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Results for “multiplex PCR”

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At least 19 recordsLinked to original sources

PubMed1998

Detection of microbial pathogens in shellfish with multiplex PCR.

Multiplex PCR amplification of uidA, cth, invA, ctx, and tl genes was developed enabling simultaneous detection in shellfish of Escherichia coli, an indicator of fecal contamination and microbial pathogens, Salmonella typhimurium, Vibrio vulnificus, V. cholerae, and V. parahaemolyticus, respectively. Each of the five pairs of oligonucleotide primers was found to support PCR amplifications of only its targeted gene. The optimized multiplex PCR reaction utilized a PCR reaction buffer containing 2.5 mM MgCl2 and primer annealing temperature of 55 degrees C. Oyster tissue homogenate seeded with these microbial pathogens was subjected to DNA purification by the Chelextrade mark 100 (BioRad) method. The sensitivity of detection for each of the microbial pathogens was </=10(1)-10(2) cells following a "double" multiplex PCR amplification approach. Amplified target genes in a multiplex PCR reaction were subjected to a colorimetric GeneCombtrade mark (BioRad) DNA-DNA hybridization assay. This assay was rapid and showed sensitivity of detection comparable to the agarose gel electrophoresis method. The colorimetric GeneCombtrade mark assay avoids use of hazardous materials inherent in conventional gel electrophoresis and radioactive-based hybridization methods. Multiplex PCR amplification, followed by colorimetric GeneCombtrade mark DNA-DNA hybridization, has been shown to be an effective, sensitive, and rapid method to detect microbial pathogens in shellfish.

Animals↗

PubMed2004

DNA differential diagnosis of taeniasis and cysticercosis by multiplex PCR.

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.

Animals↗

PubMed2005

Detection of porcine circovirus type 2, porcine parvovirus and porcine pseudorabies virus from pigs with postweaning multisystemic wasting syndrome by multiplex PCR.

Multiplex PCR was established to detect porcine circovirus type 2 (PCV-2), porcine parvovirus (PPV) and porcine pseudorabies virus (PRV) and applied to samples from 137 piglets exhibiting clinical signs of postweaning multisystemic wasting syndrome (PMWS). PCV-2 DNA was detected from all samples. Moreover, 43 samples were positive for PPV but negative for PRV; 11 samples were positive for PRV but negative for PPV; and 35 samples were positive both for PPV and PRV. These results suggests that PCV-2 co-infection with PRV and PPV may play an important role in PMWS. Also, multiplex PCR is an appropriate candidate method for diagnosis of PCV-2, PRV and PPV simultaneously in field cases.

Animals↗

PubMed2004

Detection and identification of Actinobacillus pleuropneumoniae serotypes 1, 2, and 8 by multiplex PCR.

Multiplex PCR assays were developed to identify Actinobacillus pleuropneumoniae serotypes 1, 2, and 8. Primers designed for the conserved capsular polysaccharide (CP) export region amplified a 489-bp DNA fragment from all serotypes. Primers specific to the CP biosynthesis regions of serotypes 1, 2, and 8 amplified fragments of 1.6 kb, 1.7 kb, and 970 bp from only their respective serotypes.

Actinobacillus pleuropneumoniae↗

PubMed2007

Comparison of GenoType MTBC with RFLP-PCR and multiplex PCR to identify Mycobacterium tuberculosis complex species.

The aims of the work presented here were to evaluate a commercial system based on DNA hybridization technology on nitrocellulose strips (GenoType MTBC; Hain Diagnostika, Nehren, Germany) for identifying Mycobacterium tuberculosis complex isolates to the species level and to compare the results with those obtained using the RFLP-PCR and multiplex PCR molecular techniques, biochemical tests and susceptibility testing. The commercial system correctly identified 102 of 103 (99.03%) strains studied, indicating it is capable of discriminating among the tuberculosis complex species.

DNA Probes↗

PubMed2001

Three molecular methods to identify Salmonella enterica serotype Typhimurium DT104: PCR fingerprinting, multiplex PCR and rapid PFGE.

