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PubMed2004

A multiplex polymerase chain reaction assay for genus-, group- and species-specific detection of mycobacteria.

We developed and evaluated a single-step, multiplex polymerase chain reaction (PCR) assay for distinguishing (1) between the Mycobacterium tuberculosis complex (MTBC) and mycobacteria other than tuberculosis (MOTT) and (2) between M. tuberculosis and M. bovis species. The assay targeted the 16S and the 23S rDNA to distinguish between MTBC and MOTT species, and the oxyR gene to distinguish between M. tuberculosis and M. bovis strains. Clinical samples and reference strains (N = 156) comprised 93 strains of M. tuberculosis, 44 of M. bovis, 1 M. africanum strain, and 18 strains representing 9 different species of MOTTs. MOTTs generated only a single PCR product of about 2.5 kilobase; however, all of the MTBC strains produced a 118 base pair (bp) fragment and an additional 270 bp fragment was obtained for M. tuberculosis and M. africanum when the primer pair oxyRTB-2.1/oxyRMT-1 was used. When oxyRTB-2.1/oxyRMB-1 primers were used, the 270 bp fragment was obtained for only M. bovis. The assay needed as little as 1 pg of purified genomic DNA to make a positive identification.

Animals↗
PubMed2005

Simultaneous detection of isoniazid, rifampin, and ethambutol resistance of Mycobacterium tuberculosis by a single multiplex allele-specific polymerase chain reaction (PCR) assay.

Prompt detection of drug resistance of Mycobacterium tuberculosis is essential for effective control of tuberculosis (TB). We developed a multiplex allele-specific polymerase chain reaction (MAS-PCR) that detects the most commonly observed isoniazid (INH), rifampin (RIF), and ethambutol resistance-associated mutations in a single assay. The usefulness of the newly developed method was evaluated with 174 clinical isolates of M. tuberculosis obtained from Turkey. Distinct PCR banding patterns were observed for different mutation profiles and the correlation between MAS-PCR results and DNA sequencing findings was 99.4%. With culture-based phenotypic drug susceptibility testing as a reference standard, the sensitivity and specificity of the newly developed MAS-PCR assay for drug resistance-related genetic mutation detection were determined to be 81.1% and 97.5% for INH, 93.0% and 98.9 % for RIF, and 54.5% and 68.0 % for ethambutol. MAS-PCR provides a rapid, potentially more cost-effective, method of detecting multidrug-resistant TB.

Alleles↗
PubMed2006

Improved multiplex PCR method for the rapid detection of beta-lactamase genes in Escherichia coli of animal origin.

We developed 2 variants, A and B, of a multiplex polymerase chain reaction method for detecting the beta-lactam resistance genes bla(TEM), bla(SHV), and bla(OXA) in 122 uropathogenic Escherichia coli animal strains. Method B yielded 98% specificity and 100% sensitivity, and method A yielded 100% and 89%, respectively. Variant B was more accurate (99%) than A (94%).

Animals↗
PubMed2006

Detection and differentiation between pathogenic and saprophytic Leptospira spp. by multiplex polymerase chain reaction.

A multiplex polymerase chain reaction (PCR) was developed for diagnosing leptospirosis and differentiating pathogenic and saprophytic leptospires. Specific primers were designed to amplify 23S rDNA from pathogenic Leptospira and saprophytic Leptospira spp. PCR products from 27 pathogenic and 5 (including 1 intermediate) saprophytic serovars were 615 and 316 base pairs (bp), respectively. After the restriction enzyme's digestion of PCR products, the fragments by SacI of pathogenic serovars and by PstI of saprophytic serovars were 339 and 276 bp and 202 and 114 bp, respectively. The PCR primers enabled amplification of DNA from L. meyeri serovar Ranarum as a pathogenic Leptospira spp. The PCR assay could detect 1 to 2 cells of leptospires and not amplify DNA from other 18 bacterial species. The sensitivity and specificity of this PCR in rat kidney, using isolation as gold standard, were 98.6% and 100%, respectively. The most appropriate sample preparation of blood for detecting DNA was buffy coat. Among the sample preparations from 7 laboratory-confirmed leptospirosis cases, leptospiral DNA was detected in all 7 buffy coat preparations, whereas leptospiral DNA was detected in only 3 plasma or serum samples. The PCR assay may be useful as a diagnostic tool for leptospirosis.

