods.

PubMed

Panda, Rakhi; Boyer, Marc; Garber, Eric A E

2017-12-01

A novel competitive ELISA was developed utilizing the G12, R5, 2D4, MIoBS, and Skerritt antibody-HRP conjugates employed in nine commercial ELISA test kits that are routinely used for gluten detection. This novel multiplex competitive ELISA simultaneously measures gliadin-, deamidated gliadin-, and glutenin-specific epitopes. The assay was used to evaluate 20 wheat beers, 20 barley beers, 6 barley beers processed to reduce gluten, 15 soy sauces, 6 teriyaki sauces, 6 Worcestershire sauces, 6 vinegars, and 8 sourdough breads. For wheat beers, the apparent gluten concentration values obtained by the G12 and Skerritt antibodies were typically higher than those obtained using the R5 antibodies. The sourdough bread samples resulted in higher apparent gluten concentration values with the Skerritt antibody, while the values generated by the G12 and R5 antibodies were comparable. Although the soy-based sauces showed non-specific inhibition with the multiple R5 and G12 antibodies, their overall profile was distinguishable from the other categories of fermented foods. Cluster analysis of the apparent gluten concentration values obtained by the multiplex competitive ELISA, as well as the relative response of the nine gluten-specific antibodies used in the assay to different gluten proteins/peptides, distinguishes among the different categories of fermented-hydrolyzed foods by recognizing the differences in the protein/peptide profiles characteristic of each product. This novel gluten-based multiplex competitive ELISA provides insight into the extent of proteolysis resulting from various fermentation processes, which is essential for accurate gluten quantification in fermented-hydrolyzed foods. Graphical abstract A novel multiplex competitive ELISA for the detection and characterization of gluten in fermented-hydrolyzed foods.

Detection and Serotyping of Dengue Virus in Serum Samples by Multiplex Reverse Transcriptase PCR-Ligase Detection Reaction Assay▿ †

PubMed Central

Das, S.; Pingle, M. R.; Muñoz-Jordán, J.; Rundell, M. S.; Rondini, S.; Granger, K.; Chang, G.-J. J.; Kelly, E.; Spier, E. G.; Larone, D.; Spitzer, E.; Barany, F.; Golightly, L. M.

2008-01-01

The detection and successful typing of dengue virus (DENV) from patients with suspected dengue fever is important both for the diagnosis of the disease and for the implementation of epidemiologic control measures. A technique for the multiplex detection and typing of DENV serotypes 1 to 4 (DENV-1 to DENV-4) from clinical samples by PCR-ligase detection reaction (LDR) has been developed. A serotype-specific PCR amplifies the regions of genes C and E simultaneously. The two amplicons are targeted in a multiplex LDR, and the resultant fluorescently labeled ligation products are detected on a universal array. The assay was optimized using 38 DENV strains and was evaluated with 350 archived acute-phase serum samples. The sensitivity of the assay was 98.7%, and its specificity was 98.4%, relative to the results of real-time PCR. The detection threshold was 0.017 PFU for DENV-1, 0.004 PFU for DENV-2, 0.8 PFU for DENV-3, and 0.7 PFU for DENV-4. The assay is specific; it does not cross-react with the other flaviviruses tested (West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Kunjin virus, Murray Valley virus, Powassan virus, and yellow fever virus). All but 1 of 26 genotypic variants of DENV serotypes in a global DENV panel from different geographic regions were successfully identified. The PCR-LDR assay is a rapid, sensitive, specific, and high-throughput technique for the simultaneous detection of all four serotypes of DENV. PMID:18685000

Near-infrared fluorescence-based multiplex lateral flow immunoassay for the simultaneous detection of four antibiotic residue families in milk.

PubMed

Chen, Yiqiang; Chen, Qian; Han, Miaomiao; Liu, Jiangyang; Zhao, Peng; He, Lidong; Zhang, Yuan; Niu, Yiming; Yang, Wenjun; Zhang, Liying

2016-05-15

In this study, we developed a novel near-infrared fluorescence based multiplex lateral flow immunoassay by conjugating a near-infrared label to broad-specificity monoclonal antibody/receptor as detection complexes. Different antigens were dispensed onto separate test zones of nitrocellulose membrane to serve as capture reagents. This assay format allowed the simultaneous detection of four families of antibiotics (β-lactams, tetracyclines, quinolones and sulfonamides) in milk within 20 min. Qualitative and quantitative analysis of target antibiotics were realized by imaging the fluorescence intensity of the near-infrared label captured on respective test lines. For qualitative analysis, the cut-off values of β-lactams, tetracyclines, quinolones and sulfonamides were determined to be 8 ng/mL, 2 ng/mL, 4 ng/mL and 8 ng/mL respectively, which were much lower than the conventional gold nanoparticle based lateral flow immunoassay. For quantitative analysis, the detection ranges were 0.26-3.56 ng/mL for β-lactams, 0.04-0.98 ng/mL for tetracyclines, 0.08-2.0 ng/mL for quinolones, and 0.1-3.98 ng/mL for sulfonamides, with linear correlation coefficients higher than 0.97. The mean spiked recoveries ranged from 93.7% to 108.2% with coefficient of variations less than 16.3%. These results demonstrated that this novel immunoassay is a promising approach for rapidly screening the four families of antibiotic residues in milk. Copyright © 2015 Elsevier B.V. All rights reserved.

Quantitative multiplexed proteomics of Taenia solium cysts obtained from the skeletal muscle and central nervous system of pigs

PubMed Central

Navarrete-Perea, José; Isasa, Marta; Paulo, Joao A.; Corral-Corral, Ricardo; Flores-Bautista, Jeanette; Hernández-Téllez, Beatriz; Bobes, Raúl J.; Fragoso, Gladis; Sciutto, Edda; Soberón, Xavier; Gygi, Steven P.; Laclette, Juan P.

2017-01-01

In human and porcine cysticercosis caused by the tapeworm Taenia solium, the larval stage (cysts) can infest several tissues including the central nervous system (CNS) and the skeletal muscles (SM). The cyst’s proteomics changes associated with the tissue localization in the host tissues have been poorly studied. Quantitative multiplexed proteomics has the power to evaluate global proteome changes in response to different conditions. Here, using a TMT-multiplexed strategy we identified and quantified over 4,200 proteins in cysts obtained from the SM and CNS of pigs, of which 891 were host proteins. To our knowledge, this is the most extensive intermixing of host and parasite proteins reported for tapeworm infections.Several antigens in cysticercosis, i.e., GP50, paramyosin and a calcium-binding protein were enriched in skeletal muscle cysts. Our results suggested the occurrence of tissue-enriched antigen that could be useful in the improvement of the immunodiagnosis for cysticercosis. Using several algorithms for epitope detection, we selected 42 highly antigenic proteins enriched for each tissue localization of the cysts. Taking into account the fold changes and the antigen/epitope contents, we selected 10 proteins and produced synthetic peptides from the best epitopes. Nine peptides were recognized by serum antibodies of cysticercotic pigs, suggesting that those peptides are antigens. Mixtures of peptides derived from SM and CNS cysts yielded better results than mixtures of peptides derived from a single tissue location, however the identification of the ‘optimal’ tissue-enriched antigens remains to be discovered. Through machine learning technologies, we determined that a reliable immunodiagnostic test for porcine cysticercosis required at least five different antigenic determinants. PMID:28945737

Quantitative multiplexed proteomics of Taenia solium cysts obtained from the skeletal muscle and central nervous system of pigs.

PubMed

Navarrete-Perea, José; Isasa, Marta; Paulo, Joao A; Corral-Corral, Ricardo; Flores-Bautista, Jeanette; Hernández-Téllez, Beatriz; Bobes, Raúl J; Fragoso, Gladis; Sciutto, Edda; Soberón, Xavier; Gygi, Steven P; Laclette, Juan P

2017-09-01

In human and porcine cysticercosis caused by the tapeworm Taenia solium, the larval stage (cysts) can infest several tissues including the central nervous system (CNS) and the skeletal muscles (SM). The cyst's proteomics changes associated with the tissue localization in the host tissues have been poorly studied. Quantitative multiplexed proteomics has the power to evaluate global proteome changes in response to different conditions. Here, using a TMT-multiplexed strategy we identified and quantified over 4,200 proteins in cysts obtained from the SM and CNS of pigs, of which 891 were host proteins. To our knowledge, this is the most extensive intermixing of host and parasite proteins reported for tapeworm infections.Several antigens in cysticercosis, i.e., GP50, paramyosin and a calcium-binding protein were enriched in skeletal muscle cysts. Our results suggested the occurrence of tissue-enriched antigen that could be useful in the improvement of the immunodiagnosis for cysticercosis. Using several algorithms for epitope detection, we selected 42 highly antigenic proteins enriched for each tissue localization of the cysts. Taking into account the fold changes and the antigen/epitope contents, we selected 10 proteins and produced synthetic peptides from the best epitopes. Nine peptides were recognized by serum antibodies of cysticercotic pigs, suggesting that those peptides are antigens. Mixtures of peptides derived from SM and CNS cysts yielded better results than mixtures of peptides derived from a single tissue location, however the identification of the 'optimal' tissue-enriched antigens remains to be discovered. Through machine learning technologies, we determined that a reliable immunodiagnostic test for porcine cysticercosis required at least five different antigenic determinants.

SERS-based inverse molecular sentinel (iMS) nanoprobes for multiplexed detection of microRNA cancer biomarkers in biological samples

NASA Astrophysics Data System (ADS)

Crawford, Bridget M.; Wang, Hsin-Neng; Fales, Andrew M.; Bowie, Michelle L.; Seewaldt, Victoria L.; Vo-Dinh, Tuan

2017-02-01

The development of sensitive and selective biosensing techniques is of great interest for clinical diagnostics. Here, we describe the development and application of a surface enhanced Raman scattering (SERS) sensing technology, referred to as "inverse Molecular Sentinel (iMS)" nanoprobes, for the detection of nucleic acid biomarkers in biological samples. This iMS nanoprobe involves the use of plasmonic-active nanostars as the sensing platform for a homogenous assay for multiplexed detection of nucleic acid biomarkers, including DNA, RNA and microRNA (miRNA). The "OFF-to-ON" signal switch is based on a non-enzymatic strand-displacement process and the conformational change of stem-loop (hairpin) oligonucleotide probes upon target binding. Here, we demonstrate the development of iMS nanoprobes for the detection of DNA sequences as well as a modified design of the nanoprobe for the detection of short (22-nt) microRNA sequences. The application of iMS nanoprobes to detect miRNAs in real biological samples was performed with total small RNA extracted from breast cancer cell lines. The multiplex capability of the iMS technique was demonstrated using a mixture of the two differently labeled nanoprobes to detect miR-21 and miR-34a miRNA biomarkers for breast cancer. The results of this study demonstrate the feasibility of applying the iMS technique for multiplexed detection of nucleic acid biomarkers, including short miRNAs molecules.

Multiplex Lateral Flow Immunoassays Based on Amorphous Carbon Nanoparticles for Detecting Three Fusarium Mycotoxins in Maize.

PubMed

Zhang, Xiya; Yu, Xuezhi; Wen, Kai; Li, Chenglong; Mujtaba Mari, Ghulam; Jiang, Haiyang; Shi, Weimin; Shen, Jianzhong; Wang, Zhanhui

2017-09-13

The detecting labels used for lateral flow immunoassays (LFAs) have been traditionally gold nanoparticles (GNPs) and, more recently, luminescent nanoparticles, such as quantum dots (QDs). However, these labels have low sensitivity and are costly, in particular, for trace detection of mycotoxins in cereals. Here, we provided a simple preparation procedure for amorphous carbon nanoparticles (ACNPs) and described multiplex LFAs employing ACNPs as labels (ACNP-LFAs) for detecting three Fusarium mycotoxins. The analytical performance of ACNPs in LFA was compared to GNPs and QDs using the same immunoreagents, except for the labels, allowing for their analytical characteristics to be objectively compared. The visual limit of detection for ACNP-LFAs in buffer was 8-fold better than GNPs and 2-fold better than QDs. Under optimized conditions, the quantitative limit of detection of ACNP-LFAs in maize was as low as 20 μg/kg for deoxynivalenol, 13 μg/kg for T-2 toxin, and 1 μg/kg for zearalenone. These measurements were much lower than the action level of these mycotoxins in maize. The accuracy and precision of the ACNP-LFAs were evaluated by analysis of spiked and incurred maize samples with recoveries of 84.6-109% and coefficients of variation below 13%. The results of ACNP-LFAs using naturally incurred maize samples showed good agreement with results from high-performance liquid chromatography-tandem mass spectrometry, indicating that ACNPs were more sensitive labels than and a promising alternative to GNPs used in LFAs for detecting mycotoxins in cereals.

Turbulence mitigation scheme based on multiple-user detection in an orbital-angular-momentum multiplexed system

NASA Astrophysics Data System (ADS)

Zou, Li; Wang, Le; Zhao, Sheng-Mei; Chen, Han-Wu

2016-11-01

Atmospheric turbulence (AT) induced crosstalk can significantly impair the performance of a free-space optical (FSO) communication link using orbital angular momentum (OAM) multiplexing. In this paper, we propose a multiple-user detection (MUD) turbulence mitigation scheme in an OAM-multiplexed FSO communication link. First, we present a MUD equivalent communication model for an OAM-multiplexed FSO communication link under AT. In the equivalent model, each input bit stream represents one user’s information. The deformed OAM spatial modes caused by AT, instead of the pure OAM spatial modes, are used as information carriers, and the overlapping between the deformed OAM spatial modes are computed as the correlation coefficients between the users. Then, we present a turbulence mitigation scheme based on MUD idea to enhance AT tolerance of the OAM-multiplexed FSO communication link. In the proposed scheme, the crosstalk caused by AT is used as a useful component to deduce users’ information. The numerical results show that the performance of the OAM-multiplexed communication link has greatly improved by the proposed scheme. When the turbulence strength is 1 × 10-15 m-2/3, the transmission distance is 1000 m and the channel signal-to-noise ratio (SNR) is 26 dB, the bit-error-rate (BER) performance of four spatial multiplexed OAM modes lm = +1,+2,+3,+4 are all close to 10-5, and there is a 2-3 fold increase in the BER performance in comparison with those results without the proposed scheme. In addition, the proposed scheme is more effective for an OAM-multiplexed FSO communication link with a larger OAM mode topological charge interval. The proposed scheme is a promising direction for compensating the interference caused by AT in the OAM-multiplexed FSO communication link. Project supported by the National Natural Science Foundation of China (Grant Nos. 61271238 and 61475075), the Open Research Fund of Key Lab of Broadband Wireless Communication and Sensor Network

Real-time multiplex PCR assay for detection of Yersinia pestis and Yersinia pseudotuberculosis.

PubMed

Matero, Pirjo; Pasanen, Tanja; Laukkanen, Riikka; Tissari, Päivi; Tarkka, Eveliina; Vaara, Martti; Skurnik, Mikael

2009-01-01

A multiplex real-time polymerase chain reaction (PCR) assay was developed for the detection of Yersinia pestis and Yersinia pseudotuberculosis. The assay includes four primer pairs, two of which are specific for Y. pestis, one for Y. pestis and Y. pseudotuberculosis and one for bacteriophage lambda; the latter was used as an internal amplification control. The Y. pestis-specific target genes in the assay were ypo2088, a gene coding for a putative methyltransferase, and the pla gene coding for the plasminogen activator. In addition, the wzz gene was used as a target to specifically identify both Y. pestis and the closely related Y. pseudotuberculosis group. The primer and probe sets described for the different genes can be used either in single or in multiplex PCR assays because the individual probes were designed with different fluorochromes. The assays were found to be both sensitive and specific; the lower limit of the detection was 10-100 fg of extracted Y. pestis or Y. pseudotuberculosis total DNA. The sensitivity of the tetraplex assay was determined to be 1 cfu for the ypo2088 and pla probe labelled with FAM and JOE fluorescent dyes, respectively.

Multiplex Immunoassay Profiling.

PubMed

Stephen, Laurie

2017-01-01

Multiplex immunoassays allow for the rapid profiling of biomarker proteins in biological fluids, using less sample and labor than single immunoassays. This chapter details the methods to develop and manufacture multiplex assays for the Luminex ® platform. Although assay development is not included here, the same methods can be used to covalently couple antibodies to the Luminex beads and to label antibodies for the screening of sandwich pairs, if needed. The assay optimization, detection of cross-reactivity, and minimizing antibody interactions and matrix interferences will be addressed.

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

PubMed Central

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

2008-01-01

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

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

PubMed

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

2008-01-01

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

Evaluation of an Internally Controlled Multiplex Tth Endonuclease Cleavage Loop-Mediated Isothermal Amplification (TEC-LAMP) Assay for the Detection of Bacterial Meningitis Pathogens.

PubMed

Higgins, Owen; Clancy, Eoin; Cormican, Martin; Boo, Teck Wee; Cunney, Robert; Smith, Terry J

2018-02-09

Bacterial meningitis infection is a leading global health concern for which rapid and accurate diagnosis is essential to reduce associated morbidity and mortality. Loop-mediated isothermal amplification (LAMP) offers an effective low-cost diagnostic approach; however, multiplex LAMP is difficult to achieve, limiting its application. We have developed novel real-time multiplex LAMP technology, TEC-LAMP, using Tth endonuclease IV and a unique LAMP primer/probe. This study evaluates the analytical specificity, limit of detection (LOD) and clinical application of an internally controlled multiplex TEC-LAMP assay for detection of leading bacterial meningitis pathogens: Streptococcus pneumoniae , Neisseria meningitidis and Haemophilus influenzae . Analytical specificities were established by testing 168 bacterial strains, and LODs were determined using Probit analysis. The TEC-LAMP assay was 100% specific, with LODs for S. pneumoniae , N. meningitidis and H. influenzae of 39.5, 17.3 and 25.9 genome copies per reaction, respectively. Clinical performance was evaluated by testing 65 archived PCR-positive samples. Compared to singleplex real-time PCR, the multiplex TEC-LAMP assay demonstrated diagnostic sensitivity and specificity of 92.3% and 100%, respectively. This is the first report of a single-tube internally controlled multiplex LAMP assay for bacterial meningitis pathogen detection, and the first report of Tth endonuclease IV incorporation into nucleic acid amplification diagnostic technology.

Multiplexed protein detection using antibody-conjugated microbead arrays in a microfabricated electrophoretic device

PubMed Central

Barbee, Kristopher D.; Hsiao, Alexander P.; Roller, Eric E.; Huang, Xiaohua

2011-01-01

We report the development of a microfabricated electrophoretic device for assembling high-density arrays of antibody-conjugated microbeads for chip-based protein detection. The device consists of a flow cell formed between a gold-coated silicon chip with an array of microwells etched in a silicon dioxide film and a glass coverslip with a series of thin gold counter electrode lines. We have demonstrated that 0.4 and 1 μm beads conjugated with antibodies can be rapidly assembled into the microwells by applying a pulsed electric field across the chamber. By assembling step-wise a mixture of fluorescently labeled antibody-conjugated microbeads, we incorporated both spatial and fluorescence encoding strategies to demonstrate significant multiplexing capabilities. We have shown that these antibody-conjugated microbead arrays can be used to perform on-chip sandwich immunoassays to detect test antigens at concentrations as low as 40 pM (6 ng/mL). A finite element model was also developed to examine the electric field distribution within the device for different counter electrode configurations over a range of line pitches and chamber heights. This device will be useful for assembling high-density, encoded antibody arrays for multiplexed detection of proteins and other types of protein-conjugated microbeads for applications such as the analysis of protein-protein interactions. PMID:20820631

Quantitative secondary electron detection

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

Agrawal, Jyoti; Joy, David C.; Nayak, Subuhadarshi

Quantitative Secondary Electron Detection (QSED) using the array of solid state devices (SSD) based electron-counters enable critical dimension metrology measurements in materials such as semiconductors, nanomaterials, and biological samples (FIG. 3). Methods and devices effect a quantitative detection of secondary electrons with the array of solid state detectors comprising a number of solid state detectors. An array senses the number of secondary electrons with a plurality of solid state detectors, counting the number of secondary electrons with a time to digital converter circuit in counter mode.

Detection of Neisseria meningitidis in cerebrospinal fluid using a multiplex PCR and the Luminex detection technology.

PubMed

Møller, Jens Kjølseth

2012-01-01

Rapid clinical and laboratory diagnoses are the foundation for a successful management of serious infections with Neisseria meningitidis. A species-specific multiplex polymerase chain reaction (PCR) coupled with fluidic microarrays using microbeads (the Luminex xMAP™ Technology) can detect pathogens most frequently found in the cerebrospinal fluid of patients. The Luminex suspension array system uniquely combines flow cytometry, microspheres, laser technology, digital signal processing, and traditional chemistry. In this method, the reaction is carried out in one vessel, in which distinctly color-coded bead sets, each conjugated with a different specific nucleic acid reactant, are hybridized with the PCR products, and a reporter molecule is used to quantify the interaction. The flow-based Luminex array reader identifies each reaction (bead set) after excitation by a red classification laser. Reporter signals from each reaction are simultaneously quantified by fluorescence generated by a green reporter laser. This nonculture, multiplex assay may prove to be an important tool for optimal laboratory diagnosis, not only of meningococcal meningitis, but also of meningitis caused by other bacterial or viral pathogens.

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.

One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples.

PubMed

Gautam, Rashi; Mijatovic-Rustempasic, Slavica; Esona, Mathew D; Tam, Ka Ian; Quaye, Osbourne; Bowen, Michael D

2016-01-01

Background. Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8-100% sensitivity, 99.7-100% specificity, 85-95% efficiency and a limit of detection of 4-60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81-92% efficiency and limit of detection of 150-600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98

One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples

PubMed Central

Mijatovic-Rustempasic, Slavica; Esona, Mathew D.; Tam, Ka Ian; Quaye, Osbourne; Bowen, Michael D.

2016-01-01

Background. Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8–100% sensitivity, 99.7–100% specificity, 85–95% efficiency and a limit of detection of 4–60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81–92% efficiency and limit of detection of 150–600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8�

Development of a Multiplexed Bead-Based Suspension Array for the Detection and Discrimination of Pospiviroid Plant Pathogens

PubMed Central

van Brunschot, Sharon L.; Bergervoet, Jan H. W.; Pagendam, Daniel E.; de Weerdt, Marjanne; Geering, Andrew D. W.; Drenth, André; van der Vlugt, René A. A.

2014-01-01

Efficient and reliable diagnostic tools for the routine indexing and certification of clean propagating material are essential for the management of pospiviroid diseases in horticultural crops. This study describes the development of a true multiplexed diagnostic method for the detection and identification of all nine currently recognized pospiviroid species in one assay using Luminex bead-based suspension array technology. In addition, a new data-driven, statistical method is presented for establishing thresholds for positivity for individual assays within multiplexed arrays. When applied to the multiplexed array data generated in this study, the new method was shown to have better control of false positives and false negative results than two other commonly used approaches for setting thresholds. The 11-plex Luminex MagPlex-TAG pospiviroid array described here has a unique hierarchical assay design, incorporating a near-universal assay in addition to nine species-specific assays, and a co-amplified plant internal control assay for quality assurance purposes. All assays of the multiplexed array were shown to be 100% specific, sensitive and reproducible. The multiplexed array described herein is robust, easy to use, displays unambiguous results and has strong potential for use in routine pospiviroid indexing to improve disease management strategies. PMID:24404188

Quantitative detection method for Roundup Ready soybean in food using duplex real-time PCR MGB chemistry.

PubMed

Samson, Maria Cristina; Gullì, Mariolina; Marmiroli, Nelson

2010-07-01

Methodologies that enable the detection of genetically modified organisms (GMOs) (authorized and non-authorized) in food and feed strongly influence the potential for adequate updating and implementation of legislation together with labeling requirements. Quantitative polymerase chain reaction (qPCR) systems were designed to boost the sensitivity and specificity on the identification of GMOs in highly degraded DNA samples; however, such testing will become economically difficult to cope with due to increasing numbers of approved genetically modified (GM) lines. Multiplexing approaches are therefore in development to provide cost-efficient solution. Construct-specific primers and probe were developed for quantitative analysis of Roundup Ready soybean (RRS) event glyphosate-tolerant soybean (GTS) 40-3-2. The lectin gene (Le1) was used as a reference gene, and its specificity was verified. RRS- and Le1-specific quantitative real-time PCR (qRTPCR) were optimized in a duplex platform that has been validated with respect to limit of detection (LOD) and limit of quantification (LOQ), as well as accuracy. The analysis of model processed food samples showed that the degradation of DNA has no adverse or little effects on the performance of quantification assay. In this study, a duplex qRTPCR using TaqMan minor groove binder-non-fluorescent quencher (MGB-NFQ) chemistry was developed for specific detection and quantification of RRS event GTS 40-3-2 that can be used for practical monitoring in processed food products.