Here we report the accuracy with which three molecular techniques (PCR fingerprinting, multiplex PCR and macrorestriction profiling) distinguished Salmonella enterica Typhimurium DT104 (hereafter referred to as DT104) from other related strains of Salmonella. Each technique was tested by screening a set of 20 isolates (10 DT104, eight non-DT104 Typhimurium, one S. enterica Agona, one S. enterica Newport) and each consistently differentiated DT104 from non-DT104 isolates based on visual inspection of band patterns. The accuracy of each technique was confirmed by computer analysis. As such, these data indicate that each technique could be used to presumptively identify DT104 isolates as a precursor to phage typing.

Bacterial Typing Techniques↗

PubMed2003

PCR-multiplexes for a genome-wide framework of simple sequence repeat marker loci in cultivated sunflower.

Simple sequence repeat (SSR) and other DNA sequence-tagged site markers can be genotyped more rapidly and cost efficiently by simultaneously amplifying multiple loci (multiplex PCR). The development of PCR-multiplexes for a nearly genome-wide framework of 78 SSR marker loci in cultivated sunflower ( Helianthus annuus L.) is described herein. The most outstanding single-locus SSR markers in the public collection (300 out of 1,089) were identified and screened for polymorphisms among 24 elite inbred lines, preparatory to selecting SSR markers for testing in multiplex PCRs. The selected SSR markers produced robust PCR products, amplified a single locus each, were polymorphic among elite inbred lines (minimum, mean and maximum heterozygosities were 0.08, 0.53 and 0.85, respectively), and supply a dense genome-wide framework of predominantly or completely codominant, single-locus DNA markers for molecular breeding and genomics research in sunflower. Thirteen six-locus multiplex PCRs were developed for 78 SSR marker loci strategically positioned throughout the sunflower genome (three to five per linkage group) by identifying compatible SSR primer combinations and optimizing multiplex PCR protocols. The multiplexed SSR markers, when coupled with 17 complementary SSR marker loci, create a 'standard genotyping' set ideal for first-pass scans of the genome, as are often needed when screening bulked-segregant DNA samples or mapping phenotypic trait loci. The minimum, mean and maximum heterozygosities of the multiplexed SSR markers were 0.38, 0.62 and 0.83, respectively. The PCR-multiplexes increase genotyping throughput, reduce reagent costs, and are ideal for repetitive genotyping applications where common sets of SSR marker loci are required or advantageous.

Chromosome Mapping↗

PubMed2004

Development of a 13-locus PCR multiplex system for paternity testing.

In this study the development of a 13-locus multiplex-PCR system fitting the updated demands for paternity testing in Germany is described. For this purpose an existing multiplex PCR system that allows the simultaneous amplification of eight different STR loci together with the sex-specific locus amelogenin ( genRESMPX-2, Serac, Germany) was extended. Whereas some of the primers were taken from the underlying multiplex system, suitable primer sequences were chosen for the STR loci D19S433, TPOX, TH01, D16S539, D5S818, D2S1338 and FGA. Primers of loci resulting in potentially overlapping fragment sizes were labelled with the fluorescent dyes 6-FAM, JOE and NED. Reaction conditions, such as annealing temperature, concentrations of primers and polymerase or buffer conditions were optimised to obtain a robust amplification and reproducible genotype analysis for various sample sources. Full DNA profiles from single source samples were reliably typed from template DNA amounts of as low as 120 pg, suggesting a potential use of this system also in forensic casework analysis. With a mean exclusion chance (MEC) of 99.9989% and a power of discrimination (P(D)) of about 1x10(14) (Caucasians), the new multiplex PCR system provides a significant and sensitive system for forensic DNA analysis. On the basis of these studies, a commercial kit system is now provided by Serac (Bad Homburg, Germany, genRESMPX-3).

Allelic Imbalance↗

PubMed2006

[Simultaneous detection by multiplex PCR of atypical bacterial pathogens involved in infections of respiratory tract. Is it useful for the microbiological diagnosis of respiratory infections].

