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PubMed2004

Application of a multiplex PCR for the detection of protozoan pathogens of the eastern oyster Crassostrea virginica in field samples.

Populations of eastern oysters Crassostrea virginica along the east coast of North America have repeatedly experienced epizootic mass mortality due to infections by protozoan parasites, and molecular diagnostic methodologies are fast becoming more widely available for the diagnosis of protozoan diseases of oysters. In this study we applied a modified version of an existing multiplex polymerase chain reaction (PCR) for detection of the eastern oyster parasites Haplosporidium nelsoni, H. costale and Perkinsus marinus from field-collected samples. We incorporated primers for DNA quality control based on the large subunit ribosomal RNA (LSU rRNA) gene of C. virginica. The multiplex PCR (MPCR) simultaneously amplified genomic DNA of C. virginica, and cloned DNA of H. nelsoni, P. marinus and H. costale. In field trial applications, we compared the performance of the MPCR to that of the conventional diagnostic techniques of histopathological tissue examination and the Ray/Mackin fluid thioglycollate medium (RMFT) assay. A total of 530 oysters were sampled from 18 sites at 12 locations along the east coast of the United States from the Gulf of Mexico to southern New England. The modified MPCR detected 21% oysters with H. nelsoni, 2% oysters with H. costale, and 40% oysters with P. marinus infections. In comparison, histopathological examination detected H. nelsoni and H. costale infections in 6 and 0.8% oysters, respectively, and the RMFT assay detected P. marinus infection in 31% oysters. The MPCR is a more sensitive diagnostic assay for detection of H. nelsoni, H. costale, and P. marinus, and incorporation of an oyster quality control product limits false negative results.

Animals↗

PubMed1999

Evaluation of a multiplex PCR assay for simultaneous identification of Salmonella sp., Salmonella enteritidis and Salmonella typhimurium from environmental swabs of poultry houses.

A Multiplex PCR-based assay (m-PCR) with three sets of primers was developed for the detection of all serotypes of Salmonella enterica and the identification of Salmonella Enteritidis and Salmonella Typhimurium. This method was evaluated against a bacteriological method for the analysis of environmental swabs of poultry houses. Samples were preenriched in phosphate-buffered peptone water for 24 h and subjected to three different protocols prior to PCR: (i) an immunomagnetic separation using Dynabeads anti-Salmonella (Dynal); (ii) a DNA extraction procedure using the Instagene matrix; (iii) an additional step of culture on an MSRV medium. With protocols 1 and 2, eight positive results were found by PCR and 20 with the bacteriological method. Protocol 3 combining MSRV and PCR gave similar results to those obtained from bacteriological methods and allowed Salmonella detection within 2 days.

Animals↗

PubMed2002

Identification of Escherichia coli O157:H7 by multiplex PCR with primers specific to the hlyA, eaeA, stx1, stx2, fliC and rfb genes.

Escherichia coli O157:H7 can cause fatal diseases such as hemolytic-uremic syndrome and thrombotic thrombocytopenic purpura. The early symptoms of E. coli O157:H7 infection are similar to those of infection with other gastrointestinal bacteria, so the availability of a rapid and precise method to identify E. coli O157:H7 is important for early recognition. We report a multiplex polymerase chain reaction (PCR) with primers to detect the presence of the E. coli O157:H7 genes hlyA, which produces enterohemolysin, eaeA (intimin), stx1 (Shiga-like toxin I), stx2 (Shiga-like toxin II), fliC (flagella H-antigen), and rfb (surface O-antigen). The technique improves specificity compared to general PCR when used with > or = 10(3) CFU/assay. Specificity was examined using 32 E. coli O157:H7 strains from the USA, Japan, Canada and Taiwan, all of which were positive for hlyA, eaeA, fliC, rfb, and stx1 and/or stx2. Thirty-five non-O157 enterovirulent E. coli strains, two Shigella sp strains, and two Salmonella sp strains were also examined and none gave the positive gene expression results described above. This multiplex PCR was highly specific in identifying E. coli O157 and was more useful than general PCR in early clinical detection of this pathogen.

Bacterial Proteins↗

PubMed1995

Multiplex PCR analysis and genotype-phenotype correlations of frequent APC mutations.