Animals↗
PubMed2006

Detection of herpes simplex virus type 1 in addition to Epstein-Bar virus in tonsils using a new multiplex polymerase chain reaction assay.

HSV-1, HSV-2, CMV, EBV, which are the members of the herpes virus family colonize and establish latent infection in human. Although EBV is a well known virus most involved in recurrent bouts of acute tonsillitis, the role and possibility of HSV-1, HSV-2, and CMV for establishing infection in tonsils are not clear. The purpose of this study is to verify whether the tonsils might harbor the HSV-1, HSV-2, and CMV, in addition to EBV, in chronically hyperplastic nasopharyngeal lymphoid tissue. To accomplish the purpose, we developed a new Multiplex Polymerase Chain Reaction (M-PCR) assay using a single consensus forward primer and virus specific reverse primers for DNA polymerase gene of HSV-1, and 2, EBV, and CMV, and investigated its efficiency for detecting HSV1, HSV2, CMV, and EBV. The sample of 52 patients underwent tonsillectomy or adenectomy because of chronic lymphoid hyperplasia without any evidence of acute infections and were investigated for presence of HSV-1, HSV-2, CMV, and EBV. Of the 54 samples, 11 (20.4%) of them were positive for EBV, 4 of them (7.4%) were positive for HSV-1, and none of the samples were positive for HSV-2 and CMV. To the best of our knowledge, this is the first report that tonsils may be the reservoir for HSV-1 in addition to EBV, and HSV-1 may have a role in recurrent tonsillitis and systemic diseases. The MC-PCR assay presented in this study can provide a rapid, sensitive, and economical method for detection of HSV-1, HSV-2, EBV, and CMV in a single PCR tube.

Adolescent↗
PubMed2006

Diagnostic challenges for multiplexed protein microarrays.

Multiplexed protein analysis using planar microarrays or microbeads is growing in popularity for simultaneous assays of antibodies, cytokines, allergens, drugs and hormones. However, this new assay format presents several new operational issues for the clinical laboratory, such as the quality control of protein-microarray-based assays, the release of unrequested test data and the use of diagnostic algorithms to transform microarray data into diagnostic results.

Algorithms↗
PubMed2006

Multiplex fluorescence in situ hybridization identifies novel rearrangements of chromosomes 6, 15, and 18 in primary uveal melanoma.

Uveal melanomas are the commonest ocular tumour of adults and are characterized by reproducible alterations of chromosomes 1, 3, 6 and 8. These alterations are of prognostic relevance and have also be shown to correlate to high risk and low risk metastatic categories of uveal melanoma as defined by micro-array analysis. It is, however, possible that a catalogue of relevant genetic alterations, involving gene rearrangement rather than amplification, have as yet eluded identification. To address this point we examined 14 primary uveal melanomas, using 24 colour multiplex fluorescence in situ hybridization (M-FISH). All tumours were karyotyped following G-Banding, and M-FISH was performed to confirm and clarify the identity of abnormal chromosomes. M-FISH data were obtained from all tumours and was able to establish the nature of most abnormalities not fully characterized by cytogenetics. Abnormalities of chromosome 6 were far more frequent than previously indicated, in approximately 70% of cases, indicating they have been substantially underrepresented in past studies of uveal melanoma. Spindle melanomas were found to have novel rearrangements affecting in particular chromosomes 6, 15 and 18, suggesting that juxtaposition of genes through translocational events may play a role in the development of some uveal melanomas. In conclusion, this study is the largest of primary uveal melanoma analysed by M-FISH and indicates that alterations of chromosome 6 have previously been underestimated. Furthermore spindle melanomas are prone to rearrangements affecting chromosomes 6, 15 and 18, which may relate to early changes in uveal melanoma development or associate with those melanomas of a more differentiated status.