Multiplexed Colorimetric Solid-Phase Extraction

NASA Technical Reports Server (NTRS)

Gazda, Daniel B.; Fritz, James S.; Porter, Marc D.

2009-01-01

Multiplexed colorimetric solid-phase extraction (MC-SPE) is an extension of colorimetric solid-phase extraction (C-SPE) an analytical platform that combines colorimetric reagents, solid phase extraction, and diffuse reflectance spectroscopy to quantify trace analytes in water. In CSPE, analytes are extracted and complexed on the surface of an extraction membrane impregnated with a colorimetric reagent. The analytes are then quantified directly on the membrane surface using a handheld diffuse reflectance spectrophotometer. Importantly, the use of solid-phase extraction membranes as the matrix for impregnation of the colorimetric reagents creates a concentration factor that enables the detection of low concentrations of analytes in small sample volumes. In extending C-SPE to a multiplexed format, a filter holder that incorporates discrete analysis channels and a jig that facilitates the concurrent operation of multiple sample syringes have been designed, enabling the simultaneous determination of multiple analytes. Separate, single analyte membranes, placed in a readout cartridge create unique, analyte-specific addresses at the exit of each channel. Following sample exposure, the diffuse reflectance spectrum of each address is collected serially and the Kubelka-Munk function is used to quantify each water quality parameter via calibration curves. In a demonstration, MC-SPE was used to measure the pH of a sample and quantitate Ag(I) and Ni(II).

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

PubMed

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

2017-08-01

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

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

PubMed Central

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

2017-01-01

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

Ratiometric biosensor array for multiplexed detection of microRNAs based on electrochemiluminescence coupled with cyclic voltammetry.

PubMed

Feng, Xiaobin; Gan, Ning; Zhang, Huairong; Li, Tianhua; Cao, Yuting; Hu, Futao; Jiang, Qianli

2016-01-15

A novel multiplexed ratiometric biosensor array was fabricated on a homemade screen-printed carbon electrode (SPCE) for near-simultaneous detection of microRNA (miRNA)-21 and miRNA-141 based on electrochemiluminescence (ECL) coupled with cyclic voltammetry (CV) method. In the detection system, the ECL signal tags (Ru-SiO2@PLL-Au) were fabricated using poly-l-lysine (PLL) as bridging agent and co-reactant to connect Ru-SiO2 (Ru(bpy)3(2+)-doped silica) and gold nanoparticles (Au NPs), which were respectively modified on two spatial resolved working electrodes (WE1 and WE2) of SPCE. Then the ferrocene (Fc)-labeled hairpin DNA (Fc-HDNA1 and Fc-HDNA2) as CV signal tags and ECL quenching material were immobilized on Ru-SiO2@PLL-Au. Upon miRNA-21 and miRNA-141 adding, the target miRNAs could hybridize with corresponding Fc-HDNA, which could lead to Fc away from Ru-SiO2@PLL-Au. Such conformational changes could recover the ECL of Ru-SiO2@PLL-Au and decreased the CV current of Fc, respectively. This "signal-on" of ECL and "signal-off" of CV were employed for dual-signal ratiometric readout. With the help of a multiplexed switch, two dual-signals from WE1 and WE2 were used for multiplexed detection of miRNA-21 and miRNA-141 down to 6.3 and 8.6fM, respectively. This approach was used in real sample analysis and has significant potential for miRNA biomarkers detection in a clinical laboratory setting. Copyright © 2015 Elsevier B.V. All rights reserved.

A Multiplex PCR for Simultaneous Detection of Three Zoonotic Parasites Ancylostoma ceylanicum, A. caninum, and Giardia lamblia Assemblage A

PubMed Central

Hu, Wei; Wu, Sheng; Yu, Xingang; Abullahi, Auwalu Yusuf; Song, Meiran; Tan, Liping; Wang, Zhen; Jiang, Biao; Li, Guoqing

2015-01-01

Ancylostoma ceylanicum, A. caninum, and Giardia lamblia assemblage A are common intestinal parasites of dogs and cats; they can also infect humans, causing parasitic zoonoses. In this study, a multiplex PCR method was developed for simultaneous identification and detection of those three zoonotic parasites. Three pairs of specific primers were designed based on ITS sequence of A. ceylanicum and A. caninum and TPI gene of G. lamblia available in the GenBank. The multiplex PCR reaction system was established by optimizing the reaction condition, and a series of tests on the sensitivity, specificity, and clinical application were also conducted. Results showed that three target fragments were amplified specifically; the detection limit was 10 eggs for both A. ceylanicum and A. caninum, 72 pg DNA for G. lamblia. Of 112 clinical fecal samples, 34.8% and 17.8% samples were positive for A. caninum and A. ceylanicum, respectively, while only 2.7% samples were positive for G. lamblia assemblage A. It is concluded that the established multiplex PCR assay is a convenient, rapid, cost-effective, and high-efficiency method for molecular detection and epidemiological investigation of three zoonotic parasites. PMID:26447336

Optical Interference-Free Surface-Enhanced Raman Scattering CO-Nanotags for Logical Multiplex Detection of Vascular Disease-Related Biomarkers.

PubMed

Gong, Tianxun; Hong, Zi-Yao; Chen, Ching-Hsiang; Tsai, Cheng-Yen; Liao, Lun-De; Kong, Kien Voon

2017-03-28

Matrix metalloproteinases (MMPs), specifically MMP-2, MMP-7, and MMP-9, have been discovered to be linked to many forms of vascular diseases such as stroke, and their detection is crucial to facilitate clinical diagnosis. In this work, we prepared a class of optical interference-free SERS nanotags (CO-nanotags) that can be used for the purpose of multiplex sensing of different MMPs. Multiplex detection with the absence of cross-talk was achieved by using CO-nanotags with individual tunable intrinsic Raman shifts of CO in the 1800-2200 cm -1 region determined by the metal core and ligands of the metal carbonyl complex. Boolean logic was used as well to simultaneously probe for two proteolytic inputs. Such nanotags offer the advantages of convenient detection of target nanotags and high sensitivity as validated in the ischemia rat model.

Multiplex polymerase chain reaction assay for the detection of minute virus of mice and mouse parvovirus infections in laboratory mice.

PubMed

Wang, K W; Chueh, L L; Wang, M H; Huang, Y T; Fang, B H; Chang, C Y; Fang, M C; Chou, J Y; Hsieh, S C; Wan, C H

2013-04-01

Mouse parvoviruses are among the most prevalent infectious pathogens in contemporary mouse colonies. To improve the efficiency of routine screening for mouse parvovirus infections, a multiplex polymerase chain reaction (PCR) assay targeting the VP gene was developed. The assay detected minute virus of mice (MVM), mouse parvovirus (MPV) and a mouse housekeeping gene (α-actin) and was able to specifically detect MVM and MPV at levels as low as 50 copies. Co-infection with the two viruses with up to 200-fold differences in viral concentrations can easily be detected. The multiplex PCR assay developed here could be a useful tool for monitoring mouse health and the viral contamination of biological materials.

A System for Multiplexed Direct Electrical Detection of DNA Synthesis.

PubMed

Anderson, Erik P; Daniels, Jonathan S; Yu, Heng; Karhanek, Miloslav; Lee, Thomas H; Davis, Ronald W; Pourmand, Nader

2008-01-29

An electronic system for the multiplexed detection of DNA polymerization is designed and characterized. DNA polymerization is detected by the measurement of small transient currents arising from ion diffusion during polymerization. A transimpedance amplifier is used to detect these small currents; we implemented a twenty-four channel recording system on a single printed circuit board. Various contributions to the input-referred current noise are analyzed and characterized, as it limits the minimum detectable current and thus the biological limit of detection. We obtained 8.5 pA RMS mean noise current (averaged over all 24 channels) over the recording bandwidth (DC to 2 kHz). With digital filtering, the input-referred current noise of the acquisition system is reduced to 2.4 pA, which is much lower than the biological noise. Electrical crosstalk between channels is measured, and a model for the crosstalk is presented. Minimizing the crosstalk is critical because it can lead to erroneous microarray data. With proper precautions, crosstalk is reduced to a negligible value (less than 1.4%). Using a micro-fabricated array of 24 gold electrodes, we demonstrated system functionality by detecting the presence of a target DNA oligonucleotide which hybridized onto its corresponding target.

Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria, Cylindrospermopsis and Microcystis.

PubMed

Te, Shu Harn; Chen, Enid Yingru; Gin, Karina Yew-Hoong

2015-08-01

The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques-qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Elucidating the Burden of HIV in Tissues Using Multiplexed Immunofluorescence and In Situ Hybridization: Methods for the Single-Cell Phenotypic Characterization of Cells Harboring HIV In Situ.

PubMed

Vasquez, Joshua J; Hussien, Rajaa; Aguilar-Rodriguez, Brandon; Junger, Henrik; Dobi, Dejan; Henrich, Timothy J; Thanh, Cassandra; Gibson, Erica; Hogan, Louise E; McCune, Joseph; Hunt, Peter W; Stoddart, Cheryl A; Laszik, Zoltan G

2018-06-01

Persistent tissue reservoirs of HIV present a major barrier to cure. Defining subsets of infected cells in tissues is a major focus of HIV cure research. Herein, we describe a novel multiplexed in situ hybridization (ISH) (RNAscope) protocol to detect HIV-DNA (vDNA) and HIV-RNA (vRNA) in formalin-fixed paraffin-embedded (FFPE) human tissues in combination with immunofluorescence (IF) phenotyping of the infected cells. We show that multiplexed IF and ISH (mIFISH) is suitable for quantitative assessment of HIV vRNA and vDNA and that multiparameter IF phenotyping allows precise identification of the cellular source of the ISH signal. We also provide semi-quantitative data on the impact of various tissue fixatives on the detectability of vDNA and vRNA with RNAscope technology. Finally, we describe methods to quantitate the ISH signal on whole-slide digital images and validation of the quantitative ISH data with quantitative real-time PCR for vRNA. It is our hope that this approach will provide insight into the biology of HIV tissue reservoirs and to inform strategies aimed at curing HIV.

Development and Validation of a Multiplex PCR for Detection of Scedosporium spp. in Respiratory Tract Specimens from Patients with Cystic Fibrosis▿

PubMed Central

Harun, Azian; Blyth, Christopher C.; Gilgado, Felix; Middleton, Peter; Chen, Sharon C.-A.; Meyer, Wieland

2011-01-01

The emergence of Scedosporium infections in diverse groups of individuals, which are often treatment refractory, warrants timely and accurate laboratory diagnosis. Species- or group-specific primers based on internal transcribed spacer (ITS) sequence polymorphisms were designed for Scedosporium aurantiacum, Scedosporium dehoogii, Scedosporium prolificans, Pseudallescheria boydii species complex (former clade 5)/Pseudallescheria apiosperma (formerly classified as S. apiospermum sensu lato) and Pseudallescheria minutispora. Primers for S. aurantiacum, S. prolificans, and P. boydii species complex/P. apiosperma were incorporated into a multiplex PCR assay for the detection and identification of the three major clinically important Scedosporium species and validated using sputum specimens collected from patients seen at a major Australian cystic fibrosis clinic. The multiplex PCR assay showed 100% specificity in identifying the three major clinically relevant Scedosporium species from pure culture. When evaluated using DNA extracts from sputa, sensitivity and specificity of the multiplex PCR assay were 62.1% and 97.2%, respectively. This highly species-specific multiplex PCR assay offers a rapid and simple method of detection of the most clinically important Scedosporium species in respiratory tract specimens. PMID:21325557

Development and validation of a multiplex PCR for detection of Scedosporium spp. in respiratory tract specimens from patients with cystic fibrosis.

PubMed

Harun, Azian; Blyth, Christopher C; Gilgado, Felix; Middleton, Peter; Chen, Sharon C-A; Meyer, Wieland

2011-04-01

The emergence of Scedosporium infections in diverse groups of individuals, which are often treatment refractory, warrants timely and accurate laboratory diagnosis. Species- or group-specific primers based on internal transcribed spacer (ITS) sequence polymorphisms were designed for Scedosporium aurantiacum, Scedosporium dehoogii, Scedosporium prolificans, Pseudallescheria boydii species complex (former clade 5)/Pseudallescheria apiosperma (formerly classified as S. apiospermum sensu lato) and Pseudallescheria minutispora. Primers for S. aurantiacum, S. prolificans, and P. boydii species complex/P. apiosperma were incorporated into a multiplex PCR assay for the detection and identification of the three major clinically important Scedosporium species and validated using sputum specimens collected from patients seen at a major Australian cystic fibrosis clinic. The multiplex PCR assay showed 100% specificity in identifying the three major clinically relevant Scedosporium species from pure culture. When evaluated using DNA extracts from sputa, sensitivity and specificity of the multiplex PCR assay were 62.1% and 97.2%, respectively. This highly species-specific multiplex PCR assay offers a rapid and simple method of detection of the most clinically important Scedosporium species in respiratory tract specimens.

Thermally multiplexed polymerase chain reaction.

PubMed

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

2015-07-01

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

Rate of Detection of Multiple Organisms and Clostridium difficile with Stool Multiplex PCR Detection Test in Pediatrics

PubMed Central

Mangla, Saisho; Villalobos, Tibisay

2017-01-01

Abstract Background New multiplex molecular assays have been developed to determine the etiology of infectious gastroenteritis. Unfortunately, these assays can detect multiple organisms simultaneously along with Clostridium difficile (C.diff), making it difficult to differentiate true pathogen vs. colonization. In January 2015, our institution switched from traditional testing methods to a multiplex polymerase chain reaction (PCR) detection test (FilmArrayTM Gastrointestinal Panel. BioFireDX, Salt Lake City, Utah). The objective of our study was to determine the number of FilmArrayTMpanels that detected C.diff and/or multiple organisms. Methods We conducted a retrospective data review of FilmArray™ panels in pediatric patients 18 years and younger from January 2015 to December 2016. Stool samples were received from both inpatient and outpatient setting. Results In 2016, 495 FilmArray™ panels were reviewed and 300 (61%) isolated at least one organism. Among the positives panels, 206 (69%) detected one organism, 73 (24%) detected 2 organisms and 21 (7.0%) detected 3 or more organisms. No more than 4 organisms were detected in a single panel. C.diff was most commonly isolated, found 105 times (25%), and 34 (31%) of these were in children younger than 2 years. Amongst the 105 C.diff isolates, 64% were alone and 35% with another organism. Amongst children younger than 2, C.diff was isolated alone in 13 (38%) samples and with another organism in 21 (62%) samples. In 2015, 353 panels were reviewed with a detection rate of 60.3%. C.Diff was isolated 70 times (24% of total isolates) and 22 (31%) were in children younger than 2 years. Amongst those C.diff isolates, 49% were alone and 51% with another organism. Amongst children younger than 2, C.diff was isolated alone in 8 (38%) samples and with another organism in 14 (62%) samples. Conclusion Although the FilmArray™ Gastrointestinal Panel is a useful single modality for determining the etiology of infectious

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

PubMed

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

2011-01-01

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

Quantitation of Marek's disease and chicken anemia viruses in organs of experimentally infected chickens and commercial chickens by multiplex real-time PCR.

PubMed

Davidson, Irit; Raibshtein, I; Al-Touri, A

2013-06-01

The worldwide distribution of chicken anemia virus (CAV) and Marek's disease virus (MDV) is well documented. In addition to their economic significance in single- or dual-virus infections, the two viruses can often accompany various other pathogens and affect poultry health either directly, by causing tumors, anemia, and delayed growth, or indirectly, by aggravating other diseases, as a result of their immunosuppressive effects. After a decade of employing the molecular diagnosis of those viruses, which replaced conventional virus isolation, we present the development of a real-time multiplex PCR for the simultaneous detection of both viruses. The real-time PCRs for MDV and for CAV alone are more sensitive than the respective end-point PCRs. In addition, the multiplex real-time shows a similar sensitivity when compared to the single real-time PCR for each virus. The newly developed real-time multiplex PCR is of importance in terms of the diagnosis and detection of low copies of each virus, MDV and CAV in single- and in multiple-virus infections, and its applicability will be further evaluated.

Combination of Mass Signal Amplification and Isotope-Labeled Alkanethiols for the Multiplexed Detection of miRNAs.

PubMed

Kang, Hyunook; Hong, Seol-Hye; Sung, Jiha; Yeo, Woon-Seok

2017-08-04

We report a fast and sensitive method for the multiplexed detection of miRNAs by combining mass signal amplification and isotope-labeled signal reporter molecules. In our strategy, target miRNAs are captured specifically by immobilized DNAs on gold nanoparticles (AuNPs), which carry a large number of small molecules, called amplification tags (Am-tags), as the reporter for the detection of target miRNAs. For multiplexed detection, we designed and synthesized four Am-tags containing 0, 4, 8, 12 isotopes so that they had same molecular properties but different molecular weights. By observing the mass signals of the Am-tags on AuNPs decorated along with different probe DNAs, four types of miRNAs in a sample could be easily discriminated, and the relative amounts of these miRNAs could be quantified. The practicability of our strategy was further verified by measuring the expression levels of two miRNAs in HUVECs in response to different CuSO 4 concentrations. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparison of three human papillomavirus DNA detection methods: Next generation sequencing, multiplex-PCR and nested-PCR followed by Sanger based sequencing.

PubMed

da Fonseca, Allex Jardim; Galvão, Renata Silva; Miranda, Angelica Espinosa; Ferreira, Luiz Carlos de Lima; Chen, Zigui

2016-05-01

To compare the diagnostic performance for HPV infection using three laboratorial techniques. Ninty-five cervicovaginal samples were randomly selected; each was tested for HPV DNA and genotypes using 3 methods in parallel: Multiplex-PCR, the Nested PCR followed by Sanger sequencing, and the Next_Gen Sequencing (NGS) with two assays (NGS-A1, NGS-A2). The study was approved by the Brazilian National IRB (CONEP protocol 16,800). The prevalence of HPV by the NGS assays was higher than that using the Multiplex-PCR (64.2% vs. 45.2%, respectively; P = 0.001) and the Nested-PCR (64.2% vs. 49.5%, respectively; P = 0.003). NGS also showed better performance in detecting high-risk HPV (HR-HPV) and HPV16. There was a weak interobservers agreement between the results of Multiplex-PCR and Nested-PCR in relation to NGS for the diagnosis of HPV infection, and a moderate correlation for HR-HPV detection. Both NGS assays showed a strong correlation for detection of HPVs (k = 0.86), HR-HPVs (k = 0.91), HPV16 (k = 0.92) and HPV18 (k = 0.91). NGS is more sensitive than the traditional Sanger sequencing and the Multiplex PCR to genotype HPVs, with promising ability to detect multiple infections, and may have the potential to establish an alternative method for the diagnosis and genotyping of HPV. © 2015 Wiley Periodicals, Inc.

Suspension arrays based on nanoparticle-encoded microspheres for high-throughput multiplexed detection

PubMed Central

Leng, Yuankui

2017-01-01

Spectrometrically or optically encoded microsphere based suspension array technology (SAT) is applicable to the high-throughput, simultaneous detection of multiple analytes within a small, single sample volume. Thanks to the rapid development of nanotechnology, tremendous progress has been made in the multiplexed detecting capability, sensitivity, and photostability of suspension arrays. In this review, we first focus on the current stock of nanoparticle-based barcodes as well as the manufacturing technologies required for their production. We then move on to discuss all existing barcode-based bioanalysis patterns, including the various labels used in suspension arrays, label-free platforms, signal amplification methods, and fluorescence resonance energy transfer (FRET)-based platforms. We then introduce automatic platforms for suspension arrays that use superparamagnetic nanoparticle-based microspheres. Finally, we summarize the current challenges and their proposed solutions, which are centered on improving encoding capacities, alternative probe possibilities, nonspecificity suppression, directional immobilization, and “point of care” platforms. Throughout this review, we aim to provide a comprehensive guide for the design of suspension arrays, with the goal of improving their performance in areas such as multiplexing capacity, throughput, sensitivity, and cost effectiveness. We hope that our summary on the state-of-the-art development of these arrays, our commentary on future challenges, and some proposed avenues for further advances will help drive the development of suspension array technology and its related fields. PMID:26021602

Parallel separations using capillary electrophoresis on a multilane microchip with multiplexed laser-induced fluorescence detection.

PubMed

Nikcevic, Irena; Piruska, Aigars; Wehmeyer, Kenneth R; Seliskar, Carl J; Limbach, Patrick A; Heineman, William R

2010-08-01

Parallel separations using CE on a multilane microchip with multiplexed LIF detection is demonstrated. The detection system was developed to simultaneously record data on all channels using an expanded laser beam for excitation, a camera lens to capture emission, and a CCD camera for detection. The detection system enables monitoring of each channel continuously and distinguishing individual lanes without significant crosstalk between adjacent lanes. Multiple analytes can be determined in parallel lanes within a single microchip in a single run, leading to increased sample throughput. The pK(a) determination of small molecule analytes is demonstrated with the multilane microchip.

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

PubMed

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

2015-02-01

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

Evaluation of an Internally Controlled Multiplex Tth Endonuclease Cleavage Loop-Mediated Isothermal Amplification (TEC-LAMP) Assay for the Detection of Bacterial Meningitis Pathogens

PubMed Central

Clancy, Eoin; Cormican, Martin; Boo, Teck Wee; Cunney, Robert

2018-01-01

Bacterial meningitis infection is a leading global health concern for which rapid and accurate diagnosis is essential to reduce associated morbidity and mortality. Loop-mediated isothermal amplification (LAMP) offers an effective low-cost diagnostic approach; however, multiplex LAMP is difficult to achieve, limiting its application. We have developed novel real-time multiplex LAMP technology, TEC-LAMP, using Tth endonuclease IV and a unique LAMP primer/probe. This study evaluates the analytical specificity, limit of detection (LOD) and clinical application of an internally controlled multiplex TEC-LAMP assay for detection of leading bacterial meningitis pathogens: Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae. Analytical specificities were established by testing 168 bacterial strains, and LODs were determined using Probit analysis. The TEC-LAMP assay was 100% specific, with LODs for S. pneumoniae, N. meningitidis and H. influenzae of 39.5, 17.3 and 25.9 genome copies per reaction, respectively. Clinical performance was evaluated by testing 65 archived PCR-positive samples. Compared to singleplex real-time PCR, the multiplex TEC-LAMP assay demonstrated diagnostic sensitivity and specificity of 92.3% and 100%, respectively. This is the first report of a single-tube internally controlled multiplex LAMP assay for bacterial meningitis pathogen detection, and the first report of Tth endonuclease IV incorporation into nucleic acid amplification diagnostic technology. PMID:29425124

Optimizing diffusion in multiplexes by maximizing layer dissimilarity

NASA Astrophysics Data System (ADS)

Serrano, Alfredo B.; Gómez-Gardeñes, Jesús; Andrade, Roberto F. S.

2017-05-01

Diffusion in a multiplex depends on the specific link distribution between the nodes in each layer, but also on the set of the intralayer and interlayer diffusion coefficients. In this work we investigate, in a quantitative way, the efficiency of multiplex diffusion as a function of the topological similarity among multiplex layers. This similarity is measured by the distance between layers, taken among the pairs of layers. Results are presented for a simple two-layer multiplex, where one of the layers is held fixed, while the other one can be rewired in a controlled way in order to increase or decrease the interlayer distance. The results indicate that, for fixed values of all intra- and interlayer diffusion coefficients, a large interlayer distance generally enhances the global multiplex diffusion, providing a topological mechanism to control the global diffusive process. For some sets of networks, we develop an algorithm to identify the most sensitive nodes in the rewirable layer, so that changes in a small set of connections produce a drastic enhancement of the global diffusion of the whole multiplex system.

A nucleic acid strand displacement system for the multiplexed detection of tuberculosis-specific mRNA using quantum dots

NASA Astrophysics Data System (ADS)

Gliddon, H. D.; Howes, P. D.; Kaforou, M.; Levin, M.; Stevens, M. M.

