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PubMed2006

Combined sigB allelic typing and multiplex PCR provide improved discriminatory power and reliability for Listeria monocytogenes molecular serotyping.

Conventional serotyping has traditionally been used to subtype Listeria monocytogenes, but has several limitations, including low discriminatory power and poor reproducibility. Molecular serotyping methods have been developed for L. monocytogenes, but generally show limited discriminatory power and high misclassification rates. We selected 157 Listeria isolates to evaluate a combination of a previously described multiplex PCR assay and sigB allelic typing as an alternative molecular serotyping and subtyping strategy for L. monocytogenes. While the multiplex PCR assay differentiated five L. monocytogenes subtypes (Simpson's Index of Discrimination [SID]=0.78), including classification of the most common disease-associated serotypes (1/2a, 1/2b, 1/2c, and lineage I 4b) into four distinct groups, it misclassified 3.8% of the isolates studied here. sigB allelic typing differentiated 29 subtypes (SID=0.87) and also allowed identification of lineage III L. monocytogenes, which could not be differentiated from the other Listeria spp. by the multiplex PCR assay. sigB allelic typing failed to differentiate serotype 1/2c and 1/2a isolates and one sigB allelic type included serotype 4b and 1/2b isolates. A molecular serotyping approach that combines multiplex PCR and sigB sequence data showed increased discriminatory power (SID=0.91) over either method alone as well as conventional serotyping (SID=0.87) and classifies the four major serotypes (i.e., 1/2a, 1/2b, 1/2c, and 4b) into unique subgroups with a lower misclassification rate as compared to the multiplex PCR assay. This combined approach also differentiates lineage I serotype 4b isolates from the genetically distinct serotype 4b isolates classified into lineage III.

Alleles↗

PubMed2004

Simultaneous detection of Dialister pneumosintes and Filifactor alocis in endodontic infections by 16S rDNA-directed multiplex PCR.

Dialister pneumosintes and Filifactor alocis have been recently considered as candidate endodontic pathogens. In this study, we devised a 16S rDNA-directed multiplex PCR protocol for simultaneous detection of these two bacterial species in endodontic infections. Samples were taken from infected root canals associated with asymptomatic periradicular lesions as well as from cases of acute periradicular abscesses. DNA extracted from the samples was used as template for simultaneous detection of D. pneumosintes and F. alocis through a multiplex PCR assay. Two fragments of the expected sizes, one specific for D. pneumosintes and the other for F. alocis, were simultaneously amplified from a mixture of reference genomic DNA containing DNA from both species. Clinical samples that were positive for the target species showed a single band of the predicted size for each species. D. pneumosintes was detected by multiplex PCR in 11 samples (7 asymptomatic and 4 abscesses) and F. alocis was identified in 9 cases (6 asymptomatic and 3 abscesses). Six samples (3 asymptomatic and 3 abscesses) shared the two species. Data from the present study confirmed that D. pneumosintes and F. alocis are common members of the microbiota present in primary endodontic infections and thereby may participate in the pathogenesis of periradicular lesions. The proposed multiplex PCR assay is a simple, rapid, and accurate method for the simultaneous detection of these two candidate endodontic pathogens.

Adult↗

PubMed2005

Computing TaqMan probes for multiplex PCR detection of E. coli O157 serotypes in water.

