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Results for “Multiplex PCR”

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At least 361 records · Page 20Linked to original sources

PubMed2001

Sequential monitoring of chimerism and detection of minimal residual disease after allogeneic blood stem cell transplantation (BSCT) using multiplex PCR amplification of short tandem repeat-markers.

Sequential analysis of chimerism after allogeneic blood stem cell transplantation (BSCT) has been shown to be predictive for graft failure and relapse. We have explored the impact of a novel approach for the quantitative determination of chimerism using a commercial PCR assay with multiplex amplification of nine STR-loci and fluorescence detection. The feasibility was studied in 121 patients transplanted from related or unrelated donors. Follow-up investigation was performed in 88 patients. Twenty-eight of these patients had received a transplantation after dose-reduced conditioning therapy. Results were compared to data obtained by FISH analysis in a subgroup of patients receiving grafts from sex-mismatched donors. The analysis was possible in all patients, the median number of informative alleles was 4 (range 1-8) compared to 7 (range 1-9) in the related and unrelated situation, respectively. A good correlation was seen in 84 samples from 14 patients analyzed in parallel with STR-PCR and FISH. Decreasing values of donor chimerism were detected prior to or concomitantly with the occurrence of graft failure and relapse of disease in all patients investigated prospectively. Using FACS-sorted material, eg peripheral blood CD34+ cells, the assay permitted the detection of residual recipient cells with high sensitivity (down to one CD34+ Kasumi cell in 40,000 normal WBC). Evaluation of the inter-laboratory reproducibility revealed that in 20 samples analyzed in three different centers, the median coefficient of variation was 2.1% (range 0.7-9.6%). Taken together, the results support the use of the test as a valuable tool in the follow-up of patients undergoing allogeneic BSCT. In cases lacking PCR-detectable disease-specific gene products, this assay may represent an alternative to recently established real-time PCR methods.

Adolescent↗

PubMed2004

Development of a multiplex PCR assay for the identification of pathogenic genes of Escherichia coli in milk and milk products.

A multiplex PCR for the simultaneous detection of some pathogenic genes of enteropathogenic, enterotoxigenic and verocytotoxin-producing Escherichia coli was developed. In this study primers found in literature as well as primers to the purpose designed were used. In this way, it was possible to generate specific fragments of 96, 170, 229, 285, 348, 414 and 510 bp for Hlya, St, EaeA, Lt, Vt1, UidA and Vt2 genes, respectively. When applied to bacterial strains experimentally inoculated in milk and milk products, the proposed PCR showed a detection limit of 5 x 10(4)CFU/ml for Hyla, St, Eaea, Vt1 primers, while for Lt and Vt2 primers the limit resulted of 10(6)CFU/ml.

Animals↗

PubMed2005

A multiplex PCR for detection of Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, and Bordetella pertussis in clinical specimens.

A multiplex PCR was developed that is capable of detecting four of the most important bacterial agents of atypical pneumonia, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, and Bordetella pertussis in uncultured patient specimens. These organisms cause similar symptomologies and are often not diagnosed because they are difficult to identify with classical methods such as culture and serology. Given this, the overall impact of these pathogens on public health may be grossly underestimated. The molecular test presented here provides a simple method for identification of four common, yet diagnostically challenging, pathogens.

Bordetella pertussis↗

PubMed2005

Identification of naturally infected Lutzomyia intermedia and Lutzomyia migonei with Leishmania (Viannia) braziliensis in Rio de Janeiro (Brazil) revealed by a PCR multiplex non-isotopic hybridisation assay.

To identify naturally infected Lutzomyia spp. by Leishmania (Viannia) braziliensis, a PCR multiplex non-isotopic hybridisation assay was developed for the analysis of insect samples collected in distinct areas of the municipality of Rio de Janeiro (Brazil), from March to December 2003. Data from experimental infection indicate that the method can detect one individual infected insect out of ten. Wild sand flies were classified and grouped into pools of 10 specimens each, reaching a total of 40 female groups. Positive results were obtained with pools of Lu. intermedia (5/32) and Lu. migonei (3/5) collected in two areas from the district of Jacarepaguá presenting recent cases of human and canine leishmaniasis. Considering eight infected groups (8/40) with at least one positive insect in each, it was possible to infer an infection rate of 2%. This technique permits the synchronous processing of a large number of samples, in order to investigate infection rates in sand fly populations and to identify potential insect vectors. The results presented here represent the first molecular approach used to infer the natural infection index in both Lutzomyia spp. and constitute essential data to the understanding of leishmaniasis ecoepidemiology in endemic areas from Rio de Janeiro.