2016-05-01

The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. We have developed an assay for the detection of multiple mRNA molecules that combines DNA nanotechnology with fluorescent nanomaterials. The core switching mechanism is toehold-mediated strand displacement. We have used fluorescent quantum dots (QDs) as signal transducers in this assay, as they bring many benefits including bright fluorescence and multiplexing abilities. The resulting assay is capable of multiplexed detection of long RNA targets against a high concentration of background non-target RNA, with high sensitivity and specificity and limits of detection in the nanomolar range using only a standard laboratory plate reader. We demonstrate the utility of our QD-based system for the detection of two genes selected from a microarray-derived tuberculosis-specific gene expression signature. Levels of up- and downregulated gene transcripts comprising this signature can be combined to give a disease risk score, making the signature more amenable for use as a diagnostic marker. Our QD-based approach to detect these transcripts could pave the way for novel diagnostic assays for tuberculosis.The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. We have developed an assay for the detection of multiple mRNA molecules that combines DNA nanotechnology with fluorescent nanomaterials. The core switching mechanism is toehold-mediated strand displacement. We have used fluorescent quantum dots (QDs) as signal transducers in this assay, as they bring many benefits including bright fluorescence and multiplexing abilities. The resulting assay is capable of multiplexed detection of long RNA targets against a high concentration of background non-target RNA, with high sensitivity and specificity and limits of detection in the nanomolar

Simultaneous detection and differentiation of three Potyviridae viruses by a multiplex TaqMan real time RT-PCR assay

USDA-ARS?s Scientific Manuscript database

A multiplex TaqMan real time RT-PCR was developed for detection and differentiation of Sweet potato virus G, Sweet potato latent virus and Sweet potato mild mottle virus in one tube. Amplification and detection of a fluorogenic cytochrome oxidase gene was included as an internal control. The assay w...

Simultaneous aptasensor for multiplex pathogenic bacteria detection based on multicolor upconversion nanoparticles labels.

PubMed

Wu, Shijia; Duan, Nuo; Shi, Zhao; Fang, Congcong; Wang, Zhouping

2014-03-18

A highly sensitive and specific multiplex method for the simultaneous detection of three pathogenic bacteria was fabricated using multicolor upconversion nanoparticles (UCNPs) as luminescence labels coupled with aptamers as the molecular recognition elements. Multicolor UCNPs were synthesized via doping with various rare-earth ions to obtain well-separated emission peaks. The aptamer sequences were selected using the systematic evolution of ligands by exponential enrichment (SELEX) strategy for Staphylococcus aureus, Vibrio parahemolyticus, and Salmonella typhimurium. When applied in this method, aptamers can be used for the specific recognition of the bacteria from complex mixtures, including those found in real food matrixes. Aptamers and multicolor UCNPs were employed to selectively capture and simultaneously quantify the three target bacteria on the basis of the independent peaks. Under optimal conditions, the correlation between the concentration of three bacteria and the luminescence signal was found to be linear from 50-10(6) cfu mL(-1). Improved by the magnetic separation and concentration effect of Fe3O4 magnetic nanoparticles, the limits of detection of the developed method were found to be 25, 10, and 15 cfu mL(-1) for S. aureus, V. parahemolyticus, and S. typhimurium, respectively. The capability of the bioassay in real food samples was also investigated, and the results were consistent with experimental results obtained from plate-counting methods. This proposed method for the detection of various pathogenic bacteria based on multicolor UCNPs has great potential in the application of food safety and multiplex nanosensors.

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2017-09-01

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

Engineered nanoconstructs for the multiplexed and sensitive detection of high-risk pathogens

NASA Astrophysics Data System (ADS)

Seo, Youngmin; Kim, Ji-Eun; Jeong, Yoon; Lee, Kwan Hong; Hwang, Jangsun; Hong, Jongwook; Park, Hansoo; Choi, Jonghoon

2016-01-01

Many countries categorize the causative agents of severe infectious diseases as high-risk pathogens. Given their extreme infectivity and potential to be used as biological weapons, a rapid and sensitive method for detection of high-risk pathogens (e.g., Bacillus anthracis, Francisella tularensis, Yersinia pestis, and Vaccinia virus) is highly desirable. Here, we report the construction of a novel detection platform comprising two units: (1) magnetic beads separately conjugated with multiple capturing antibodies against four different high-risk pathogens for simple and rapid isolation, and (2) genetically engineered apoferritin nanoparticles conjugated with multiple quantum dots and detection antibodies against four different high-risk pathogens for signal amplification. For each high-risk pathogen, we demonstrated at least 10-fold increase in sensitivity compared to traditional lateral flow devices that utilize enzyme-based detection methods. Multiplexed detection of high-risk pathogens in a sample was also successful by using the nanoconstructs harboring the dye molecules with fluorescence at different wavelengths. We ultimately envision the use of this novel nanoprobe detection platform in future applications that require highly sensitive on-site detection of high-risk pathogens.

Detection of Gastrointestinal Pathogens from Stool Samples on Hemoccult Cards by Multiplex PCR

PubMed Central

Schlenker, Nicklas; Bauer, Malkin; Helfrich, Kerstin; Mengele, Carolin; Löscher, Thomas; Nothdurft, Hans Dieter; Bretzel, Gisela; Beissner, Marcus

2017-01-01

Purpose. Up to 30% of international travelers are affected by travelers' diarrhea (TD). Reliable data on the etiology of TD is lacking. Sufficient laboratory capacity at travel destinations is often unavailable and transporting conventional stool samples to the home country is inconvenient. We evaluated the use of Hemoccult cards for stool sampling combined with a multiplex PCR for the detection of model viral, bacterial, and protozoal TD pathogens. Methods. Following the creation of serial dilutions for each model pathogen, last positive dilution steps (LPDs) and thereof calculated last positive sample concentrations (LPCs) were compared between conventional stool samples and card samples. Furthermore, card samples were tested after a prolonged time interval simulating storage during a travel duration of up to 6 weeks. Results. The LPDs/LPCs were comparable to testing of conventional stool samples. After storage on Hemoccult cards, the recovery rate was 97.6% for C. jejuni, 100% for E. histolytica, 97.6% for norovirus GI, and 100% for GII. Detection of expected pathogens was possible at weekly intervals up to 42 days. Conclusion. Stool samples on Hemoccult cards stored at room temperature can be used in combination with a multiplex PCR as a reliable tool for testing of TD pathogens. PMID:28408937

Detection of Gastrointestinal Pathogens from Stool Samples on Hemoccult Cards by Multiplex PCR.

PubMed

Alberer, Martin; Schlenker, Nicklas; Bauer, Malkin; Helfrich, Kerstin; Mengele, Carolin; Löscher, Thomas; Nothdurft, Hans Dieter; Bretzel, Gisela; Beissner, Marcus

2017-01-01

Purpose . Up to 30% of international travelers are affected by travelers' diarrhea (TD). Reliable data on the etiology of TD is lacking. Sufficient laboratory capacity at travel destinations is often unavailable and transporting conventional stool samples to the home country is inconvenient. We evaluated the use of Hemoccult cards for stool sampling combined with a multiplex PCR for the detection of model viral, bacterial, and protozoal TD pathogens. Methods . Following the creation of serial dilutions for each model pathogen, last positive dilution steps (LPDs) and thereof calculated last positive sample concentrations (LPCs) were compared between conventional stool samples and card samples. Furthermore, card samples were tested after a prolonged time interval simulating storage during a travel duration of up to 6 weeks. Results . The LPDs/LPCs were comparable to testing of conventional stool samples. After storage on Hemoccult cards, the recovery rate was 97.6% for C. jejuni , 100% for E . histolytica , 97.6% for norovirus GI, and 100% for GII. Detection of expected pathogens was possible at weekly intervals up to 42 days. Conclusion . Stool samples on Hemoccult cards stored at room temperature can be used in combination with a multiplex PCR as a reliable tool for testing of TD pathogens.

Broadband Direct Detection Submillimeter Spectrometer with Multiplexed Superconducting Transition Edge Thermometer Bolometers

NASA Technical Reports Server (NTRS)

Benford, D. J.; Ames, T. A.; Chervenak, J. A.; Moseley, S. H.; Shafer, R. A.; Staguhn, J. G.; Voellmer, G. M.; Pajot, F.; Rioux, C.; Phillips, T. G.;

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

DTIC Science & Technology

2017-07-01

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

One-Step Multiplex RT-qPCR Assay for the Detection of Peste des petits ruminants virus, Capripoxvirus, Pasteurella multocida and Mycoplasma capricolum subspecies (ssp.) capripneumoniae.

PubMed

Settypalli, Tirumala Bharani Kumar; Lamien, Charles Euloge; Spergser, Joachim; Lelenta, Mamadou; Wade, Abel; Gelaye, Esayas; Loitsch, Angelika; Minoungou, Germaine; Thiaucourt, Francois; Diallo, Adama

2016-01-01

Respiratory infections, although showing common clinical symptoms like pneumonia, are caused by bacterial, viral or parasitic agents. These are often reported in sheep and goats populations and cause huge economic losses to the animal owners in developing countries. Detection of these diseases is routinely done using ELISA or microbiological methods which are being reinforced or replaced by molecular based detection methods including multiplex assays, where detection of different pathogens is carried out in a single reaction. In the present study, a one-step multiplex RT-qPCR assay was developed for simultaneous detection of Capripoxvirus (CaPV), Peste de petits ruminants virus (PPRV), Pasteurella multocida (PM) and Mycoplasma capricolum ssp. capripneumonia (Mccp) in pathological samples collected from small ruminants with respiratory disease symptoms. The test performed efficiently without any cross-amplification. The multiplex PCR efficiency was 98.31%, 95.48%, 102.77% and 91.46% whereas the singleplex efficiency was 93.43%, 98.82%, 102.55% and 92.0% for CaPV, PPRV, PM and Mccp, respectively. The correlation coefficient was greater than 0.99 for all the targets in both multiplex and singleplex. Based on cycle threshold values, intra and inter assay variability, ranged between the limits of 2%-4%, except for lower concentrations of Mccp. The detection limits at 95% confidence interval (CI) were 12, 163, 13 and 23 copies/reaction for CaPV, PPRV, PM and Mccp, respectively. The multiplex assay was able to detect CaPVs from all genotypes, PPRV from the four lineages, PM and Mccp without amplifying the other subspecies of mycoplasmas. The discriminating power of the assay was proven by accurate detection of the targeted pathogen (s) by screening 58 viral and bacterial isolates representing all four targeted pathogens. Furthermore, by screening 81 pathological samples collected from small ruminants showing respiratory disease symptoms, CaPV was detected in 17 samples

78 FR 16513 - Application of Advances in Nucleic Acid and Protein Based Detection Methods to Multiplex...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-03-15

... Transfusion-Transmissible Agents and Blood Cell Antigens in Blood Donations; Public Workshop AGENCY: Food and... Methods to Multiplex Detection of Transfusion- Transmissible Agents and Blood Cell Antigens in Blood... and the use of these tests in blood donor screening and blood cell antigen typing. The public workshop...

Detection of intestinal protozoa in paediatric patients with gastrointestinal symptoms by multiplex real-time PCR.

PubMed

Maas, L; Dorigo-Zetsma, J W; de Groot, C J; Bouter, S; Plötz, F B; van Ewijk, B E

2014-06-01

The performance of a multiplex real-time PCR for the detection of Blastocystis, Dientamoeba fragilis, Giardia lamblia, Cryptosporidium species and Entamoeba species in faecal samples was evaluated in an observational prospective study. Paediatric patients (0-18 years) presenting with gastrointestinal symptoms and suspected of having enteroparasitic disease were included. A questionnaire on gastrointestinal symptoms and the chosen treatment was completed at the start of the study and after 6 weeks. Of 163 paediatric patients (mean age, 7.8 years), 114 (70%) had a PCR-positive faecal sample. D. fragilis was detected most frequently, in 101 patients, followed by Blastocystis in 49. In faecal samples of 47 patients, more than one protozoan was detected, mainly the combination of D. fragilis and Blastocystis. Reported gastrointestinal symptoms were abdominal pain (78%), nausea (30%), and altered bowel habits (28%). Eighty-nine of the PCR-positive patients were treated with antibiotics. A significant reduction in abdominal pain was observed both in treated and in untreated patients. This study demonstrated that multiplex real-time PCR detects a high percentage of intestinal protozoa in paediatric patients with gastrointestinal symptoms. However, interpretation and determination of the clinical relevance of a positive PCR result in this population are still difficult. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

Multiplex DNA detection of food allergens on a digital versatile disk.

PubMed

Tortajada-Genaro, Luis A; Santiago-Felipe, Sara; Morais, Sergi; Gabaldón, José Antonio; Puchades, Rosa; Maquieira, Ángel

2012-01-11

The development of a DNA microarray method on a digital versatile disk (DVD) is described for the simultaneous detection of traces of hazelnut ( Corylus avellana L.), peanut ( Arachis hypogaea ), and soybean ( Glycine max ) in foods. After DNA extraction, multiplex PCR was set up using 5'-labeled specific primers for Cor a 1, Ar h 2, and Le genes, respectively. Digoxin-labeled PCR products were detected by hybridization with 5'-biotinylated probes immobilized on a streptavidin-modified DVD surface. The reaction product attenuates the signal intensity of the laser that reached the DVD drive used as detector, correlating well with the amount of amplified sequence. Analytical performances showed a detection limit of 1 μg/g and good assay reproducibility (RSD 8%), suitable for the simultaneous detection of the three targeted allergens. The developed methodology was tested with several commercially available foodstuffs, demonstrating its applicability. The results were in good agreement, in terms of sensitivity and reproducibility, with those obtained with ELISA, PCR-gel agarose electrophoresis, and RT-PCR.

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

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

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

2002-04-17

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

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

PubMed

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

2011-03-10

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

Parallel separations using capillary electrophoresis on a multilane microchip with multiplexed laser induced fluorescence detection

PubMed Central

Nikcevic, Irena; Piruska, Aigars; Wehmeyer, Kenneth R.; Seliskar, Carl J.; Limbach, Patrick A.; Heineman, William R.

2010-01-01

Parallel separations using capillary electrophoresis on a multilane microchip with multiplexed laser induced fluorescence detection is demonstrated. The detection system was developed to simultaneously record data on all channels using an expanded laser beam for excitation, a camera lens to capture emission, and a CCD camera for detection. The detection system enables monitoring of each channel continuously and distinguishing individual lanes without significant crosstalk between adjacent lanes. Multiple analytes can be analyzed on parallel lanes within a single microchip in a single run, leading to increased sample throughput. The pKa determination of small molecule analytes is demonstrated with the multilane microchip. PMID:20737446

Multiplex PCR-based DNA array for simultaneous detection of three human herpesviruses, EVB, CMV and KSHV.

PubMed

Fujimuro, Masahiro; Nakaso, Kazuhiro; Nakashima, Kenji; Sadanari, Hidetaka; Hisanori, Inoue; Teishikata, Yasuhiro; Hayward, S Diane; Yokosawa, Hideyoshi

2006-04-01

Human lymphotropic herpesviruses, Epstein-Barr virus (EBV), cytomegalovirus (CMV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are responsible for a wide variety of human diseases. Due to an increase in diseased states associated with immunosuppression, more instances of co-morbid infections with these herpesviruses have resulted in viral reactivations that have caused numerous fatalities. Therefore, the development of rapid and accurate method to detect these viruses in immunocompromised patients is vital for immediate treatment with antiviral prophylactic drugs. In this study, we developed a new multiplex PCR method coupled to DNA array hybridization, which can simultaneously detect all three human herpesviruses in one single cell sample. Multiplex PCR primers were designed to amplify specific regions of the EBV (EBER1), CMV (IE) and KSHV (LANA) viral genomes. Pre-clinical application of this method revealed that this approach is capable of detecting as few as 1 copy of the viral genomes for KSHV and CMV and 100 copies of the genome for EBV. Furthermore, this highly sensitive test showed no cross-reactivity among the three viruses and is capable of detecting both KSHV and EBV viral genomes simultaneously in the lymphoblastoid cells that have been double infected with both viruses. Thus, this array-based approach serves as a rapid and reliable diagnostic tool for clinical applications.

Universal detection of phytoplasmas and Xylella spp. by TaqMan singleplex and multiplex real-time PCR with dual priming oligonucleotides.

PubMed

Ito, Takao; Suzaki, Koichi

2017-01-01

Phytoplasmas and Xylella spp. are bacteria that cause many economically important plant diseases worldwide. TaqMan probe-based quantitative real-time polymerase chain reaction (qPCR) assays have been utilized to universally detect phytoplasmas or Xylella fastidiosa. To develop a superior universal qPCR method, we used a dual priming oligonucleotide (DPO) with two annealing sites as a reverse primer to target the well-conserved bacterial 16S rDNA. The new qPCR assays universally detected various species of phytoplasmas and subspecies of X. fastidiosa as well as Xylella taiwanensis, and generally showed superior threshold cycle values when amplifying specific or non-specific products compared to current universal qPCR assays. The proposed qPCR assays were integrated to develop a multiplex qPCR assay that simultaneously detected phytoplasmas, Xylella spp., and an internal plant DNA positive control within 1 hour. This assay could detect a minimum of ten bacterial cells and was compatible with crude extractions used in the rapid screening of various plants. The amplicons were of sufficient lengths to be directly sequenced for preliminary identification, and the primers could be used in universal conventional PCR assays. Additionally, reverse DPO primers can be utilized to improve other probe-based qPCR assays.

Universal detection of phytoplasmas and Xylella spp. by TaqMan singleplex and multiplex real-time PCR with dual priming oligonucleotides

PubMed Central

Suzaki, Koichi

2017-01-01

Phytoplasmas and Xylella spp. are bacteria that cause many economically important plant diseases worldwide. TaqMan probe-based quantitative real-time polymerase chain reaction (qPCR) assays have been utilized to universally detect phytoplasmas or Xylella fastidiosa. To develop a superior universal qPCR method, we used a dual priming oligonucleotide (DPO) with two annealing sites as a reverse primer to target the well-conserved bacterial 16S rDNA. The new qPCR assays universally detected various species of phytoplasmas and subspecies of X. fastidiosa as well as Xylella taiwanensis, and generally showed superior threshold cycle values when amplifying specific or non-specific products compared to current universal qPCR assays. The proposed qPCR assays were integrated to develop a multiplex qPCR assay that simultaneously detected phytoplasmas, Xylella spp., and an internal plant DNA positive control within 1 hour. This assay could detect a minimum of ten bacterial cells and was compatible with crude extractions used in the rapid screening of various plants. The amplicons were of sufficient lengths to be directly sequenced for preliminary identification, and the primers could be used in universal conventional PCR assays. Additionally, reverse DPO primers can be utilized to improve other probe-based qPCR assays. PMID:28957362

Detecting and Genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays

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

Call, Douglas R.; Brockman, Fred J.; Chandler, Darrell P.

2000-12-01

Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification.more » The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFU ml-1 (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products and the system is amenable to automation.« less

Detecting and genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays

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

Call, Douglas R.; Brockman, Fred J.; Chandler, Darrell P.

2001-07-05

Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification.more » The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFUs ml-1 (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products and the system is amenable to automation.« less

A novel multiplex PCR assay for simultaneous detection of nine clinically significant bacterial pathogens associated with bovine mastitis.

PubMed

Ashraf, Aqeela; Imran, Muhammad; Yaqub, Tahir; Tayyab, Muhammad; Shehzad, Wasim; Thomson, Peter C

2017-06-01

For rapid and simultaneous detection of nine bovine mastitic pathogens, a sensitive and specific multiplex PCR assay was developed. The assay was standardized using reference strains and validated on mastitic milk cultures which were identified to species level based on 16S rRNA sequencing. Multiplex PCR assay also efficiently detected the target bacterial strains directly from milk. The detection limit of the assay was up to 50 pg for DNA isolated from pure cultures and 10 4  CFU/ml for spiked milk samples. As estimated by latent class analysis, the assay was sensitive up to 88% and specific up to 98% for targeted mastitic pathogens, compared with the bacterial culture method and the 16S rRNA sequence analysis. This novel molecular assay could be useful for monitoring and maintaining the bovine udder health, ensuring the bacteriological safety of milk, and conducting epidemiological studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

Multicolor fluorescent biosensor for multiplexed detection of DNA.

PubMed

Hu, Rong; Liu, Tao; Zhang, Xiao-Bing; Huan, Shuang-Yan; Wu, Cuichen; Fu, Ting; Tan, Weihong

2014-05-20

Development of efficient methods for highly sensitive and rapid screening of specific oligonucleotide sequences is essential to the early diagnosis of serious diseases. In this work, an aggregated cationic perylene diimide (PDI) derivative was found to efficiently quench the fluorescence emission of a variety of anionic oligonucleotide-labeled fluorophores that emit at wavelengths from the visible to NIR region. This broad-spectrum quencher was then adopted to develop a multicolor biosensor via a label-free approach for multiplexed fluorescent detection of DNA. The aggregated perylene derivative exhibits a very high quenching efficiency on all ssDNA-labeled dyes associated with biosensor detection, having efficiency values of 98.3 ± 0.9%, 97 ± 1.1%, and 98.2 ± 0.6% for FAM, TAMRA, and Cy5, respectively. An exonuclease-assisted autocatalytic target recycling amplification was also integrated into the sensing system. High quenching efficiency combined with autocatalytic target recycling amplification afforded the biosensor with high sensitivity toward target DNA, resulting in a detection limit of 20 pM, which is about 50-fold lower than that of traditional unamplified homogeneous fluorescent assay methods. The quencher did not interfere with the catalytic activity of nuclease, and the biosensor could be manipulated in either preaddition or postaddition manner with similar sensitivity. Moreover, the proposed sensing system allows for simultaneous and multicolor analysis of several oligonucleotides in homogeneous solution, demonstrating its potential application in the rapid screening of multiple biotargets.

SERS based immuno-microwell arrays for multiplexed detection of foodborne pathogenic bacteria

NASA Astrophysics Data System (ADS)

Sun, Jian; Hankus, Mikella E.; Cullum, Brian M.

2009-05-01

A novel surface enhanced Raman scattering (SERS)-based immuno-microwell array has been developed for multiplexed detection of foodborne pathogenic bacteria. The immuno-microwell array was prepared by immobilizing the optical addressable immunomagnetic beads (IMB) into the microwell array on one end of a fiber optic bundle. The IMBs, magnetic beads coated with specific antibody to specific bacteria, were used for immunomagnetic separation (IMS) of corresponding bacteria. The magnetic separation by the homemade magnetic separation system was evaluated in terms of the influences of several important parameters including the beads concentration, the sample volume and the separation time. IMS separation efficiency of the model bacteria E.coli O157:H7 was 63% in 3 minutes. The microwell array was fabricated on hydrofluoric acid etched end of a fiber optic bundle containing 30,000 fiber elements. After being coated with silver, the microwell array was used as a uniform SERS substrate with the relative standard deviation of the SERS enhancement across the microwell array < 2% and the enhancement factor as high as 2.18 x 107. The antibody modified microwell array was prepared for bacteria immobilization into the microwell array, which was characterized by a sandwich immunoassay. To demonstrate the potential of multiplexed SERS detection with the immuno-microwell array, the SERS spectra of different Raman dye labeled magnetic beads as well as mixtures were measured on the mircrowell array. In bead mixture, different beads were identified by the characteristic SERS bands of the corresponding Raman label.

A multiplex method for detection of glucose-6-phosphate dehydrogenase (G6PD) gene mutations.

PubMed

Zhang, L; Yang, Y; Liu, R; Li, Q; Yang, F; Ma, L; Liu, H; Chen, X; Yang, Z; Cui, L; He, Y

2015-12-01

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect caused by G6PD gene mutations. This study aimed to develop a cost-effective, multiplex, genotyping method for detecting common mutations in the G6PD gene. We used a SNaPshot approach to genotype multiple G6PD mutations that are common to human populations in South-East Asia. This assay is based on multiplex PCR coupled with primer extension reactions. Different G6PD gene mutations were determined by peak retention time and colors of the primer extension products. We designed PCR primers for multiplex amplification of the G6PD gene fragments and for primer extension reactions to genotype 11 G6PD mutations. DNA samples from a total of 120 unrelated G6PD-deficient individuals from the China-Myanmar border area were used to establish and validate this method. Direct sequencing of the PCR products demonstrated 100% concordance between the SNaPshot and the sequencing results. The SNaPshot method offers a specific and sensitive alternative for simultaneously interrogating multiple G6PD mutations. © 2015 John Wiley & Sons Ltd.

