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PubMed2002

Identification of Salmonella enteritidis isolates by polymerase chain reaction and multiplex polymerase chain reaction.

The polymerase chain reaction and the multiplex polymerase chain reaction were developed for detection of Salmonella and for identification of the serotype enteritidis. Three sets of primers were selected from different genomic sequences amplifying a 429 bp fragment specific for the genus Salmonella within a randomly cloned sequence, including a 250 bp fragment within the spv gene, and a 310 bp fragment within the sefA gene specific for Salmonella enteritidis. The polymerase chain reaction and the multiplex polymerase chain reaction were used for detecting S. enteritidis isolated from stool samples during outbreaks of foodborne gastroenteritis between 1992 and 1998 in Taiwan. The sefA gene was detected in all 27 strains of S. enteritidis by this polymerase chain reaction method. Multiplex polymerase chain reaction could detect 3 genes in all strains, but could not detect the spv gene in 2 strains. The sensitivity of the polymerase chain reaction and the multiplex polymerase chain reaction were 10(4) and 10(5) cells/mL, respectively. In double polymerase chain reaction, the sensitivity increased to 100 cells/mL. These data indicate that the specificity and sensitivity of the polymerase chain reaction and the multiplex polymerase chain reaction make them potentially valuable tools for diagnosis of S. enteritidis infection and that they may be used for the identification of S. enteritidis responsible for sporadic enteritis cases.

Animals↗

PubMed2003

Evaluation of methods to improve detection of Escherichia coli O157:H7 in fresh produce by multiplex polymerase chain reaction.

Multiplex polymerase chain reaction (PCR) analysis was used to detect two genes encoding Shiga-like toxins (stx1 and stx2) and a universal Escherichia coli gene (gadA/B) in fresh produce spiked with E. coli O157:H7. Current U.S. Food and Drug Administration procedures for the analysis of fresh produce include the use of the rinsate from an initial rinse for the analysis of several potential pathogens, including E. coli O157:H7. In this study, several procedures were evaluated for their ability to increase the sensitivity of PCR analysis of rinsates from 15 types of produce. The procedures evaluated included the preliminary clarification and concentration of templates by centrifugation and the treatment of templates with compounds reported to facilitate nucleic acid amplification, including polyvinlypolypyrrolidone (PVPP), nonfat dry milk (NFDM), and InstaGene. The preliminary concentration of rinsates resulted in moderate improvements in detection sensitivity. The use of PVPP-treated templates in PCR reaction mixtures did not further improve sensitivity, but the inclusion of NFDM-treated templates increased sensitivity by an order of magnitude for 12 rinsates. The incorporation of InstaGene also improved the detection capability of the analysis; this procedure yielded the strongest gel bands for eight rinsates. However, for four other rinsates, the use of this reagent decreased sensitivity; these four rinsates were those for the produce varieties with the largest surface areas and were the most turbid rinsates. The use of facilitating compounds to block PCR inhibition may enable an analysis for Shiga toxin-producing E. coli in fresh produce to be completed in 1 to 2 days, rather than the 5 days required for current methods.

DNA, Bacterial↗

PubMed1992

Simultaneous detection of toxin A and toxin B genetic determinants of Clostridium difficile using the multiplex polymerase chain reaction.

A multiplex polymerase chain reaction was developed to simultaneously detect the presence of toxin A and toxin B genes of Clostridium difficile. A 1050-bp fragment of the toxin B gene and a 1217-bp fragment of the toxin A gene were amplified from 42 toxic strains of C. difficile; however, from 10 nontoxic strains the toxin gene fragments were not amplified; these data demonstrate that this multiplex polymerase chain reaction procedure can be used to differentiate between toxic and nontoxic strains. This sensitive and specific multiplex polymerase chain reaction for C. difficile toxins may prove to be a valuable diagnostic procedure.

Bacterial Proteins↗

PubMed2004

Application of extended single-reaction multiplex polymerase chain reaction for toxin typing of Staphylococcus aureus isolates in South Korea.