Animals↗

PubMed2001

Effect of routine use of a multiplex PCR for detection of vanA- and vanB- mediated enterococcal resistance on accuracy, costs and earlier reporting.

A multiplex PCR (MPCR) for detection of vanA-and vanB-mediated resistance to vancomycin was optimized and adapted for use in the routine microbiology laboratory. Consecutive specimens (1196) submitted for vancomycin resistant Enterococci (VRE) surveillance were processed by clinical technologists on Bile Esculin Azide Agar containing 6 mg/L vancomycin (BEAA/Vanco6) plates and 466 showing black colony growth were processed by conventional biochemical testing (CBT) and by MPCR. CBT identified 208 VRE positives. MPCR detected 205 of the CBT- positives plus an additional 10. Analysis of the discordant specimens determined that 5 CBT- negative/MPCR-positive specimens also contained Enterococci with vanC resistance, 3 CBT-positive/MPCR-negative specimens were true positives, and 5 CBT-negative/MPCR-positive specimens occurred due to technical error. The sensitivity and specificity of MPCR were 98.4% and 96.1%. MPCR identifications of VRE were achieved approximately 48 h earlier than CBT and at 60% of the costs.

Bacterial Proteins↗

PubMed2000

Development of a multiplex PCR assay for the simultaneous detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii.

A multiplex PCR assay with five primers targeting the 16S and 23S rRNA genes was developed for the simultaneous detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii. The selected primers amplify a 257-bp fragment from A. cryaerophilus, a 401-bp fragment from A. butzleri and a 641-bp fragment from A. skirrowii. No PCR product was generated for closely related bacteria including Campylobacter and Helicobacter species. The assay was useful to identify cultures after in vitro cultivation and to detect and identify A. butlzeri and A. cryaerophilus from poultry samples present in 24-h old enrichment in Arcobacter broth with cefoperazone, amphotericin and teicoplanin (CAT)-supplement.

Animals↗

PubMed2000

Multiplex PCR for the detection of Lactobacillus pontis and two related species in a sourdough fermentation.

A specific multiplex PCR assay based on the amplification of parts of the 16S rRNA molecule was designed. Primers derived from variable regions of the 16S rRNA provided a means of easily differentiating the species Lactobacillus pontis and Lactobacillus panis. They could be clearly discriminated from the phylogenetically related species Lactobacillus vaginalis, Lactobacillus oris, and Lactobacillus reuteri and from other lactobacilli commonly known to be present in sourdough. Other strains isolated together with L. pontis from an industrial sourdough fermentation could be clearly separated from these species by comparative sequence analysis and construction of a specific PCR primer. For a fast identification a DNA isolation protocol based on the ultrasonic lysis of cells from single colonies was developed. To demonstrate the potential of such techniques for tracking these organisms in a laboratory-scale fermentation, we combined the specific PCR assay with direct DNA extraction from the organisms in the sourdough without previous cultivation.

Bread↗

PubMed1995

Specific detection of methicillin-resistant Staphylococcus species by multiplex PCR.

In Staphylococcus aureus, mecA and femA are the genetic determinants of methicillin resistance. By using a multiplex PCR strategy, 310- and 686-bp regions of the mecA and femA genes, respectively, were coamplified to identify susceptible (lacking mecA) and resistant (mecA+) staphylococci and to differentiate S. aureus (femA+) from coagulase-negative staphylococci (lacking femA). A third staphylococcal genomic sequence, corresponding to IS431 and spanning 444 bp, was used as a PCR control. One hundred sixty-five staphylococcal strains were tested. All 72 methicillin-resistant strains were found to be mecA+, and 92 of the 93 susceptible isolates lacked mecA. Only one coagulase-negative Staphylococcus isolate carrying the mecA gene was highly susceptible to oxacillin. The femA determinant was a unique feature of S. aureus; it was found in 100% of the S. aureus strains tested but was undetectable in all of the coagulase-negative staphylococci tested. The possibility of directly detecting the mecA and femA genes in blood samples was also investigated. After two amplification steps, a sensitivity of 50 microorganisms per ml of freshly collected spiked blood was achieved. In conclusion, coamplification of mecA and femA determinants proved to be very reliable both for rapid detection of methicillin resistance and differential diagnosis between S. aureus and other staphylococci. This technique, which can be successfully performed with blood samples, could be a useful tool in the diagnosis and treatment monitoring of staphylococcal infections.

