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PubMed2002

Utility of a multiplex PCR assay for detecting herpesvirus DNA in clinical samples.

A multiplex PCR was designed to amplify herpes simplex virus types 1 and 2, cytomegalovirus, and varicella-zoster virus DNA present in a diverse range of clinical material. The susceptibility of these viruses to in vivo inhibition by at least one antiviral drug was an important consideration in their inclusion in the multiplex detection system. An aliquot of equine herpesvirus was introduced into each specimen prior to extraction and served as an indicator of potential inhibitors of the PCR and a detector of suboptimal PCR conditions. Compared to virus isolation and immunofluorescence-based antigen detection, the multiplex assay yielded higher detection rates for all viruses represented in the assay. The turnaround time for performance of the assay was markedly reduced compared to those for the other techniques used to identify these viruses. More than 21,000 tests have been performed using the assay. Overall, the multiplex PCR enabled the detection of substantially increased numbers of herpesviruses, in some cases in specimens or anatomical sites where previously they were rarely if ever identified using traditional detection methods.

Bronchoalveolar Lavage Fluid↗

PubMed1998

Rapid and specific detection of toxigenic Staphylococcus aureus: use of two multiplex PCR enzyme immunoassays for amplification and hybridization of staphylococcal enterotoxin genes, exfoliative toxin genes, and toxic shock syndrome toxin 1 gene.

Two multiplex PCR enzyme immunoassays (PCR-EIAs) were developed for Staphylococcus aureus exotoxin gene screening as an alternative to the conventional biological assays, which depend on detectable amounts of toxins produced. One set of oligonucleotide primers and probes was designed to search for enterotoxin A to E genes (entA, entB, entC, entD, and entE), and the other one was designed to detect the staphylococcal exfoliative toxin genes (eta and etb) and the toxic shock syndrome toxin 1 gene (tst). Oligonucleotide primers were used as published previously, modified or newly developed to meet the requirements of both good size-distinguishable amplification bands of multiplex PCR and the temperature limit of the uracil DNA glycosylase system for carryover protection. Amplification products were visualized by agarose gel electrophoresis, and specificity was controlled with the aid of a DNA EIA system using oligonucleotide probes derived from the sequences of the S. aureus toxin genes. PCR procedures were performed by using template nucleic acids extracted from a panel of S. aureus reference strains and from a collection of 50 clinical strains. The PCR results were compared with those of immunological toxin production assays. This multiplex PCR-EIA system offers an alternative method for the rapid, sensitive, specific, and simultaneous detection of the clinically important exotoxin potency of isolated S. aureus strains for diagnostic purposes as well as research studies.

Bacterial Toxins↗

PubMed2025

Development of a multiplex PCR for detection of pathogenic Mycobacterium orygis in cattle tissues harboring tuberculous-like lesions.

Mycobacterium orygis, a recently defined member species of Mycobacterium tubercuolsis complex (MTBC), is emerging as a major threat to zoonotic tuberculosis control, especially in the Asian Subcontinent. The dearth of low-cost diagnostic assay to differentiate M. orygis from other members of the MTBC leads to unavailability of information about the actual burden of this species in human and animal population. In this study, we developed a multiplex PCR for distinguishing M. orygis from other MTBC based on two M. orygis-specific nonsynonymous point mutations in mbtG and fadD23 genes identified by comparative genome analysis. The specificity of the assay shows that a 434 bp IS1081 fragment was amplified from common MTBC species including M. orygis while 240 bp and 181 bp mbtG and fadD23 gene fragments were amplified only from M. orygis. No amplification was observed for nontuberculous Mycobacterium (NTM) and non-Mycobacterial pathogens. The multiplex PCR assay showed a detection limit of 32 pg of M. orygis DNA. Furthermore, a total of 85 tuberculous-like lesions in the different tissues of slaughtered cattle were tested for identification of the M. orygis, and the results showed IS1081, mbtG and fadD23 amplicons in three tissue DNA extracts confirming they contain M. orygis DNA. Also, a single IS1081 amplicon was amplified from one tissue sample signifying presence of DNA of any MTBC species other than M. orygis. An established TaqMan real time PCR assay targeting region of differences (RD) in M. orygis genome was carried out to validate the result of the assay. This showed 100 % accuracy of the in-house developed multiplex PCR.