Germline mutations of the adenomatous polyposis coli (APC) gene tend to cluster in discrete regions. Some of these mutations occur frequently in familial adenomatous polyposis coli (FAP) patients, and strategies for genetic diagnosis of the disease should include simple methods for their detection. We studied a total of 48 FAP-affected or "at-risk" members from 31 unrelated FAP pedigrees. Unrelated patients were analyzed using heteroduplex analysis on agarose minigels (HAAM) and multiplex allele-specific PCR. This novel strategy readily and reliably detected the three frequently occurring APC deletions at codons 1061, 1068, and 1309, allowing identification of mutant alleles in nine unrelated patients. A targeted mutational analysis, based on HAAM and amplification refractory mutation system (ARMS), allowed the rapid identification of 11 additional subjects with germline deletions, among relatives of the patients in whom mutations had been detected by multiplex PCR and HAAM. The use of two independent PCR-based tests, employing distinct sets of primers, reduces the possibility that artifacts occurring during DNA amplification may interfere with the diagnostic evaluation. The analysis of genotype-phenotype correlations provided evidence for heterogeneity with regard to the extent of colonic and extracolonic manifestations of the disease in subjects bearing identical mutations. However, the consistent association of the deletion at codon 1309 with more severe colonic disease than that observed in patients with mutations at codons 1061 and 1068, supports a correlation between mutation site and penetrance of FAP.

Adenomatous Polyposis Coli↗

PubMed2001

Detecting and genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays.

Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification. The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFU ml(-1) (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass-based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products; the system is amenable to automation.

Animals↗

PubMed2001

Rapid detection by fluorescent multiplex PCR of exon deletions and duplications in the C1 inhibitor gene of hereditary angioedema patients.

Hereditary angioedema (HAE) is due to a variety of defects in the C1 inhibitor gene (C1NH gene), including approximately 20% of partial deletions/duplications whose boundaries are usually within Alu repeats. To ensure complete molecular characterization of C1 inhibitor deficiencies a fluorescent multiplex assay was constructed to amplify simultaneously five exons of C1NH and an exon of the BRCA1 gene. PCR protocols were optimized for these amplicons (size range between 300 and 700 bp). Forward and reverse chimeric primers that carry strand-specific 5' tags of 16 nucleotides were used to ensure similar levels of PCR products for each amplicon in the multiplex. Data were analyzed by superposing fluorescent profiles of test and control DNA and by visually comparing the normalized peak levels of corresponding amplicons, rather than by calculating the ratios of peak areas. Tests on a collection of known defects, including five different Alu-mediated deletions and a partial duplication have validated this approach. In a study of 19 sporadic cases of HAE, of which four had failed to reveal mutations upon screening all exons by fluorescent chemical cleavage, three de novo deletions were diagnosed by using this multiplex PCR approach: a deletion of exon 4, a deletion of exons 5 and 6, and an apparently complete gene deletion. Besides being suitable for the initial DNA screening of the C1NH gene in HAE patients prior to screening for point mutations, this method can be easily adapted to complex genes for the screening of rearrangements.

Angioedema↗

PubMed2005

Multiplex PCR for detection of three exfoliative toxin serotype genes in Staphylococcus aureus.

Rapid and specific detection of exfoliative toxin (ET)-producing Staphylococcus aureus strains by multiplex polymerase chain reaction (PCR) was used for identification of exfoliative toxin genes in a diverse set of 115 clinical S. aureus strains isolated in 14 Czech cities between 1998 and 2004. Fifty-nine wild-type ET-positive isolates of which 40 strains were the causative agents of toxic epidermolysis in neonates were classified into 4 PCR types. The genes coding for ETA, ETB or ETD were not detected in any of non-ET-producing isolates. The PCR method using the multiplex and specific primer set was shown to be reliable in rapid identification of the exfoliative toxin producing S. aureus and can be used as a convenient tool for hospital epidermolytic infection control.

Adult↗

PubMed2003

Real-time nested multiplex PCR for the detection of herpes simplex virus types 1 and 2 and varicella zoster virus.

One hundred forty-nine specimens were tested in a LightCycler nested multiplex polymerase chain reaction (LCnmPCR) for Herpes simplex virus (HSV)1, HSV2, and VZV. Eighty-one were from genitourinary medicine (GUM) patients and the other 68 specimens were from other patients with skin lesions. The results were compared to a conventional multiplex nested PCR (nmPCR) using agarose gel electrophoresis. Twenty-five specimens were positive in both assays for HSV1 and 29 were positive for VZV. For HSV2 there were 27 positive in the LCnmPCR and 26 positive in the nmPCR assay. The melting temperatures (Tms) of each target were different with a mean of 84.75 degrees C for HSV1, 88.57 degrees C for HSV2, and 83.62 degrees C for VZV. The melting curves of positive specimens directly overlaid the melting curves of the positive controls in the assay. The LCnmPCR assay is a convenient alternative to conventional PCR using agarose gel electrophoresis. It improves specimen turnaround time by eliminating the need for gel electrophoresis, transillumination, and gel photography. It also shows increased sensitivity for HSV2 over our standard assay. This LCnmPCR reduces further the possibility of amplicon contamination with nested PCR protocols.