The extended single-reaction multiplex PCR (esr-mPCR) developed in this study to detect staphylococcal enterotoxins (SEs), including SEA, SEB, SEC, SED, SEE, SEH, SEI, and SEJ, requires fewer sets of primers than other conventional multiplex PCRs and can be used to detect newly identified staphylococcal enterotoxins SEs more readily. Esr-mPCR analysis of 141 isolates of Staphylococcus aureus obtained from abattoir and livestock product samples revealed that 27 of the S. aureus isolates were toxigenic, and two were 2 multitoxigenic isolates. The most prevalent SE type was SEI followed by SEA and SEH. In addition, we investigated the clonal relatedness of toxigenic S. aureus isolates by arbitrarily primed PCR (AP-PCR). AP-PCR analysis of toxigenic S. aureus isolates revealed that the discriminatory power of AP-PCR was 9 (D=0.81), 8 (D=0.77), and 10 types (D=0.83) with primers AP1, ERIC2, and AP7, respectively. The combination of three each AP-PCR result could rearrange toxigenic S. aureus isolates into 10 types and five subtypes, with the D-value of 0.92. Interestingly, our data showed that toxigenic S. aureus isolates from different sources had different fingerprinting patterns although some of them carried the same types of SE genes. These data suggest that combinations of esr-mPCR and AP-PCR can provide a powerful approach for epidemiological investigation of toxigenic S. aureus isolates.

Abattoirs↗

PubMed1998

Detection of staphylococcal genes directly from cerebrospinal and peritoneal fluid samples using a multiplex polymerase chain reaction.

A multiplex polymerase chain reaction (PCR) that can simultaneously detect eubacterial isolates and the methicillin-susceptibility of staphylococcal isolates from cerebrospinal and peritoneal fluid samples was compared to conventional microbiological methods. Using conventional methods, bacteria were isolated from 8% (29/350) of the cerebrospinal fluid samples and from 5% (3/60) of the peritoneal fluid samples. All isolates except two Staphylococcus epidermidis isolates were also detected using the multiplex PCR. Coagulase-negative staphylococci and Staphylococcus aureus were correctly identified using both methods. The multiplex PCR can rapidly and simultaneously detect eubacteria, and the methicillin susceptibility of staphylococci from samples containing > or = 10(2) cfu/ml of bacteria.

Ascitic Fluid↗

PubMed2002

Detection and typing of respiratory adenoviruses in a single-tube multiplex polymerase chain reaction.

A multiplex polymerase chain reaction (PCR) assay that is capable of detecting and typing six serotypes of respiratory adenovirus (Ad) was developed, using multiple sets of type-specific primers. The detection of each different serotype depended on distinguishing different numbers and sizes of amplification products on agarose gels following PCR. The multiplex PCR was tested with 26 clinical Ad isolates and other respiratory viruses including influenza viruses, parainfluenza viruses, and respiratory syncytial viruses as well as respiratory bacterial pathogens such as Chlamydia pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, and Mycoplasma pneumoniae. The multiplex PCR for the detection and typing of Ads gave an excellent correlation with the results by conventional typing with type-specific antisera. This assay may serve as a rapid means of confirming Ad with simultaneous serotype identification of the isolates. It will also have relevance as an adjunctive tool to conventional serotyping for diagnostic and epidemiological purposes.

Adenovirus Infections, Human↗

PubMedDate not supplied

Detection of human papillomavirus types 6, 11, 16 and 18 in mucosal and cutaneous lesions by the multiplex polymerase chain reaction.

A multiplex polymerase chain reaction (PCR) based on the simultaneous amplification of human papillomavirus (HPV) types 6/11, 16 and 18 in a single-step procedure was developed, using primers chosen in the E6-E7 region. The specificity and sensitivity of this technique have been proved by amplifying mixtures or various amounts of plasmid-containing HPV DNA; it allowed the detection of as few as 5-25 HPV DNA copies. Application of the multiplex PCR to 71 clinical samples showed that HPV DNA was detected in 80% (45/57 cases) of mucosal biopsies and 35% (5/14 cases) of cutaneous specimens. HPV 16 was predominant in high-grade CIN whereas HPV 6 and 11 were detected more frequently in genital condylomas and laryngeal papillomas. In cutaneous Bowen's disease HPV 16, 18 or 6/11 + 16 were detected and in squamous cell carcinomas HPV 6/11 or 16 were found. After sequence amplification with primers of one HPV type, the clinical samples displayed the same HPV types but the frequency of positive and coinfected lesions increased. Thus, multiplex PCR is a valuable technique for typing HPV DNA but coinfections may be underestimated.