Mycobacterium orygis↗

PubMed2005

New microsatellite multiplex PCR for Candida albicans strain typing reveals microevolutionary changes.

Five new microsatellite loci were described and characterized for use as molecular markers for the identification and genetic differentiation of Candida albicans strains. Following the typing of 72 unrelated clinical isolates, the analysis revealed that they were all polymorphic, presenting from 5 to 30 alleles and 8 to 46 different genotypes. The discriminatory power obtained by combining the information generated by three microsatellites used in a multiplex PCR amplification strategy was 0.99, the highest ever reported. The multiplex PCR was later used to test a total of 114 C. albicans strains, including multiple isolates from the same patient collected from different body locations and along episodes of vulvovaginal infections. Three different scenarios for strain relatedness were identified: (i) different isolates that were revealed to be the same strain, (ii) isolates that were the same strain but that apparently underwent a process of microevolution, and (iii) isolates that corresponded to different strains. Analysis of the microevolutionary changes between isolates from recurrent infections indicated that the genotype alterations observed could be the result of events that lead to the loss of heterozygosity (LOH). In one case of recurrent infection, LOH was observed at the CAI locus, and this could have been related to exposure to fluconazole, since such strains were exposed to this antifungal during treatment. The analysis of microsatellites by a multiplex PCR strategy was found to be a highly efficient tool for the rapid and accurate differentiation of C. albicans strains and adequate for the identification of fine microevolutionary events that could be related to strain microevolution in response to environmental stress conditions.

Candida albicans↗

PubMed2004

Development of a multiplex PCR technique for detection and epidemiological typing of salmonella in human clinical samples.

We have developed a multiplex PCR assay for Salmonella detection and epidemiological typing. Six sets of primers were designed to detect the major Salmonella serotypes and phage types in Spain. An internal amplification control was designed in order to detect PCR inhibition. The different amplification profiles obtained allowed us to detect Salmonella bacteria and to distinguish the clinically prevalent Salmonella enterica serotypes Enteritidis, Typhimurium and subspecies I serotype 4,5,12:i:-. Using this method, we could detect a specific band for DT104 and U302 phage types in Salmonella serotype Typhimurium. Salmonella enterica serotype Hadar and other C2 serogroup strains showed two specific band profiles. In the validation stage, the assay was reproducible for all serotypes studied, apart from some C2 serogroup strains. When the technique was applied to clinical stool specimens, the prevalent serotypes Enteritidis and Typhimurium were detected with a sensitivity of 93%, specificity of 100%, and efficiency of 98%. Also, a low PCR inhibition rate (8%) was obtained. The overall agreement of the multiplex PCR with conventional culture-based techniques was 95% for Salmonella typing using Cohen's kappa index.

Bacterial Typing Techniques↗

PubMed2005

Multiplex PCR for rapid differentiation of three species in the "Clostridium clostridioforme group".

Clostridium clostridioforme is a relatively antimicrobial resistant, phenotypically heterogeneous anaerobe that has been involved in a variety of infections. 16S rDNA sequencing analysis revealed three principal species in what has been called Clostridium clostridioforme - Clostridium bolteae, C. clostridioforme, and Clostridium hathewayi. Based on the 16S rDNA sequence information we obtained, we developed a cost-effective, timesaving one-step multiplex PCR assay for rapid and accurate differentiation of these three species. The established multiplex PCR identification scheme was applied to the identification of 88 clinical isolates that had previously been identified phenotypically as C. clostridioforme. The identification obtained from multiplex PCR assays showed 100% agreement with 16S rDNA sequencing identification. This scheme will permit more accurate assessment of the role of these three Clostridium species in infection and of the degree of antimicrobial resistance in each of the species.