The aim was to evaluate the "strategical place" of the new commercial test Chlamylege (Argene-Biosoft-France) which allows the simultaneous detection in respiratory samples of Chlamydophila pneumoniae, Mycoplasma pneumoniae and most Legionella species using a PCR multiplex. 41 patients with an atypical pneumonia were included, all standard procedures of diagnosis were done and in addition the chamylege test. A pathogen was identified in 12 patients, an other microorganisms than the 3 targeted by our study was found in 8 patients. 4 positive PCR were obtained, 3 with M. pneumoniae and 1 with Legionella pneumophila 1. That means that for 29 patients no aetiology was found. Among them 23 clearly improved under antibiotic treatment. Though that PCR multiplex is an attractive test, easy to perform, sensitive, specific and convenient, we need further studies to approach the place of this PCR test in the diagnosis of multifaceted atypical pneumonia. We also need to know if the cost associated with the microbiological diagnosis (culture, serology, immunofluorescence, urinary antigen test, PCR...) for atypical pneumonia worth value? An algorithm as to be drawn to determine the value of intensive microbiological investigation. An other point to discuss, may be this kind of rapid and multiplex PCR technique could lead to spare the use of some antibiotics.

Atypical Bacterial Forms↗

PubMed2005

[Application of the molecular test PCR multiplex for identification of Mycobacterium bovis BCG strains].

In our last paper (18) we described the problem of proper microbiological identification of BCG strains and how important is distinguishing vaccine strain from virulent strains of Mycobacterium tuberculosis complex. We have suggested the modern algorithm of BCG strains identification including mycolic acids profile by HPLC and 14C PZA resistance methods. These methods allowed us to made fast and accurate microbiological identification of side effects of BCG vaccine in the children. Identification of BCG by HPLC is possible within one working day compared with 3-4 weeks required for conventional methods. However both methods need very expensive instruments like HPLC and/or Bactec-460 Tb radiometric system. Presently we have evaluated molecular test based on the analyzis of the region RD1 encoding a 9.5-kb fragment. This fragment is deleted in all BCG substrains (6) and present in all human and bovine virulent strains. To evaluate this method for the rapid and specific detection of BCG, a large strain collection (32 strains) representating M. bovis BCG (vaccine strains and strains isolated from the children in case of adverse reactions after vaccination) M. bovis and M. tuberculosis from own collection was analyzed. RD1 was present in all 15 M. tuberculosis and M. bovis tested strains and deleted in 17 of 18 BCG strains. The multiplex PCR method was 100% sensitive and specific for the identification of BCG among strains of the Mycobacterium tuberculosis complex. Multiplex PCR can be used as a diagnostic test and has significant advantages over existing methods.

Animals↗

PubMed2004

Identification of Pseudomonas aeruginosa, Burkholderia cepacia complex, and Stenotrophomonas maltophilia in respiratory samples from cystic fibrosis patients using multiplex PCR.

A multiplex PCR method was developed to identify P. aeruginosa, B. cepacia complex, and S. maltophilia directly in sputum and oropharyngeal samples from CF patients. One hundred and six patients (53 male, and 53 female) attending our pulmonology clinic were studied from September 2000-April 2001. Two hundred and fifty-seven samples were cultured in selective media and submitted to multiplex PCR reactions, using three primer pairs targeting specific genomic sequences of each species, with an additional primer pair targeting a stretch of ribosomal 16S DNA, universal for bacteria, to act as a control. P. aeruginosa was isolated by culture in 56% of samples, B. cepacia complex in 4.3%, and S. maltophilia in 2.7%, while multiplex PCR identified P. aeruginosa in 78.7%, B. cepacia complex in 3.9%, and S. maltophilia in 3.1% of samples. Multiplex PCR results were verified by PCR reactions using different species-specific primers described in the literature and DNA sequencing of amplicons from a few samples. Comparing to culture results, the sensitivity and specificity values of multiplex PCR for bacterial identification were, respectively, 97.2% and 45.5% for P. aeruginosa, 45.5% and 97.9% for B. cepacia complex, and 40% and 97.6% for S. maltophilia. All 10 multiplex PCR-positive results for B. cepacia complex were confirmed using other species-specific primers described in the literature, while this approach confirmed results for S. maltophilia identification in 7/8 samples (87.5%). Sequencing of amplicons from samples culture-negative but multiplex PCR-positive for P. aeruginosa and B. cepacia complex confirmed their identity, while minor nucleotide differences among amplicons ruled out the hypothesis of PCR contamination.

