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PubMed2004

Detection of cryptosporidium antibodies in sera and oral fluids using multiplex bead assay.

For the first time, a multiplex bead assay (MBA) was used to assay oral fluid and serum specimens for immunoglobulin G (IgG) antibodies to specific Cryptosporidium parvum antigens that were coupled to polystyrene beads. Recombinant C. parvum 17- and 27-kDa antigens (r17 and r27, respectively) both linked with glutathione-S-transferase (GST) fusion proteins, native 17-kDa antigen, and GST alone were each coupled to microspheres that could be differentiated based on variable amounts of internally incorporated red fluorescent dye. Initial and follow-up serum and oral fluid specimens from a 1997 cryptosporidiosis outbreak in Spokane, Washington, were incubated with the coupled beads. Antibodies bound to the coupled beads were detected using biotinylated monoclonal anti-human IgG antibody and streptavidin-labeled r-phycoerythrin. Fluorescence intensity was measured by flow cytometry. For the 3 C. parvum antigens, the median of the mean fluorescence intensity (MFI) was significantly higher (P < 0.03) in the initial specimens than in the follow-up specimens. No significant change in IgG responses to GST in oral fluids or serum specimens was observed. For all Cryptosporidium antigens, the MFI in the initial serum specimens correlated with the MFI in the initial oral fluid specimens (P < 0.001, r > 0.673). For the recombinant antigens used in the MBA, the MFI correlated with the response as measured by an enzyme-linked immunosorbent assay that used r17 and r27 expressed without the GST fusion partner (P < 0.001, r > 0.854). MBA using sera or more conveniently collected oral fluids, especially from children, may be an option for immunodiagnosis of C. parvum infection and for prospective epidemiological studies designed to monitor infection risk.

Animals↗

PubMed2001

Real-time multiplex PCR assays.

The ability to multiplex PCR by probe color and melting temperature (T(m)) greatly expands the power of real-time analysis. Simple hybridization probes with only a single fluorescent dye can be used for quantification and allele typing. Different probes are labeled with dyes that have unique emission spectra. Spectral data are collected with discrete optics or dispersed onto an array for detection. Spectral overlap between dyes is corrected by using pure dye spectra to deconvolute the experimental data by matrix algebra. Since fluorescence is temperature dependent and depends on the dye, spectral overlap and color compensation constants are also temperature dependent. Single-labeled probes are easier to synthesize and purify than more complex probes with two or more dyes. In addition, the fluorescence of single-labeled probes is reversible and depends only on hybridization of the probe to the target, allowing study of the melting characteristics of the probe. Although melting curves can be obtained during PCR, data are usually acquired at near-equilibrium rates of 0.05-0.2 degrees C/s after PCR is complete. Using rapid-cycle PCR, amplification requires about 20 min followed by a 10-min melting curve, greatly reducing result turnaround time. In addition to dye color, melting temperature can be used for a second dimension of multiplexing. Multiplexing by color and T(m) creates a "virtual" two-dimensional multiplexing array without the need for an immobilized matrix of probes. Instead of physical separation along the X and Y axes, amplification products are identified by different fluorescence spectra and melting characteristics.

Animals↗

PubMed2003

Multiplex PCR assay for simultaneous detection of nine clinically relevant antibiotic resistance genes in Staphylococcus aureus.

In this study we describe a multiplex PCR assay for the detection of nine clinically relevant antibiotic resistance genes of Staphylococcus aureus. Conditions were optimized to amplify fragments of mecA (encoding methicillin resistance), aacA-aphD (aminoglycoside resistance), tetK, tetM (tetracycline resistance), erm(A), erm(C) (macrolide-lincosamide-streptogramin B resistance), vat(A), vat(B), and vat(C) (streptogramin A resistance) simultaneously in one PCR amplification. An additional primer pair for the amplification of a fragment of the staphylococcal 16S rDNA was included as a positive control. The multiplex PCR assay was evaluated on 30 different S. aureus isolates, and the PCR results correlated with the phenotypic antibiotic resistance data obtained by the broth microdilution assay. The multiplex PCR assay offers a rapid, simple, and accurate identification of antibiotic resistance profiles and could be used in clinical diagnosis as well as for the surveillance of the spread of antibiotic resistance determinants in epidemiological studies.

DNA, Bacterial↗

PubMed2006

A novel multiplex PCR assay for the detection of Salmonella enterica serovar Enteritidis in human faeces.