Bacteriological Techniques↗

PubMed2001

Rapid identification of yeasts in positive blood cultures by a multiplex PCR method.

Yeasts are emerging as important etiological agents of nosocomial bloodstream infections. A multiplex PCR method was developed to rapidly identify clinically important yeasts that cause fungemia. The method amplified the internal transcribed spacer 1 (ITS1) region between the 18S and 5.8S rRNA genes and a specific DNA fragment within the ITS2 region of Candida albicans. With this method, C. albicans produced two amplicons, whereas other species produced only one. Through sequence analysis, the precise lengths of the PCR products were found to be as follows: C. glabrata (482 or 483 bp), C. guilliermondii (248 bp), C. parapsilosis (229 bp), C. albicans (218 or 219 and 110 bp), C. tropicalis (218 bp), Cryptococcus neoformans (201 bp), and C. krusei (182 bp). The PCR products could be effectively separated by disk polyacrylamide gel electrophoresis. The method was used to test 249 positive blood cultures (255 isolates), from which the following species (strain number) were isolated: C. albicans (128), C. tropicalis (51), C. glabrata (28), C. parapsilosis (23), C. neoformans (9), C. krusei (5), C. guilliermondii (3), and other, minor species (8). The test sensitivity of the method was 96.9% (247 of 255 isolates). The eight minor species were either misidentified (one strain) or not identified (seven strains). From the time at which a positive bottle was found, the multiplex PCR could be completed within 8 h; the present method is simpler than any previously reported molecular method for the identification of blood yeasts.

Blood↗

PubMed1997

A multiplex PCR for identifying Shiga-like toxin-producing Escherichia coli O157:H7.

A multiplex PCR assay specifically detecting Escherichia coli O157:H7 was developed by employing primers amplifying a DNA sequence upstream of E. coli O157:H7 eaeA gene and genes encoding Shiga-like toxins (SLT) I and II. Analysis of 151 bacterial strains revealed that all E. coli O157:H7 strains were identified simultaneously with the SLT types and could be distinguished from E. coli O55:H7 and E. coli O55:NM, and other non-O157 SLT-producing E. coli strains. Primer design, reaction composition (in particular, primer quantity and ratios), and amplification profile were most important in development of this multiplex PCR. This assay can serve not only as a confirmation test but also potentially can be applied to detect the pathogen in food.

Adhesins, Bacterial↗

PubMed2002

RPE65 gene: multiplex PCR and mutation screening in patients from India with retinal degenerative diseases.

We used multiplex PCR follwed by sequencing to screen for mutations in the 14 exons of the RPE65 gene in early-childhood-onset autosomal recessive retinitis pigmentosa (arRP) and Leber's congenital amaurosis (LCA) patients. The RPE65 protein is believed to play an important role in the metabolism of vitamin A in the visual cycle and mutations identified in the gene could have implications for vitamin A-based therapeutic intervention. We were able to identify a homozygous mutation (AAT --> AAG) in exon 9 in an arRP patient and a heterozygous missense transversion (AAT --> AAG) also in exon 9 of an LCA patient. We also identified a polymorphism in exon 10 (GAG --> GAA) in an arRP as well as an LCA patient. Mutation screening would be greatly facilitated by multiplex PCR which could cut down costs, labour and time involved. The nucleotide changes observed in this study could be de novo. Though a larger study has been undertaken, from the preliminary results it appears that in India the RPE65 gene seems to be less involved in causation of LCA.

Carrier Proteins↗

PubMed2000

Multiplex PCR for detection of trait and virulence factors in enterohemorrhagic Escherichia coli serotypes.