Multiplexed Lateral Flow Test for Detection and Differentiation of Cronobacter sakazakii Serotypes O1 and O2

PubMed Central

Scharinger, Eva J.; Dietrich, Richard; Wittwer, Tobias; Märtlbauer, Erwin; Schauer, Kristina

2017-01-01

The ubiquitous and opportunistic pathogen Cronobacter sakazakii is responsible for severe meningitis, sepsis, and necrotizing enterocolitis in neonates and infants associated with ingestion of contaminated powdered infant formula (PIF). The current ISO method for isolation and detection of Cronobacter spp. is laborious, time-consuming and expensive. In this study, a multiplexed lateral flow test strip was developed to rapidly detect and simultaneously serotype O1 and O2 C. sakazakii serotypes. The assay is based on two monoclonal antibodies (MAb) that specifically bind to the lipopolysaccharides (LPS) of these pathogens. The test strip provides results very quickly; C. sakazakii could be detected in pure culture within 15 min with a sensitivity of 107 CFU/ml. After non-selective enrichment for 18 h as low as one Cronobacter cell per g PIF could be detected. Moreover, the established lateral flow assay (LFA) offers excellent specificity showing no cross-reactivity with other C. sakazakii serotypes, Cronobacter species or Enterobacteriaceae tested. These characteristics, together with several advantages such as speed, simplicity in performance, low analysis cost, and no requirement of specialized skills or sophisticated equipment make the developed multiplexed LFA suitable for reliable detection and serotyping of C. sakazakii serotypes O1 and O2. PMID:28979257

Multiplexed Lateral Flow Test for Detection and Differentiation of Cronobacter sakazakii Serotypes O1 and O2.

PubMed

Scharinger, Eva J; Dietrich, Richard; Wittwer, Tobias; Märtlbauer, Erwin; Schauer, Kristina

2017-01-01

The ubiquitous and opportunistic pathogen Cronobacter sakazakii is responsible for severe meningitis, sepsis, and necrotizing enterocolitis in neonates and infants associated with ingestion of contaminated powdered infant formula (PIF). The current ISO method for isolation and detection of Cronobacter spp. is laborious, time-consuming and expensive. In this study, a multiplexed lateral flow test strip was developed to rapidly detect and simultaneously serotype O1 and O2 C. sakazakii serotypes. The assay is based on two monoclonal antibodies (MAb) that specifically bind to the lipopolysaccharides (LPS) of these pathogens. The test strip provides results very quickly; C. sakazakii could be detected in pure culture within 15 min with a sensitivity of 10 7 CFU/ml. After non-selective enrichment for 18 h as low as one Cronobacter cell per g PIF could be detected. Moreover, the established lateral flow assay (LFA) offers excellent specificity showing no cross-reactivity with other C. sakazakii serotypes, Cronobacter species or Enterobacteriaceae tested. These characteristics, together with several advantages such as speed, simplicity in performance, low analysis cost, and no requirement of specialized skills or sophisticated equipment make the developed multiplexed LFA suitable for reliable detection and serotyping of C. sakazakii serotypes O1 and O2.

Quantitative chemical imaging with background-free multiplex coherent anti-Stokes Raman scattering by dual-soliton Stokes pulses

PubMed Central

Chen, Kun; Wu, Tao; Wei, Haoyun; Zhou, Tian; Li, Yan

2016-01-01

Coherent anti-Stokes Raman microscopy (CARS) is a quantitative, chemically specific, and label-free optical imaging technique for studying inhomogeneous systems. However, the complicating influence of the nonresonant response on the CARS signal severely limits its sensitivity and specificity and especially limits the extent to which CARS microscopy has been used as a fully quantitative imaging technique. On the basis of spectral focusing mechanism, we establish a dual-soliton Stokes based CARS microspectroscopy and microscopy scheme capable of quantifying the spatial information of densities and chemical composition within inhomogeneous samples, using a single fiber laser. Dual-soliton Stokes scheme not only removes the nonresonant background but also allows robust acquisition of multiple characteristic vibrational frequencies. This all-fiber based laser source can cover the entire fingerprint (800-2200 cm−1) region with a spectral resolution of 15 cm−1. We demonstrate that quantitative degree determination of lipid-chain unsaturation in the fatty acids mixture can be achieved by the characterization of C = C stretching and CH2 deformation vibrations. For microscopy purposes, we show that the spatially inhomogeneous distribution of lipid droplets can be further quantitatively visualized using this quantified degree of lipid unsaturation in the acyl chain for contrast in the hyperspectral CARS images. The combination of compact excitation source and background-free capability to facilitate extraction of quantitative composition information with multiplex spectral peaks will enable wider applications of quantitative chemical imaging in studying biological and material systems. PMID:27867704

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.

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

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

PubMed

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

2015-03-01

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

A real-time multi-gases detection and concentration measurements based-on time-division multiplexed-lasers

NASA Astrophysics Data System (ADS)

Yazdandoust, Fatemeh; Tatenguem Fankem, Hervé; Milde, Tobias; Jimenez, Alvaro; Sacher, Joachim

2018-02-01

We report the development of a platform, based-on a Field-Programmable Gate Arrays (FPGAs) and suitable for Time-Division-Multiplexed DFB lasers. The designed platform is subsequently combined with a spectroscopy setup, for detection and quantification of species in a gas mixture. The experimental results show a detection limit of 460 ppm, an uncertainty of 0.1% and a computation time of less than 1000 clock cycles. The proposed system offers a high level of flexibility and is applicable to arbitrary types of gas-mixtures.

In Vivo 18-FDG/18-Choline-Mediated Cerenkov Radiation Energy Transfer (CRET) Multiplexed Optical Imaging for Human Prostate Carcinoma Detection and Staging

DTIC Science & Technology

2014-10-01

Transfer ( CRET ) Multiplexed Optical Imaging for Human Prostate Carcinoma Detection and Staging PRINCIPAL INVESTIGATOR: Susan L. Deutscher...SUBTITLE 5a. CONTRACT NUMBER In Vivo 18-FDG/18-Choline-Mediated Cerenkov Radiation Energy Transfer ( CRET ) Multiplexed Optical Imaging for Human...internal illumination via 18F-fluorocholine Cerenkov radiation energy transfer ( CRET ) coupled with TF- and ErbB2/3- molecularly targeted near-infrared

Development of a multiplex assay for genus and species-specific detection of Phytophthora based on differences in mitochondrial gene order

USDA-ARS?s Scientific Manuscript database

The availability of a molecular diagnostic assay for Phytophthora that is specific, sensitive, has both genus and species specific detection capabilities multiplexed and can be used to systematically develop markers for detection of a wide range of species would facilitate research and regulatory ef...

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

PubMed Central

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

2011-01-01

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

Comparative Evaluation of Multiplex PCR and Routine Laboratory Phenotypic Methods for Detection of Carbapenemases among Gram Negative Bacilli.

PubMed

Solanki, Rachana; Vanjari, Lavanya; Subramanian, Sreevidya; B, Aparna; E, Nagapriyanka; Lakshmi, Vemu

2014-12-01

Carbapenem resistant pathogens cause infections associated with significant morbidity and mortality. This study evaluates the use of Multiplex PCR for rapid detection of carbapenemase genes among carbapenem resistant Gram negative bacteria in comparison with the existing phenotypic methods like modified Hodge test (MHT), combined disc test (CDT) and automated methods. A total of 100 Carbapenem resistant clinical isolates, [Escherichia coli (25), Klebsiella pneumoniae (35) P. aeruginosa (18) and Acinetobacter baumannii (22)] were screened for the presence of carbapenemases (bla NDM-1, bla VIM , blaIMP and blaKPC genes) by phenotype methods such as the modified Hodge test (MHT) and combined disc test (CDT) and the molecular methods such as Multiplex PCR. Seventy of the 100 isolates were MHT positive while, 65 isolates were positive by CDT. All the CDT positive isolates with EDTA and APB were Metallo betalactamase (MBL) and K. pneumoniae carbapenemase (KPC) producers respectively. bla NDM-1 was present as a lone gene in 44 isolates. In 14 isolates bla NDM-1 gene was present with blaKPC gene, and in one isolate bla NDM-1 gene was present with blaVIM , gene. Only one E. coli isolate had a lone blaKPC gene. We didn't find bla IMP gene in any of the isolates. Neither of the genes could be detected in 35 isolates. Accurate detection of the genes related with carbapenemase production by Molecular methods like Multiplex PCR overcome the limitations of the phenotypic methods and Automated systems.

A new methodology for rapid detection of Lactobacillus delbrueckii subsp. bulgaricus based on multiplex PCR.

PubMed

Nikolaou, Anastasios; Saxami, Georgia; Kourkoutas, Yiannis; Galanis, Alex

2011-02-01

In this study we present a novel multiplex PCR assay for rapid and efficient detection of Lactobacillus delbrueckii subsp. bulgaricus. The accuracy of our method was confirmed by the successful identification of L. delbrueckii subsp. bulgaricus in commercial yoghurts and food supplements and it may be readily applied to the food industry. Copyright © 2010 Elsevier B.V. All rights reserved.

Multiplex Polymerase Chain Reaction for Detection of Gastrointestinal Pathogens in Migrant Workers in Qatar.

PubMed

Humphrey, John M; Ranbhise, Sanjay; Ibrahim, Emad; Al-Romaihi, Hamad E; Farag, Elmoubasher; Abu-Raddad, Laith J; Glesby, Marshall J

2016-12-07

The causes of infectious diarrhea among the migrant worker population in Qatar are not well understood. We conducted a prospective observational study to understand the demographic and clinical characteristics and infectious causes of diarrhea among migrant workers in Doha, Qatar. A total of 126 male workers presenting to the Qatar Red Crescent Worker's Health Center outpatient clinic or emergency department were studied over a 5-month period in 2015-2016. Epidemiologic surveys were administered to all subjects and the prevalence of 22 different stool pathogens was determined using multiplex polymerase chain reaction (PCR) (FilmArray ® Gastrointestinal PCR). A target pathogen was identified in 62.7% of subjects. Enteropathogenic Escherichia coli was the most prevalent pathogen and was detected in 24.6% of subjects, followed by Salmonella (22.2%), enteroaggregative E. coli (15.1%), Giardia lamblia (9.5%), and enterotoxigenic E. coli (8.7%). Multiple pathogens were identified in 49.3% of positive stool samples. In a multivariable analysis, the presence of a heart rate ≥ 90 (adjusted odds ratio [OR] = 3.7, 95% confidence interval [CI] = 1.4-10.0) and > 5 fecal leukocytes/high-power field (adjusted OR = 2.8, 95% CI = 1.2-7.0) were significant predictors of detecting an acute inflammatory pathogen by PCR. Use of multiplex PCR enabled the detection of gastrointestinal pathogens in a high proportion of cases, illustrating the utility of this diagnostic tool in epidemiologic studies of infectious diarrhea. © The American Society of Tropical Medicine and Hygiene.

Multiplex, quantitative cellular analysis in large tissue volumes with clearing-enhanced 3D microscopy (Ce3D)

PubMed Central

Li, Weizhe; Germain, Ronald N.

2017-01-01

Organ homeostasis, cellular differentiation, signal relay, and in situ function all depend on the spatial organization of cells in complex tissues. For this reason, comprehensive, high-resolution mapping of cell positioning, phenotypic identity, and functional state in the context of macroscale tissue structure is critical to a deeper understanding of diverse biological processes. Here we report an easy to use method, clearing-enhanced 3D (Ce3D), which generates excellent tissue transparency for most organs, preserves cellular morphology and protein fluorescence, and is robustly compatible with antibody-based immunolabeling. This enhanced signal quality and capacity for extensive probe multiplexing permits quantitative analysis of distinct, highly intermixed cell populations in intact Ce3D-treated tissues via 3D histo-cytometry. We use this technology to demonstrate large-volume, high-resolution microscopy of diverse cell types in lymphoid and nonlymphoid organs, as well as to perform quantitative analysis of the composition and tissue distribution of multiple cell populations in lymphoid tissues. Combined with histo-cytometry, Ce3D provides a comprehensive strategy for volumetric quantitative imaging and analysis that bridges the gap between conventional section imaging and disassociation-based techniques. PMID:28808033

Quantitative surface-enhanced resonance Raman scattering of phthalocyanine-labelled oligonucleotides

PubMed Central

Macaskill, A.; Chernonosov, A. A.; Koval, V. V.; Lukyanets, E. A.; Fedorova, O. S.; Smith, W. E.; Faulds, K.; Graham, D.

2007-01-01

The evaluation of phthalocyanine labels for the surface-enhanced resonance Raman scattering (SERRS) detection of oligonucleotides is reported. Three phthalocyanine-labelled oligonucleotides were assessed, each containing a different metal centre. Detection limits for each labelled oligonucleotide were determined using two excitation frequencies where possible. Limits of detection as low as 2.8 × 10−11 mol. dm−3 were obtained which are comparable to standard fluorescently labelled probes used in previous SERRS studies. The identification of two phthalocyanine-labelled oligonucleotides without separation was also demonstrated indicating their suitability for multiplexing. This study extends the range of labels suitable for quantitative surface-enhanced resonance Raman scattering with silver nanoparticles and offers more flexibility and choice when considering SERRS for quantitative DNA detection. PMID:17289751

Influence of enrichment broths on multiplex PCR detection of total coliform bacteria, Escherichia coli and Clostridium perfringens, in spiked water samples.

PubMed

Worakhunpiset, S; Tharnpoophasiam, P

2009-07-01

Although multiplex PCR amplification condition for simultaneous detection of total coliform bacteria, Escherichia coli and Clostridium perfringens in water sample has been developed, results with high sensitivity are obtained when amplifying purified DNA, but the sensitivity is low when applied to spiked water samples. An enrichment broth culture prior PCR analysis increases sensitivity of the test but the specific nature of enrichment broth can affect the PCR results. Three enrichment broths, lactose broth, reinforced clostridial medium and fluid thioglycollate broth, were compared for their influence on sensitivity and on time required with multiplex PCR assay. Fluid thioglycollate broth was the most effective with shortest enrichment time and lowest detection limit.

TiO2 Nanolayer-Enhanced Fluorescence for Simultaneous Multiplex Mycotoxin Detection by Aptamer Microarrays on a Porous Silicon Surface.

PubMed

Liu, Rui; Li, Wei; Cai, Tingting; Deng, Yang; Ding, Zhi; Liu, Yan; Zhu, Xuerui; Wang, Xin; Liu, Jie; Liang, Baowen; Zheng, Tiesong; Li, Jianlin

2018-05-02

A new aptamer microarray method on the TiO 2 -porous silicon (PSi) surface was developed to simultaneously screen multiplex mycotoxins. The TiO 2 nanolayer on the surface of PSi can enhance the fluorescence intensity 14 times than that of the thermally oxidized PSi. The aptamer fluorescence signal recovery principle was performed on the TiO 2 -PSi surface by hybridization duplex strand DNA from the mycotoxin aptamer and antiaptamer, respectively, labeled with fluorescence dye and quencher. The aptamer microarray can simultaneously screen for multiplex mycotoxins with a dynamic linear detection range of 0.1-10 ng/mL for ochratoxin A (OTA), 0.01-10 ng/mL for aflatoxins B 1 (AFB 1 ), and 0.001-10 ng/mL for fumonisin B 1 (FB 1 ) and limits of detection of 15.4, 1.48, and 0.21 pg/mL for OTA, AFB 1 , and FB 1 , respectively. The newly developed method shows good specificity and recovery rates. This method can provide a simple, sensitive, and cost-efficient platform for simultaneous screening of multiplex mycotoxins and can be easily expanded to the other aptamer-based protocol.

Detection of nucleotide-specific CRISPR/Cas9 modified alleles using multiplex ligation detection

PubMed Central

KC, R.; Srivastava, A.; Wilkowski, J. M.; Richter, C. E.; Shavit, J. A.; Burke, D. T.; Bielas, S. L.

2016-01-01

CRISPR/Cas9 genome-editing has emerged as a powerful tool to create mutant alleles in model organisms. However, the precision with which these mutations are created has introduced a new set of complications for genotyping and colony management. Traditional gene-targeting approaches in many experimental organisms incorporated exogenous DNA and/or allele specific sequence that allow for genotyping strategies based on binary readout of PCR product amplification and size selection. In contrast, alleles created by non-homologous end-joining (NHEJ) repair of double-stranded DNA breaks generated by Cas9 are much less amenable to such strategies. Here we describe a novel genotyping strategy that is cost effective, sequence specific and allows for accurate and efficient multiplexing of small insertion-deletions and single-nucleotide variants characteristic of CRISPR/Cas9 edited alleles. We show that ligation detection reaction (LDR) can be used to generate products that are sequence specific and uniquely detected by product size and/or fluorescent tags. The method works independently of the model organism and will be useful for colony management as mutant alleles differing by a few nucleotides become more prevalent in experimental animal colonies. PMID:27557703

Multiplex Detection of IgG and IgM to Rift Valley Fever Virus Nucleoprotein, Nonstructural Proteins, and Glycoprotein in Ovine and Bovine.

PubMed

Hossain, Mohammad M; Wilson, William C; Faburay, Bonto; Richt, Jürgen; McVey, David S; Rowland, Raymond R

2016-08-01

A multiplex fluorescence microsphere immunoassay (FMIA) was used to detect bovine and ovine IgM and IgG antibodies to several Rift Valley fever virus (RVFV) proteins, including the major surface glycoprotein, Gn; the nonstructural proteins, NSs and NSm; and the nucleoprotein, N. Target antigens were assembled into a multiplex and tested in serum samples from infected wild-type RVFV or MP12, a modified live virus vaccine. As expected, the N protein was immunodominant and the best target for early detection of infection. Antibody activity against the other targets was also detected. The experimental results demonstrate the capabilities of FMIA for the detection of antibodies to RVFV structural and nonstructural proteins, which can be applied to future development and validation of diagnostic tests that can be used to differentiate vaccinated from infected animals.

DNA Differential Diagnosis of Taeniasis and Cysticercosis by Multiplex PCR

PubMed Central

Yamasaki, Hiroshi; Allan, James C.; Sato, Marcello Otake; Nakao, Minoru; Sako, Yasuhito; Nakaya, Kazuhiro; Qiu, Dongchuan; Mamuti, Wulamu; Craig, Philip S.; Ito, Akira

2004-01-01

Multiplex PCR was established for differential diagnosis of taeniasis and cysticercosis, including their causative agents. For identification of the parasites, multiplex PCR with cytochrome c oxidase subunit 1 gene yielded evident differential products unique for Taenia saginata and Taenia asiatica and for American/African and Asian genotypes of Taenia solium with molecular sizes of 827, 269, 720, and 984 bp, respectively. In the PCR-based detection of tapeworm carriers using fecal samples, the diagnostic markers were detected from 7 of 14 and 4 of 9 T. solium carriers from Guatemala and Indonesia, respectively. Test sensitivity may have been reduced by the length of time (up to 12 years) that samples were stored and/or small sample volumes (ca. 30 to 50 mg). However, the diagnostic markers were detected by nested PCR in five worm carriers from Guatemalan cases that were found to be negative by multiplex PCR. It was noteworthy that a 720 bp-diagnostic marker was detected from a T. solium carrier who was egg-free, implying that it is possible to detect worm carriers and treat before mature gravid proglottids are discharged. In contrast to T. solium carriers, 827-bp markers were detected by multiplex PCR in all T. saginata carriers. The application of the multiplex PCR would be useful not only for surveillance of taeniasis and cysticercosis control but also for the molecular epidemiological survey of these cestode infections. PMID:14766815

Detection of diarrheagenic Escherichia coli by use of melting-curve analysis and real-time multiplex PCR.

PubMed

Guion, Chase E; Ochoa, Theresa J; Walker, Christopher M; Barletta, Francesca; Cleary, Thomas G

2008-05-01

Diarrheagenic Escherichia coli strains are important causes of diarrhea in children from the developing world and are now being recognized as emerging enteropathogens in the developed world. Current methods of detection are too expensive and labor-intensive for routine detection of these organisms to be practical. We developed a real-time fluorescence-based multiplex PCR for the detection of all six of the currently recognized classes of diarrheagenic E. coli. The primers were designed to specifically amplify eight different virulence genes in the same reaction: aggR for enteroaggregative E. coli, stIa/stIb and lt for enterotoxigenic E. coli, eaeA for enteropathogenic E. coli and Shiga toxin-producing E. coli (STEC), stx(1) and stx(2) for STEC, ipaH for enteroinvasive E. coli, and daaD for diffusely adherent E. coli (DAEC). Eighty-nine of ninety diarrheagenic E. coli and 36/36 nonpathogenic E. coli strains were correctly identified using this approach (specificity, 1.00; sensitivity, 0.99). The single false negative was a DAEC strain. The total time between preparation of DNA from E. coli colonies on agar plates and completion of PCR and melting-curve analysis was less than 90 min. The cost of materials was low. Melting-point analysis of real-time multiplex PCR is a rapid, sensitive, specific, and inexpensive method for detection of diarrheagenic E. coli.

Multiplexed nanoplasmonic biosensor for one-step simultaneous detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine.

PubMed

Soler, Maria; Belushkin, Alexander; Cavallini, Andrea; Kebbi-Beghdadi, Carole; Greub, Gilbert; Altug, Hatice

2017-08-15

Development of rapid and multiplexed diagnostic tools is a top priority to address the current epidemic problem of sexually transmitted diseases. Here we introduce a novel nanoplasmonic biosensor for simultaneous detection of the two most common bacterial infections: Chlamydia trachomatis and Neisseria gonorrhoeae. Our plasmonic microarray is composed of gold nanohole sensor arrays that exhibit the extraordinary optical transmission (EOT), providing highly sensitive analysis in a label-free configuration. The integration in a microfluidic system and the precise immobilization of specific antibodies on the individual sensor arrays allow for selective detection and quantification of the bacteria in real-time. We achieved outstanding sensitivities for direct immunoassay of urine samples, with a limit of detection of 300 colony forming units (CFU)/mL for C. trachomatis and 1500CFU/mL for N. gonorrhoeae. The multiplexing capability of our biosensor was demonstrated by analyzing different urine samples spiked with either C. trachomatis or N. gonorrhoeae, and also containing both bacteria. We could successfully detect, identify and quantify the levels of the two bacteria in a one-step assay, without the need for DNA extraction or amplification techniques. This work opens up new possibilities for the implementation of point-of-care biosensors that enable fast, simple and efficient diagnosis of sexually transmitted infections. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

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

Detection and Identification of Probiotic Lactobacillus plantarum Strains by Multiplex PCR Using RAPD-Derived Primers

PubMed Central

Galanis, Alex; Kourkoutas, Yiannis; Tassou, Chrysoula C.; Chorianopoulos, Nikos

2015-01-01

Lactobacillus plantarum 2035 and Lactobacillus plantarum ACA-DC 2640 are two lactic acid bacteria (LAB) strains that have been isolated from Feta cheese. Both display significant potential for the production of novel probiotic food products. The aim of the present study was the development of an accurate and efficient method for the molecular detection and identification of the above strains in a single reaction. A multiplex PCR assay was designed for each strain, based on specific primers derived from Random Amplified Polymorphic DNA (RAPD) Sequenced Characterized Amplified Region (SCAR) analysis. The specificity of the assay was tested with a total of 23 different LAB strains, for L. plantarum 2035 and L. plantarum ACA-DC 2640. The multiplex PCR assay was also successfully applied for the detection of the above cultures in yogurt samples prepared in our lab. The proposed methodology may be applied for monitoring the presence of these strains in food products, thus evaluating their probiotic character. Moreover, our strategy may be adapted for other novel LAB strains with probiotic potential, thus providing a powerful tool for molecular discrimination that could be invaluable to the food industry. PMID:26506345

Detection and Identification of Probiotic Lactobacillus plantarum Strains by Multiplex PCR Using RAPD-Derived Primers.

PubMed

Galanis, Alex; Kourkoutas, Yiannis; Tassou, Chrysoula C; Chorianopoulos, Nikos

2015-10-22

Lactobacillus plantarum 2035 and Lactobacillus plantarum ACA-DC 2640 are two lactic acid bacteria (LAB) strains that have been isolated from Feta cheese. Both display significant potential for the production of novel probiotic food products. The aim of the present study was the development of an accurate and efficient method for the molecular detection and identification of the above strains in a single reaction. A multiplex PCR assay was designed for each strain, based on specific primers derived from Random Amplified Polymorphic DNA (RAPD) Sequenced Characterized Amplified Region (SCAR) analysis. The specificity of the assay was tested with a total of 23 different LAB strains, for L. plantarum 2035 and L. plantarum ACA-DC 2640. The multiplex PCR assay was also successfully applied for the detection of the above cultures in yogurt samples prepared in our lab. The proposed methodology may be applied for monitoring the presence of these strains in food products, thus evaluating their probiotic character. Moreover, our strategy may be adapted for other novel LAB strains with probiotic potential, thus providing a powerful tool for molecular discrimination that could be invaluable to the food industry.

Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations.