Diarrheagenic E. coli strains contribute to water related diseases in urban and rural environment in developing and developed world. E. coli pathotype and pathogenicity varies due to complex multifactorial mechanism involving a large number of virulence factors. Rapid assessment of the virulence pattern of E. coli isolates is possible by Real-Time PCR probes like TaqMan. For designing TaqMan probes and primers for multiplex PCR selected E. coli gene sequences: stx1, stx2, hlyA, chuA, eae, lacZ, lamB and fimA were retrieved from NCBI's GenBank database. The alignment of the multiple sequences and analysis of conserved sequences was carried out using ClustalW and BLAST programs. The primers and Taqmen probes were designed using Beacon Designer software version 2.1 for two multiplexed PCR assays. In silico PCR simulation of these assays showed PCR products for stx2 (248bp) stx1 (102 bp), lacZ (228bp) and lamB (86 bp) in multiplex #1 and eae (200bp), chuA (147 bp), hlyA (141bp) and fimA (79 bp) in multiplex #2, respectively. These multiplexed PCR amplification products and probes can be used to identify and confirm presence of O157:H7/ H7-, O157:H43/45 and O26:H-/H11 serotypes. In conclusion, multiplex Real-Time Polymerase Chain Reaction oligomers and TaqMan probes designed and validated in silico will be helpful in management of water quality and outbreaks, by improving specificity and minimizing time needed for in vitro verification work.

Base Sequence↗

PubMed1999

Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl, tdh and trh.

Vibrio parahaemolyticus is an important human pathogen which can cause gastroenteritis when consumed in raw or partially-cooked seafood. A multiplex PCR amplification-based detection of total and virulent strains of V. parahaemolyticus was developed by targeting thermolabile hemolysin encoded by tl, thermostable direct hemolysin encoded by tdh, and thermostable direct hemolysin-related trh genes. Following optimization using oligonucleotide primers targeting tl, tdh and trh genes, the multiplex PCR was applied to V. parahaemolyticus from 27 clinical, 43 seafood, 15 environmental, 7 strains obtained from various laboratories and 19 from oyster plants. All 111 V. parahaemolyticus isolates showed PCR amplification of the tl gene; however, only 60 isolates showed amplification of tdh, and 43 isolates showed amplification of the trh gene. Also, 18 strains showed amplification of the tdh gene, but these strains did not show amplification of the trh gene. However, one strain exhibited amplification for the trh but not the tdh gene, suggesting both genes need to be targeted in a PCR amplification reaction to detect all hemolysin-producing strains of this pathogen. The multiplex PCR approach was successfully used to detect various strains of V parahaemolyticus in seeded oyster tissue homogenate. Sensitivity of detection for all three target gene segments was at least between 10(1)-10(2) cfu per 10 g of alkaline peptone water enriched seeded oyster tissue homogenate. This high level of sensitivity of detection of this pathogen within 8 h of pre-enrichment is well within the action level (10(4) cfu per 1 g of shell stock) suggested by the National Seafood Sanitation Program guideline. Compared to conventional microbiological culture methods, this multiplex PCR approach is rapid and reliable for accomplishing a comprehensive detection of V. parahaemolyticus in shellfish.

Animals↗

PubMed1998

Use of a multiplex PCR system for the simultaneous detection of heat labile toxin I and heat stable toxin II genes of enterotoxigenic Escherichia coil in skim milk and porcine stool.

Enterotoxigenic Escherichia coli (ETEC) strains which produce heat labile and/or heat stable toxins (LT and ST) may cause diarrhea in humans and farm animals. Using PCR primers specific for the LT I and ST II genes, a multiplex PCR system which allows detection of LT 1-and ST 11-producing ETEC strains was developed. When skim milk was used for a PCR assay, it was found that if target cells in the sample were precultured in MacConkey broth for 8 h prior to PCR as few as 10(0) cells per ml of the sample could be detected. Without the preculture step, 10(4) CFU of target cells per 0.2 g of porcine stool specimen were required to generate visible PCR products. The multiplex PCR system can be used for rapid testing of fecal specimens, food and possibly environmental samples for the presence of ETEC strains.

Animals↗

PubMed2002

[Application of multiplex PCR in genotyping of hepatitis B virus prevailing in Guangdong Province of China].

