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Results for “Multiplex PCR”

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PubMed2002

Comparison of multiplex PCR, and an immunochromatographic method sensitivity for the detection of Escherichia coli O157:H7 in minced beef.

In this study, the sensitivities of multiplex PCR and an immuno-chromatographic methods to detect Escherichia coli O157:H7 in minced beef were compared. The detection of Escherichia coli O157:H7 in minced beef inoculated with 1-100 cells of this bacterium was possible after enrichment of culture and subsequent analysis by either of the two methods. Enrichment conditions were eight hours of incubation at 37 degrees C or 42 degrees C in a non-selective medium (Buffered Peptone Water). Multiplex PCR analysis was performed using three primer sets with analysis by gel electrophoresis. The Quix immuno-chromatographic assay which is a new kit being marketed by New Horizons Diagnostics, Columbia, MD, was used for immunological analysis of the enriched broths.The sensitivity of both tests was similar. The results depended on the concentration of the specific bacterium in the culture since the influence of the proportion of other bacteria to the E. coli O157:H7 was not observed. The data suggests that either method or used together, when coupled with an enrichment technique, could provide a rapid mean to detect the presence of this pathogen in minced meat samples.

Animals↗

PubMed1998

Rapid detection of Actinobacillus actinomycetemcomitans, Prevotella intermedia and Porphyromona gingivalis by multiplex PCR.

The identification of specific periodontal pathogens by conventional methods, mainly anaerobic cultivation, is difficult, time consuming and even sometimes unreliable. Therefore, a multiplex PCR method for simultaneous detection of Actinobacillus actinomycetemcomitans (A.a.), Porphyromona gingivalis (P.g.) and Prevotella intermedia (P.i.) was developed for rapid and easy identification of these specific bacterial pathogens in subgingival plaque samples. In this paper, there is a detailed description of the oligonucleotide primer selection, DNA extraction and PCR conditions and the sequencing of the amplified products. The locus chosen to be amplified is a highly variable region in the 16S ribosomal DNA. For the development of this technique ATCC cultures and pure cultures from subgingival plaque samples taken from periodontitis patients were used. As an internal positive control a recombinant plasmid was developed. This simple DNA extraction procedure and the DNA amplification and visualization of the amplified product permits the detection of the bacteria in a working day. Thus, this multiplex PCR method is a rapid and effective detection method for specific periodontal pathogens.

Aggregatibacter actinomycetemcomitans↗

PubMed2001

Prenatal diagnosis of Pelizaeus-Merzbacher disease: detection of proteolipid protein gene duplication by quantitative fluorescent multiplex PCR.

A prenatal diagnosis of Pelizaeus-Merzbacher disease (PMD) resulting from proteolipid protein gene (PLP) duplication was performed by a quantitative fluorescent multiplex PCR method. PLP gene copy number was determined in the proband, the pregnant mother, the male fetus and two aunts. Small amounts of genomic DNA extracted from peripheral blood and from chorionic villi were used. The fetus, in common with the proband, was identified as PMD-affected being a carrier of the PLP gene duplication, inherited from the mother, while the two aunts were non-carriers. The data obtained were confirmed by segregation analysis of a PLP-associated dinucleotide-repeat polymorphism amplified by the same multiplex PCR.

Case-Control Studies↗

PubMed1997

Multiplex PCR for the identification of Arcobacter and differentiation of Arcobacter butzleri from other arcobacters.

A multiplex polymerase chain reaction (PCR) assay to identify Arcobacter isolates and to distinguish A. butzleri from other arcobacters is described. The test uses two primer sets. Set I targets a section of the 16S rRNA genes of Arcobacter spp. Set II amplifies a portion of the 23S rRNA genes unique to A. butzleri. Specificity of the primer sets was evaluated using ATCC reference strains of A. butzleri, A. cryaerophilus, A. skirrowii, Bacteroides spp., Campylobacter spp., Helicobacter spp. and Wolinella succinogenes. Upon PCR amplification, all of the Arcobacter isolates yielded a 1223 bp product, whereas A. butzleri ATCC 49616 exhibited both a 1223 bp and a 686 bp product. No PCR product was observed for other closely related ATCC strains (n = 37). We next analyzed by multiplex PCR field strains of Arcobacter spp. (n = 108) which had been previously characterized to the species level by either DNA-DNA hybridization, dot blot hybridization, ribotyping or by serology. The 1223 bp multiplex PCR product identified all of the isolates as Arcobacter. The presence of both the 1223 and 686 bp amplicons identified 66 strains as A. butzleri. Speciation by multiplex PCR agreed with results obtained by the other methods. The multiplex PCR assay is specific, rapid and easy to interpret and, thus, will aid in elucidating the prevalence, epidemiology and zoonotic potential of Arcobacter.