Base Sequence↗

PubMed2003

Rapid detection of vanA and vanB genes directly from clinical specimens and enrichment broths by real-time multiplex PCR assay.

A real-time PCR assay previously developed for use on the Roche LightCycler platform was investigated as an alternative to culture for the direct detection of vancomycin-resistant enterococci (VRE) in clinical specimens. PCR primers and fluorescence resonance energy transfer hybridization probes specific for the vanA and vanB genes were combined in a multiplex real-time PCR assay performed directly with fecal material obtained by rectal swabbing and with enrichment broth samples. DNA was prepared from the rectal swabs and enrichment broths with a commercially available DNA preparation column designed specifically for use with fecal specimens. One hundred eighty duplicate rectal swabs were obtained from 42 patients who were previously found to be positive for VRE and who were being monitored for carriage of VRE. Direct and enrichment broth cultures were performed with one swab, while PCR was performed with the other swab as well as any corresponding presumptive positive enrichment broth. In total, 100 specimens from 30 patients remained positive for VRE by at least one method. The multiplex real-time PCR was positive for 88 enrichment broths of rectal swabs from 27 patients but for only 45 rectal swabs from 15 patients. Direct culture was positive for VRE for only 43 specimens from 11 patients, while enrichment broth culture was positive for VRE for 75 specimens from 22 patients. Inhibition studies for the multiplex real-time PCR assay, performed by spiking the DNA extracts from 50 negative rectal swabs and the corresponding enrichment broths with between 1 and 10 CFU of a VanB Enterococcus faecium strain, detected inhibition rates of 55.1 and 10%, respectively. PCR performed directly with enrichment broths was found to be significantly more sensitive than enrichment broth culture (P < 0.025). Negative samples were identified significantly earlier by PCR than by culture alone.

Anti-Bacterial Agents↗

PubMed2003

[Determination of ureaplasma DNA by the competitive and multiplex PCR].

Quantities of Ureaplasma spp DNA were determined by the methods of competitive and multiplex polymerase chain reactions (PCR). The results obtained by each of the methods were shown to be compatible. It was established that the number of Ureaplasma spp. cells per one epithelial cell in the female cervical canal varied from 0.05 to 10.

DNA Primers↗

PubMed2001

A multiplex-PCR for the diagnosis of contagious agalactia of sheep and goats.

A multiplex polymerase chain reaction (m-PCR) assay was optimized for the simultaneous detection of several species of small ruminant mycoplasmas. Two sets of oligonucleotide primers specific for Mycoplasma agalactiae (Ma) and Mycoplasma "mycoides" cluster (M.m. cluster) were used in the test. The m-PCR was able to amplify a 375-bp fragment of Ma chromosomal DNA and a 257-260-bp fragment of M.m. cluster chromosomal DNA. Four reference strains (M. agalactiae, M. mycoides subsp. mycoides LC, M. capricolum subsp. capricolum, M. mycoides subsp. capri) and 56 samples (44 milk samples, two nasal swabs, six ocular swabs, three vaginal swabs and one sample of fibrinous exudate from carpal joint), from sheep and goats with clinical signs of contagious agalactia (CA), were examined. The m-PCR confirmed the identification of reference strains and allowed to identify, of the 43 positive samples examined, 35 Ma strains, 12 M. "mycoides" cluster strains; in four samples both Ma and mycoplasmas of M. "mycoides" cluster were revealed. The m-PCR was able to detect 1 pg of mycoplasma DNA. The specificity and sensitivity of the m-PCR suggest its use for the "routine" diagnosis of CA.

Animals↗

PubMed2000

Multiplex PCR amplification of TH01, D9S304, and D3S1744 loci.

A multiplex typing method of the tetrameric short tandem repeat (STR) loci TH11, D9S304, and D3S1744 was developed. The allelic ladder included alleles 6-11 (80-100bp) and 9.3 (95bp) for TH01, alleles 6-15 (125-161 bp) for D9S304, and alleles 13-22 (174-210bp) for D3S1744. The observed heterozygosity of D9S304 was 0.851. The combined discrimination power of the three loci was 0.991.