OBJECTIVE: To establish a convenient method for the genotyping of hepatitis B virus (HBV) using multiplex PCR. METHOD: Based on the alignment of 114 complete nucleotide sequences of HBV DNA belonging to different genotypes, acquired from the GenBank, genotype-specific sequences were identified according to which 6 pairs of primers were designed corresponding to each genotype. Subsequent genotyping of HBV was performed using these primers that were added, either alone or in conjunction with others, into a multiplex PCR reaction tube, and HBV genotype was determined according to the length of amplified DNA. RESULT: The genotyping result of multiplex PCR was consistent with that produced by PCR- restriction fragment length polymorphism as established by Lindh. We found in this study that among the HBV carriers in the vicinities Guangzhou of City, about 45% belonged to B genotype, 38.75% to C genotype and 16.75% to D genotype. CONCLUSION: This multiplex PCR method is simple, convenient and more differential.

China↗

PubMed2005

New multiplex PCR for rapid detection of isoniazid-resistant Mycobacterium tuberculosis clinical isolates.

In this study, we describe a multiplex PCR to detect a AGC-->ACC (serine to threonine) mutation in the katG gene and a -15 C-to-T substitution (inhA(C-15T)) at the 5' end of a presumed ribosome binding site in the promoter of the mabA-inhA operon. These mutations have been reported in the majority of previous studies as the most frequent mutations involved in the resistance to isoniazid (INH) of Mycobacterium tuberculosis clinical strains with high levels of resistance. The method was optimized and validated after an analysis of 30 M. tuberculosis clinical isolates with known sequences of the relevant part of the katG gene and the regulatory region of the mabA-inhA operon. We analyzed 297 INH-resistant M. tuberculosis isolates collected in Spain from 1996 to 2003 by PCR-restriction fragment length polymorphism (using the katG gene), DNA sequencing, and the newly developed multiplex PCR. The results were concordant for all 297 isolates tested. The analysis revealed that 204 (68.7%) of the isolates carried one or both of the mutations. This finding suggests that with further development this multiplex PCR will be able to detect the majority of the INH-resistant M. tuberculosis clinical isolates from Spain and other countries where a high frequency of similar mutations occur.

Antitubercular Agents↗

PubMed1998

Application of multiplex PCR using species-specific primers within the 16S rRNA gene for rapid identification of Nocardioides strains.

For the rapid identification of Nocardioides strains, multiplex PCR, using 16S rDNA as target gene, was used and its value was evaluated. Forward primers specific for Nocardioides albus, Nocardioides jensenii, Nocardioides plantarum and Nocardiodes simplex, among the five validly described Nocardioides species, were designed from the alignment of 165S rDNA sequences. Nocardioides luteus has been shown to be a member of the same species as N. albus by recent molecular systematic studies and preliminary DNA-DNA relatedness tests. Therefore, N. albus and N. luteus were considered as members of the same species in this study. Each primer was found to be species-specific by specificity testing. N. albus NSP01T, N. jensenii NSP19T, N. plantarum NSP21T and N. simplex NSP22T could be clearly differentiated by PCR products characteristics for each species in the multiplex PCR assays. N. luteus gave an identical results to N. albus NSP01. The additional 17 strains of N. albus and the additional four stains of N. simplex gave PCR products identical to those of N. albus NSP01T and N. simplex NSP22T, respectively. Multiplex PCR was found to be rapid, species-specific and reproducible. The technique evaluated in this study proved to be effective for rapidly identifying Nocardioides strains to species level.

Actinomycetales↗

PubMed2002

Detection of Thelohania solenopsae (Microsporidia: Thelohaniidae) in Solenopsis invicta (Hymenoptera: Formicidae) by multiplex PCR.