Animals↗

PubMed2000

Establishment of a multiplex PCR system to diagnose tuberculosis and other bacterial infections.

In order to rapidly diagnose and differentiate tuberculosis from other bacterial infections, a 16S rRNA gene (16s rDNA)-directed multiplex PCR system was developed. In this system, a pair of universal primers and a tubercle bacillus (Tb)-specific primer were designed based on highly conserved regions and Tb species-specific variable region of bacterial 16s rDNA. A 360 bp fragment was detected in all bacteria tested, and a 210 bp fragment was found only in Tb. 19 species of known bacteria including Tb were used for evaluating specificity, universality and sensitivity of the PCR. Candida albicans and human diploid cell served as controls. It was found that both 210 bp and 360 bp fragments were amplified only in Tb, and only 360 bp fragment was detected in other 18 species of general bacteria. Candida albicans and human cells were negative for both 360 bp and 210 bp fragments. The lowest detectable level of the PCR was 10 fg of DNA for Escherichia coli and 100 fg of DNA for Tb. The results indicated that this multiplex PCR system for the simultaneous detection of Tb and other common bacteria had higher specificity and sensitivity, as well as good universality and might be useful to rapidly diagnose bacterial infections and effectively distinguish tuberculosis from other bacterial involvement.

Amidohydrolases↗

PubMed1995

Multiplex PCR for identifying mycobacterial isolates.

AIMS: To develop a multiplex polymerase chain reaction (PCR) method to facilitate identification of mycobacterial isolates. METHODS: Type strains of 14 species of mycobacteria and 56 clinical isolates were lysed by boiling in TE Triton. The lysate (5 microliters) was used directly in a PCR reaction incorporating three pairs of PCR primers expected to amplify fragments from the genome of (a) all mycobacteria, (b) Mycobacterium tuberculosis complex only and (c) M avium only. PCR products were visualised by electrophoresis on agarose gels. RESULTS: Multiplex PCR applied to 14 type strains yielded patterns on electrophoresis which permitted identification of the mycobacterial isolates as M tuberculosis complex, M avium or as mycobacteria other than the former. The identification of 56 clinical isolates by multiplex PCR was consistent with other methods and was accomplished in less than one working day. CONCLUSIONS: This method may facilitate rapid and convenient identification of most clinical isolates of mycobacteria by PCR and gel electrophoresis. Further evaluation is warranted.

Base Sequence↗

PubMed2005

Detection of Chromobacterium violaceum by multiplex PCR targeting the prgI, spaO, invG, and sipB genes.

Based on the recently completed genomic sequence of Chromobacterium violaceum American Type Culture Collection (ATCC) 12472 a multiplex PCR assay targeting the prgI, spaO, invG, and sipB genes of the Salmonella SPI-1 homologue type-III secretion system was developed. PCR products of 255bp (prgI), 749bp (spaO), 1685bp (invG), and 1752bp (sipB) were successfully amplified simultaneously in a single reaction with all Chr. violaceum strains investigated whereas other bacteria tested negative. The detection limit for pure cultures in multiplex PCR analysis was 100CFU. The developed assay significantly improves rapid identification of Chr. violaceum and allows its differentiation from closely related organisms.

Animals↗

PubMed1999

An efficient multiplex PCR suitable for large scale typing in linkage mapping.

The dissection of polygenic traits is made possible with the development of microsatellite markers. Linkage study of this kind involves many markers with tens of hundreds of samples. Although typing essentially contains only two steps: PCR amplification and gel electrophoresis. Such work is still heavy when a large number of samples had to be genotyped. Multiplex PCR may reduce the work, but one has to optimize the conditions from marker to marker. Here we describe a dye-compatible multiplex PCR that works under standardized condition without the need to pre-determine the combinational primer concentration and the time-consuming step to mix many samples with gel loading dye before electrophoresis. This successful protocol should greatly reduce the cost and labor for genetic study of polygenic traits.