Base Sequence↗

PubMed2006

Simultaneous detection of nine antibiotic resistance-related genes in Streptococcus agalactiae using multiplex PCR and reverse line blot hybridization assay.

Streptococcus agalactiae (group B streptococcus [GBS]) is the leading cause of neonatal and maternal sepsis. Penicillin is recommended for intrapartum prophylaxis, but erythromycin or clindamycin is used for penicillin-allergic carriers. Antibiotic resistance (AR) has increased recently and needs to be monitored. We have developed a multiplex PCR-based reverse line blot (mPCR/RLB) hybridization assay to detect, simultaneously, seven genes encoding AR--erm(A/TR), erm(B), mef(A/E), tet(M), tet(O), aphA-3, and aad-6--and two AR-related genes, int-Tn and mreA. We tested 512 GBS isolates from Asia and Australasia and compared mPCR/RLB with antibiotic susceptibility phenotype or single-gene PCR. Phenotypic resistance to tetracycline was identified in 450 (88%) isolates, of which 442 had tet(M) (93%) and/or tet(O) (6%). Of 67 (13%) erythromycin-resistant isolates, 18 were susceptible to clindamycin, i.e., had the M phenotype, encoded by mef(A/E); 39 had constitutive (cMLS(B)) and 10 inducible clindamycin resistance, and of these, 34 contained erm(B) and 12 erm(A/TR). Of four additional isolates with mef(A/E), three contained erm(B) with cMLS(B) and one was erythromycin susceptible. Of 61 (12%) clindamycin-resistant isolates, 20 were susceptible to erythromycin and two had intermediate resistance. Based on sequencing, 21 of 22 isolates with mef had mef(E), and 8 of 353 with int-Tn had an atypical sequence. Several AR genes, erm(B), tet(O), aphA-3, aad-6, and mef(A/E), were significantly more common among Asian than Australasian isolates, and there were significant differences in distribution of AR genes between GBS serotypes. Our mPCR/RLB assay is simple, rapid, and suitable for surveillance of antibiotic resistance in GBS.

Anti-Bacterial Agents↗

PubMed1998

Simultaneous detection and identification of human parainfluenza viruses 1, 2, and 3 from clinical samples by multiplex PCR.

Reverse transcription (RT)-PCR assays have been widely described for use in the diagnosis of human parainfluenza viruses (HPIVs) and other respiratory virus pathogens. However, these assays are mostly monospecific, requiring separate amplifications for each HPIV type. In the present work, we describe multiplex RT-PCR assays that detect and differentiate HPIV serotypes 1, 2, and 3 in a combined reaction. Specifically, a mixture of three pairs of primers to conserved regions of the hemagglutinin-neuraminidase gene of each HPIV serotype was used for primary amplification, yielding amplicons with similar sizes. For typing, a second amplification was performed with a mixture of nested primers, yielding amplicons with sizes easily differentiated by agarose gel electrophoresis. A modified single-amplification RT-PCR assay with fluorescence-labeled nested primers, followed by analysis of the labeled products on an automated sequencing gel, was also evaluated. Fifteen temporally and geographically diverse HPIV isolates from the Centers for Disease Control and Prevention archives and 26 of 30 (87%) previously positive nasopharyngeal specimens (8 of 10 positive for HPIV serotype 1 [HPIV1], 9 of 10 positive for HPIV2, and 9 of 10 positive for HPIV3) were positive and were correctly typed by both assays. Negative results were obtained with naso- or oropharyngeal specimens and/or culture isolates of 33 unrelated respiratory tract pathogens, including HPIV4, enterovirus, rhinovirus, respiratory syncytial virus, adenovirus, influenza virus, and Streptococcus pneumoniae. Our multiplex RT-PCR assays provide sensitive, specific, and simplified tools for the rapid diagnosis of HPIV infections.

DNA Primers↗

PubMed1992

Sex determination and milk protein genotyping of preimplantation stage bovine embryos using multiplex PCR.

A method for determining the sex and milk protein genotypes (RFLPs) of preimplantation stage bovine embryos using multiplex polymerase chain reaction (PCR) is described. Day 6 to 7 embryos were micromanipulated to isolate 5 to 6 cells. These cells were then dried in reaction tubes for transport to the laboratory. Subsequently, two sets of PCRs were performed using Y chromosome, k-casein and beta-lactoglobulin gene specific primers, followed by electrophoretic analysis of the PCR products. The presence or absence of the Y chromosome was ascertained in 90 of 92 embryos. Moreover, the k-casein specific fragment was amplified and detected in all these embryos. The PCR products were digested in order to genotype the k-casein gene. In 70% of the embryos, the beta-lactoglobulin specific fragment was amplified, although together with some unspecific fragments.