PubMed

Zonta, Eleonora; Garlan, Fanny; Pécuchet, Nicolas; Perez-Toralla, Karla; Caen, Ouriel; Milbury, Coren; Didelot, Audrey; Fabre, Elizabeth; Blons, Hélène; Laurent-Puig, Pierre; Taly, Valérie

2016-01-01

In cancer research, the accuracy of the technology used for biomarkers detection is remarkably important. In this context, digital PCR represents a highly sensitive and reproducible method that could serve as an appropriate tool for tumor mutational status analysis. In particular, droplet-based digital PCR approaches have been developed for detection of tumor-specific mutated alleles within plasmatic circulating DNA. Such an approach calls for the development and validation of a very significant quantity of assays, which can be extremely costly and time consuming. Herein, we evaluated assays for the detection and quantification of various mutations occurring in three genes often misregulated in cancers: the epidermal growth factor receptor (EGFR), the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and the Tumoral Protein p53 (TP53) genes. In particular, commercial competitive allele-specific TaqMan® PCR (castPCR™) technology, as well as TaqMan® and ZEN™ assays, have been evaluated for EGFR p.L858R, p.T790M, p.L861Q point mutations and in-frame deletions Del19. Specificity and sensitivity have been determined on cell lines DNA, plasmatic circulating DNA of lung cancer patients or Horizon Diagnostics Reference Standards. To show the multiplexing capabilities of this technology, several multiplex panels for EGFR (several three- and four-plexes) have been developed, offering new "ready-to-use" tests for lung cancer patients.

Methods for detection of GMOs in food and feed.

PubMed

Marmiroli, Nelson; Maestri, Elena; Gullì, Mariolina; Malcevschi, Alessio; Peano, Clelia; Bordoni, Roberta; De Bellis, Gianluca

2008-10-01

This paper reviews aspects relevant to detection and quantification of genetically modified (GM) material within the feed/food chain. The GM crop regulatory framework at the international level is evaluated with reference to traceability and labelling. Current analytical methods for the detection, identification, and quantification of transgenic DNA in food and feed are reviewed. These methods include quantitative real-time PCR, multiplex PCR, and multiplex real-time PCR. Particular attention is paid to methods able to identify multiple GM events in a single reaction and to the development of microdevices and microsensors, though they have not been fully validated for application.

Exploring target-specific primer extension in combination with a bead-based suspension array for multiplexed detection and typing using Streptococcus suis as a model pathogen

PubMed Central

van der Wal, Fimme J.; Achterberg, René P.; van Solt-Smits, Conny; Bergervoet, Jan H. W.; de Weerdt, Marjanne; Wisselink, Henk J.

2017-01-01

We investigated the feasibility of an assay based on target-specific primer extension, combined with a suspension array, for the multiplexed detection and typing of a veterinary pathogen in animal samples, using Streptococcus suis as a model pathogen. A procedure was established for simultaneous detection of 6 S. suis targets in pig tonsil samples (i.e., 4 genes associated with serotype 1, 2, 7, or 9, the generic S. suis glutamate dehydrogenase gene [gdh], and the gene encoding the extracellular protein factor [epf]). The procedure was set up as a combination of protocols: DNA isolation from porcine tonsils, a multiplex PCR, a multiplex target-specific primer extension, and finally a suspension array as the readout. The resulting assay was compared with a panel of conventional PCR assays. The proposed multiplex assay can correctly identify the serotype of isolates and is capable of simultaneous detection of multiple targets in porcine tonsillar samples. The assay is not as sensitive as the current conventional PCR assays, but with the correct sampling strategy, the assay can be useful for screening pig herds to establish which S. suis serotypes are circulating in a pig population. PMID:28980519

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

PubMed Central

Chavada, Ruchir; Maley, Michael

2015-01-01

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

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.

Using Next Generation Sequencing for Multiplexed Trait-Linked Markers in Wheat

PubMed Central

Bernardo, Amy; Wang, Shan; St. Amand, Paul; Bai, Guihua

2015-01-01

With the advent of next generation sequencing (NGS) technologies, single nucleotide polymorphisms (SNPs) have become the major type of marker for genotyping in many crops. However, the availability of SNP markers for important traits of bread wheat ( Triticum aestivum L.) that can be effectively used in marker-assisted selection (MAS) is still limited and SNP assays for MAS are usually uniplex. A shift from uniplex to multiplex assays will allow the simultaneous analysis of multiple markers and increase MAS efficiency. We designed 33 locus-specific markers from SNP or indel-based marker sequences that linked to 20 different quantitative trait loci (QTL) or genes of agronomic importance in wheat and analyzed the amplicon sequences using an Ion Torrent Proton Sequencer and a custom allele detection pipeline to determine the genotypes of 24 selected germplasm accessions. Among the 33 markers, 27 were successfully multiplexed and 23 had 100% SNP call rates. Results from analysis of "kompetitive allele-specific PCR" (KASP) and sequence tagged site (STS) markers developed from the same loci fully verified the genotype calls of 23 markers. The NGS-based multiplexed assay developed in this study is suitable for rapid and high-throughput screening of SNPs and some indel-based markers in wheat. PMID:26625271

Simultaneous detection of the three ilarviruses affecting stone fruit trees by nonisotopic molecular hybridization and multiplex reverse-transcription polymerase chain reaction.

PubMed

Saade, M; Aparicio, F; Sánchez-Navarro, J A; Herranz, M C; Myrta, A; Di Terlizzi, B; Pallás, V

2000-12-01

ABSTRACT The three most economically damaging ilarviruses affecting stone fruit trees on a worldwide scale are the related Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), and Apple mosaic virus (ApMV). Nonisotopic molecular hybridization and multiplex reverse-transcription polymerase chain reaction (RT-PCR) methodologies were developed that could detect all these viruses simultaneously. The latter technique was advantageous because it was discriminatory. For RT-PCR, a degenerate antisense primer was designed which was used in conjunction with three virus-specific sense primers. The amplification efficiencies for the detection of the three viruses in the multiplex RT-PCR reaction were identical to those obtained in the single RT-PCR reactions for individual viruses. This cocktail of primers was able to amplify sequences from all of the PNRSV, ApMV, and PDV isolates tested in five Prunus spp. hosts (almond, apricot, cherry, peach, and plum) occurring naturally in single or multiple infections. For ApMV isolates, differences in the electrophoretic mobilities of the PCR products were observed. The nucleotide sequence of the amplified products of two representative ApMV isolates was determined, and comparative analysis revealed the existence of a 28-nucleotide deletion in the sequence of isolates showing the faster electrophoretic mobility. To our knowledge, this is the first report on the simultaneous detection of three plant viruses by multiplex RT-PCR in woody hosts. This multiplex RT-PCR could be a useful time and cost saving method for indexing these three ilarviruses, which damage stone fruit tree yields, and for the analysis of mother plants in certification programs.

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

PubMed Central

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

2011-01-01

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

Analysis of hybrid subcarrier multiplexing of OCDMA based on single photodiode detection

NASA Astrophysics Data System (ADS)

Ahmad, N. A. A.; Junita, M. N.; Aljunid, S. A.; Rashidi, C. B. M.; Endut, R.

2017-11-01

This paper analyzes the performance of subcarrier multiplexing (SCM) of spectral amplitude coding optical code multiple access (SAC-OCDMA) by applying Recursive Combinatorial (RC) code based on single photodiode detection (SPD). SPD is used in the receiver part to reduce the effect of multiple access interference (MAI) which contributes as a dominant noise in incoherent SAC-OCDMA systems. Results indicate that the SCM OCDMA network performance could be improved by using lower data rates and higher number of weight. Total number of users can also be enhanced by adding lower data rates and higher number of subcarriers.

Rapid detection of Shigella and enteroinvasive Escherichia coli in produce enrichments by a conventional multiplex PCR assay.

PubMed

Binet, Rachel; Deer, Deanne M; Uhlfelder, Samantha J

2014-06-01

Faster detection of contaminated foods can prevent adulterated foods from being consumed and minimize the risk of an outbreak of foodborne illness. A sensitive molecular detection method is especially important for Shigella because ingestion of as few as 10 of these bacterial pathogens can cause disease. The objectives of this study were to compare the ability of four DNA extraction methods to detect Shigella in six types of produce, post-enrichment, and to evaluate a new and rapid conventional multiplex assay that targets the Shigella ipaH, virB and mxiC virulence genes. This assay can detect less than two Shigella cells in pure culture, even when the pathogen is mixed with background microflora, and it can also differentiate natural Shigella strains from a control strain and eliminate false positive results due to accidental laboratory contamination. The four DNA extraction methods (boiling, PrepMan Ultra [Applied Biosystems], InstaGene Matrix [Bio-Rad], DNeasy Tissue kit [Qiagen]) detected 1.6 × 10(3)Shigella CFU/ml post-enrichment, requiring ∼18 doublings to one cell in 25 g of produce pre-enrichment. Lower sensitivity was obtained, depending on produce type and extraction method. The InstaGene Matrix was the most consistent and sensitive and the multiplex assay accurately detected Shigella in less than 90 min, outperforming, to the best of our knowledge, molecular assays currently in place for this pathogen. Published by Elsevier Ltd.

Development of a multiplex assay for genus- and species-specific detection of Phytophthora based on differences in mitochondrial gene order

Treesearch

G. J. Bilodeau; F. N. Martin; M. D. Coffey; C. L. Blomquist

2014-01-01

A molecular diagnostic assay for Phytophthora spp. that is specific, sensitive, has both genus- and species-specific detection capabilities multiplexed, and can be used to systematically develop markers for detection of a wide range of species would facilitate research and regulatory efforts. To address this need, a marker system was developed...

Multiplexed direct genomic selection (MDiGS): a pooled BAC capture approach for highly accurate CNV and SNP/INDEL detection.

PubMed

Alvarado, David M; Yang, Ping; Druley, Todd E; Lovett, Michael; Gurnett, Christina A

2014-06-01

Despite declining sequencing costs, few methods are available for cost-effective single-nucleotide polymorphism (SNP), insertion/deletion (INDEL) and copy number variation (CNV) discovery in a single assay. Commercially available methods require a high investment to a specific region and are only cost-effective for large samples. Here, we introduce a novel, flexible approach for multiplexed targeted sequencing and CNV analysis of large genomic regions called multiplexed direct genomic selection (MDiGS). MDiGS combines biotinylated bacterial artificial chromosome (BAC) capture and multiplexed pooled capture for SNP/INDEL and CNV detection of 96 multiplexed samples on a single MiSeq run. MDiGS is advantageous over other methods for CNV detection because pooled sample capture and hybridization to large contiguous BAC baits reduces sample and probe hybridization variability inherent in other methods. We performed MDiGS capture for three chromosomal regions consisting of ∼ 550 kb of coding and non-coding sequence with DNA from 253 patients with congenital lower limb disorders. PITX1 nonsense and HOXC11 S191F missense mutations were identified that segregate in clubfoot families. Using a novel pooled-capture reference strategy, we identified recurrent chromosome chr17q23.1q23.2 duplications and small HOXC 5' cluster deletions (51 kb and 12 kb). Given the current interest in coding and non-coding variants in human disease, MDiGS fulfills a niche for comprehensive and low-cost evaluation of CNVs, coding, and non-coding variants across candidate regions of interest. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Enhanced sensitivity and multiplexing with 2D LC/MRM-MS and labeled standards for deeper and more comprehensive protein quantitation.

PubMed

Percy, Andrew J; Simon, Romain; Chambers, Andrew G; Borchers, Christoph H

2014-06-25

Mass spectrometry (MS)-based protein quantitation is increasingly being employed to verify candidate protein biomarkers. Multiple or selected reaction monitoring-mass spectrometry (MRM-MS or SRM-MS) with isotopically labeled internal standards has proven to be a successful approach in that regard, but has yet to reach its full potential in terms of multiplexing and sensitivity. Here, we report the development of a new MRM method for the quantitation of 253 disease-associated proteins (represented by 625 interference-free peptides) in 13 LC fractions. This 2D RPLC/MRM-MS approach extends the depth and breadth of the assay by 2 orders of magnitude over pre-fractionation-free assays, with 31 proteins below 10 ng/mL and 41 proteins above 10 ng/mL now quantifiable. Standard flow rates are used in both chromatographic dimensions, and up-front depletion or antibody-based enrichment is not required. The LC separations utilize high and low pH conditions, with the former employing an ammonium hydroxide-based eluent, instead of the conventional ammonium formate, resulting in improved LC column lifetime and performance. The high sensitivity (determined concentration range: 15 mg/mL to 452 pg/mL) and robustness afforded by this method makes the full MRM panel, or subsets thereof, useful for the verification of disease-associated plasma protein biomarkers in patient samples. The described research extends the breadth and depth of protein quantitation in undepleted and non-enriched human plasma by employing standard-flow 2D RPLC/MRM-MS in conjunction with a complex mixture of isotopically labeled peptide standards. The proteins quantified are mainly putative biomarkers of non-communicable (i.e., non-infectious) disease (e.g., cardiovascular or cancer), which require pre-clinical verification and validation before clinical implementation. Based on the enhanced sensitivity and multiplexing, this quantitative plasma proteomic method should prove useful in future candidate biomarker

Highly multiplexed simultaneous detection of RNAs and proteins in single cells.

PubMed

Frei, Andreas P; Bava, Felice-Alessio; Zunder, Eli R; Hsieh, Elena W Y; Chen, Shih-Yu; Nolan, Garry P; Gherardini, Pier Federico

2016-03-01

To enable the detection of expression signatures specific to individual cells, we developed PLAYR (proximity ligation assay for RNA), a method for highly multiplexed transcript quantification by flow and mass cytometry that is compatible with standard antibody staining. When used with mass cytometry, PLAYR allowed for the simultaneous quantification of more than 40 different mRNAs and proteins. In primary cells, we quantified multiple transcripts, with the identity and functional state of each analyzed cell defined on the basis of the expression of a separate set of transcripts or proteins. By expanding high-throughput deep phenotyping of cells beyond protein epitopes to include RNA expression, PLAYR opens a new avenue for the characterization of cellular metabolism.

Quantum-dot-based suspension microarray for multiplex detection of lung cancer markers: preclinical validation and comparison with the Luminex xMAP® system

NASA Astrophysics Data System (ADS)

Bilan, Regina; Ametzazurra, Amagoia; Brazhnik, Kristina; Escorza, Sergio; Fernández, David; Uríbarri, María; Nabiev, Igor; Sukhanova, Alyona

2017-03-01

A novel suspension multiplex immunoassay for the simultaneous specific detection of lung cancer markers in bronchoalveolar lavage fluid (BALF) clinical samples based on fluorescent microspheres having different size and spectrally encoded with quantum dots (QDEM) was developed. The designed suspension immunoassay was validated for the quantitative detection of three lung cancer markers in BALF samples from 42 lung cancer patients and 10 control subjects. Tumor markers were detected through simultaneous formation of specific immune complexes consisting of a capture molecule, the target antigen, and biotinylated recognition molecule on the surface of the different QDEM in a mixture. The immune complexes were visualized by fluorescently labeled streptavidin and simultaneously analyzed using a flow cytometer. Preclinical validation of the immunoassay was performed and results were compared with those obtained using an alternative 3-plex immunoassay based on Luminex xMAP® technology, developed on classical organic fluorophores. The comparison showed that the QDEM and xMAP® assays yielded almost identical results, with clear discrimination between control and clinical samples. Thus, developed QDEM technology can become a good alternative to xMAP® assays permitting analysis of multiple protein biomarkers using conventional flow cytometers.

Quantum-dot-based suspension microarray for multiplex detection of lung cancer markers: preclinical validation and comparison with the Luminex xMAP® system

PubMed Central

Bilan, Regina; Ametzazurra, Amagoia; Brazhnik, Kristina; Escorza, Sergio; Fernández, David; Uríbarri, María; Nabiev, Igor; Sukhanova, Alyona

2017-01-01

A novel suspension multiplex immunoassay for the simultaneous specific detection of lung cancer markers in bronchoalveolar lavage fluid (BALF) clinical samples based on fluorescent microspheres having different size and spectrally encoded with quantum dots (QDEM) was developed. The designed suspension immunoassay was validated for the quantitative detection of three lung cancer markers in BALF samples from 42 lung cancer patients and 10 control subjects. Tumor markers were detected through simultaneous formation of specific immune complexes consisting of a capture molecule, the target antigen, and biotinylated recognition molecule on the surface of the different QDEM in a mixture. The immune complexes were visualized by fluorescently labeled streptavidin and simultaneously analyzed using a flow cytometer. Preclinical validation of the immunoassay was performed and results were compared with those obtained using an alternative 3-plex immunoassay based on Luminex xMAP® technology, developed on classical organic fluorophores. The comparison showed that the QDEM and xMAP® assays yielded almost identical results, with clear discrimination between control and clinical samples. Thus, developed QDEM technology can become a good alternative to xMAP® assays permitting analysis of multiple protein biomarkers using conventional flow cytometers. PMID:28300171

Validating multiplexes for use in conjunction with modern interpretation strategies.

PubMed

Taylor, Duncan; Bright, Jo-Anne; McGoven, Catherine; Hefford, Christopher; Kalafut, Tim; Buckleton, John

2016-01-01

In response to requests from the forensic community, commercial companies are generating larger, more sensitive, and more discriminating STR multiplexes. These multiplexes are now applied to a wider range of samples including complex multi-person mixtures. In parallel there is an overdue reappraisal of profile interpretation methodology. Aspects of this reappraisal include 1. The need for a quantitative understanding of allele and stutter peak heights and their variability, 2. An interest in reassessing the utility of smaller peaks below the often used analytical threshold, 3. A need to understand not just the occurrence of peak drop-in but also the height distribution of such peaks, and 4. A need to understand the limitations of the multiplex-interpretation strategy pair implemented. In this work we present a full scheme for validation of a new multiplex that is suitable for informing modern interpretation practice. We predominantly use GlobalFiler™ as an example multiplex but we suggest that the aspects investigated here are fundamental to introducing any multiplex in the modern interpretation environment. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Electrochemical primer extension based on polyoxometalate electroactive labels for multiplexed detection of single nucleotide polymorphisms.

PubMed

Chahin, Nassif; Uribe, Laura A; Debela, Ahmed M; Thorimbert, Serge; Hasenknopf, Bernold; Ortiz, Mayreli; Katakis, Ioannis; O'Sullivan, Ciara K

2018-06-07

Polyoxymetalates (POMs) ([SiW 11 O 39 {Sn(CH 2 ) 2 CO)}] 4- and [P 2 W 17 O 61 {Sn(CH 2 ) 2 CO)}] 6- ) were used to modify dideoxynucleotides (ddNTPs) through amide bond formation, and applied to the multiplexed detection of single nucleotide polymorphisms (SNPs) in an electrochemical primer extension reaction. Each gold electrode of an array was functionalised with a short single stranded thiolated DNA probe, specifically designed to extend with the POM-ddNTP at the SNP site to be interrogated. The system was applied to the simultaneous detection of 4 SNPs within a single stranded 103-mer model target generated using asymmetric PCR, highlighting the potential of POM-ddNTPs for targeted, multiplexed SNP detection. The four DNA bases were successfully labelled with both ([SiW 11 O 39 {Sn(CH 2 ) 2 CO)}] 4- and [P 2 W 17 O 61 {Sn(CH 2 ) 2 CO)}] 6- ), and [SiW 11 O 39 {Sn(CH 2 ) 2 CO)}] 4- demonstrated to be the more suitable due to its single oxidation peak, which provides an unequivocal signal. The POM-ddNTP enzymatically incorporated to the DNA anchored to the surface was visualised by AFM using gold coated mica. The developed assay has been demonstrated to be highly reproducible, simple to carry out and with very low non-specific background signals. Future work will focus on applying the developed platform to the detection of SNPs associated with rifampicin resistance in real samples from patients suffering from tuberculosis. Copyright © 2018. Published by Elsevier B.V.

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

PubMed

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

2011-12-01

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

Multiplex PCR assay for detection of recombinant genes encoding fatty acid desaturases fused with lichenase reporter protein in GM plants.

PubMed

Berdichevets, Iryna N; Shimshilashvili, Hristina R; Gerasymenko, Iryna M; Sindarovska, Yana R; Sheludko, Yuriy V; Goldenkova-Pavlova, Irina V

2010-07-01

Thermostable lichenase encoded by licB gene of Clostridium thermocellum can be used as a reporter protein in plant, bacterial, yeast, and mammalian cells. It has important advantages of high sensitivity and specificity in qualitative and quantitative assays. Deletion variants of LicB (e.g., LicBM3) retain its enzymatic activity and thermostability and can be expressed in translational fusion with target proteins without compromising with their properties. Fusion with the lichenase reporter is especially convenient for the heterologous expression of proteins whose analysis is difficult or compromised by host enzyme activities, as it is in case of fatty acid desaturases occurring in all groups of organisms. Recombinant desaturase-lichenase genes can be used for creating genetically modified (GM) plants with improved chill tolerance. Development of an analytical method for detection of fused desaturase-lichenase transgenes is necessary both for production of GM plants and for their certification. Here, we report a multiplex polymerase chain reaction method for detection of desA and desC desaturase genes of cyanobacteria Synechocystis sp. PCC6803 and Synechococcus vulcanus, respectively, fused to licBM3 reporter in GM plants.

Quantitative polarized light microscopy using spectral multiplexing interferometry.

PubMed

Li, Chengshuai; Zhu, Yizheng

2015-06-01

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

Multiplex real-time PCR assay for detection of Escherichia coli O157:H7 and screening for non-O157 Shiga toxin-producing E. coli.

PubMed

Li, Baoguang; Liu, Huanli; Wang, Weimin

2017-11-09

Shiga toxin-producing Escherichia coli (STEC), including E. coli O157:H7, are responsible for numerous foodborne outbreaks annually worldwide. E. coli O157:H7, as well as pathogenic non-O157:H7 STECs, can cause life-threating complications, such as bloody diarrhea (hemolytic colitis) and hemolytic-uremic syndrome (HUS). Previously, we developed a real-time PCR assay to detect E. coli O157:H7 in foods by targeting a unique putative fimbriae protein Z3276. To extend the detection spectrum of the assay, we report a multiplex real-time PCR assay to specifically detect E. coli O157:H7 and screen for non-O157 STEC by targeting Z3276 and Shiga toxin genes (stx1 and stx2). Also, an internal amplification control (IAC) was incorporated into the assay to monitor the amplification efficiency. The multiplex real-time PCR assay was developed using the Life Technology ABI 7500 System platform and the standard chemistry. The optimal amplification mixture of the assay contains 12.5 μl of 2 × Universal Master Mix (Life Technology), 200 nM forward and reverse primers, appropriate concentrations of four probes [(Z3276 (80 nM), stx1 (80 nM), stx2 (20 nM), and IAC (40 nM)], 2 μl of template DNA, and water (to make up to 25 μl in total volume). The amplification conditions of the assay were set as follows: activation of TaqMan at 95 °C for 10 min, then 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 60 s. The multiplex assay was optimized for amplification conditions. The limit of detection (LOD) for the multiplex assay was determined to be 200 fg of bacterial DNA, which is equivalent to 40 CFU per reaction which is similar to the LOD generated in single targeted PCRs. Inclusivity and exclusivity determinants were performed with 196 bacterial strains. All E. coli O157:H7 (n = 135) were detected as positive and all STEC strains (n = 33) were positive for stx1, or stx2, or stx1 and stx2 (Table 1). No cross reactivity was detected with Salmonella

Cross-reactivity profiles of legumes and tree nuts using the xMAP® multiplex food allergen detection assay.

PubMed

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

2017-10-01

The homology between proteins in legumes and tree nuts makes it common for individuals with food allergies to be allergic to multiple legumes and tree nuts. This propensity for allergenic and antigenic cross-reactivity means that commonly employed commercial immunodiagnostic assays (e.g., dipsticks) for the detection of food allergens may not always accurately detect, identify, and quantitate legumes and tree nuts unless additional orthogonal analytical methods or secondary measures of analysis are employed. The xMAP ® Multiplex Food Allergen Detection Assay (FADA) was used to determine the cross-reactivity patterns and the utility of multi-antibody antigenic profiling to distinguish between legumes and tree nuts. Pure legumes and tree nuts extracted using buffered detergent displayed a high level of cross-reactivity that decreased upon dilution or by using a buffer (UD buffer) designed to increase the stringency of binding conditions and reduce the occurrence of false positives due to plant-derived lectins. Testing for unexpected food allergens or the screening for multiple food allergens often involves not knowing the identity of the allergen present, its concentration, or the degree of modification during processing. As such, the analytical response measured may represent multiple antigens of varying antigenicity (cross-reactivity). This problem of multiple potential analytes is usually unresolved and the focus becomes the primary analyte, the antigen the antibody was raised against, or quantitative interpretation of the content of the analytical sample problematic. The alternative solution offered here to this problem is the use of an antigenic profile as generated by the xMAP FADA using multiple antibodies (bead sets). By comparing the antigenic profile to standards, the allergen may be identified along with an estimate of the concentration present. Cluster analysis of the xMAP FADA data was also performed and agreed with the known phylogeny of the legumes

Quantitative and simultaneous detection of four foodborne bacterial pathogens with a multi-channel SPR sensor.