A multiplex PCR assay was developed which allowed the simultaneous detection of five trait genes or virulence markers in enterohemorrhagic Escherichia coli (EHEC) serotypes. A primer pair, designed to detect a single base-pair mutation in the uidA gene, is specific only for the prototypic EHEC of O157:H7 serotype and its toxigenic, non-motile variants. In a similar way, primers to the eaeA gene of the gamma-intimin derivative specifically detects strains in the EHEC 1 clonal group, which consists mostly of O157:H7 and some O55:H7 serotypes. The other three primer pairs, specific for stx1, stx2 and both variants of ehxA genes, will detect the presence of these virulence genes in all EHEC serotypes. Analysis of 34 strains, including various serotypes of EHEC, Shiga toxin-producing E. coli and enteropathogenic E. coli, confirmed that the multiplex PCR assay detected the presence of these genes in a manner consistent with the known genotype of each respective strains.

Bacterial Proteins↗

PubMed1996

A multiplex PCR procedure for polymorphic analysis of GSTM1 and GSTT1 genes in population studies.

A deletion polymorphism in glutathione S-transferase theta (GSTT1) gene was recently discovered in humans. Similar to the GSTM1 gene, GSTT1 is also recognized as a risk modifier in exposed populations. To evaluate the role of genetic polymorphism in health effects, the combined genetic polymorphism of different genes should be taken into consideration. In the present study, we have developed a multiplex PCR approach for simultaneous replication of both genes for molecular analysis. The multiplex PCR protocol was validated using donor DNA with different polymorphic combinations for both genes from two different ethnic populations (North Americans and Egyptians). The prevalence of the GSTM1 null genotype was 51% among North Americans and 44% among Egyptians. The prevalence of the GSTT1 null genotype was 15% among North Americans and 14.7% among Egyptians. Combined polymorphism analysis of both genes revealed that 6.3% of North Americans harbor the deleted genotype of both genes compared to 8.8% of the Egyptians. The data indicate that there is no major difference in allelic distribution of both genes between the ethnic populations. The multiplex PCR assay used in this study has the advantage of reducing the time, effort and cost required to carry out such analysis. It will also significantly enhance the ability to use genetic screening techniques as a potential tool for early detection of health outcomes in exposed populations.

Egypt↗

PubMed2006

Novel multiplex PCR approaches for the simultaneous detection of human pathogens: Escherichia coli 0157:H7 and Listeria monocytogenes.

Escherichia coli 0157:H7 and Listeria monocytogenes are the two most important food-borne human pathogens. To develop a single, rapid and sensitive PCR based test for simultaneous detection of both the organisms, fliCh7 and iap gene specific primers were used respectively for E. coli 0157:H7 and L. monocytogenes. Initially, with equal quantities of purified genomic DNAs of these organisms a multiplex PCR reaction was standardized to yield uniform amplification of both targets. Although, this assay detected E. coli 0157:H7 with high sensitivity, it failed to pick up L. monocytogenes after several hours of enrichment in broth medium initially spiked with equal numbers of live cells. This was found to be due to unequal growth of these organisms leading to disparity in the amount of template DNAs represented in the DNA preparation applied for conventional multiplex PCR amplification. To circumvent this, we have developed a modified method of enrichment and harvesting leading to highly sensitive and rapid single reaction PCR detection of both pathogens. We have also successfully developed two novel multiplex PCR formats for the generation of uniform PCR signals. Some of these methods might find broader application for the simultaneous detection of different combinations of multiple pathogens.

Bacterial Proteins↗

PubMed2004

Use of a genus- and species-specific multiplex PCR for identification of enterococci.