Animals↗

PubMed2003

A multiplex PCR assay for the simultaneous detection and discrimination of the seven Eimeria species that infect domestic fowl.

This study reports the development of a novel multiplex PCR assay based on SCAR (Sequence-Characterised Amplified Region) markers for the simultaneous diagnosis of the 7 Eimeria species that infect domestic fowl. Primer pairs specific for each species were designed in order to generate a ladder of amplification products ranging from 200 to 811 bp. Sensitivity tests for each species were carried out, showing a detection threshold of 1-5 pg, which corresponds approximately to 2-8 sporulated oocysts. Distinct isolates of the 7 Eimeria species from different geographical sources were tested and successfully detected by the assay. All the species were amplified homogeneously, whether or not one of them was present in a high quantity, indicating that there was no cross-interference. The assay was also tested with different sources of Taq DNA polymerase and thermocycler models, confirming the high reproducibility of the reaction. The economy of consumables and labour represented by a single-tube reaction greatly facilitates the molecular diagnosis of a large number of samples, making it appropriate for field epizootiological surveys. We propose the use of this multiplex PCR assay as a rapid and cost-effective diagnostic method for the detection and discrimination of the 7 Eimeria species that infect domestic fowl.

Animals↗

PubMed2004

Development of a multiplex PCR (m-PCR) test for rapid identification of genes encoding heat-labile (LTI) and heat-stable (STI and STII) toxins of enterotoxigenic Escherichia coli (ETEC) with internal control of amplification.

A multiplex PCR system was developed for specific identification of genes encoding heat-labile (LTI) and heat-stable (STI and STII) toxins of enterotoxigenic Escherichia coli (ETEC) strains. In addition, primers specific for the E. coli gene coding for 16S rRNA were used as an internal control of the DNA amplification. The specificity of the method was validated by single PCR tests performed with reference to E. coli strains as well as pig-isolated bacteria and 100% correlation was observed. The developed multiplex PCR allowed rapid and specific identification of enterotoxin-positive E. coli and may be used as a sensitive and specific method for a direct determination of ETEC and to differentiate them from other E. coli isolates.

Bacterial Toxins↗

PubMed1997

A simple filtration technique to detect enterohemorrhagic Escherichia coli O157:H7 and its toxins in beef by multiplex PCR.

Primers, specific for a unique base substitution in uidA of Escherichia coli O157:H7, were coupled with oligonucleotides for the shiga-like toxin I (SLT-I) and SLT-II genes in a multiplex PCR assay. A minimum of 10(2) CFU per PCR (10 microliters) was necessary to amplify E. coli O157:H7-specific bands by multiplex PCR. Food particles as well as various unknown metabolic by-products of bacteria inhibited the PCR, but a simple two-step filtration procedure eliminated this inhibition. To reliably generate PCR products, an E. coli inoculum of 10(3) CFU g of food slurry-1 in a nonspecific medium was required with 6 h of enrichment at 37 degrees C. However, when the food homogenate was incubated overnight, E. coli O157:H7 at an initial inoculum of even 1 CFU g-1 was detected.

Animals↗

PubMed1999

Viral diagnosis of neurological infection by RT multiplex PCR: a search for entero- and herpesviruses in a prospective study.

The diagnosis of a wide range of different neurological syndromes was established by a reverse transcription multiplex PCR assay. The presence of enterovirus and herpesviruses was studied in cerebrospinal fluid samples collected prospectively from 200 patients hospitalized with neurological diseases suspected of viral infection. Positive PCR results for enterovirus and neurotropic herpesvirus (herpes simplex, HSV, and varicella zoster, VZV) were obtained among the immunocompetent patients (55/156, 35%) who presented aseptic meningitis or encephalitis. Among immunocompromised patients the yield of positive PCR results was 41% (18/44), predominantly lymphotropic herpesviruses (15/44, 34%). Cytomegalovirus (CMV) DNA was detected in patients with several clinical syndromes, including encephalitis, chronic meningitis, retinitis, ventriculitis, polyradiculomyelitis, and myeloradiculitis. Epstein-Barr (EBV) and VZV-specific DNA sequences were detected in patients with either encephalitis, aseptic meningitis, and chronic meningitis. Dual infections of CMV and HSV or CMV and EBV were established in two AIDS patients with encephalitis and polyradiculomyelitis, respectively. The applications of this RT multiplex PCR assay are extensive and may prove to be particularly valuable for the rapid and sensitive diagnosis of neurological diseases in both immunocompetent and immunocompromised patients.