PubMed

Taylor, Allen D; Ladd, Jon; Yu, Qiuming; Chen, Shengfu; Homola, Jirí; Jiang, Shaoyi

2006-12-15

We report the quantitative and simultaneous detection of four species of bacteria, Escherichia coli O157:H7, Salmonella choleraesuis serotype typhimurium, Listeria monocytogenes, and Campylobacter jejuni, using an eight-channel surface plasmon resonance (SPR) sensor based on wavelength division multiplexing. Detection curves showing SPR response versus analyte concentration were established for each species of bacteria in buffer at pH 7.4, apple juice at native pH 3.7, and apple juice at an adjusted pH of 7.4, as well as for a mixture containing all four species of bacteria in buffer. Control experiments were performed to show the non-fouling characteristics of the sensor surface as well as the specificity of the amplification antibodies used in this study. The limit of detection (LOD) for each of the four species of bacteria in the tested matrices ranges from 3.4 x 10(3) to 1.2 x 10(5) cfu/ml. Detection curves in buffer of an individual species of bacteria in a mixture of all four species of bacteria correlated well with detection curves of the individual species of bacteria alone. SPR responses were higher for bacteria in apple juice at pH 7.4 than in apple juice at pH 3.7. This difference in sensor response could be partly attributed to the pH dependence of antibody-antigen binding.

Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations

PubMed Central

Zonta, Eleonora; Garlan, Fanny; Pécuchet, Nicolas; Perez-Toralla, Karla; Caen, Ouriel; Milbury, Coren; Didelot, Audrey; Fabre, Elizabeth; Blons, Hélène; Laurent-Puig, Pierre; Taly, Valérie

2016-01-01

In cancer research, the accuracy of the technology used for biomarkers detection is remarkably important. In this context, digital PCR represents a highly sensitive and reproducible method that could serve as an appropriate tool for tumor mutational status analysis. In particular, droplet-based digital PCR approaches have been developed for detection of tumor-specific mutated alleles within plasmatic circulating DNA. Such an approach calls for the development and validation of a very significant quantity of assays, which can be extremely costly and time consuming. Herein, we evaluated assays for the detection and quantification of various mutations occurring in three genes often misregulated in cancers: the epidermal growth factor receptor (EGFR), the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and the Tumoral Protein p53 (TP53) genes. In particular, commercial competitive allele-specific TaqMan® PCR (castPCR™) technology, as well as TaqMan® and ZEN™ assays, have been evaluated for EGFR p.L858R, p.T790M, p.L861Q point mutations and in-frame deletions Del19. Specificity and sensitivity have been determined on cell lines DNA, plasmatic circulating DNA of lung cancer patients or Horizon Diagnostics Reference Standards. To show the multiplexing capabilities of this technology, several multiplex panels for EGFR (several three- and four-plexes) have been developed, offering new "ready-to-use" tests for lung cancer patients. PMID:27416070

Pre-capture multiplexing improves efficiency and cost-effectiveness of targeted genomic enrichment.

PubMed

Shearer, A Eliot; Hildebrand, Michael S; Ravi, Harini; Joshi, Swati; Guiffre, Angelica C; Novak, Barbara; Happe, Scott; LeProust, Emily M; Smith, Richard J H

2012-11-14

Targeted genomic enrichment (TGE) is a widely used method for isolating and enriching specific genomic regions prior to massively parallel sequencing. To make effective use of sequencer output, barcoding and sample pooling (multiplexing) after TGE and prior to sequencing (post-capture multiplexing) has become routine. While previous reports have indicated that multiplexing prior to capture (pre-capture multiplexing) is feasible, no thorough examination of the effect of this method has been completed on a large number of samples. Here we compare standard post-capture TGE to two levels of pre-capture multiplexing: 12 or 16 samples per pool. We evaluated these methods using standard TGE metrics and determined the ability to identify several classes of genetic mutations in three sets of 96 samples, including 48 controls. Our overall goal was to maximize cost reduction and minimize experimental time while maintaining a high percentage of reads on target and a high depth of coverage at thresholds required for variant detection. We adapted the standard post-capture TGE method for pre-capture TGE with several protocol modifications, including redesign of blocking oligonucleotides and optimization of enzymatic and amplification steps. Pre-capture multiplexing reduced costs for TGE by at least 38% and significantly reduced hands-on time during the TGE protocol. We found that pre-capture multiplexing reduced capture efficiency by 23 or 31% for pre-capture pools of 12 and 16, respectively. However efficiency losses at this step can be compensated by reducing the number of simultaneously sequenced samples. Pre-capture multiplexing and post-capture TGE performed similarly with respect to variant detection of positive control mutations. In addition, we detected no instances of sample switching due to aberrant barcode identification. Pre-capture multiplexing improves efficiency of TGE experiments with respect to hands-on time and reagent use compared to standard post-capture TGE

Solution-based circuits enable rapid and multiplexed pathogen detection.

PubMed

Lam, Brian; Das, Jagotamoy; Holmes, Richard D; Live, Ludovic; Sage, Andrew; Sargent, Edward H; Kelley, Shana O

2013-01-01

Electronic readout of markers of disease provides compelling simplicity, sensitivity and specificity in the detection of small panels of biomarkers in clinical samples; however, the most important emerging tests for disease, such as infectious disease speciation and antibiotic-resistance profiling, will need to interrogate samples for many dozens of biomarkers. Electronic readout of large panels of markers has been hampered by the difficulty of addressing large arrays of electrode-based sensors on inexpensive platforms. Here we report a new concept--solution-based circuits formed on chip--that makes highly multiplexed electrochemical sensing feasible on passive chips. The solution-based circuits switch the information-carrying signal readout channels and eliminate all measurable crosstalk from adjacent, biomolecule-specific microsensors. We build chips that feature this advance and prove that they analyse unpurified samples successfully, and accurately classify pathogens at clinically relevant concentrations. We also show that signature molecules can be accurately read 2  minutes after sample introduction.

A multiplex PCR method of detecting recombinant DNAs from five lines of genetically modified maize.

PubMed

Matsuoka, T; Kuribara, H; Akiyama, H; Miura, H; Goda, Y; Kusakabe, Y; Isshiki, K; Toyoda, M; Hino, A

2001-02-01

Seven lines of genetically modified (GM) maize have been authorized in Japan as foods and feeds imported from the USA. We improved a multiplex PCR method described in the previous report in order to distinguish the five lines of GM maize. Genomic DNA was extracted from GM maize with a silica spin column kit, which could reduce experimental time and improve safety in the laboratory and potentially in the environment. We sequenced recombinant DNA (r-DNA) introduced into GM maize, and re-designed new primer pairs to increase the specificity of PCR to distinguish five lines of GM maize by multiplex PCR. A primer pair for the maize intrinsic zein gene (Ze1) was also designed to confirm the presence of amplifiable maize DNA. The lengths of PCR products using these six primer pairs were different. The Ze1 and the r-DNAs from the five lines of GM maize were qualitatively detected in one tube. The specific PCR bands were distinguishable from each other on the basis of the expected length. The r-DNA could be detected from maize samples containing 0.5% of each of the five lines of GM maize. The sensitivity would be acceptable to secure the verification of non-GMO materials and to monitor the reliability of the labeling system.

Rapid and simultaneous detection of Salmonella spp., Escherichia coli O157, and Listeria monocytogenes by magnetic capture hybridization and multiplex real-time PCR.

PubMed

Carloni, Elisa; Rotundo, Luca; Brandi, Giorgio; Amagliani, Giulia

2018-05-25

The application of rapid, specific, and sensitive methods for pathogen detection and quantification is very advantageous in diagnosis of human pathogens in several applications, including food analysis. The aim of this study was the evaluation of a method for the multiplexed detection and quantification of three significant foodborne pathogenic species (Escherichia coli O157, Salmonella spp., and Listeria monocytogenes). The assay combines specific DNA extraction by multiplex magnetic capture hybridization (mMCH) with multiplex real-time PCR. The amplification assay showed linearity in the range 10 6 -10 genomic units (GU)/PCR for each co-amplified species. The sensitivity corresponded to 1 GU/PCR for E. coli O157 and L. monocytogenes, and 10 GU/PCR for Salmonella spp. The immobilization process and the hybrid capture of the MCH showed good efficiency and reproducibility for all targets, allowing the combination in equal amounts of the different nanoparticle types in mMCH. MCH and mMCH efficiencies were similar. The detection limit of the method was 10 CFU in samples with individual pathogens and 10 2  CFU in samples with combination of the three pathogens in unequal amounts (amount's differences of 2 or 3 log). In conclusion, this multiplex molecular platform can be applied to determine the presence of target species in food samples after culture enrichment. In this way, this method could be a time-saving and sensitive tool to be used in routine diagnosis.

Development of a multiplex PCR assay for rapid and simultaneous detection of four genera of fish pathogenic bacteria.

PubMed

Zhang, D F; Zhang, Q Q; Li, A H

2014-11-01

Species of genus Aeromonas, Vibrio, Edwardsiella and Streptococcus are the most common fish pathogenic bacteria that cause economically devastating losses in aquaculture. A multiplex polymerase chain reaction (mPCR) was developed for the simultaneous detection and differentiation of the four genera of fish pathogenic bacteria. Through the use of genus-specific primers instead of species-specific ones, the current mPCR covered much more target bacterial species compared with previously reported species-specific mPCR methods. The specificity of the four putative genus-specific primers was validated experimentally while used exclusively (uniplex PCR) or combined (mPCR) against bacterial genomic DNA templates of the target bacteria and nontarget bacteria. The PCR amplicons for the following genera were obtained as expected: Aeromonas (875 bp), Vibrio (524 bp), Edwardsiella (302 bp) and Streptococcus (197 bp), and the fragments could be separated clearly on the agarose gel electrophoresis. The mPCR did not produce nonspecific amplification products when used to amplify 21 nontarget species of bacteria. The mPCR detection limits for each target bacterial genera were 50 colony-forming units (CFU) in pure culture and 100 CFU in fish tissue samples. In conclusion, the mPCR assay was proven to be a powerful alternative to the conventional culture-based method, given its rapid, specific, sensitive and reliable detection of target pathogens. The fish pathogenic bacteria of genus Aeromonas, Vibrio, Edwardsiella and Streptococcus frequently cause severe outbreaks of diseases in cultured fish, and the genus-specific multiplex PCR assay developed in this study can detect the bacteria of the four genera when present in the samples either alone or mixed. The mPCR assay is expected to identify the causative agents more efficiently than uniplex PCR or species-specific multiplex PCR for clinical diagnosis, resulting in the earlier implementation of control measures. This m

An experimental study for rapid detection and quantification of endodontic microbiota following photo-activated disinfection via new multiplex real-time PCR assay.

PubMed

Pourhajibagher, Maryam; Raoofian, Reza; Ghorbanzadeh, Roghayeh; Bahador, Abbas

2018-03-01

The infected root canal system harbors one of the highest accumulations of polymicrobial infections. Since the eradication of endopathogenic microbiota is a major goal in endodontic infection therapy, photo-activated disinfection (PAD) can be used as an alternative therapeutic method in endodontic treatment. Compared to cultivation-based approaches, molecular techniques are more reliable for identifying microbial agents associated with endodontic infections. The purpose of this study was to evaluate the ability of designed multiplex real-time PCR protocol for the rapid detection and quantification of six common microorganisms involved in endodontic infection before and after the PAD. Samples were taken from the root canals of 50 patients with primary and secondary/persistent endodontic infections using sterile paper points. PAD with toluidine blue O (TBO) plus diode laser was performed on root canals. Resampling was then performed, and the samples were transferred to transport medium. Then, six target microorganisms were detected using multiplex real-time PCR before and after the PAD. Veillonella parvula was found using multiplex real-time PCR to have the highest frequency among samples collected before the PAD (29.4%), followed by Porphyromonas gingivalis (23.1%), Aggregatibacter actinomycetemcomitans (13.6%), Actinomyces naeslundii (13.0%), Enterococcus faecalis (11.5%), and Lactobacillus rhamnosus (9.4%). After TBO-mediated PAD, P. gingivalis strains, the most resistance microorganisms, were recovered in 41.7% of the samples using molecular approach (P > 0.05). As the results shown, multiplex real-time PCR as an accurate detection approach with high-throughput and TBO-mediated PAD as an efficient antimicrobial strategy due to the significant reduction of the endopathogenic count can be used for detection and treatment of microbiota involved in infected root canals, respectively. Copyright © 2018 Elsevier B.V. All rights reserved.

Multiplexed detection of mycotoxins in foods with a regenerable array.

PubMed

Ngundi, Miriam M; Shriver-Lake, Lisa C; Moore, Martin H; Ligler, Frances S; Taitt, Chris R

2006-12-01

The occurrence of different mycotoxins in cereal products calls for the development of a rapid, sensitive, and reliable detection method that is capable of analyzing samples for multiple toxins simultaneously. In this study, we report the development and application of a multiplexed competitive assay for the simultaneous detection of ochratoxin A (OTA) and deoxynivalenol (DON) in spiked barley, cornmeal, and wheat, as well as in naturally contaminated maize samples. Fluoroimmunoassays were performed with the Naval Research Laboratory array biosensor, by both a manual and an automated version of the system. This system employs evanescent-wave fluorescence excitation to probe binding events as they occur on the surface of a waveguide. Methanolic extracts of the samples were diluted threefold with buffer containing a mixture of fluorescent antibodies and were then passed over the arrays of mycotoxins immobilized on a waveguide. Fluorescent signals of the surface-bound antibody-antigen complexes decreased with increasing concentrations of free mycotoxins in the extract. After sample analysis was completed, surfaces were regenerated with 6 M guanidine hydrochloride in 50 mM glycine, pH 2.0. The limits of detection determined by the manual biosensor system were as follows: 1, 180, and 65 ng/g for DON and 1, 60, and 85 ng/g for OTA in cornmeal, wheat, and barley, respectively. The limits of detection in cornmeal determined with the automated array biosensor were 15 and 150 ng/g for OTA and DON, respectively.

Simple and fast multiplex PCR method for detection of species origin in meat products.

PubMed

Izadpanah, Mehrnaz; Mohebali, Nazanin; Elyasi Gorji, Zahra; Farzaneh, Parvaneh; Vakhshiteh, Faezeh; Shahzadeh Fazeli, Seyed Abolhassan

2018-02-01

Identification of animal species is one of the major concerns in food regulatory control and quality assurance system. Different approaches have been used for species identification in animal origin of feedstuff. This study aimed to develop a multiplex PCR approach to detect the origin of meat and meat products. Specific primers were designed based on the conserved region of mitochondrial Cytochrome C Oxidase subunit I ( COX1 ) gene. This method could successfully distinguish the origin of the pig, camel, sheep, donkey, goat, cow, and chicken in one single reaction. Since PCR products derived from each species represent unique molecular weight, the amplified products could be identified by electrophoresis and analyzed based on their size. Due to the synchronized amplification of segments within a single PCR reaction, multiplex PCR is considered to be a simple, fast, and inexpensive technique that can be applied for identification of meat products in food industries. Nowadays, this technique has been considered as a practical method to identify the species origin, which could further applied for animal feedstuffs identification.

Nanoparticle Enhancement Cascade for Sensitive Multiplex Measurements of Biomarkers in Complex Fluids with Surface Plasmon Resonance Imaging.

PubMed

Hendriks, Jan; Stojanovic, Ivan; Schasfoort, Richard B M; Saris, Daniël B F; Karperien, Marcel

2018-06-05

There is a large unmet need for reliable biomarker measurement systems for clinical application. Such systems should meet challenging requirements for large scale use, including a large dynamic detection range, multiplexing capacity, and both high specificity and sensitivity. More importantly, these requirements need to apply to complex biological samples, which require extensive quality control. In this paper, we present the development of an enhancement detection cascade for surface plasmon resonance imaging (SPRi). The cascade applies an antibody sandwich assay, followed by neutravidin and a gold nanoparticle enhancement for quantitative biomarker measurements in small volumes of complex fluids. We present a feasibility study both in simple buffers and in spiked equine synovial fluid with four cytokines, IL-1β, IL-6, IFN-γ, and TNF-α. Our enhancement cascade leads to an antibody dependent improvement in sensitivity up to 40 000 times, resulting in a limit of detection as low as 50 fg/mL and a dynamic detection range of more than 7 logs. Additionally, measurements at these low concentrations are highly reliable with intra- and interassay CVs between 2% and 20%. We subsequently showed this assay is suitable for multiplex measurements with good specificity and limited cross-reactivity. Moreover, we demonstrated robust detection of IL-6 and IL-1β in spiked undiluted equine synovial fluid with small variation compared to buffer controls. In addition, the availability of real time measurements provides extensive quality control opportunities, essential for clinical applications. Therefore, we consider this method is suitable for broad application in SPRi for multiplex biomarker detection in both research and clinical settings.

Multiplexed chemiluminescent assays in ArrayPlates for high-throughput measurement of gene expression

NASA Astrophysics Data System (ADS)

Martel, Ralph R.; Rounseville, Matthew P.; Botros, Ihab W.; Seligmann, Bruce E.

2002-06-01

Multiplexed Molecular Profiling (MMP) assays for drug discovery are performed in ArrayPlates. ArrayPlates are 96- well microtiter plates that contain a 16-element array at the bottom of each well. Each element within an array measures one analyte in a sample. A CCD imager records the quantitative chemiluminescent readout of all 1,536 elements in a 96-well plate simultaneously. Since array elements are reagent modifiable by the end-user, ArrayPlates can be adapted to a broad range of nucleic acid- and protein-based assays. Such multiplexed assays are rapidly established, flexible, robust, automation-friendly and cost-effective. Nucleic acid assays in ArrayPlates can detect DNA and RNA, including SNPs and ESTs. A multiplexed mRNA assay to measure the expression of 16 genes is described. The assay combines a homogeneous nuclease protection assay with subsequent probe immobilization to the array by means of a sandwich hybridization followed with chemiluminescent detection. This assay was used to examine cells grown and treated in microplates and avoided cloning, transfection, RNA insolation, reverse transcription, amplification and fluorochrome labeling. Standard deviations for the measurement of 16 genes ranged from 3 percent to 13 percent in samples of 30,000 cells. Such ArrayPlates transcription assays are useful in drug discovery and development for target validation, screening, lead optimization, metabolism and toxicity profiling. Chemiluminescent detection provides ArrayPlates assays with high signal-to-noise readout and simplifies imager requirements. Imaging a 2D surface that contains arrays simplifies lens requirements relative to imaging columns of liquid in microtiter plate wells. The Omix imager for ArrayPlates is described.

Multiplexing of spatial modes in the mid-IR region

NASA Astrophysics Data System (ADS)

Gailele, Lucas; Maweza, Loyiso; Dudley, Angela; Ndagano, Bienvenu; Rosales-Guzman, Carmelo; Forbes, Andrew

2017-02-01

Traditional optical communication systems optimize multiplexing in polarization and wavelength both trans- mitted in fiber and free-space to attain high bandwidth data communication. Yet despite these technologies, we are expected to reach a bandwidth ceiling in the near future. Communications using orbital angular momentum (OAM) carrying modes offers infinite dimensional states, providing means to increase link capacity by multiplexing spatially overlapping modes in both the azimuthal and radial degrees of freedom. OAM modes are multiplexed and de-multiplexed by the use of spatial light modulators (SLM). Implementation of complex amplitude modulation is employed on laser beams phase and amplitude to generate Laguerre-Gaussian (LG) modes. Modal decomposition is employed to detect these modes due to their orthogonality as they propagate in space. We demonstrate data transfer by sending images as a proof-of concept in a lab-based scheme. We demonstrate the creation and detection of OAM modes in the mid-IR region as a precursor to a mid-IR free-space communication link.

Multiplexed Analysis of Serum Breast and Ovarian Cancer Markers by Means of Suspension Bead-quantum Dot Microarrays

NASA Astrophysics Data System (ADS)

Brazhnik, Kristina; Sokolova, Zinaida; Baryshnikova, Maria; Bilan, Regina; Nabiev, Igor; Sukhanova, Alyona

Multiplexed analysis of cancer markers is crucial for early tumor diagnosis and screening. We have designed lab-on-a-bead microarray for quantitative detection of three breast cancer markers in human serum. Quantum dots were used as bead-bound fluorescent tags for identifying each marker by means of flow cytometry. Antigen-specific beads reliably detected CA 15-3, CEA, and CA 125 in serum samples, providing clear discrimination between the samples with respect to the antigen levels. The novel microarray is advantageous over the routine single-analyte ones due to the simultaneous detection of various markers. Therefore the developed microarray is a promising tool for serum tumor marker profiling.

Fluorescence-Raman Dual Modal Endoscopic System for Multiplexed Molecular Diagnostics

NASA Astrophysics Data System (ADS)

Jeong, Sinyoung; Kim, Yong-Il; Kang, Homan; Kim, Gunsung; Cha, Myeong Geun; Chang, Hyejin; Jung, Kyung Oh; Kim, Young-Hwa; Jun, Bong-Hyun; Hwang, Do Won; Lee, Yun-Sang; Youn, Hyewon; Lee, Yoon-Sik; Kang, Keon Wook; Lee, Dong Soo; Jeong, Dae Hong

2015-03-01

Optical endoscopic imaging, which was recently equipped with bioluminescence, fluorescence, and Raman scattering, allows minimally invasive real-time detection of pathologies on the surface of hollow organs. To characterize pathologic lesions in a multiplexed way, we developed a dual modal fluorescence-Raman endomicroscopic system (FRES), which used fluorescence and surface-enhanced Raman scattering nanoprobes (F-SERS dots). Real-time, in vivo, and multiple target detection of a specific cancer was successful, based on the fast imaging capability of fluorescence signals and the multiplex capability of simultaneously detected SERS signals using an optical fiber bundle for intraoperative endoscopic system. Human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR) on the breast cancer xenografts in a mouse orthotopic model were successfully detected in a multiplexed way, illustrating the potential of FRES as a molecular diagnostic instrument that enables real-time tumor characterization of receptors during routine endoscopic procedures.

Multiplexed operation of a micromachined ultrasonic droplet ejector array.

PubMed

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

2007-10-01

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

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.

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

PubMed Central

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

2016-01-01

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

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

PubMed

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

2015-07-01

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

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. Copyright © 2014 Elsevier B.V. All rights reserved.

Evaluation of selective control information detection scheme in orthogonal frequency division multiplexing-based radio-over-fiber and visible light communication links

NASA Astrophysics Data System (ADS)

Dalarmelina, Carlos A.; Adegbite, Saheed A.; Pereira, Esequiel da V.; Nunes, Reginaldo B.; Rocha, Helder R. O.; Segatto, Marcelo E. V.; Silva, Jair A. L.

2017-05-01

Block-level detection is required to decode what may be classified as selective control information (SCI) such as control format indicator in 4G-long-term evolution systems. Using optical orthogonal frequency division multiplexing over radio-over-fiber (RoF) links, we report the experimental evaluation of an SCI detection scheme based on a time-domain correlation (TDC) technique in comparison with the conventional maximum likelihood (ML) approach. When compared with the ML method, it is shown that the TDC method improves detection performance over both 20 and 40 km of standard single mode fiber (SSMF) links. We also report a performance analysis of the TDC scheme in noisy visible light communication channel models after propagation through 40 km of SSMF. Experimental and simulation results confirm that the TDC method is attractive for practical orthogonal frequency division multiplexing-based RoF and fiber-wireless systems. Unlike the ML method, another key benefit of the TDC is that it requires no channel estimation.