Adult↗
PubMed2005

Improved multiplex-PCR method for the simultaneous detection of food bacteria producing biogenic amines.

This study describes a simple and rapid multiplex-PCR method to determine the ability to produce histamine, tyramine and putrescine by bacteria. The assay is an improved method based on an assay designed for lactic acid bacteria. This improved method includes a pair of primers based on sequences from histidine decarboxylases from Gram-negative bacteria. Under the optimised conditions, the assay yielded a 367-bp DNA fragment from histidine decarboxylases of Gram-positive bacteria, 534-bp fragment from histidine decarboxylases of Gram-negative bacteria, 924-bp from bacterial tyrosine decarboxylases, and 1446-bp fragment from bacterial ornithine decarboxylases. The method was successfully applied to several biogenic amine-producing bacterial strains, even when DNAs of several target organisms were included in the same reaction. This simple method could be easily incorporated in food control laboratories to detect potentially biogenic amine-producing bacteria in foods.

Bacteriological Techniques↗
PubMed2005

Rapid identification of potentially probiotic Bifidobacterium species by multiplex PCR using species-specific primers based on the region extending from 16S rRNA through 23S rRNA.

This study aimed at developing a novel multiplex polymerase chain reaction (PCR) primer set for identification of the potentially probiotic Bifidobacterium species B. adolescentis, B. animalis subsp. animalis (B. animalis), B. bifidum, B. breve, B. longum biovar infantis (B. infantis), B. animalis subsp. lactis B. lactis, B. longum biovar longum (B. longum) and B. pseudolongum. The primer set comprised specific and conserved primers and was derived from the integrated sequences of 16S and 23S rRNA genes and the rRNA intergenic spacer region (ISR) of each species. It could detect and identify type strains and isolates from pharmaceuticals or dairy products corresponding to the eight Bifidobacterium species with high specificity. It was also useful for screening of the related strains from natural sources such as the gastro-intestinal tract and feces. We suggest that the assay system from this study is an efficient tool for simple, rapid and reliable identification of Bifidobacterium species for which probiotic strains are known.

Base Sequence↗
PubMed2004

Identification of meiotic anomalies with multiplex fluorescence in situ hybridization: Preliminary results.

OBJECTIVE: To characterize meiotic anomalies in infertile men by multiplex fluorescence in situ hybridization (M-FISH) and to determine whether synaptic problems affect specific bivalents or whether anomalies are random. DESIGN: Analysis of meiotic preparations with standard techniques and M-FISH. SETTING: Assisted reproduction centers and Universitat Autònoma de Barcelona. PATIENT(S): Three fertile men undergoing vasectomy, four sterile patients with oligoasthenoteratozoospermia, and one patient with a Robertsonian translocation t(13;14). INTERVENTION(S): Unilateral testicular biopsy in controls and patients with oligoasthenoteratozoospermia and collection of a semen sample from the translocation carrier. MAIN OUTCOME MEASURE(S): Identification of bivalents in metaphase I and chromosomes in metaphase II and characterization of chromosome abnormalities. RESULT(S): All bivalents in metaphase I and all chromosomes in metaphase II could be identified. In controls and in one patient with oligoasthenoteratozoospermia, meiosis was normal. Other patients with oligoasthenoteratozoospermia showed different types of anomaly: desynapsis, breaks, precocious XY separation, or cryptic reorganizations. The Robertsonian translocation t(13;14) was easily identified. CONCLUSION(S): Results confirm the high incidence of synaptic errors in oligoasthenoteratozoospermia patients. Bivalents in metaphase I and chromosomes in metaphase II were individually identifiable. Nondisjunctional errors or small reorganizations overlooked in classic meiotic preparations were identified. Synaptic anomalies seem to affect meiotic bivalents at random.

Chromosome Mapping↗
PubMed2006

Multiplex PCR for the detection and identification of dairy bacteriophages in milk.

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.

Animals↗
PubMed2006

Developmental validation of a multiplex qPCR assay for assessing the quantity and quality of nuclear DNA in forensic samples.