Oligonucleotide primer pairs were designed to unique areas of the small subunit (16S) rRNA gene of Thelohania solenopsae and a region of the Gp-9 gene of Solenopsis invicta. Multiplex PCR resulted in sensitive and specific detection of T. solenopsae infection of S. invicta. The T. solenopsae-specific primer pair only amplified DNA from T. solenopsae and T. solenopsae-infected S. invicta. This primer pair did not produce any amplification products from DNA preparations from uninfected S. invicta, seven additional species of microsporidia (including Vairimorpha invictae), or Mattesia spp. The Gp-9-specific primers recognized and amplified DNA from Solenopsis xyloni, Solenopsis richteri, Solenopsis geminata, the invicta/richteri hybrid, and monogyne and polygyne S. invicta, but not from T. solenopsae, and, as such, served as a positive control verifying successful DNA preparation. Multiplex PCR detected T. solenopsae in worker fire ants infected with as few as 5000 spores. Furthermore, multiplex PCR detected T. solenopsae in all developmental stages of S. invicta. However, detection could be made more sensitive by using only the T. solenopsae-specific primer pair; ants infected with as few as 10 spores were able to be discerned. Multiplex PCR detection of T. solenopsae offers the advantages of a positive control, a single PCR amplification, detection of all developmental stages, and increased sensitivity and specificity compared with microscopy.

Animals↗

PubMed2003

Detection of pathogenic bacteria in shellfish using multiplex PCR followed by CovaLink NH microwell plate sandwich hybridization.

Outbreak of diseases associated with consumption of raw shellfish especially oysters is a major concern to the seafood industry and public health agencies. A multiplex PCR amplification of targeted gene segments followed by DNA-DNA sandwich hybridization was optimized to detect the etiologic agents. First, a multiplex PCR amplification of hns, spvB, vvh, ctx and tl was developed enabling simultaneous detection of total Salmonella enterica serotype Typhimurium, Vibrio vulnificus, Vibrio cholerae and Vibrio parahaemolyticus from both pure cultures and seeded oysters. Amplicons were then subjected to a colorimetric CovaLink NH microwell plate sandwich hybridization using phosphorylated and biotinlylated oligonucleotide probes, the nucleotide sequences of which were located internal to the amplified DNA. The results from the hybridization with the multiplexed PCR amplified DNA exhibited a high signal/noise ratio ranging between 14.1 and 43.2 measured at 405 nm wavelength. The sensitivity of detection for each pathogen was 10(2) cells/g of oyster tissue homogenate. The results from this study showed that the combination of the multiplex PCR with a colorimetric microwell plate sandwich hybridization assay permits a specific, sensitive, and reproducible system for the detection of the microbial pathogens in shellfish, thereby improving the microbiological safety of shellfish to consumers.

Animals↗

PubMed2005

[Utility of multiplex PCR for diagnosis of neurological disorders in HIV-infected patients and patients with AIDS].

OBJECTIVE: Evaluation of multiplex PCR utility for diagnosis of central nervous system disorders in HIV-infected patients. METHODS: Multiplex PCR assay for simultaneous detection of HSV-1, HSV-2, VZV, HHV-6, CMV, EBV, JCV, T. gondii DNA in 27 CSF samples from HIV-infected patients was used. PCR results were compared with clinical diagnosis and histopathological examination of brain obtained at autopsy. MAIN OBSERVATIONS: Five of seven positive results were corresponding with clinical diagnosis. In two cases also with autopsy. RESULTS: DNA of one or more pathogens was found in 6 out of 27 (22%) samples: CMV DNA in four patients, JCV DNA and T. gondii DNA in one patients each. In one case co-infections of CMV and EBV was found. CONCLUSIONS: Multiplex PCR is a useful diagnostic method. LIPA-CNS test has high specificity. Lack of nesting step of the reaction and PCR positive control verification could cause a relatively low sensitivity of the test.

AIDS-Related Opportunistic Infections↗

PubMed2005

Comparison of three in-house multiplex PCR assays for the detection of Neisseria gonorrhoeae and Chlamydia trachomatis using real-time and conventional detection methodologies.