Adult↗

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↗

PubMed1996

Detection of pathogenic Yersinia enterocolitica using the multiplex polymerase chain reaction.

A multiplex polymerase chain reaction (PCR) was developed to detect the presence of the ail, yst, and virF genes of Yersinia enterocolitica simultaneously, quickly and accurately. The amplified fragment sizes were 356 base-pairs (bp) for the ail gene, 134 bp for the yst gene, and 231 bp for the virF gene. The specificity of the amplified products was confirmed by hybridization with digoxigenin-labelled oligonucleotide probes. Amplification was successful whether the template was derived from a single colony of bacteria, aliquots of boiled bacterial suspensions, from DNA extracted from pure or mixed cultures or from stool specimens. Amplification of the virF gene was also achieved from strains of Y. pseudotuberculosis carrying the 70 kb plasmid but not with preparations from other related Yersinia species or from other members of the family Enterobacteriaceae. The detection limit we established was 5-10 colony forming units per millilitre (cfu/ml) and 1.0 pg of DNA.

Animals↗

PubMed1994

Detection of dystrophin gene deletion in Chinese Duchenne/Becker muscular dystrophy patients utilizing multiplex polymerase chain reaction.

Multiplex PCR was utilized to detect deletions at the hot spot regions of dystrophin gene. Thirty-four cases of DMD and 7 cases of BMD, based on their clinical manifestations, were examined. Eight normal male adults were included as control. Eleven DNA fragments coding the exons 1, 3, 6, 8, 13, 17, 43, 47, 50, 52 and 60 were chosen and primers were prepared with oligonucleotide synthesizer. Multiplex PCR was performed under the condition of 94 degrees C 1 minute for denaturation, 65 degrees C 3 minutes for annealing, 72 degrees C 3 minutes for extension. Thirty cycles were followed by a last cycle with 7 minutes extension. Clinically, there were no significant differences between Chinese patients and those reported in other areas except the low rate of positive family history (32%). The results from multiplex PCR showed that 15 cases (37%), 11 DMD and 4 BMD, proved to have deletions in the exons studied; 1 located at exons 6 and 8, 1 at exon 8, 1 at exons 8, 13 and 17, 1 at exons 13 and 17, 1 at exon 17, 3 at exon 43, 1 at exon 50, and 3 at exons 50 and 52, after false negatives were excluded. No difference in size or location was noticed between DMD and BMD in the sample-limited result.

Adult↗

PubMed1997

Identification of methicillin-resistant staphylococci by multiplex polymerase chain reaction assay.

A multiplex polymerase chain reaction (PCR) assay using oligonucleotide primers to detect mecA and 16S ribosomal RNA gene was developed to aid in identification of methicillin-resistant staphylococci. Validation included 99 isolates of staphylococcus grouped into one of five categories: methicillin-susceptible coagulase-negative staphylococcus (MSCNS), methicillin-resistant coagulase-negative staphylococcus (MRCNS), methicillin-susceptible Staphylococcus aureus (MSSA), high beta-lactamase producing S aureus (HiBSA), and methicillin-resistant S aureus (MRSA). mecA was detected in MRSA (21/21), and in MRCNS (20/20), but not in MSSA (0/20). mecA was occasionally detected in HiBSA (1/19) and MSCNS (3/19). This multiplex PCR assay was also used to test 30 clinical isolates of coagulase-negative staphylococci with discrepancies between results of in vitro tests for susceptibility to oxacillin and was found to be valuable when a more definitive determination of intrinsic methicillin-resistance was desired.

DNA, Bacterial↗

PubMed1996

Differentiation between Mycobacterium tuberculosis and Mycobacterium bovis by a multiplex-polymerase chain reaction.