Adolescent↗

PubMed1999

Identification of cecidophyopsis mites (Acari: Eriophyidae) based on variable simple sequence repeats of ribosomal DNA internal transcribed spacer-1 sequences via multiplex PCR.

A PCR multiplex technique was developed for identifying Cecidophyopsis mites using species-specific differences in rDNA ITS-1 sequences. Four PCR primers derived from ITS-1 were used for the simultaneous amplification (multiplex PCR) of interspecifically variable simple sequence repeats (vSSRs). Mites were identified by electrophoresing PCR products alongside those obtained from plasmids containing ITS copies of known mite species. The multiplex PCR assay was rapid, reproducible and had a sensitivity comparable to sequencing. It was used to identify mite specimens on Ribes from around the world. It also identified a profile from mites on R. rubrum that had no equivalent amongst the known Cecidophyopsis species. Sequence and ecological analysis of this mite suggest that it is a new species of nongall-forming Cecidophyopsis mite.

Animals↗

PubMed1999

Rapid detection of methicillin-resistant staphylococci by multiplex PCR.

A multiplex PCR was developed to detect the coagulase gene (coa; pathognomic of Staphylococcus aureus) and the mecA gene (characteristically encoding for methicillin resistance in staphylococci) in a single, rapid test. Suitable primers for the gene targets and an internal, amplification control were incorporated into a multiplex PCR assay, which was then optimized on a capillary air thermal cycler to improve the turnaround time of the test to approximately 1.5 hours. The assay was evaluated with 111 fresh clinical isolates of staphylococci. The multiplex PCR correctly distinguished between isolates of S. aureus, which were sensitive to methicillin (MSSA) and those resistant to it (MRSA). It also correctly differentiated between similar isolates of coagulase negative staphylococci (MSSE and MRSE respectively). It was concluded that this multiplex PCR was a rapid and reliable method for the detection of methicillin-resistant staphylococci.

Coagulase↗

PubMed2002

Rapid identification of pathogenic fungi directly from cultures by using multiplex PCR.

A multiplex PCR method was developed to identify simultaneously multiple fungal pathogens in a single reaction. Five sets of species-specific primers were designed from the internal transcribed spacer (ITS) regions, ITS1 and ITS2, of the rRNA gene to identify Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, and Aspergillus fumigatus. Another set of previously published ITS primers, CN4 and CN5, were used to identify Cryptococcus neoformans. Three sets of primers were used in one multiplex PCR to identify three different species. Six different species of pathogenic fungi can be identified with two multiplex PCRs. Furthermore, instead of using templates of purified genomic DNA, we performed the PCR directly from yeast colonies or cultures, which simplified the procedure and precluded contamination during the extraction of DNA. A total of 242 fungal isolates were tested, representing 13 species of yeasts, four species of Aspergillus, and three zygomycetes. The multiplex PCR was tested on isolated DNA or fungal colonies, and both provided 100% sensitivity and specificity. However, DNA from only about half the molds could be amplified directly from mycelial fragments, while DNA from every yeast colony was amplified. This multiplex PCR method provides a rapid, simple, and reliable alternative to conventional methods to identify common clinical fungal isolates.

Culture Media↗

PubMed2003

Species identification and detection of vancomycin resistance genes in enterococci of animal origin by multiplex PCR.

A multiplex PCR assay for the differentiation of species and detection of vancomycin (van) resistance genes in enterococci was established. Three hundred sixty-seven enterococcal strains isolated from food were selected from a sample collection and were examined with regards to the existence of four vancomycin resistance genes (vanA, vanB, vanC1 and vanC2) and to species differentiation (E. faecalis, E. faecium, E. casseliflavus and E. gallinarum) by multiplex PCR. Apart from unambiguous results for the majority of strains, the E. faecium specific primers used here, showed weak points in the specificity and sensitivity, respectively. Despite these rare instances, we succeeded in optimising the multiplex PCR assay by variation of annealing temperature and primer combination. The assay presented here, with its optimised parameters, is therefore suitable for routine use and because of time and material saving, it is an alternative and rapid method in comparison to conventional tests.