AIMS: To develop a multiplex PCR assay for the detection of Salmonella enterica serovar Enteritidis in human faeces. METHODS AND RESULTS: A total of 54 Salmonella strains representing 19 serovars and non-Salmonella strains representing 11 different genera were used. Five primer pairs were employed in the assay. Three of them targeted to the genes hilA, spvA and invA that encode virulence-associated factors. A fourth primer pair amplified a fragment of a unique sequence within S. enterica serovar Enteritidis genomes. An internal amplification control (a fragment of a conservative sequence within the 16S rRNA genes) was targeted by a fifth primer pair. The assay produced two or three amplicons from the invA, hilA and 16S rRNA genes for 19 Salmonella serovars. All Salmonella and non-Salmonella strains yielded a band of an internal amplification control. For S. enterica serovar Typhimurium, four products (the fourth from the spvA gene), and for S. enterica serovar Enteritidis five amplicons (the fifth from the sdf gene) were observed. S. enterica serovar Enteritidis was cultured from three of 71 rectal swabs from diarrhoeal patients. Five specific amplicons were generated with the multiplex PCR assay only from culture-positive faecal samples. CONCLUSION: The multiplex PCR assay specifically detects S. enterica serovar Enteritidis. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a novel multiplex PCR assay, which contains an internal amplification control and enables concurrent survey for Salmonella virulence genes.

Feces↗

PubMed2004

[Multiplex PCR assay against E. coli O157 in foodstuffs].

A multiplex PCR assay for detection of Escherichia coli O157:H7 was developed with 4 pairs primers which focus on its specific fliC, rfbE, SLT I and SLT II genes. The target genes fragments of the PCR assay were 560 bp, 678 bp, 210 bp and 484 bp. The multiplex PCR had very high specificity to match all the 71 tested strains including 58 E. coli strains and other 18 strains. The strain of E. coli O157:H7 was identified in one step, and the type of SLT was also identified as well. With the enrichment procedure, the strain of E. coli O157:H7 were recovered at the detection limit of 0.1 cfu/ml from artificial inoculated pasteurized milk.

Animals↗

PubMed2001

Identification of herpes simplex virus genital infection: comparison of a multiplex PCR assay and traditional viral isolation techniques.

Genital herpes simplex virus (HSV) is of major public health importance, as indicated by the marked increase in the prevalence of genital herpes over the past two decades. Viral culture has traditionally been regarded as the gold standard for diagnosis. In this study, we compared viral culture and the amplification of HSV DNA by the polymerase chain reaction (PCR) with respect to sensitivity, cost, clinical utility, and turnaround time. Patient sample swabs from 100 individuals were inoculated onto MRC-5 cells for isolation. Positive results were confirmed via a direct fluorescent antibody technique, and serotyping, when requested, was performed using HSV-1 and -2-type-specific sera. PCR techniques employed an extraction step of the same initial swab specimen, followed by PCR amplification, using a multiplex assay for HSV-1, 2 DNA. HSV-positive results were found in 32/100 samples via culture and in 36/100 samples via PCR. PCR-positive results yielded 16 (44%) patients infected with HSV-1 and 20 (56%) patients infected with HSV-2. Turnaround time for viral culture averaged 108 hours for positive results and 154 hours for negative results; PCR turnaround time averaged 24--48 hours. Laboratory cost using viral culture was $3.22 for a negative result and $6.49 for a positive result (including direct fluorescent antibody). Serotyping added $10.88 to each culture-positive test. Although laboratory costs for PCR were higher at $8.20/sample, reimbursement levels were also higher. We propose a multiplex PCR assay for diagnosis of HSV-1 and HSV-2 from patient swabs for use in a routine clinical laboratory setting. This assay offers increased sensitivity, typing, and improved turnaround time when compared with traditional viral culture techniques. Although it appears that PCR testing in a routine clinical laboratory setting is cost prohibitive compared with the case of nonserotyped viral culture, it may be very useful when clinical utility warrants distinguishing between HSV 1 and 2 and may be cost effective when reimbursement issues are examined.

Cells, Cultured↗

PubMed2002

Multiplex PCR assays for simultaneous detection of six major serotypes and two virulence-associated phenotypes of Streptococcus suis in tonsillar specimens from pigs.