Multiplex and high-throughput DNA detection using surface plasmon mediated fluorescence

NASA Astrophysics Data System (ADS)

Mei, Zhong

The overall objective of this research project was to develop a user-friendly and sensitive biosensor for nucleic acid aptamers with multiplexing and high-throughput capability. The sensing was based on the fluorescence signals emitted by the fluorophores coupling with plamonic nanoparticle (gold nanorod) deposited on a patterned substrate. Gold nanorods (GNRs) were synthesized using a binary mixture of hexadecyltrimethylammonium bromide (CTAB) and sodium oleate (NaOL) in seed mediated growth method. Polytetrafluoroethylene (PTFE) printed glass slides were selectively coated with a gold thin-film to define hydrophilic areas for GNR deposition. Due to the wettablity contrast, GNR solution dropped on the slide was induced to assemble exclusively in the hydrophilic spots. By controlling temperature and humidity of the evaporation process, vertically-standing GNR arrays were achieved on the pattered slide. Fluorescence was conjugated to GNR surface via DNA double strand with tunable length. Theoretical simulation predicted a flat layer ( 30 nm thick) of uniform "hot spots" presented on the GNR tips, which could modify the nearby fluorescence. Experimentally, the vertical GNR arrays yielded metallic enhanced fluorescence (MEF) effect, which was dependent on the spectrum overlap and GNR-fluorophore distance. Specifically, the maximum enhancement of Quasar 670 and Alexa 750 was observed when it was coupled with GNR664 (plasmonic wavelength 664 nm) and GNR778 respectively at a distance of 16 nm, while the carboxyfluorescein (FAM) was at maximal intensity when attached to gold nanosphere520. This offers an opportunity for multiplexed DNA sensing. Based on this, we developed a novel GNR mediated fluorescence biosensor for DNA detection. Fluorescence labeled haipin-DNA probes were introduced to designated spots of GNR array with the matching LSPR wavelengths on the substrate. The fluorescence was quenched originally because of Forster resonance energy transfer (FRET) effect

Detection of Nicotiana DNA in Tobacco Products Using a Novel Multiplex Real-Time PCR Assay.

PubMed

Korchinski, Katie L; Land, Adrian D; Craft, David L; Brzezinski, Jennifer L

2016-07-01

Establishing that a product contains tobacco is a requirement for the U.S. Food and Drug Administration's regulation and/or prosecution of tobacco products. Therefore, a multiplex real-time PCR method was designed to determine if Nicotiana (tobacco) DNA is present in tobacco products. The PCR method simultaneously amplifies a 73 bp fragment of the cytochrome P450 monoxygenase CYP82E4 gene and 66 bp fragment in the nia-1 gene for nitrate reductase, which are detected using dual-labeled TaqMan probes. The assay is capable of detecting approximately 7.8 pg purified tobacco DNA, with a similar sensitivity for either gene target while incorporating an internal positive control (IPC). DNA was extracted from prepared tobacco products-including chewing tobacco, pipe tobacco, and snuff-or from the cut fill (no wrapper) of cigarettes and cigars. Of the 13 products analyzed, 12 were positive for both tobacco-specific markers and the IPC. DNA was also extracted from the fill of five varieties of herbal cigarettes, which were negative for both tobacco-specific gene targets and positive for the IPC. Our method expands on current assays by introducing a multiplex reaction, targeting two sequences in two different genes of interest, incorporating an IPC into the reaction, and lowering the LOD and LOQ while increasing the efficiency of the PCR.

A multiplex PCR assay for the rapid and sensitive detection of methicillin-resistant Staphylococcus aureus and simultaneous discrimination of Staphylococcus aureus from coagulase-negative staphylococci.

PubMed

Xu, Benjin; Liu, Ling; Liu, Li; Li, Xinping; Li, Xiaofang; Wang, Xin

2012-11-01

Methicillin-resistant Staphylococcus aureus (MRSA) is a global health concern, which had been detected in food and food production animals. Conventional testing for detection of MRSA takes 3 to 5 d to yield complete information of the organism and its antibiotic sensitivity pattern. So, a rapid method is needed to diagnose and treat the MRSA infections. The present study focused on the development of a multiplex PCR assay for the rapid and sensitive detection of MRSA. The assay simultaneously detected 4 genes, namely, 16S rRNA of the Staphylococcus genus, femA of S. aureus, mecA that encodes methicillin resistance, and one internal control. It was rapid and yielded results within 4 h. The analytical sensitivity and specificity of the multiplex PCR assay was evaluated by comparing it with the conventional method. The analytical sensitivity of the multiplex PCR assay at the DNA level was 10 ng DNA. The analytical specificity was evaluated with 10 reference staphylococci strains and was 100%. The diagnostic evaluation of MRSA was carried out using 360 foodborne staphylococci isolates, and showed 99.1% of specificity, 96.4% of sensitivity, 97.5% of positive predictive value, and 97.3% of negative predictive value compared to the conventional method. The inclusion of an internal control in the multiplex PCR assay is important to exclude false-negative cases. This test can be used as an effective diagnostic and surveillance tool to investigate the spread and emergence of MRSA. © 2012 Institute of Food Technologists®

Perspectives on using a multiplex human kidney safety biomarker panel to detect cisplatin-induced tubular toxicity in male and female Cynomolgus monkeys.

PubMed

Chen, Yafei; Dale Thurman, J; Kinter, Lewis B; Bialecki, Russell; Eric McDuffie, J

2017-12-01

Multiplex biomarker panel assays would enable early de-risking of discovery compound related kidney safety liabilities. The objective of this study was to evaluate the usefulness of the Myriad RBM Human KidneyMAP (Multi-Analyte Profile)® v.1.0 panel to detect experimental nephrotoxicity in Cynomolgus monkeys following a single intravenous administration of cisplatin (2.5mg/kg). Urine samples were collected at baseline on day -2; at approximately 4hr post-dose on day 1; and on days 4, 9, 15 and/or 20. Blood samples were collected at predose on day -2; at 4hr post-dose on day 1; and on days 2, 5, 10 and/or 21. Changes in toxicokinetic and biochemistry parameters in plasma, qualitative/quantitative urinalysis parameters, and urinary kidney safety biomarkers were assessed. Kidney tissues were collected on days 2, 5, 10 and 21 for routine microscopy. Cisplatin-induced tubular alterations were characterized by acute and progressive cortical tubular degeneration/necrosis, regeneration, tubular dilation and proteinaceous cast in the absence of statistically significant changes in traditional plasma biochemistry and urinalysis parameters. When normalized to urinary creatinine, cisplatin-induced significant increases in urinary levels of kidney injury molecule 1 (females on day 4), increases in calbindin D28k (males and females on day 4), decreases in Tamm-Horsfall glycoprotein (males on days 1, 4 and 9), and increases in clusterin (females and males on days 15 and 20, respectively), when compared to concurrent controls. This study revealed the usefulness of the Human KidneyMAP® multiplex panel when measuring changes in urine-based biomarkers to reliably detect cisplatin-induced acute/progressive cortical tubular injury in male and female Cynomolgus monkeys. Copyright © 2017 Elsevier Inc. All rights reserved.

Multiplex autoantibody detection for autoimmune liver diseases and autoimmune gastritis.

PubMed

Vanderlocht, Joris; van der Cruys, Mart; Stals, Frans; Bakker-Jonges, Liesbeth; Damoiseaux, Jan

2017-09-01

Autoantibody detection for autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC) and autoimmune gastritis (AIG) is traditionally performed by IIF on a combination of tissues. Multiplex line/dot blots (LIA/DIA) offer multiple advantages, i.e. automation, objective reading, no interfering reactivities, no coincidental findings. In the current study we evaluated automated DIA (D-Tek) for detecting autoantibodies related to autoimmune diseases of the gastrointestinal tract. We tested samples of the Dutch EQC program and compared the results with the consensus of the participating labs. For the autoimmune liver diseases and AIG, respectively, 64 and 36 samples were tested. For anti-mitochondrial and anti-smooth muscle antibodies a concordance rate of 97% and 88% was observed, respectively. The concordance rate for anti-parietal cell antibodies was 92% when samples without EQC consensus (n=15) were excluded. For antibodies against intrinsic factor a concordance of 96% was observed. For all these antibodies discrepancies were identified that relate to the different test characteristics and the preponderance of IIF utilizing labs in the EQC program. In conclusion, we observed good agreement of the tested DIA blots with the consensus results of the Dutch EQC program. Taken together with the logistic advantages these blots are a good alternative for autoantibody detection in the respective diseases. A large prospective multicenter study is warranted to position these novel tests further in the whole spectrum of assays for the detection of these antibodies in a routine autoimmune laboratory. Copyright © 2017 Elsevier B.V. All rights reserved.

A PDMS/paper/glass hybrid microfluidic biochip integrated with aptamer-functionalized graphene oxide nano-biosensors for one-step multiplexed pathogen detection.

PubMed

Zuo, Peng; Li, XiuJun; Dominguez, Delfina C; Ye, Bang-Ce

2013-10-07

Infectious pathogens often cause serious public health concerns throughout the world. There is an increasing demand for simple, rapid and sensitive approaches for multiplexed pathogen detection. In this paper we have developed a polydimethylsiloxane (PDMS)/paper/glass hybrid microfluidic system integrated with aptamer-functionalized graphene oxide (GO) nano-biosensors for simple, one-step, multiplexed pathogen detection. The paper substrate used in this hybrid microfluidic system facilitated the integration of aptamer biosensors on the microfluidic biochip, and avoided complicated surface treatment and aptamer probe immobilization in a PDMS or glass-only microfluidic system. Lactobacillus acidophilus was used as a bacterium model to develop the microfluidic platform with a detection limit of 11.0 cfu mL(-1). We have also successfully extended this method to the simultaneous detection of two infectious pathogens - Staphylococcus aureus and Salmonella enterica. This method is simple and fast. The one-step 'turn on' pathogen assay in a ready-to-use microfluidic device only takes ~10 min to complete on the biochip. Furthermore, this microfluidic device has great potential in rapid detection of a wide variety of different other bacterial and viral pathogens.

A PDMS/paper/glass hybrid microfluidic biochip integrated with aptamer-functionalized graphene oxide nano-biosensors for one-step multiplexed pathogen detection

PubMed Central

Zuo, Peng; Dominguez, Delfina C.; Ye, Bang-Ce

2014-01-01

Infectious pathogens often cause serious public health concerns throughout the world. There is an increasing demand for simple, rapid and sensitive approaches for multiplexed pathogen detection. In this paper we have developed a polydimethylsiloxane (PDMS)/paper/glass hybrid microfluidic system integrated with aptamer-functionalized graphene oxide (GO) nano-biosensors for simple, one-step, multiplexed pathogen detection. The paper substrate used in this hybrid microfluidic system facilitated the integration of aptamer biosensors on the microfluidic biochip, and avoided complicated surface treatment and aptamer probe immobilization in a PDMS or glass-only microfluidic system. Lactobacillus acidophilus was used as a bacterium model to develop the microfluidic platform with a detection limit of 11.0 cfu mL−1. We have also successfully extended this method to the simultaneous detection of two infectious pathogens - Staphylococcus aureus and Salmonella enterica. This method is simple and fast. The one-step ‘turn on’ pathogen assay in a ready-to-use microfluidic device only takes ~10 min to complete on the biochip. Furthermore, this microfluidic device has great potential in rapid detection of a wide variety of different other bacterial and viral pathogens. PMID:23929394

A multiplex PCR-based method for the detection and early identification of wood rotting fungi in standing trees.

PubMed

Guglielmo, F; Bergemann, S E; Gonthier, P; Nicolotti, G; Garbelotto, M

2007-11-01

The goal of this research was the development of a PCR-based assay to identify important decay fungi from wood of hardwood tree species in northern temperate regions. Eleven taxon-specific primers were designed for PCR amplification of either nuclear or mitochondrial ribosomal DNA regions of Armillaria spp., Ganoderma spp., Hericium spp., Hypoxylon thouarsianum var. thouarsianum, Inonotus/Phellinus-group, Laetiporus spp., Perenniporia fraxinea, Pleurotus spp., Schizophyllum spp., Stereum spp. and Trametes spp. Multiplex PCR reactions were developed and optimized to detect fungal DNA and identify each taxon with a sensitivity of at least 1 pg of target DNA in the template. This assay correctly identified the agents of decay in 82% of tested wood samples. The development and optimization of multiplex PCRs allowed for reliable identification of wood rotting fungi directly from wood. Early detection of wood decay fungi is crucial for assessment of tree stability in urban landscapes. Furthermore, this method may prove useful for prediction of the severity and the evolution of decay in standing trees.

Dual-channel-coded microbeads for multiplexed detection of biomolecules using assembling of quantum dots and element coding nanoparticles.

PubMed

Lu, Bangrong; He, Qinghua; He, Yonghong; Chen, Xuejing; Feng, Guangxia; Liu, Siyu; Ji, Yanhong

2018-09-18

To achieve the dual-channel (analog and digital) encoding, microbeads assembled with quantum dots (QDs) and element coding nanoparticles (ECNPs) have been prepared. Dual-spectra, including fluorescence generated from quantum dots (QDs) and laser induced breakdown spectrum obtained from the plasma of ECNPs, including AgO, MgO and ZnO nanoparticles, has been adopted to provide more encoding amounts and more accurate dual recognition for encoded microbeads in multiplexed utilization. The experimental results demonstrate that the single microbead can be decoded in two optical channels. Multiplexed analysis and contrast adsorption experiment of anti-IgG verified the availability and specificity of dual-channel-coded microbeads in bioanalysis. In gradient detection of anti-IgG, we obtained the linear concentration response to target biomolecules from 3.125 × 10 -10  M to 1 × 10 -8  M, and the limit of detection was calculated to be 2.91 × 10 -11  M. Copyright © 2018 Elsevier B.V. All rights reserved.

Metric projection for dynamic multiplex networks.

PubMed

Jurman, Giuseppe

2016-08-01

Evolving multiplex networks are a powerful model for representing the dynamics along time of different phenomena, such as social networks, power grids, biological pathways. However, exploring the structure of the multiplex network time series is still an open problem. Here we propose a two-step strategy to tackle this problem based on the concept of distance (metric) between networks. Given a multiplex graph, first a network of networks is built for each time step, and then a real valued time series is obtained by the sequence of (simple) networks by evaluating the distance from the first element of the series. The effectiveness of this approach in detecting the occurring changes along the original time series is shown on a synthetic example first, and then on the Gulf dataset of political events.

Multiplexed MRM-based assays for the quantitation of proteins in mouse plasma and heart tissue.

PubMed

Percy, Andrew J; Michaud, Sarah A; Jardim, Armando; Sinclair, Nicholas J; Zhang, Suping; Mohammed, Yassene; Palmer, Andrea L; Hardie, Darryl B; Yang, Juncong; LeBlanc, Andre M; Borchers, Christoph H

2017-04-01

The mouse is the most commonly used laboratory animal, with more than 14 million mice being used for research each year in North America alone. The number and diversity of mouse models is increasing rapidly through genetic engineering strategies, but detailed characterization of these models is still challenging because most phenotypic information is derived from time-consuming histological and biochemical analyses. To expand the biochemists' toolkit, we generated a set of targeted proteomic assays for mouse plasma and heart tissue, utilizing bottom-up LC/MRM-MS with isotope-labeled peptides as internal standards. Protein quantitation was performed using reverse standard curves, with LC-MS platform and curve performance evaluated by quality control standards. The assays comprising the final panel (101 peptides for 81 proteins in plasma; 227 peptides for 159 proteins in heart tissue) have been rigorously developed under a fit-for-purpose approach and utilize stable-isotope labeled peptides for every analyte to provide high-quality, precise relative quantitation. In addition, the peptides have been tested to be interference-free and the assay is highly multiplexed, with reproducibly determined protein concentrations spanning >4 orders of magnitude. The developed assays have been used in a small pilot study to demonstrate their application to molecular phenotyping or biomarker discovery/verification studies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Detecting Nonvolatile Life- and Nonlife-Derived Organics in a Carbonaceous Chondrite Analogue with a New Multiplex Immunoassay and Its Relevance for Planetary Exploration.

PubMed

Moreno-Paz, Mercedes; Gómez-Cifuentes, Ana; Ruiz-Bermejo, Marta; Hofstetter, Oliver; Maquieira, Ángel; Manchado, Juan M; Morais, Sergi; Sephton, Mark A; Niessner, Reinhard; Knopp, Dietmar; Parro, Victor

2018-04-11

Potential martian molecular targets include those supplied by meteoritic carbonaceous chondrites such as amino acids and polycyclic aromatic hydrocarbons and true biomarkers stemming from any hypothetical martian biota (organic architectures that can be directly related to once living organisms). Heat extraction and pyrolysis-based methods currently used in planetary exploration are highly aggressive and very often modify the target molecules making their identification a cumbersome task. We have developed and validated a mild, nondestructive, multiplex inhibitory microarray immunoassay and demonstrated its implementation in the SOLID (Signs of Life Detector) instrument for simultaneous detection of several nonvolatile life- and nonlife-derived organic molecules relevant in planetary exploration and environmental monitoring. By utilizing a set of highly specific antibodies that recognize D- or L- aromatic amino acids (Phe, Tyr, Trp), benzo[a]pyrene (B[a]P), pentachlorophenol, and sulfone-containing aromatic compounds, respectively, the assay was validated in the SOLID instrument for the analysis of carbon-rich samples used as analogues of the organic material in carbonaceous chondrites or even Mars samples. Most of the antibodies enabled sensitivities at the 1-10 ppb level and some even at the ppt level. The multiplex immunoassay allowed the detection of B[a]P as well as aromatic sulfones in a water/methanol extract of an Early Cretaceous lignite sample (c.a., 140 Ma) representing type IV kerogen. No L- or D-aromatic amino acids were detected, reflecting the advanced diagenetic stage and the fossil nature of the sample. The results demonstrate the ability of the liquid extraction by ultrasonication and the versatility of the multiplex inhibitory immunoassays in the SOLID instrument to discriminate between organic matter derived from life and nonlife processes, an essential step toward life detection outside Earth. Key Words: Planetary exploration

A Portable Immunoassay Platform for Multiplexed Detection of Biotoxins in Clinical and Environmental Samples

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

Koh, Chung-Yan; Piccini, Matthew Ernest; Schaff, Ulrich Y.

Multiple cases of attempted bioterrorism events using biotoxins have highlighted the urgent need for tools capable of rapid screening of suspect samples in the field (e.g., mailroom and public events). We present a portable microfluidic device capable of analyzing environmental (e.g., white powder), food (e.g., milk) and clinical (e.g., blood) samples for multiplexed detection of biotoxins. The device is rapid (<15-30 min sample-to-answer), sensitive (< 0.08 pg/mL detection limit for botulinum toxin), multiplexed (up to 64 parallel assays) and capable of analyzing small volume samples (< 20 μL total sample input). The immunoassay approach (SpinDx) is based on binding ofmore » toxins in a sample to antibody-laden capture particles followed by sedimentation of particles through a density-media in a microfluidic disk and quantification using a laser-induced fluorescence detector. A direct, blinded comparison with a gold standard ELISA revealed a 5-fold more sensitive detection limit for botulinum toxin while requiring 250-fold less sample volume and a 30 minute assay time with a near unity correlation. A key advantage of the technique is its compatibility with a variety of sample matrices with no additional sample preparation required. Ultrasensitive quantification has been demonstrated from direct analysis of multiple clinical, environmental and food samples, including white powder, whole blood, saliva, salad dressing, whole milk, peanut butter, half and half, honey, and canned meat. We believe that this device can met an urgent need in screening both potentially exposed people as well as suspicious samples in mail-rooms, airports, public sporting venues and emergency rooms. The general-purpose immunodiagnostics device can also find applications in screening of infectious and systemic diseases or serve as a lab device for conducting rapid immunoassays.« less

The Design of a Quantitative Western Blot Experiment

PubMed Central

Taylor, Sean C.; Posch, Anton

2014-01-01

Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. In the past, western blotting was used simply to detect a specific target protein in a complex mixture, but now journal editors and reviewers are requesting the quantitative interpretation of western blot data in terms of fold changes in protein expression between samples. The calculations are based on the differential densitometry of the associated chemiluminescent and/or fluorescent signals from the blots and this now requires a fundamental shift in the experimental methodology, acquisition, and interpretation of the data. We have recently published an updated approach to produce quantitative densitometric data from western blots (Taylor et al., 2013) and here we summarize the complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting. PMID:24738055

[Multiplex PCR strategy for the simultaneous identification of Staphylococcus aureus and detection of staphylococcal enterotoxins in isolates from food poisoning outbreaks].

PubMed

Brizzio, Aníbal A; Tedeschi, Fabián A; Zalazar, Fabián E

2013-01-01

Staphylococcal food poisoning is the most frequent type of food poisoning around the world. Staphylococcus aureus enterotoxins cause significant loss of water in the intestinal lumen, followed by vomiting and diarrhea. To report a fast, reliable and inexpensive strategy based on multiplex PCR for the simultaneous identification of S. aureus and detection of five classical S. aureus enterotoxin genes ( sea, seb, sec, sed, see ) in Staphylococcus spp. strains isolated from food poisoning outbreaks. We analyzed isolates from 12 food poisoning outbreaks occurred in Santa Fe province (Argentina). Isolation and phenotypic characterization were carried out by standard procedures. Genotypic analysis was performed by multiplex PCR, using primers for nuc , sea-see and 16S rRNA genes simultaneously. Of all the strains tested, 58% were found to carry toxigenic genes. Sea and seb toxins were found at the same percentage (29%) while sec, sed and see genes were found in a lower and identical proportion (14%). We did not find more than one different type of S. aureus enterotoxin in the isolates analyzed. The multiplex PCR strategy designed in this work has enabled us to identify strains of S. aureus and detect -at the same time- their enterotoxigenic ability. At present, our efforts are devoted to the detection of genes encoding enterotoxins other than the classical ones, in order to know their impact on staphylococcal food poisoning, as well as to investigate their relevance to our country's public health.

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

PubMed

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

2015-04-24

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

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

PubMed Central

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

2014-01-01

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

Simplified Real-Time Multiplex Detection of Loop-Mediated Isothermal Amplification Using Novel Mediator Displacement Probes with Universal Reporters.

PubMed

Becherer, Lisa; Bakheit, Mohammed; Frischmann, Sieghard; Stinco, Silvina; Borst, Nadine; Zengerle, Roland; von Stetten, Felix

2018-04-03

A variety of real-time detection techniques for loop-mediated isothermal amplification (LAMP) based on the change in fluorescence intensity during DNA amplification enable simultaneous detection of multiple targets. However, these techniques depend on fluorogenic probes containing target-specific sequences. That complicates the adaption to different targets leading to time-consuming assay optimization. Here, we present the first universal real-time detection technique for multiplex LAMP. The novel approach allows simple assay design and is easy to implement for various targets. The innovation features a mediator displacement probe and a universal reporter. During amplification of target DNA the mediator is displaced from the mediator displacement probe. Then it hybridizes to the reporter generating a fluorescence signal. The novel mediator displacement (MD) detection was validated against state-of-the-art molecular beacon (MB) detection by means of a HIV-1 RT-LAMP: MD surpassed MB detection by accelerated probe design (MD: 10 min, MB: 3-4 h), shorter times to positive (MD 4.1 ± 0.1 min shorter than MB, n = 36), improved signal-to-noise fluorescence ratio (MD: 5.9 ± 0.4, MB: 2.7 ± 0.4; n = 15), and showed equally good or better analytical performance parameters. The usability of one universal mediator-reporter set in different multiplex assays was successfully demonstrated for a biplex RT-LAMP of HIV-1 and HTLV-1 and a biplex LAMP of Haemophilus ducreyi and Treponema pallidum, both showing good correlation between target concentration and time to positive. Due to its simple implementation it is suggested to extend the use of the universal mediator-reporter sets to the detection of various other diagnostic panels.

Clinical evaluation of a new single-tube multiplex reverse transcription PCR assay for simultaneous detection of 11 respiratory viruses, Mycoplasma pneumoniae and Chlamydia in hospitalized children with acute respiratory infections.

PubMed

Zhao, Meng-Chuan; Li, Gui-Xia; Zhang, Dan; Zhou, Hang-Yu; Wang, Hao; Yang, Shuo; Wang, Le; Feng, Zhi-Shan; Ma, Xue-Jun

2017-06-01

Respiratory Pathogen 13 Detection Kit (13× kit) is able to simultaneously detect 11 respiratory viruses, Mycoplasma pneumoniae (MP) and Chlamydia in a single reaction. Using 572 Nasopharyngeal aspirates collected from hospitalized children, the clinical performance of 13× kit for detecting 11 respiratory viruses was evaluated in comparison with a routinely used 2-tube multiplex reverse transcription PCR assay (2-tube assay) at provincial Centers for Disease Control and Prevention in China. The clinical performance of 13× kit for detecting MP and Chlamydia was evaluated by commercial real-time quantitative PCR (qPCR) kits or sequencing. For tested viruses, the assay concordance was 95.98% and the kappa coefficient was 0.89. All the MP and Chlamydia positive samples detected by 13× kit were confirmed as true positives. The utilization of the 13× kit in clinical settings will be helpful for doctors to assess clinical outcome according to virus type or multiple infections, and to limit the use of antibiotics. Copyright © 2017 Elsevier Inc. All rights reserved.