Forensic scientists are constantly searching for better, faster, and less expensive ways to increase the first-pass success rate of forensic sample analysis. Technological advances continue to increase the sensitivity of analysis methods to enable genotyping of samples containing minimal amounts of DNA, yet few tools are available that can simultaneously alert the analyst to both the presence of inhibition and level of degradation in samples prior to genotyping to allow analysts the opportunity to make appropriate modifications to their protocols and, consequently, to use less sample. Our laboratory developed a multiplex quantitative PCR assay that amplifies two human nuclear DNA target sequences of different length to assess DNA degradation and a third amplification target, a synthetic oligonucleotide internal PCR control (IPC), to allow for the assessment of PCR inhibition. We chose the two nuclear targets to provide quantity and fragment-length information relevant to the STR amplification targets commonly used for forensic genotyping. The long target (nuTH01, 170-190 bp) spans the TH01 STR locus and uses a FAM-labeled TaqMan probe for detection. The short nuclear target (nuCSF, 67 bp) is directed at the upstream flanking region of the CSF1PO STR locus and is detected using a VIC-labeled TaqManMGB probe. The IPC target sequence is detected using a NED-labeled TaqManMGB probe. The assay was validated on the Applied Biosystems 7500 Real-Time PCR system, which is optimized for NED detection. We report the results of a developmental validation in which the assay was rigorously tested, in accordance with the current SWGDAM guidelines, for precision, sensitivity, accuracy, reproducibility, species specificity, and stability.

DNA↗
PubMed2003

Multiplex single nucleotide extension: a robust and high throughput method for HLA-A locus typing.

This report describes a typing method that can identify all known human leukocyte antigen A (HLA-A) alleles by determining nucleotides present at polymorphic sites using single nucleotide extension. Allele specific primers are bound to capture oligonucleotides which allows for a multiplex approach during single nucleotide extension (SNE) reactions. Eleven group-specific polymerase chain reaction amplifications were performed to obtain the templates to be analyzed with sets of primers designed to investigate the polymorphisms. Extension of biotin-labeled ddNTPs onto allele-specific primers was catalyzed by a DNA polymerase and each primer was hybridized to a specific capture oligonucleotide covalently bound to a bead. After staining with streptavidin-PE, incorporated fluorescence was determined with a flow cytometer. Fluorescence intensities were interpreted by computer and the nucleotide sequence was translated into HLA-A genotypes. Group-specific amplification reactions and primer sets for SNE were validated with 42 reference samples of known HLA-A alleles. In addition, 296 samples from three populations (N. A. Caucasian, African-American, Terena S. A. Indian) were analyzed and results compared to previous typing by SSOP. Reproducibility between repeated typings was 100% and ambiguities were quite rare. The method has been found to be accurate, relatively simple to perform and fast. It is our method of choice for high resolution clinical HLA-A typing.

Alleles↗
PubMed2006

Multiplex PCR for genes encoding prevalent OXA carbapenemases in Acinetobacter spp.

Carbapenem resistance in Acinetobacter baumannii is a growing public health concern and is most often mediated by OXA carbapenemases. We describe a novel multiplex polymerase chain reaction (PCR) assay able to detect and distinguish alleles encoding three subgroups of acquired OXA carbapenemases (OXA-23-like, OXA-24-like and OXA-58-like) that are scattered in Acinetobacter spp., and a fourth subgroup, OXA-51-like, which appears to be intrinsic to Acinetobacter baumannii. Isolates belonging to two prevalent UK A. baumannii 'OXA' clones (OXA-23 clones 1 and 2) had alleles encoding both an intrinsic OXA-51-like and an acquired OXA-23 enzyme, whereas isolates of the 'SE clone' had only an intrinsic bla(OXA-51-like) allele. Genes encoding OXA-58 were detected (with bla(OXA-51-like)) in a cluster of related isolates from a single hospital. This simple assay will assist in monitoring the mechanisms responsible for carbapenem resistance in Acinetobacter spp.

Acinetobacter↗
PubMed2004

Detection of Cryptosporidium and Giardia in molluscan shellfish by multiplexed nested-PCR.