Animals↗

PubMed2004

Preliminary study on the detection of the SARS-CoV specific target cDNA fragments by multiplex PCR.

The multiplex polymerase chain reaction (PCR) technique was applied to detect the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) specific target cDNA fragments in the present study. The target cDNA fragments of SARS-CoV were synthesized artificially according to the genome sequence of SARS-CoV in GenBank submitted by The Chinese University of Hong Kong, and were used as simulated positive samples. Five primers recommended by World Health Organization (WHO) were used to amplify the fragments by single PCR and multiplex PCR. Three target cDNA fragments (121, 182 and 302 bp), as well as the three different combinations of any two of these fragments, were amplified by single PCR. The combination of these three fragments was amplified by multiplex PCR. The results indicated that the multiplex PCR technique could be applied to detect the SARS-CoV specific target cDNA fragments successfully.

Base Sequence↗

PubMed2006

Detection of respiratory herpesviruses in foals and adult horses determined by nested multiplex PCR.

A nested multiplex PCR was developed as a rapid (<12h), sensitive test for the simultaneous identification of equine herpesviruses (EHV1, EHV4, EHV2 and EHV5) in clinical samples from horses. Peripheral blood and nasal swab (NS) samples from 205 weanling Thoroughbred foals on 6 different studs over 3 consecutive seasons and from 92 adult horses without clinical signs of respiratory disease were examined using direct multiplex PCR of clinical samples (direct PCR) and conventional cell culture with differentiation of EHV in cell cultures by multiplex PCR. Multiplex PCR proved a sensitive and specific technique for the detection of EHV in cell culture and clinical samples. The technique described appeared equally sensitive as one using a single set of primers for individual EHV but reduced labour and reagent costs. Cell cultures showing cytopathic effect (CPE) were always positive for EHV on PCR. EHV were also detected by multiplex PCR in 11 samples which failed to show CPE. By a combination of multiplex PCR and cell culture or direct multiplex PCR, the presence of up to three EHV in the same sample was detected. Overall, EHV5 was detected by direct multiplex PCR of peripheral blood mononuclear cells (PBMC) and/or NS samples from 78% of foals and 47% of adult horses. Repeated sampling or cell culture in combination with multiplex PCR and with the incorporation of IL-2 in culture medium increased the sensitivity for detection of EHV in PBMC and demonstrated that EHV5 DNA could be identified in PBMC from 89% of foals and 100% of adult horses. EHV2 was identified from approximately 30% of foals, but was more frequently identified in samples from 17 foals with mild respiratory disease and was isolated infrequently from adult horses. EHV1 and EHV4 were identified uncommonly in any population in the current study.

Animals↗

PubMed1999

Typing of porcine circovirus in clinical specimens by multiplex PCR.

A multiplex PCR assay was developed to detect and differentiate between the porcine circovirus (PCV) infecting persistently the PK 15 cell line (PCV type I) and the PCV associated with postweaning multisystemic wasting syndrome (PMWS) (PCV type II). DNA products with unique sizes characteristic of each type of PCV were obtained. Sequencing of these products demonstrated that the nucleotide sequences were type-specific. Tissue samples from a total of 42 field cases from Québec were studied, among which 41 were collected in 1997-1998 and one which had been previously collected in 1994. These 42 cases found previously to be PCV-positive by PCR were tested in the present study by a multiplex PCR assay to determine the type of PCV in each case. From these 42 field cases, 40 cases were PCV type II-positive, one case was PCV type I-positive and one case was positive for both PCV types I and II. PCV type II was identified in typical PMWS field cases, but also in field cases submitted for various clinical histories, some of which were not suggestive of PMWS. In the field case where PCV type I was detected, there was no clinical evidence nor histological lesions suggestive of PMWS. The demonstration of PCV type II in a total of 41/42 field cases in the present study suggests that PCV type II may be the main type of PCV circulating in pigs. Furthermore the detection of PCV type II in a field case dating back to 1994 indicates that this PCV type was circulating in pigs in Québec several years before the report of clinical PMWS in this province.

Animals↗