The 16S rRNA gene has previously been used to develop genus-specific PCR primers for identification of enterococci. In addition, the superoxide dismutase gene (sodA) has been identified as a potential target for species differentiation of enterococci. In this study, Enterococcus genus-specific primers developed by Deasy et al. (E1/E2) were incorporated with species-specific primers based upon the superoxide dismutase (sodA) gene for development of a multiplex PCR. This assay provides simultaneous genus and species identification of 23 species of enterococci using seven different reaction mixtures. Accuracy of identification of the multiplex PCR was determined by comparisons to standard biochemical testing, the BBL Crystal kit, VITEK, and API Rapid ID 32 Strep. Isolates from swine feces, poultry carcasses, environmental sources, and retail food were evaluated and, overall, results for 90% of the isolates tested by PCR agreed with results obtained using standard biochemical testing and VITEK. Eighty-five percent and 82% of PCR results agreed with results from the API Rapid ID 32 Strep and BBL Crystal tests, respectively. With the exception of concurrence between identification using standard biochemical testing and VITEK (85%) and between BBL Crystal and VITEK (83%), the percent agreement for PCR was higher than or equal to any other pairwise comparison. Multiplex PCR for genus and species determination of enterococci provides an improved, rapid method for identification of this group of bacteria.

Animals↗

PubMed2001

Detection of Escherichia coli O157:H7 in soil and water using multiplex PCR.

AIMS: To evaluate the suitability of a multiplex PCR-based assay for sensitive and rapid detection of Escherichia coli O157:H7 in soil and water. METHODS AND RESULTS: Soil and water samples were spiked with E. coli O157:H7 and subjected to two stages of enrichment prior to multiplex PCR. Detection sensitivities were as high as 1 cfu ml(-1) drinking water and 2 cfu g(-1) soil. Starvation of E. coli O157:H7 for 35 d prior to addition to soil did not affect the ability of the assay to detect initial cell numbers as low as 10 cfu g(-1) soil. Use of an 8-h primary enrichment enabled detection of as few as 6 cfu g(-1) soil, and 10(4) cfu g(-1) soil with a 6-h primary enrichment. When soil was inoculated with 10(5) cfu g(-1), the PCR assay indicated persistence of E. coli O157:H7 during a 35 d incubation. However, when soil was inoculated with lower numbers of pathogen, PCR amplification signals indicated survival to be dependent on cell concentration. CONCLUSIONS: A multiplex PCR-based assay, in combination with an enrichment strategy enabled sensitive and rapid detection of E. coli O157:H7 in soil and water. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to sensitively detect E.coli O157:H7 in environmental material within one working day represents a considerable advancement over alternative more time-consuming methods for detection of this pathogen.

DNA, Bacterial↗

PubMed1996

Multiplex PCR assay for the routine detection of Listeria in food.

The development and validation of a multiplex PCR assay for the detection of Listeria that can be employed in routine investigation of food samples are described. The assay, which employs a short culture enrichment step followed by isolation of bacterial cells and detection by multiplex PCR reaction, is highly sensitive and specific for the detection of Listeria monocytogenes and all other Listeria species. Over 350 food samples were tested in parallel by standard cultural procedures and the PCR assay, with no false-positive or false-negative results obtained with the PCR assay. Compared to the standard cultural methods the PCR assay is highly sensitive, cost effective and extremely rapid with results obtained within 48 h from sample receipt.

Food Microbiology↗

PubMed1996

Multiplex PCR using conserved and species-specific 16S rRNA gene primers for simultaneous detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis.

Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis are strongly associated with periodontitis. However, little is known about their distribution in periodontally healthy individuals, because culturing techniques are not sufficiently sensitive. A modified multiplex PCR was developed to address that question. Our method uses two species-specific forward primers in combination with a single reverse primer. These primers target variable and conserved regions of the 16S rRNA gene. Sensitivity was determined by testing serial dilutions of A. actinomycetemcomitans and P. gingivalis cells. Primer specificity was tested against (i) six A. actinomycetemcomitans strains and four P. gingivalis strains, (ii) seven different species of oral bacteria, and (iii) supra- and subgingival plaque from 20 subjects. The multiplex PCR had a lower limit of detection of 2 A. actinomycetemcomitans and 30 P. gingivalis cells. Species-specific amplicons were obtained for all A. actinomycetemcomitans and P. gingivalis strains tested and did not occur with seven other bacterial species unless A. actinomycetemcomitans and P. gingivalis were added. Neither target species was detected in supragingival plaque; A. actinomycetemcomitans was detected in one subgingival specimen, and P. gingivalis was detected in another. When plaque samples were spiked with 10 A. actinomycetemcomitans cells and 100 P. gingivalis cells, species-specific amplicons were detected. These findings show our multiplex PCR to be highly sensitive and specific while allowing simultaneous detection of A. actinomycetemcomitans and P. gingivalis. This assay has potential applications in epidemiological studies, diagnosis, treatment planning, and monitoring of periodontal pathogens.

Aggregatibacter actinomycetemcomitans↗

PubMed2001

Multiplex PCR using internal transcribed spacer 1 and 2 regions for rapid detection and identification of yeast strains.

Multiplex PCR amplification followed by either agarose gel electrophoresis (PCR-AGE) or microchip electrophoresis (PCR-ME) was used to test a total of 120 fungal strains. The internal transcribed spacer 1 (ITS1) and ITS2 regions and the 5.8S ribosomal DNA (rDNA) region of the fungi were amplified by using universal primers ITS1 and ITS4. The ITS2 region was simultaneously amplified by using universal primers ITS3 and ITS4. Since Trichosporon asahi and T. asteroides showed similar lengths for two amplicons, 29 different gel patterns were demonstrated for 30 yeast species tested on the basis of differences in the lengths of one or two amplicons. Of 75 yeast isolates from clinical materials, 5 isolates (6.8%) which were incompletely identified or not identified by the phenotypic method were identified with our PCR-based method (2 isolates as Candida guilliermondii, 2 as C. krusei, and 1 as C. zeylanoides). No differences in discriminating power or sensitivity were observed between the PCR-AGE method and the PCR-ME method. These methods, prospectively applied to 24 yeast-positive blood culture bottles (16 patients), resulted in the correct detection of 24 yeast strains. In conclusion, multiplex PCR followed by electrophoresis seems to be a promising tool for the rapid identification of common and uncommon yeast strains from culture colonies and from yeast-positive blood culture bottles (5.5 h for the PCR-AGE method and 3 h for the PCR-ME method).

DNA, Fungal↗

PubMed2006

Improved and rapid detection of methicillin-resistant Staphylococcus aureus nasal carriage using selective broth and multiplex PCR.

To improve efficiency in detecting nasal methicillin-resistant Staphylococcus aureus (MRSA), we evaluated a multiplex PCR using pre-enrichment of the specimen in selective broths, and compared it with detection performed by routine tests in hospital laboratories. Nasal swab specimens from 311 patients were inoculated onto mannitol-salt agar (MSA) at the hospital laboratories and in two Mueller-Hinton broths with 7% NaCl containing oxacillin at concentrations of 2 and 4 micro g/ml. Isolates on MSA were identified as MRSA by classical laboratory tests (coagulase and oxacillin disk diffusion tests). Oxacillin broth cultures were subcultured on blood agar and MRSA isolates were identified by coagulase and susceptibility tests, including agar dilution and the oxacillin-screening method (gold standard method). Simultaneously, multiplex-PCR was performed from the selective broths to detect S. aureus species-specific and mecA gene segments (OxMPCR method). Thirty-two S. aureus isolates were recovered: 29 (90.6%) were MRSA strains and 3 (9.4%) were oxacillin-susceptible isolates. Twenty-eight (96.5%) MRSA isolates were detected by OxMPCR, while 17 (58.6%) were identified by routine tests (P=0.002). This new method for detection of MRSA nasal carriers showed higher sensitivity and led to faster reporting--i.e., within 24 h--of results.

Bacterial Proteins↗