Multiplex PCR assays for the detection and identification of various Streptococcus suis strains in tonsillar specimens from pigs were developed and evaluated. In two separate reactions, five distinct DNA targets were amplified. Three targets, based on the S. suis capsular polysaccharide (cps) genes specific for serotypes 1 (and 14), 7, and 9, were amplified in multiplex PCR I. Two other targets, based on the serotype 2- (and 1/2-) specific cps gene and the epf gene, encoding the EF proteins of virulent serotype 2 and highly virulent serotype 1 strains, were amplified in multiplex PCR II. To identify false-negative results, firefly luciferase (luc) DNA and primers based on the luc gene were included in the assay. The multiplex PCR assays were evaluated with tonsillar specimens from pigs infected with S. suis strains. The results obtained with the PCR assays were compared with the results obtained with a bacteriological examination. Most (94%) of the results obtained with multiplex PCR assays were confirmed by the bacteriological examination. The PCR method seems to be more sensitive compared to the bacteriological method, since the remaining 6% of the samples were positive by PCR and negative by bacteriological examination. These results indicate that the PCR method is highly specific for the detection of S. suis strains most frequently involved in clinical disease in infected pig herds. The serotypes found by PCR in tonsillar specimens from diseased pigs were compared with the serotypes of the strains isolated from the affected tissues of the same pigs. The results showed that there is significant association between carriership and clinical illness for S. suis serotype 9 and EF-positive serotype 2 strains and not for serotype 7 and EF-negative serotype 2 (or 1/2) strains.

Animals↗

PubMed2004

Multiplex GPCR assay in reverse transfection cell microarrays.

G protein-coupled receptors (GPCRs) are a superfamily of proteins that include some of the most important drug targets in the pharmaceutical industry. Despite the success of this group of drugs, there remains a need to identify GPCR-targeted drugs with greater selectivity, to develop screening assays for validated targets, and to identify ligands for orphan receptors. To address these challenges, the authors have created a multiplexed GPCR assay that measures greater than 3000 receptor: ligand interactions in a single microplate. The multiplexed assay is generated by combining reverse transfection in a 96-well plate format with a calcium flux readout. This assay quantitatively measures receptor activation and inhibition and permits the determination of compound potency and selectivity for entire families of GPCRs in parallel. To expand the number of GPCR targets that may be screened in this system, receptors are cotransfected with plasmids encoding a promiscuous G protein, permitting the analysis of receptors that do not normally mobilize intracellular calcium upon activation. The authors demonstrate the utility of reverse transfection cell microarrays to GPCR-targeted drug discovery with examples of ligand selectivity screening against a panel of GPCRs as well as dose-dependent titrations of selected agonists and antagonists.

Dose-Response Relationship, Drug↗

PubMed1998

Single-step multiplex PCR assay for characterization of New World Leishmania complexes.

We have developed a PCR assay for one-step differentiation of the three complexes of New World Leishmania (Leishmania braziliensis, Leishmania mexicana, and Leishmania donovani). This multiplex assay is targeted to the spliced leader RNA (mini-exon) gene repeats of these organisms and can detect all three complexes simultaneously, generating differently sized products for each complex. The assay is specific to the Leishmania genus and does not recognize related kinetoplastid protozoa, such as Trypanosoma cruzi, Trypanosoma brucei, and Crithidia fasciculata. It correctly identified Leishmania species with a broad geographic distribution in Central and South America. The sensitivity of the PCR amplification ranged from 1 fg to 10 pg of DNA (0.01 to 100 parasites), depending on the complex detected. Crude extracts of cultured parasites, prepared simply by boiling diluted cultures, served as excellent templates for amplification. Crude preparations of clinical material were also tested. The assay detected L. braziliensis in dermal scrapings from cutaneous leishmanial lesions, Leishmania chagasi in dermal scrapings of atypical cutaneous leishmaniasis, and L. mexicana from lesion aspirates from infected hamsters. We have minimized the material requirements and maximized the simplicity, rapidity, and informative content of this assay to render it suitable for use in laboratories in countries where leishmaniasis is endemic. This assay should be useful for rapid in-country identification of Leishmania parasites, particularly where different Leishmania complexes are found in the same geographical area.

Animals↗

PubMed2004

Multiplex opsonophagocytosis assay (MOPA): a useful tool for the monitoring of the 7-valent pneumococcal conjugate vaccine.