Multiplex PCR for the detection and identification of dairy bacteriophages in milk.

PubMed

del Rio, B; Binetti, A G; Martín, M C; Fernández, M; Magadán, A H; Alvarez, M A

2007-02-01

Bacteriophage infections of starter lactic acid bacteria are a serious risk in the dairy industry. Phage infection can lead to slow lactic acid production or even the total failure of fermentation. The associated economic losses can be substantial. Rapid and sensitive methods are therefore required to detect and identify phages at all stages of the manufacture of fermented dairy products. This study describes a simple and rapid multiplex PCR method that, in a single reaction, detects the presence of bacteriophages infecting Streptococcus thermophilus and Lactobacillus delbrueckii, plus three genetically distinct 'species' of Lactococcus lactis phages commonly found in dairy plants (P335, 936 and c2). Available bacteriophage genome sequences were examined and the conserved regions used to design five pairs of primers, one for each of the above bacteriophage species. These primers were designed to generate specific fragments of different size depending on the species. Since this method can detect the above phages in untreated milk and can be easily incorporated into dairy industry routines, it might be readily used to earmark contaminated milk for use in processes that do not involve susceptible starter organisms or for use in those that involve phage-deactivating conditions.

Multiplexed operation of a micromachined ultrasonic droplet ejector array

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

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

2007-10-15

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

Rapid, sensitive, and simultaneous detection of three foodborne pathogens using magnetic nanobead-based immunoseparation and quantum dot-based multiplex immunoassay.

PubMed

Wang, Hong; Li, Yanbin; Wang, Andrew; Slavik, Michael

2011-12-01

Losses caused by foodborne diseases are enormous in terms of human life, illness, medical costs, and food product recalls. Rapid detection of multiple bacterial pathogens in foods is extremely important to ensure food safety. The objective of this research was to develop a multiplex immunoassay by integrating magnetic nanobeads (MNBs) for immunoseparation with quantum dots (QDs) as fluorescent labels for rapid, sensitive, and simultaneous detection of three major pathogenic bacteria, Salmonella Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes, in food products. In this research, both streptavidin-conjugated MNBs (30- and 150-nm diameter) and QDs (530-, 580-, and 620-nm emission wavelength) were separately coated with biotinylated anti-Salmonella, anti-E. coli, and anti-Listeria antibodies. The immuno-MNBs were mixed with a food sample to capture the three target bacteria. After being magnetically separated from the sample, the MNB-cell conjugates were mixed with the immuno-QDs to form the MNB-cell-QD complexes, and unattached QDs were removed. The fluorescence intensity of the MNB-cell-QD complexes was measured at wavelengths of 530, 580, and 620 nm to determine the populations of Salmonella Typhimurium, E. coli O157:H7, and L. monocytogenes, respectively. This multiplex immunoassay simultaneously detected Salmonella Typhimurium, E. coli O157:H7, and L. monocytogenes at levels as low as 20 to 50 CFU/ml in food samples in less than 2 h without enrichment. The change in fluorescence intensity was linearly correlated (R(2) > 0.96) with the logarithmic value of bacterial level in the range of 10 to 10(3) CFU/ml. More than 85% of the three target pathogens could be simultaneously separated from food samples. The multiplex immunoassay could be expanded to detect more target pathogens, depending on the availability of specific antibodies and QDs with different emission wavelengths.

Burst noise in the HAWAII-1RG multiplexer

NASA Astrophysics Data System (ADS)

Bacon, Candice M.; McMurtry, Craig W.; Pipher, Judith L.; Forrest, William J.; Garnett, James D.

2005-08-01

Burst noise (also known as popcorn noise and random telegraph signal/noise) is a phenomenon that is understood to be a result of defects in the vicinity of a p-n junction. It is characterized by rapid level shifts in both positive and negative directions and can have varying magnitudes. This noise has been seen in both HAWAII-1RG and HAWAII-2RG multiplexers and is under investigation. We have done extensive burst noise testing on a HAWAII-1RG multiplexer, where we have determined a significant percentage of pixels exhibit the phenomenon. In addition, the prevalence of small magnitude transitions make sensitivity of detection the main limiting factor. Since this is a noise source for the HAWAII-1RG multiplexer, its elimination would make the HAWAII-1RG and the HAWAII-2RG even lower noise multiplexers.

Single-Cell, Multiplexed Protein Detection of Rare Tumor Cells Based on a Beads-on-Barcode Antibody Microarray.

PubMed

Yang, Liu; Wang, Zhihua; Deng, Yuliang; Li, Yan; Wei, Wei; Shi, Qihui

2016-11-15

Circulating tumor cells (CTCs) shed from tumor sites and represent the molecular characteristics of the tumor. Besides genetic and transcriptional characterization, it is important to profile a panel of proteins with single-cell precision for resolving CTCs' phenotype, organ-of-origin, and drug targets. We describe a new technology that enables profiling multiple protein markers of extraordinarily rare tumor cells at the single-cell level. This technology integrates a microchip consisting of 15000 60 pL-sized microwells and a novel beads-on-barcode antibody microarray (BOBarray). The BOBarray allows for multiplexed protein detection by assigning two independent identifiers (bead size and fluorescent color) of the beads to each protein. Four bead sizes (1.75, 3, 4.5, and 6 μm) and three colors (blue, green, and yellow) are utilized to encode up to 12 different proteins. The miniaturized BOBarray can fit an array of 60 pL-sized microwells that isolate single cells for cell lysis and the subsequent detection of protein markers. An enclosed 60 pL-sized microchamber defines a high concentration of proteins released from lysed single cells, leading to single-cell resolution of protein detection. The protein markers assayed in this study include organ-specific markers and drug targets that help to characterize the organ-of-origin and drug targets of isolated rare tumor cells from blood samples. This new approach enables handling a very small number of cells and achieves single-cell, multiplexed protein detection without loss of rare but clinically important tumor cells.

Application of a multiplex PCR assay for Campylobacter fetus detection and subspecies differentiation in uncultured samples of aborted bovine fetuses.

PubMed

Iraola, Gregorio; Hernández, Martín; Calleros, Lucía; Paolicchi, Fernando; Silveyra, Silvia; Velilla, Alejandra; Carretto, Luis; Rodríguez, Eliana; Pérez, Ruben

2012-12-01

Campylobacter (C.) fetus (epsilonproteobacteria) is an important veterinary pathogen. This species is currently divided into C. fetus subspecies (subsp.) fetus (Cff) and C. fetus subsp. venerealis (Cfv). Cfv is the causative agent of bovine genital Campylobacteriosis, an infectious disease that leads to severe reproductive problems in cattle worldwide. Cff is a more general pathogen that causes reproductive problems mainly in sheep although cattle can also be affected. Here we describe a multiplex PCR method to detect C. fetus and differentiate between subspecies in a single step. The assay was standardized using cultured strains and successfully used to analyze the abomasal liquid of aborted bovine fetuses without any pre-enrichment step. Results of our assay were completely consistent with those of traditional bacteriological diagnostic methods. Furthermore, the multiplex PCR technique we developed may be easily adopted by any molecular diagnostic laboratory as a complementary tool for detecting C. fetus subspecies and obtaining epidemiological information about abortion events in cattle.

Evaluation of a multiplex PCR assay for detection of cytomegalovirus in stool samples from patients with ulcerative colitis

PubMed Central

Nahar, Saifun; Iraha, Atsushi; Hokama, Akira; Uehara, Ayako; Parrott, Gretchen; Ohira, Tetsuya; Kaida, Masatoshi; Kinjo, Tetsu; Kinjo, Takeshi; Hirata, Tetsuo; Kinjo, Nagisa; Fujita, Jiro

2015-01-01

AIM: To evaluate a multiplex PCR assay for the detection of bacterial and viral enteropathogens in stool samples from patients with ulcerative colitis (UC). METHODS: We prospectively analyzed 300 individuals, including immunocompetent patients, immunocompromised patients, and patients with UC. Stool samples were collected from the recto-sigmoid region of the colon by endoscopy. The samples were qualitatively analyzed for bacterial and viral enteropathogens with a multiplex PCR assay using a Seeplex® Kit. Additional clinical and laboratory data were collected from the medical records. RESULTS: A multiplex PCR assay detected 397 pathogens (191 bacteria and 206 viruses) in 215 samples (71.7%). The most frequently detected bacteria were Escherichia coli H7, 85 (28.3%); followed by Aeromonas spp., 43 (14.3%); and Clostridium perfringens, 36 (12.0%) samples. The most prevalent viruses were Epstein-Barr virus (EBV), 90 (30.0%); followed by human herpes virus-6 (HHV-6), 53 (17.7%); and cytomegalovirus (CMV), 37 (12.3%) samples. The prevalence rate of CMV infection was significantly higher in the immunocompromised group than in the immunocompetent group (P < 0.01). CMV infection was more common in patients with UC (26/71; 36.6%) than in the immunocompetent patients excluding UC (6/188; 3.2%) (P < 0.01). CMV infection was more prevalent in UC active patients (25/58; 43.1%) than in UC inactive patients (1/13; 7.7%) (P < 0.05). Among 4 groups which defined by the UC activity and immunosuppressive drugs, the prevalence rate of CMV infection was highest in the UC active patients with immunosuppressive drugs (19/34; 55.8%). Epstein-Barr virus (EBV) infection was more common in the immunocompromised patients excluding UC (18/41; 43.9%) than in the immunocompetent patients excluding UC (47/188; 25.0%) (P < 0.05). The simultaneous presence of CMV and EBV and/or HHV6 in UC active patients (14/58; 24.1%) was greater than in immunocompromised patients excluding UC (5/41; 12.2%) (P < 0

Multiplex PCR assay for the detection of five meat species forbidden in Islamic foods.

PubMed

Ali, Md Eaqub; Razzak, Md Abdur; Hamid, Sharifah Bee Abd; Rahman, Md Mahfujur; Amin, Md Al; Rashid, Nur Raifana Abd; Asing

2015-06-15

Food falsification has direct impact on public health, religious faith, fair-trades and wildlife. For the first time, here we described a multiplex polymerase chain reaction assay for the accurate identification of five meat species forbidden in Islamic foods in a single assay platform. Five pairs of species-specific primers were designed targeting mitochondrial ND5, ATPase 6, and cytochrome b genes to amplify 172, 163, 141, 129 and 108 bp DNA fragments from cat, dog, pig, monkey and rat meats, respectively. All PCR products were identified in gel-images and electrochromatograms obtained from Experion Bioanalyzer. Species-specificity checking against 15 important meat and fish and 5 plant species detected no cross-species amplification. Screening of target species in model and commercial meatballs reflected its application to detect target species in process foods. The assay was tested to detect 0.01-0.02 ng DNA under raw states and 1% suspected meats in meatball formulation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Spectroscopic optical coherence tomography based on wavelength de-multiplexing and smart pixel array detection

NASA Astrophysics Data System (ADS)

Laubscher, Markus; Bourquin, Stéphane; Froehly, Luc; Karamata, Boris; Lasser, Theo

2004-07-01

Current spectroscopic optical coherence tomography (OCT) methods rely on a posteriori numerical calculation. We present an experimental alternative for accessing spectroscopic information in OCT without post-processing based on wavelength de-multiplexing and parallel detection using a diffraction grating and a smart pixel detector array. Both a conventional A-scan with high axial resolution and the spectrally resolved measurement are acquired simultaneously. A proof-of-principle demonstration is given on a dynamically changing absorbing sample. The method's potential for fast spectroscopic OCT imaging is discussed. The spectral measurements obtained with this approach are insensitive to scan non-linearities or sample movements.

Improved serotype-specific dengue virus detection in Trinidad and Tobago using a multiplex, real-time RT-PCR.

PubMed

Waggoner, Jesse J; Sahadeo, Nikita S D; Brown, Arianne; Mohamed-Hadley, Alisha; Hadley, Dexter; Carrington, Leslie; Carrington, Christine V F; Pinsky, Benjamin A

2015-02-01

Dengue virus (DENV) transmission occurs throughout the Caribbean, though laboratory confirmation and epidemiologic surveillance are limited by the availability of serotype-specific molecular diagnostics. In this study, we show that a serotype-specific DENV multiplex, real-time reverse transcriptase-PCR (RT-PCR) detected DENV RNA in significantly more samples (82/182) than a reference hemi-nested RT-PCR (57/182; P=0.01). Copyright © 2015 Elsevier Inc. All rights reserved.

Allele-specific methylated multiplex real-time quantitative PCR (ASMM RTQ-PCR), a powerful method for diagnosing loss of imprinting of the 11p15 region in Russell Silver and Beckwith Wiedemann syndromes.

PubMed

Azzi, Salah; Steunou, Virginie; Rousseau, Alexandra; Rossignol, Sylvie; Thibaud, Nathalie; Danton, Fabienne; Le Jule, Marilyne; Gicquel, Christine; Le Bouc, Yves; Netchine, Irène

2011-02-01

Many human syndromes involve a loss of imprinting (LOI) due to a loss (LOM) or a gain of DNA methylation (GOM). Most LOI occur as mosaics and can therefore be difficult to detect with conventional methods. The human imprinted 11p15 region is crucial for the control of fetal growth, and LOI at this locus is associated with two clinical disorders with opposite phenotypes: Beckwith-Wiedemann syndrome (BWS), characterized by fetal overgrowth and a high risk of tumors, and Russell-Silver syndrome (RSS), characterized by intrauterine and postnatal growth restriction. Until recently, we have been using Southern blotting for the diagnosis of RSS and BWS. We describe here a powerful quantitative technique, allele-specific methylated multiplex real-time quantitative PCR (ASMM RTQ-PCR), for the diagnosis of these two complex disorders. We first checked the specificity of the probes and primers used for ASMM RTQ-PCR. We then carried out statistical validation for this method, on both retrospective and prospective populations of patients. This analysis demonstrated that ASMM RTQ-PCR is more sensitive than Southern blotting for detecting low degree of LOI. Moreover, ASMM RTQ-PCR is a very rapid, reliable, simple, safe, and cost effective method. © 2011 Wiley-Liss, Inc.

A gold nanoparticle-based semi-quantitative and quantitative ultrasensitive paper sensor for the detection of twenty mycotoxins

NASA Astrophysics Data System (ADS)

Kong, Dezhao; Liu, Liqiang; Song, Shanshan; Suryoprabowo, Steven; Li, Aike; Kuang, Hua; Wang, Libing; Xu, Chuanlai

2016-02-01

A semi-quantitative and quantitative multi-immunochromatographic (ICA) strip detection assay was developed for the simultaneous detection of twenty types of mycotoxins from five classes, including zearalenones (ZEAs), deoxynivalenols (DONs), T-2 toxins (T-2s), aflatoxins (AFs), and fumonisins (FBs), in cereal food samples. Sensitive and specific monoclonal antibodies were selected for this assay. The semi-quantitative results were obtained within 20 min by the naked eye, with visual limits of detection for ZEAs, DONs, T-2s, AFs and FBs of 0.1-0.5, 2.5-250, 0.5-1, 0.25-1 and 2.5-10 μg kg-1, and cut-off values of 0.25-1, 5-500, 1-10, 0.5-2.5 and 5-25 μg kg-1, respectively. The quantitative results were obtained using a hand-held strip scan reader, with the calculated limits of detection for ZEAs, DONs, T-2s, AFs and FBs of 0.04-0.17, 0.06-49, 0.15-0.22, 0.056-0.49 and 0.53-1.05 μg kg-1, respectively. The analytical results of spiked samples were in accordance with the accurate content in the simultaneous detection analysis. This newly developed ICA strip assay is suitable for the on-site detection and rapid initial screening of mycotoxins in cereal samples, facilitating both semi-quantitative and quantitative determination.A semi-quantitative and quantitative multi-immunochromatographic (ICA) strip detection assay was developed for the simultaneous detection of twenty types of mycotoxins from five classes, including zearalenones (ZEAs), deoxynivalenols (DONs), T-2 toxins (T-2s), aflatoxins (AFs), and fumonisins (FBs), in cereal food samples. Sensitive and specific monoclonal antibodies were selected for this assay. The semi-quantitative results were obtained within 20 min by the naked eye, with visual limits of detection for ZEAs, DONs, T-2s, AFs and FBs of 0.1-0.5, 2.5-250, 0.5-1, 0.25-1 and 2.5-10 μg kg-1, and cut-off values of 0.25-1, 5-500, 1-10, 0.5-2.5 and 5-25 μg kg-1, respectively. The quantitative results were obtained using a hand-held strip scan

A multiplex PCR for detection of knockdown resistance mutations, V1016G and F1534C, in pyrethroid-resistant Aedes aegypti.

PubMed

Saingamsook, Jassada; Saeung, Atiporn; Yanola, Jintana; Lumjuan, Nongkran; Walton, Catherine; Somboon, Pradya

2017-10-10

Mutation of the voltage-gated sodium channel (VGSC) gene, or knockdown resistance (kdr) gene, is an important resistance mechanism of the dengue vector Aedes aegypti mosquitoes against pyrethroids. In many countries in Asia, a valine to glycine substitution (V1016G) and a phenylalanine to cysteine substitution (F1534C) are common in Ae. aegypti populations. The G1016 and C1534 allele frequencies have been increasing in recent years, and hence there is a need to have a simple and inexpensive tool to monitor the alleles in large scale. A multiplex PCR to detect V1016G and F1534C mutations has been developed in the current study. This study utilized primers from previous studies for detecting the mutation at position 1016 and newly designed primers to detect variants at position 1534. The PCR conditions were validated and compared with DNA sequencing using known kdr mutant laboratory strains and field collected mosquitoes. The efficacy of this method was also compared with allele-specific PCR (AS-PCR). The results of our multiplex PCR were in complete agreement with sequencing data and better than the AS-PCR. In addition, the efficiency of two non-toxic DNA staining dyes, Ultrapower™ and RedSafe™, were evaluated by comparing with ethidium bromide (EtBr) and the results were satisfactory. Our multiplex PCR method is highly reliable and useful for implementing vector surveillance in locations where the two alleles co-occur.

21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Respiratory viral panel multiplex nucleic acid... § 866.3980 Respiratory viral panel multiplex nucleic acid assay. (a) Identification. A respiratory viral... simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or...

21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Respiratory viral panel multiplex nucleic acid... § 866.3980 Respiratory viral panel multiplex nucleic acid assay. (a) Identification. A respiratory viral... simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or...

21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Respiratory viral panel multiplex nucleic acid... § 866.3980 Respiratory viral panel multiplex nucleic acid assay. (a) Identification. A respiratory viral... simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or...

21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Respiratory viral panel multiplex nucleic acid... § 866.3980 Respiratory viral panel multiplex nucleic acid assay. (a) Identification. A respiratory viral... simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or...

21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Respiratory viral panel multiplex nucleic acid... § 866.3980 Respiratory viral panel multiplex nucleic acid assay. (a) Identification. A respiratory viral... simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or...

Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections

PubMed Central

Kishen, Ria E. B.; Kluth, David C.; Bellamy, Christopher O. C.

2016-01-01

The recent availability of novel dyes and alternative light sources to facilitate complex tissue immunofluorescence studies such as multiplex labelling has not been matched by reports critically evaluating the considerations and relative benefits of these new tools, particularly in combination. Product information is often limited to wavelengths used for older fluorophores (FITC, TRITC & corresponding Alexa dyes family). Consequently, novel agents such as Quantum dots are not widely appreciated or used, despite highly favourable properties including extremely bright emission, stability and potentially reduced tissue autofluorescence at the excitation wavelength. Using spectral analysis, we report here a detailed critical appraisal and comparative evaluation of different light sources and fluorophores in multiplex immunofluorescence of clinical biopsy sections. The comparison includes mercury light, metal halide and 3 different LED-based systems, using 7 Qdots (525, 565, 585, 605, 625, 705), Cy3 and Cy5. We discuss the considerations relevant to achieving the best combination of light source and fluorophore for accurate multiplex fluorescence quantitation. We highlight practical limitations and confounders to quantitation with filter-based approaches. PMID:27632367

Rapid detection and typing of pathogenic nonpneumophila Legionella spp. isolates using a multiplex real-time PCR assay.

PubMed

Benitez, Alvaro J; Winchell, Jonas M

2016-04-01

We developed a single tube multiplex real-time PCR assay that allows for the rapid detection and typing of 9 nonpneumophila Legionella spp. isolates that are clinically relevant. The multiplex assay is capable of simultaneously detecting and discriminating L. micdadei, L. bozemanii, L. dumoffii, L. longbeachae, L. feeleii, L. anisa, L. parisiensis, L. tucsonensis serogroup (sg) 1 and 3, and L. sainthelensis sg 1 and 2 isolates. Evaluation of the assay with nucleic acid from each of these species derived from both clinical and environmental isolates and typing strains demonstrated 100% sensitivity and 100% specificity when tested against 43 other Legionella spp. Typing of L. anisa, L. parisiensis, and L. tucsonensis sg 1 and 3 isolates was accomplished by developing a real-time PCR assay followed by high-resolution melt (HRM) analysis targeting the ssrA gene. Further typing of L. bozemanii, L. longbeachae, and L. feeleii isolates to the serogroup level was accomplished by developing a real-time PCR assay followed by HRM analysis targeting the mip gene. When used in conjunction with other currently available diagnostic tests, these assays may aid in rapidly identifying specific etiologies associated with Legionella outbreaks, clusters, sporadic cases, and potential environmental sources. Published by Elsevier Inc.

Multiplex PCR for the detection of genes encoding aminoglycoside modifying enzymes and methicillin resistance among Staphylococcus species.

PubMed Central

Choi, Su Mi; Kim, Seung-Han; Kim, Hee-Jung; Lee, Dong-Gun; Choi, Jung-Hyun; Yoo, Jin-Hong; Kang, Jin-Han; Shin, Wan-Shik; Kang, Moon-Won

2003-01-01

We developed multiplex polymerase chain reaction (PCR) to detect aac(6 ')/aph(2 "), aph(3 ')-IIIa, and ant(4 ')-Ia, the genes encoding the most clinically relevant amino-glycoside modifying enzymes (AME), and simultaneously, the methicillin resistant gene, mecA, in Staphylococcus species. Clinical isolates of 45 S. aureus and 47 coagulase negative staphylococci (CNS) from tertiary university hospitals were tested by conventional susceptibility testing, using the agar dilution method and by multiplex PCR. Of a total of 92 isolates, 61 isolates were found to be methicillin-resistant. Of these, 54 isolates (89%) were found to be harboring mecA. Seventy-five percent of the 92 isolates demonstrated resistance to at least one of the aminoglycosides tested. Moreover, resistance to aminoglycosides was closely associated with methicillin-resistance (p<0.05). The most prevalent AME gene was aac(6 ')/aph(2 ") which was found in 65% of the isolates, and ant(4 ')-Ia and aph(3 ')-IIIa were present in 41% and 9% of the isolates, respectively. The concordance between methicillin-resistance and the presence of mecA gene was 98% in S. aureus and 81% in CNS. The concordance between gentamicin resistance and the presence of aac(6 ')/aph(2 ") gene was 100% in S. aureus and 85% in CNS. The multiplex PCR method that we developed appears to be both a more rapid and reliable than conventional method. PMID:14555812

All Inkjet-Printed Amperometric Multiplexed Biosensors Based on Nanostructured Conductive Hydrogel Electrodes.

PubMed

Li, Lanlan; Pan, Lijia; Ma, Zhong; Yan, Ke; Cheng, Wen; Shi, Yi; Yu, Guihua

2018-06-13

Multiplexing, one of the main trends in biosensors, aims to detect several analytes simultaneously by integrating miniature sensors on a chip. However, precisely depositing electrode materials and selective enzymes on distinct microelectrode arrays remains an obstacle to massively produced multiplexed sensors. Here, we report on a "drop-on-demand" inkjet printing process to fabricate multiplexed biosensors based on nanostructured conductive hydrogels in which the electrode material and several kinds of enzymes were printed on the electrode arrays one by one by employing a multinozzle inkjet system. The whole inkjet printing process can be finished within three rounds of printing and only one round of alignment. For a page of sensor arrays containing 96 working electrodes, the printing process took merely ∼5 min. The multiplexed assays can detect glucose, lactate, and triglycerides in real time with good selectivity and high sensitivity, and the results in phosphate buffer solutions and calibration serum samples are comparable. The inkjet printing process exhibited advantages of high efficiency and accuracy, which opens substantial possibilities for massive fabrication of integrated multiplexed biosensors for human health monitoring.