Journal Article↗

PubMed1999

The identification of Cryptosporidium species and Cryptosporidium parvum directly from whole faeces by analysis of a multiplex PCR of the 18S rRNA gene and by PCR/RFLP of the Cryptosporidium outer wall protein (COWP) gene.

A multiplex polymerase chain reaction (PCR) procedure to amplify 18S rRNA gene fragments has been developed. Amplified DNA fragments of the expected size were obtained which were specific for Cryptosporidium parvum and Cryptosporidium wrairi (422 bp), Cryptosporidium baileyi (11106 bp) and Cryptosporidium muris (1346 bp). Criptosporidium parvum and C. wrairi can be distinguished using a PCR/restriction fragment length polymorphism (RFLP) analysis of the Cryptosporidium outer wall protein (COWP) gene, and these two techniques were applied to DNA extracted from whole faeces using a simple and rapid procedure. Cryptosporidium parvum DNA was detected in the faeces of 72 humans and 24 calves where cryptosporidial oocysts were demonstrated using conventional light microscopy. The specific DNA fragments were not amplified using extracts of material containing other lower eukaryotic parasites.

Animals↗

PubMed1994

Multiplex PCR analysis of in vivo-arising deletion mutations in the hprt gene of human T-lymphocytes.

A multiplex polymerase chain reaction (PCR) procedure was adapted for the rapid and efficient evaluation of deletions of the hypoxanthine guanine phosphoribosyltransferase (hprt) gene in human T-lymphocytes. The hprt clonal assay was used to isolate in vivo-arising hprt-deficient T-cells from six healthy males. Mutant frequencies ranged from 9-27 x 10(-6). Simple crude cellular extracts from 223 mutants were analyzed for hprt gene deletion. Sixteen (7.2%) were found to be due to total gene deletion and 22 (9.9%) were due to partial gene deletion. The relatively high frequency of total gene deletions was caused by replicate isolates of a single mutational event as shown by single-strand conformation polymorphism (SSCP) analysis of rearranged T-cell receptor (TCR)-gamma genes. Eighteen of the 22 partial hprt gene deletion mutants were determined to be of independent origin based on a unique hprt mutation or SSCP-TCR -gamma pattern. One-half (9/18) of the partial deletion mutants involved all or part of exon 4 alone, suggesting that this region of the hprt gene is prone to deletion. The small deletions effecting exon 1 (1 mutant), exon 2 (2 mutants), and exon 4 (6 mutants) would not have been detected by conventional Southern blot analysis and may represent a new, previously unrecognized class of mutations. The ready isolation of such intragenic deletions will allow the characterization of breakpoint junctions and may provide insights into the important processes of DNA breakage and rejoining.

Adult↗

PubMed1998

Multiplex PCR assay for detection of Clostridium perfringens in feces and intestinal contents of pigs and in swine feed.

A multiplex polymerase chain reaction (PCR) assay, developed to detect the alpha-toxin and enterotoxin genes (cpa and cpe, respectively) of Clostridium perfringens, was used to identify enterotoxigenic isolates of this organism from feces and intestinal contents of pigs and from feed samples from pig farms in Iowa. The organism was grown on tryptose-sulfite-cycloserine (TSC) agar, TSC agar without egg-yolk, sheep blood agar, or in brain heart infusion broth or cooked meat medium. DNA was extracted by boiling and the PCR assay was carried out using reagents from a commercial kit. The 319 bp amplification product of cpa and the 364 bp product of cpe were visualized under UV light after electrophoresis in a 2% agarose gel containing ethidium bromide. The average sensitivity of the assay, determined on artificially contaminated feces, was 9.2 x 10(4) colony forming units per gram. Assay of 97 isolates from feces and intestinal contents revealed cpa in 89, but all were negative for cpe. While 28% of the 442 total samples cultured yielded C. perfringens, only 5% of 298 fecal or intestinal contents samples were positive upon direct examination by the PCR assay. Ninety-one and eight-tenths % of isolates with the phenotype of C. perfringens were cpa positive by PCR. Forty-three percent of feed samples were culture positive, while 48.3% were PCR positive for cpa. None of these were cpe positive. We conclude that PCR is a useful assay for rapid detection of C. perfringens in feed, and for confirmation of the identity of isolates presumed to be C. perfringens.

Animal Feed↗