Pneumococcal conjugate vaccination is highly efficacious against invasive diseases in young children. Since host protection is mainly mediated by opsonin-dependent phagocytosis, the in vitro measurement of opsonophagocytic activity of the anti-capsular antibodies is assumed to be a reliable correlate of protection to monitor vaccine efficacy. Unfortunately, the methods used so far are all tedious to perform and material-consuming. Therefore, we modified the multi-specificity opsonophagocytosis killing assay (MSOPKA) into a high-throughput method, which simultaneously measures the opsonophagocytosis against the seven serotypes covered by the current conjugate vaccine in a single assay. In the so-called multiplex opsonophagocytosis assay (MOPA), a mixture containing equal numbers of colony forming units (CFUs) of chloramphenicol-resistant serotype 4, spectinomycin-resistant serotype 6B, streptomycin-resistant serotype 9V, erythromycin-resistant serotype 14, rifampicin-resistant serotype 18C, tetracycline-resistant serotype 19F, and trimethoprim-resistant serotype 23F pneumococci was used as a target mixture and incubated with serial dilutions of test serum. After opsonophagocytosis by differentiated HL-60 cells in the presence of complement, the samples were spotted onto different blood agar plates containing the seven selective antibiotics, respectively. Opsonophagocytosis was calculated as the highest serum dilution resulting in 90% or more reduction in CFUs. The data obtained by this assay correlated well with the data obtained by the MSOPKA. In conclusion, the MOPA simultaneously measures opsonophagocytosis capacity of serum against the capsular serotypes included in the 7-valent pneumococcal conjugate vaccine in a high-throughput fashion, requiring low volumes of patient sera.

Antibodies, Bacterial↗

PubMed1998

Development and evaluation of a multiplex PCR assay for rapid detection of toxigenic Vibrio cholerae O1 and O139.

A multiplex polymerase chain reaction assay was developed for concurrent detection of rfb sequences specific for the O1 and the O139 serogroups of Vibrio cholerae and for ctxA specific sequences. The multiplex PCR assay was found to be highly specific and sensitive and was capable of detecting 65 cfu and 200 cfu per assay of V. cholerae O1 and O139, respectively. Evaluation of the multiplex PCR assay using 121 stool samples from patients admitted to the Infectious Diseases Hospital, Calcutta, showed the assay to be 100% sensitive and 95.2% specific when the culture method was taken as the standard. In addition to the 38 PCR positive stool samples, an additional four samples which were negative by culture method but positive by PCR assay belonged to the O139 serogroup. All the 38 stool samples positive for either O1 or O139 serogroup by PCR assay were also positive for the ctxA amplicon indicating that the O1 and O139 V. cholerae strains have the genetic potential of producing cholera toxin.

Bacterial Proteins↗

PubMed2003

Multiplex PCR assay for identification of human diarrheagenic Escherichia coli.

A multiplex PCR assay for the identification of human diarrheagenic Escherichia coli was developed. The targets selected for each category were eae for enteropathogenic E. coli, stx for Shiga toxin-producing E. coli, elt and est for enterotoxigenic E. coli, ipaH for enteroinvasive E. coli, and aggR for enteroaggregative E. coli. This assay allowed the categorization of a diarrheagenic E. coli strain in a single reaction tube.

Diarrhea↗

PubMed2003

Molecular typing of Salmonella enterica serovar typhi isolates from various countries in Asia by a multiplex PCR assay on variable-number tandem repeats.

A multiplex PCR method incorporating primers flanking three variable-number tandem repeat (VNTR) loci (arbitrarily labeled TR1, TR2, and TR3) in the CT18 strain of Salmonella enterica serovar Typhi has been developed for molecular typing of S. enterica serovar Typhi clinical isolates from several Asian countries, including Singapore, Indonesia, India, Bangladesh, Malaysia, and Nepal. We have demonstrated that the multiplex PCR could be performed on crude cell lysates and that the VNTR banding profiles produced could be easily analyzed by visual inspection after conventional agarose gel electrophoresis. The assay was highly discriminative in identifying 49 distinct VNTR profiles among 59 individual isolates. A high level of VNTR profile heterogeneity was observed in isolates from within the same country and among countries. These VNTR profiles remained stable after the strains were passaged extensively under routine laboratory culture conditions. In contrast to the S. enterica serovar Typhi isolates, an absence of TR3 amplicons and a lack of length polymorphisms in TR1 and TR2 amplicons were observed for other S. enterica serovars, such as Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Paratyphi A, B, and C. DNA sequencing of the amplified VNTR regions substantiated these results, suggesting the high stability of the multiplex PCR assay. The multiplex-PCR-based VNTR profiling developed in this study provides a simple, rapid, reproducible, and high-resolution molecular tool for the epidemiological analysis of S. enterica serovar Typhi strains.