A multiplexed nested-PCR procedure (ABC-PCR) previously developed to detect Cryptosporidium spp. and Giardia duodenalis assemblages A and B in whole human faeces was applied to DNA extracted from filter-feeding molluscs. Species of Cryptosporidium and G. duodenalis were identified by restriction fragment analysis of the PCR products and by DNA sequencing. The extraction and ABC-PCR procedures were shown to be suitable for application to shellfish by amplification of specific target sequences using DNA from Cryptosporidium parvum genotype 2 and G. duodenalis assemblages A and B which were spiked into DNA extracted from mussels. Using 49 molluscan shellfish specimens (18 clam, 22 mussel and 9 oyster samples) from Spain, cryptosporidial oocysts were detected in 56% by immunofluorescence microscopy, and in 44% by ABC-PCR. For detection of Cryptosporidium, there was a significant association, but not total agreement, between the results of microscopy and PCR. G. duodenalis assemblage B was detected from one oyster sample by PCR. Amongst 38 specimens (20 mussel and 18 cockle samples) collected in the UK and tested by the ABC-PCR, G. duodenalis was not detected, and Cryptosporidium was detected in 11% of the samples. Overall, the 26 samples where Cryptosporidium was detected, C. hominis/C. parvum genotype 1 was detected in 1, C. parvum genotype 2 in 22, and the remaining three samples contained either sequences similar to C. parvum genotype 2 or heterogeneous mixtures of Cryptosporidium species. There was no significant association between the level of Escherichia coli detected by conventional microbiological methods and the presence of Cryptosporidium detected by ABC-PCR.

Animals↗
PubMed2005

Detection of cdtA, cdtB, and cdtC genes in Campylobacter jejuni by multiplex PCR.

A multiplex PCR was developed for simultaneous detection of the cytolethal distending toxin (cdt) genes of Campylobacter jejuni. Three primer pairs targeting each one of the cdtA, cdtB and cdtC genes were designed and combined in the same PCR reaction. The assay was evaluated with 100 C. jejuni strains recovered from humans and animals and it was found to be rapid and specific. Two isolates presented several deletions affecting both cdtA and cdtB genes. High prevalence (98%) of the three cdt genes was found among isolates of different geographic origins.

Animals↗
PubMed2006

Presence of perineuritis in a case of papular purpuric gloves and socks syndrome associated with mononeuritis multiplex attributable to B19 parvovirus.

A previously healthy 16-year-old boy developed papular purpuric gloves and socks syndrome concomitant with mononeuritis multiplex attributable to acute infection with parvovirus B19. A cutaneous biopsy showed lymphocytic lichenoid vasculitis and perineuritis. We discuss the relevance of these pathologic findings, and we suggest a correlation with the patient's neurologic condition. This case illustrates the difficulties in differentiating dysesthesia accompanying cutaneous manifestations of exanthem from neurologic disease on the basis of the clinical appearance alone.

Adolescent↗
PubMed2003

Multiplex polymerase chain reaction/membrane hybridization assay for detection of genetically modified organisms.

To improve detection efficiency and result accuracy, four screening primer pairs, four identifying primer pairs, one common primer pair and corresponding probes were designed for the development of multiplex polymerase chain reaction/membrane hybridization assay (MPCR-MHA) for detection of the foreign genes insert in genetically modified organisms (GMOs). After detecting condition and parameter were optimized and determined, MPCR reactions were developed for amplifying several target genes simultaneously in one tube. Primers were labeled with biotin at the 5'-end; biotinylated MPCR products were detected by hybridization to the oligonucleotide probes immobilized on a membrane with subsequent colorimetric detection to confirm hybridization. The testing of screening primers can judge whether the sample contains GMOs, and that of identifying primers can further judge what kinds of trait genes are contained in the sample. We detected nine soybean samples, six maize samples, seven potato samples and two rice samples by the MPCR-MHA method; at the same time we also detected them with single PCR-MHA method. The results between two methods have good consistency.

DNA Primers↗