A multiplex-polymerase chain reaction (PCR) assay, based on one-step amplification and detection of three different mycobacterial genomic fragments, was designed for differentiation between Mycobacterium bovis and Mycobacterium tuberculosis. The oligonucleotide primers were chosen from the groEL gene, present in the genus Mycobacterium sp., from the IS6110 insertion sequence, present in Myco. tuberculosis complex and from the mtp40 gene, identified as a specific-species Myco. tuberculosis genomic fragment. This amplification method allowed the detection of two fragments of 576 and 317 base pairs in Myco. bovis and three fragments of 576, 396 and 317 base pairs in Myco. tuberculosis strains, including atypical strains of Myco. tuberculosis where the copy number of the IS6110 element is low. The multiplex-PCR assay described may be a very useful tool for the rapid and specific differentiation of these related mycobacteria and easy to use in medical and veterinary microbiology laboratories.

Base Sequence↗

PubMed1999

Detection and typing of lymphotropic herpesviruses by multiplex polymerase chain reaction.

A multiplex nested-polymerase chain reaction (PCR) method was developed for the simultaneous detection and typing of all human lymphotropic herpesviruses described to date, including Ebstein Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6, variants A and B (HHV6-A, HHV6-B), human herpesvirus 7 (HHV7) and human herpesvirus 8 (HHV8). Oligonucleotide primers were designed to amplify a highly conserved region within the DNA polymerase gene. Each reaction component and thermal cycling parameters were thoroughly standardized to achieve optimal specificity and sensitivity for the PCR assay, which was estimated at about 10-100 molecules for each virus. An internal control, consisting of 100 molecules of a cloned fragment of the porcine pseudorabies herpesvirus (PrV) genome, was included to detect false negative results. To assess suitability and clinical application of the multiplex PCR method, a total of 35 well-characterized specimens, including Kaposi's sarcoma skin lesions, serum, cerebrospinal fluid, saliva and urine samples, were tested. Results obtained suggest this technique could be applied as a sole diagnostic tool in several clinical settings in which herpesviral infection is suspected and differential diagnosis required, avoiding the need to test specimens by separate PCR methods.

Adult↗

PubMed2005

Rapid detection of mecA and nuc genes in staphylococci by real-time multiplex polymerase chain reaction.

A multiplex real-time polymerase chain reaction (RT-PCR) targeting the mecA and nuc genes was developed for the detection of methicillin resistance and identification of Staphylococcus aureus. Novel mecA and nuc primers and fluorescence resonance energy transfer hybridization probes specific for the mecA and nuc genes were evaluated. The assay was performed using the LightCycler system (Roche Molecular Biochemicals, Mannheim, Germany) and evaluated against the traditional gel-based multiplex PCR (PCR-gel) method currently used at Royal Perth Hospital. Clinical isolates (n = 222) and isolates from a culture collection library (n = 206) were tested by both assays in parallel. The RT-PCR assay was 100% sensitive and specific for the detection of methicillin resistance and for the identification of S. aureus when compared with the PCR-gel assay. Results from the RT-PCR assay showed 5 isolates with lower efficiency fluorescence curves for the nuc gene PCR fragment. DNA sequencing showed mutations within the region of the probe-binding sites compared with the reference strain. The results of the RT-PCR assay were available within 2 h. This rapid mecA/nuc RT-PCR assay is a suitable and practical tool for the routine detection of methicillin resistance and identification of S. aureus, which can be easily incorporated into the diagnostic molecular microbiology laboratory work flow.

Bacterial Proteins↗

PubMed2003

Detection of novel Bartonella strains and Yersinia pestis in prairie dogs and their fleas (Siphonaptera: Ceratophyllidae and Pulicidae) using multiplex polymerase chain reaction.