Alleles↗

PubMed1996

Detection of enterotoxigenic Escherichia coli, Shigella and Campylobacter spp. by multiplex PCR assay.

Three oligonucleotide primers were used in a polymerase chain reaction (PCR) assay for the simultaneous amplification of regions of the invasive plasmid antigen (ipaH) of Shigella spp., flagellin gene (flaA) of Campylobacter spp., and heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC). The multiplex assay was performed using DNA extracted by a chaotropic method directly from diarrhoeal stools. The diagnostic efficacy of the assay was analyzed by agarose gel electrophoresis. This assay shows a novel approach for the diagnosis of diarrhoea caused by Shigella spp., ETEC, and Campylobacter spp.

Campylobacter↗

PubMed2003

Simultaneous quantitation of antibodies to neutralizing epitopes on virus-like particles for human papillomavirus types 6, 11, 16, and 18 by a multiplexed luminex assay.

Several different methods have been developed to quantitate neutralizing antibody responses to human papillomaviruses (HPVs), including in vivo neutralization assays, in vitro pseudoneutralization assays, competitive radioimmunoassays (cRIAs), and enzyme-linked immunosorbent assays. However, each of these techniques possesses one or more limitations that preclude testing large numbers of patient sera for use in natural history studies and large vaccine clinical trials. We describe here a new multiplexed assay, by using the Luminex Laboratory MultiAnalyte Profiling (LabMAP3) assay system, that can simultaneously quantitate neutralizing antibodies to human papillomavirus types 6, 11, 16, and 18 in 50 micro l of serum. The HPV-Luminex competitive immunoassay measures titers of polyclonal antibodies in serum capable of displacing phycoerythrin-labeled detection monoclonal antibodies binding to conformationally sensitive, neutralizing epitopes on the respective virus-like particles. This competitive Luminex immunoassay was found to be as sensitive, accurate, and precise as the currently used cRIAs. An effective HPV vaccine will most likely require several distinct genotypes to protect against multiple cancer causing papillomaviruses. The HPV-Luminex immunoassay should prove to be a useful tool in simultaneously quantitating antibody immune responses to multiple HPV genotypes for natural history infection studies and for monitoring the efficacy of prospective vaccines.

Adolescent↗

PubMed2005

Multiplex PCR assay for toxinotyping Clostridium perfringens isolates obtained from Finnish broiler chickens.

AIMS: The aim of the study was to determine the presence of genes coding for alpha (cpa), beta (cpb), epsilon (etx), iota (iA) and enterotoxin (cpe) from Clostridium perfringens broiler chicken isolates, using multiplex PCR assay established in the study. METHODS AND RESULTS: The multiplex PCR assay was shown to be specific when tested with 10 C. perfringens strains representing different toxin types, and 15 strains of other bacterial species. All 118 broiler chicken C. perfringens isolates were shown to carry the cpa gene but not cpb, etx, iap or cpe genes, signifying that all isolates represented type A and were cpe-negative. CONCLUSIONS: The assay established in the study enables the simultaneous detection of the major toxin genes and the cpe gene from C. perfringens isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study offers a new primer pair for detecting cpa, combined with a multiplex PCR assay. In addition, the study provides data of the presence of different toxin genes in C. perfringens isolates obtained from broiler chickens.

ADP Ribose Transferases↗

PubMed2004

Multiplex PCR assay for rapid detection and genotyping of Helicobacter pylori directly from biopsy specimens.

We developed and evaluated a simple, novel multiplex PCR assay for rapid detection of Helicobacter pylori infection and for the determination of vacA and cagA genotypes directly from gastric biopsy specimens. This assay did not require culturing of strains or extraction of DNA from biopsy samples. This multiplex PCR assay would be of particularly great value for laboratories in developing countries.

Antigens, Bacterial↗

PubMed2006

Development and implementation of multiplexed cell-based imaging assays.

Fluorescence microscopy, image analysis, and automated screening technologies are some of the most powerful tools enabling cell biologists to investigate complex signaling pathways and compound or siRNA effects on cellular function in individual cells. Researchers can now use multiple fluorescent probes to quantify effects on intracellular molecular events, measure phenotypic changes, and provide contextual information about cellular pathways not discernible by traditional single-parameter, end point experiments. This chapter focuses on fluorescent labeling techniques and methods for designing image-based assays, multiplexed readouts, and image analysis routines. Case studies are presented describing the use of cell-based imaging assays for monitoring cell proliferation, cell cycle stage, and apoptosis.

Animals↗