AIMS: To develop and evaluate multiplex PCR assays for Chlamydia trachomatis and Neisseria gonorrhoeae, using real-time and conventional PCR detection methodologies. METHODS: Two real-time multiplex PCR assays, using nuclease (TaqMan-ABI7500) and hybridisation (LightCycler) probe formats, and a third assay using conventional PCR with solid-phase hybridisation and colour detection, were developed. The porA pseudogene was targeted for N. gonorrhoeae, and the major outer membrane protein gene for C. trachomatis. A total of 145 urogenital specimens were tested in all assays, and the results were compared with the Roche Cobas Amplicor assay. RESULTS: There was little difference in clinical sensitivity and specificity, result discrimination and test cost for the three in-house assays. Our results showed that competitive inhibition of the PCR occurred in some samples that were positive for both organisms. CONCLUSIONS: These results highlight the suitability and versatility of three multiplex PCR methods for the detection of C. trachomatis and N. gonorrhoeae.

Adolescent↗

PubMed2004

Multiple-locus variable-number tandem-repeats analysis of Salmonella enterica subsp. enterica serovar Typhimurium using PCR multiplexing and multicolor capillary electrophoresis.

The multiple-locus variable-number tandem-repeats analysis (MLVA) method is currently being used as the primary typing tool for Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) isolates in our laboratory. Our published initial MLVA was performed using a single fluorescent dye and the different patterns were assigned from gel images. Here we present a new and significantly improved assay using multiple dye colors, enhanced PCR multiplexing and the introduction of two new loci for better adaptation to capillary electrophoresis with increased speed. The different MLVA patterns are now based on allele sizes entered as character values, thus removing the uncertainties introduced when analyzing band patterns from gel images. We additionally propose an easy numbering scheme for the identification of separate isolates that will facilitate exchange of typing data. A total of 106 human, bird, animal and food isolates of S. Typhimurium, including 16 with definite type (DT) 104, were used for the development of the improved MLVA. The method is based on capillary separation of multiplexed PCR products from five VNTR loci in the S. Typhimurium genome labeled with multiple fluorescent dyes. The different alleles at each locus were then assigned allele numbers, which were used for strain comparison.

Alleles↗

PubMed2004

Multiple-Locus Variable-Number Tandem-Repeats Analysis of Escherichia coli O157 using PCR multiplexing and multi-colored capillary electrophoresis.

The Multiple-Locus Variable-Number Tandem-Repeats Analysis (MLVA) method is currently being used as the primary typing tool for Shiga-toxin-producing Escherichia coli (STEC) O157 isolates in our laboratory. The initial assay was performed using a single fluorescent dye and the different patterns were assigned using a gel image. Here, we present a significantly improved assay using multiple dye colors and enhanced PCR multiplexing to increase speed, and ease the interpretation of the results. The different MLVA patterns are now based on allele sizes entered as character values, thus removing the uncertainties introduced when analyzing band patterns from the gel image. We additionally propose an easy numbering scheme for the identification of separate isolates that will facilitate exchange of typing data. Seventy-two human and animal strains of Shiga-toxin-producing E. coli O157 were used for the development of the improved MLVA assay. The method is based on capillary separation of multiplexed PCR products of VNTR loci in the E. coli O157 genome labeled with multiple fluorescent dyes. The different alleles at each locus were then assigned to allele numbers, which were used for strain comparison.

Animals↗

PubMed1999

Multiplex PCR--a rapid screening method for detection of gene rearrangements in childhood acute lymphoblastic leukemia.

Chromosomal rearrangements in childhood acute lymphoblastic leukemia (ALL) play an important role in the identification of clinical relevant subgroups. For rapid and easy detection of the clinically most important gene rearrangements, a nested multiplex reverse transcriptase polymerase chain reaction (multiplex PCR) was developed. This multiplex PCR enables the detection of M-BCR/ABL, m-BCR/ABL, TEL/ AML1, and MLL/AF4 fusion transcripts in one PCR reaction. However, the existence of splicing variants and different breakpoints on the DNA level hampers the discrimination of the rearrangements by their fragment size on an agarose gel. Therefore, one of the internal primers of each translocation (ABL-2, TEL-2, AF4-2) was labeled with a characteristic fluorescent dye, and an automatic fluorescence-based DNA fragment analysis was performed. The sensitivity of this multiplex PCR is in the same range as that of the corresponding single PCR reaction and allows a fast screening for the detection of therapy-relevant rearrangements, with a high turnover of samples.