We developed a multiplex polymerase chain reaction (PCR) assay that simultaneously detects three types of flea-associated microorganisms. Targets for the assay were sequences encoding portions of the gltA, a 17-kDa antigen, and pla genes of Bartonella spp. Strong et al., Rickettsia spp. da Rocha-Lima, and Yersinia pestis Yersin, respectively. A total of 260 flea samples containing bloodmeal remnants were analyzed from fleas collected from abandoned prairie dog (Cynomys ludovicianus) burrows at the site of an active plague epizootic in Jefferson County, CO. Results indicated that 34 (13.1%) fleas were positive for Bartonella spp., 0 (0%) were positive for Rickettsia spp., and 120 (46.2%) were positive for Y. pestis. Twenty-three (8.8%) of these fleas were coinfected with Bartonella spp. and Y. pestis. A second group of 295 bloodmeal-containing fleas was collected and analyzed from abandoned burrows in Logan County, CO, where a prairie dog die-off had occurred 2-4 mo before the time of sampling. Of these 295 fleas, 7 (2.4%) were positive for Bartonella spp., 0 (0%) were positive for Rickettsia spp., and 46 (15.6%) were positive for Y. pestis. Coinfections were not observed in fleas from the Logan County epizootic site. The multiplex PCR also was used to identify Y. pestis and Bartonella in prairie dog blood and tissues. This report represents the first identification of Bartonella from prairie dogs and their fleas. Prairie dog fleas were tested with PCR, and the Bartonella PCR amplicons produced were sequenced and found to be closely related to similar sequences amplified from Bartonella that had been isolated from prairie dog blood samples. Phylogenetic analyses indicate that the sequences of bartonellae from prairie dogs and prairie dog fleas cluster tightly within a clade that is distinct from those containing other known Bartonella genotypes.

Animals↗

PubMed2000

Detection and typing of HSV-1, HSV-2, and VZV by a multiplex polymerase chain reaction.

The development of a multiplex polymerase chain reaction method for the rapid and accurate detection and typing of HSV-1, HSV-2, and VZV from clinical specimens is described. A sensitive multiplex polymerase chain reaction was achieved by optimization of parameters such as the primers, magnesium, and dNTPs concentrations. False-negative results that sometimes arise due to inhibitors of DNA amplification or failure of DNA extraction procedure used may be avoided by assaying each specimen with alpha-tubulin primers. Multiplex PCR amplified viral sequences from all 55 specimens obtained from patients with clinical evidence of HSV or VZV infection indicated 100% sensitivity. From 55 patients who were investigated by multiplex PCR, HSV-1 was detected in 28, HSV-2 in 20, and VZV in 7 specimens. The reported results indicate that the present multiplex PCR assay has a potential application in clinical diagnosis when a rapid and accurate detection and typing of involved viruses HSV-1, HSV-2, or VZV is needed.

Adult↗

PubMed1998

Detection of verotoxin-producing Escherichia coli O157:H7 by multiplex polymerase chain reaction.

We constructed primers for multiplex polymerase chain reaction (PCR) to detect verotoxin-producing Escherichia coli (VTEC) O157:H7. The multiplex PCR primers were designed from the sequence of the flagellin structural gene of Escherichia coli flagellar type H7 (GenBank under accession number L07388), and from the sequence of the rfbE gene of Escherichia coli O157:H7 (GenBank under accession number S83460). In addition to these primers, we used a primer pair reported by Karch and Meyer (J. Clin. Microbiol. 27: 2751-2757, 1989) to amplify various VT genes from VTEC. All of the examined specimens (18 isolates) of VT-producing E. coli O157:H7 showed a positive result by the multiplex PCR test with the three sets of primers. The sensitivity of detection for VT-producing E. coli O157:H7 was shown to be at least 3,000 cells per PCR tube.

Animals↗

PubMed1991

[Analysis of deletion mutations of the dystrophin gene by the multiplex polymerase chain reaction method in the diagnosis of Duchenne muscular dystrophy].

The 33 patients suffering from the Duchenne muscular dystrophy (DMD), 7 healthy donors and a DMD risk family were studied by means of polymerase multiplex chain reaction (MPCR) with 6 oligoprimer pairs for 6 different exons of dystrophin gene. The deletions varying in sizes from 1 to 6 exons were detected in 12 out of 33 DMD patients studied (36.3%). The prenatal diagnosis of DMD was carried out by chorionic villus biopsy on the 1st trimester of pregnancy. Contrary to earlier findings, in elder brother with sever DMD manifestation, no visible deletion was detected in the DNA sample from the male foetus and thus the diagnosis of DMD in foetus was rejected. The perspectives of MPCR in pre and postnatal diagnosis of DMD are discussed.

Chromosome Deletion↗