Adult↗

PubMed2002

A modified multiplex PCR assay for detection of large deletions in MSH2 and MLH1.

A method for detection of large genomic deletions in the MSH2 and MLH1 genes based on multiplex PCR and quantitative evaluation of PCR products is presented. All 35 exons of MSH2 and MLH1 were screened simultaneously in seven PCR reactions, each of them including primers for both genes. The method is reliable for uncovering large genomic deletions in patients suspected of HNPCC. With this method, six novel deletions were identified, two in MSH2: EX1_10del and EX1_16del (representing deletion of the entire MSH2 gene); and four in MLH1: EX1_10del in two unrelated patients, EX3_5del, and EX4del. The deletions were detected in 18 unrelated patients in whom no germline mutation had been identified by SSCP and DHPLC. These results indicate that our modified multiplex PCR assay is suited for the detection of large deletions both in the MSH2 and MLH1 gene and therefore represents an additional valuable tool for mutation screening in HNPCC families.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Multiplex PCR method for use in real-time PCR for identification of fish fillets from grouper (Epinephelus and Mycteroperca species) and common substitute species.

Mitochondrial 16S rRNA sequences from morphological validated grouper (Epinephelus aeneus, E. caninus, E. costae, and E. marginatus; Mycteroperca fusca and M. rubra), Nile perch (Lates niloticus), and wreck fish (Polyprion americanus) were used to develop an analytical system for group diagnosis based on two alternative Polymerase Chain Reaction (PCR) approaches. The first includes conventional multiplex PCR in which electrophoretic migration of different sizes of bands allowed identification of the fish species. The second approach, involving real-time PCR, produced a single amplicon from each species that showed different Tm values allowing the fish groups to be directly identified. Real-time PCR allows the quick differential diagnosis of the three groups of species and high-throughput screening of multiple samples. Neither PCR system cross-reacted with DNA samples from 41 common marketed fish species, thus conforming to standards for species validation. The use of these two PCR-based methods makes it now possible to discriminate grouper from substitute fish species.

Animals↗

PubMed2004

Simultaneous and sensitive detection of three foodborne pathogens by multiplex PCR, capillary gel electrophoresis, and laser-induced fluorescence.

The simultaneous detection of Staphylococcus aureus, Listeria monocytogenes, and Salmonella spp. has been approached by a new multiplex PCR-based procedure followed by capillary gel electrophoresis with laser-induced fluorescence detection (multiplex-PCR-CGE-LIF). As compared to slab gel electrophoresis, the use of CGE-LIF improved from 10- to 1000-fold the sensitivity of the multiplex PCR analysis, allowing the detection of 2.6 x 10(3) cfu mL(-1) of S. aureus, 570 cfu mL(-1) of L. monocytogenes, and 790 cfu mL(-1) of Salmonella in artificially inoculated food, without enrichment. Following 6 h of enrichment, as low as 260, 79, and 57 cfu mL(-1) of S. aureus, L. monocytogenes, and Salmonella, respectively, were detected. The CGE-LIF method is shown to be reproducible, providing relative standard deviation (RSD) values lower than 0.8% for analysis time and lower than 5.8% for peak areas. The multiplex-PCR-CGE-LIF proved a powerful analytical tool to detect various food pathogens simultaneously in a fast, reproducible, and sensitive way.

Electrophoresis, Capillary↗

PubMed2004

Development of a multiplex PCR for the detection of asa1, gelE, cylA, esp, and hyl genes in enterococci and survey for virulence determinants among European hospital isolates of Enterococcus faecium.