Base Sequence↗

PubMed2005

Multiplex PCR for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in meat samples.

A multiplex PCR method was developed for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in meat samples. DNA detection sensitivity for this method was 10(3) CFU/ml for each pathogen. When this protocol was used for the detection of each of the above pathogenic bacteria in spiked pork samples, 1 cell per 25 g of inoculated sample could be detected within 30 h. In the samples of naturally contaminated meat, Salmonella spp., L. monocytogenes, and E. coli O157:H7 were detected over the same time period. Excellent agreement was obtained for the results of multiplex PCR and the conventional culture method, which suggests that the multiplex PCR is a reliable and useful method for rapid screening of meat products for Salmonella spp., L. monocytogenes, and E. coli O157:H7 contamination.

Animals↗

PubMed2000

Direct genomic multiplex PCR for BRCA1 and application to mutation detection by single-strand conformation and heteroduplex analysis.

Most mutation detection methods are based on analysis of PCR amplified segments and the application of multiplex PCR is one central approach to improving screening efficiency. Genes like the breast-ovarian cancer susceptibility gene BRCA1 pose a difficult challenge to efficient mutation screening because of large coding regions, numerous exons, and complex mutational spectra. The application to BRCA1 of a general approach to effective multiplex PCR is described here. Fifteen triplex PCRs and a single PCR reaction condition were used for amplification of all BRCA1 coding regions and the BRCA1-specific segments from the duplicated promoter region. SSCP/HDX gel analysis of the multiplex products detected mobility distinctions for 34/34 sets of allelic BRCA1 fragments. A novel polymorphism was found, CTTCT(4)CT(10)CT(12) >CT(4)CT(11), a compound deletion in a region beginning at the +33 position of IVS7 and resulting in a net deletion of 15 bp. This change was shown to be one of the common polymorphisms that define the two major haplotypes of the BRCA1-RNU2 region in a large proportion of the world population. A triplex PCR for SSCP detection of this deletion and two other distantly located common polymorphisms may be used to screen haplotype content and facilitate comparison of samples with similar haplotypes in subsequent mutation screening. The approach for robust multiplex amplification is generally applicable and allows rapid development of efficient testing for a wide variety of mutations in any gene(s) encompassing a large coding region or numerous exons and including as many as 50 different genomic PCR products.

Breast Neoplasms↗

PubMed2005

Distribution of virulence related genes among enteroaggregative Escherichia coli isolates: using multiplex PCR and hybridization.

The importance of enteroaggregative Escherichia coli (EAEC) strains in public health around the world is becoming increasingly clear. EAEC diagnosis has long been problematic. In this study, the recently designed multiplex PCR based on three plasmid-borne genes (AA probe, aap, and aggR) and DNA hybridization assay with plasmid-derived DNA probes were used for detection of HEp-2 adherent strains. These were isolated from an epidemiologic study of diarrhea in Iran. Using AA and DA probes revealed that 32.4%, 16.2%, 23%, and 28.4% of these isolates were AA, DA, AA/DA, and non-AA/DA, respectively. However, employing multiplex PCR for detection of these isolates, showed that 51.3% of the strains were AA and the rest 48.7% were not AA. While presence of other genes (pet, shf, aggA, aafA) considered to be specific for EAEC were checked among these isolates. The data obtained revealed that except for AA, aap, and aggR, the rest of the virulence related genes are not specific for EAEC isolates and are randomly distributed among adherent isolates. Over all the results obtained here indicated that this multiplex PCR is specific and sensitive assay. Phenotypically adherent strains are divided into two main groups, by use of this multiplex PCR i.e., typical EAEC isolates that carry the three plasmid-borne genes all together and atypical EAEC isolates in which the three genes are not linked together.

Cell Line↗