A multiplex PCR for the simultaneous detection of five virulence genes (asa1, gelE, cylA, esp, and hyl) in enterococci was developed. The presence of these genes was investigated in 153 clinical and 118 fecal Enterococcus faecium isolates from inpatients at an increased risk of developing infections (such as patients in intensive care units and hematology wards) from 13 hospitals in eight European countries. Of the 271 E. faecium isolates, 135 were vancomycin resistant E. faecium (VREF) isolates and 136 were vancomycin susceptible E. faecium (VSEF) isolates. Susceptibilities to ampicillin, gentamicin, streptomycin, vancomycin, teicoplanin, ramoplanin, quinupristin-dalfopristin, and linezolid were tested by the microdilution method. Overall, the prevalence of esp was significantly higher (P = 0.03) in clinical VREF isolates (92%) than in fecal VREF isolates (73%). In Italy, the prevalence of esp was significantly higher (P = 0.02) in VREF isolates (91%) than in VSEF isolates (68%), whereas in the United Kingdom, hyl was significantly more prevalent (P = 0.01) in VREF isolates (71%) than in VSEF isolates (29%). No significant differences were found for the other countries. Pulsed-field gel electrophoresis was used to check the clonality among the strains tested and showed the spread of two center-specific (esp-positive) VREF clones in Italy and one center-specific (hyl-positive) clone in the United Kingdom. These clones were resistant to ampicillin, gentamicin, and streptomycin. The multiplex PCR reported in this study is a convenient and rapid method for the simultaneous detection of the virulence genes asa1, gelE, cylA, esp, and hyl in enterococci. Molecular analysis showed the intrahospital spread of esp-positive VREF clones (in Italy) and hyl-positive VREF clones (in the United Kingdom); the role of hyl remains to be elucidated.

Anti-Bacterial Agents↗

PubMed2003

Multiplex PCR screening of soil isolates for novel Bacillus-related lineages.

A16S rDNA multiplex PCR-based high-throughput protocol is presented to screen bacterial isolates in large amounts for the appearance of novel lineages of bacteria, especially hitherto unknown Bacillus relatives. The 16S rDNAs of 4224 isolates from a comprehensive cultivation campaign were screened for similarity to predominant uncultured soil bacteria. Soil suspensions were plated in serial dilutions on various media. After 2, 4 and 6 weeks, colonies were collected with toothpicks and transferred to microtiterplates for cell lysis and storage plates for subculture. Cell lysis was a simple freeze-heating cycle in distilled water. The multiplex PCR was adapted to operate sufficiently for Gram positives under these conditions. Approximately 10.6% of all picked colonies reacted with a primer targeting a Bacillus fraction containing novel Bacillus benzoevorans-relatives previously detected as predominant soil bacteria by culture-independent studies. From these 446 colonies detected by multiplex PCR, 363 (81.4%) could be successfully used for continued subculture and 16S rDNA sequencing. All identification was done by 16S rDNA sequencing. This revealed that more than 60% of them represented a variety of candidates for potentially new species. Twelve colonies were identified as almost identical matches to 16S rDNA sequences of hitherto uncultured but apparently predominant soil bacteria cloned from directly extracted soil DNA. Also, novel lineages of unpredicted phylogenetic diversity like novel Paenibacillus, Sporosarcina and even Xanthomonads were represented.

Bacillus↗

PubMed2000

Multiplex PCR: optimization and application in diagnostic virology.

PCR has revolutionized the field of infectious disease diagnosis. To overcome the inherent disadvantage of cost and to improve the diagnostic capacity of the test, multiplex PCR, a variant of the test in which more than one target sequence is amplified using more than one pair of primers, has been developed. Multiplex PCRs to detect viral, bacterial, and/or other infectious agents in one reaction tube have been described. Early studies highlighted the obstacles that can jeopardize the production of sensitive and specific multiplex assays, but more recent studies have provided systematic protocols and technical improvements for simple test design. The most useful of these are the empirical choice of oligonucleotide primers and the use of hot start-based PCR methodology. These advances along with others to enhance sensitivity and specificity and to facilitate automation have resulted in the appearance of numerous publications regarding the application of multiplex PCR in the diagnosis of infectious agents, especially those which target viral nucleic acids. This article reviews the principles, optimization, and application of multiplex PCR for the detection of viruses of clinical and epidemiological importance.

Humans↗