Bacterial Proteins↗

PubMed2006

Evaluation of a multiplex PCR method to detect enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAEC) has been implicated in sporadic diarrhea in children and adults and has been identified as the cause of several outbreaks worldwide. The HEp-2 test remains the gold standard for identification of this pathotype. A 60-65 MDa plasmid encodes the aggregative adherence fimbriae (AAF/I and AAF/II), a transcriptional activator (aggR gene), the enteroaggregative heat-stable enterotoxin EAST1 (astA gene) and a cytotoxin (Pet). The standard assay for EAEC is performed only in research laboratories, because it is expensive, labor intensive and time-consuming. The Polymerase Chain Reaction (PCR) offers the possibility of rapid diagnosis. In the current study, a multiplex PCR assay which checks aggR and astA genes was designed. Eigthy-eight E. coli strains, isolated from children with acute diarrhea in Mendoza, Argentina, were characterized by the reference method (HEp-2 assay), and by aggR-astA PCR. A strong correlation between the presence of the specific marker aggR and the reference test was found. The astA gene had a similar distribution between aggregative and localized strains, indicating that this gene could not be considered as a marker of EAEC. We conclude that aggR may be used to identify EAEC, using the PCR method as a screening test.

Bacterial Adhesion↗

PubMed2001

A multiplex PCR test for determination of mating type applied to the plant pathogens Tapesia yallundae and Tapesia acuformis.

A multiplex PCR test for determining mating type of the pathogens Tapesia yallundae and Tapesia acuformis is described. The test involves three primers: a "common" primer annealing to DNA sequence conserved in the flanking region of both mating-type idiomorphs and two specific primers annealing to sequence in either the MAT-1 or the MAT-2 idiomorphs. Locating the specific primers in different positions relative to the common primer yielded PCR products of 812 or 418 bp from MAT-1 and MAT-2 isolates, respectively. The test was used successfully to determine the mating type of 118 isolates of T. yallundae and T. acuformis from Europe, North America, and New Zealand. Isolates of both mating types were found on all continents for both species despite the rarely observed occurrence of sexual reproduction of T. acuformis. The multiplex test design should be applicable to other ascomycete species, of use in studies of MAT distribution and facilitating sexual crossing by identifying compatible isolates.

Ascomycota↗

PubMed2005

Detection and differentiation of Plum pox virus using real-time multiplex PCR with SYBR Green and melting curve analysis: a rapid method for strain typing.

A real-time multiplex PCR procedure with melting curve analysis, using the green fluorescence dye SYBR Green I, was developed for rapid and reliable identification of Plum pox virus (PPV) isolates of strains D and M. Members of the different strains were identified by their distinctive melting temperatures (T(m)s); 84.3-84.43 degrees C for D isolates, and 85.34-86.11 degrees C for M isolates. The associated amplicon sizes were 114 and 380 bp, respectively. The procedure was used for detection and identification of PPV in both herbaceous and woody hosts. The Tm for members of a particular strain was very similar, with a host effect that did not hinder strain identification. Universal primers included in the study detected all isolates of PPV tested, amplifying a 74 bp fragment. The Tm of this fragment varied from 80.12 to 81.52 degrees C and may have supplementary value for PPV identification. SYBR Green-based detection was compared to detection using a hybridization LUX fluorogenic primer. Better resolution of the melting peaks was observed with SYBR Green I, than with the LUX primers, hence strain identification with SYBR Green I was more reliable. This is a simple approach to PPV strain identification with the relatively inexpensive dye SYBR Green I, and eliminates any need for electrophoretic analysis of amplicons or RFLP patterns using ethidium bromide.

Benzothiazoles↗

PubMed2004

Improvement of a multiplex PCR system for DYS441, DYS442, DYS443, DYS444 and DYS445, and a population study in 340 Japanese males.

A multiplex PCR system for five Y-STRs (DYS441, DYS442, DYS443, DYS444 and DYS445) has been improved to increase the probability of obtaining a DNA typing result from aged samples. Newly designed PCR primers for amplification of the DYS441 and DYS442 loci and optimization of PCR conditions enabled successful typing from blood and semen stains that had been stored for more than seven years at room temperature. Analysis of 340 Japanese males revealed 7, 5, 6, 5 and 4 alleles at the DYS441, DYS442, DYS443, DYS444 and DYS445 loci, respectively, yielding 122 haplotypes with a cumulative haplotype diversity of 0.97.

Chromosomes, Human, Y↗

PubMed2005

Design of a multiplex PCR for Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae and Chlamydophila pneumoniae to be used on sputum samples.

A multiplex PCR (mPCR) was developed for simultaneous detection of specific genes for Streptococcus pneumoniae (lytA), Mycoplasma pneumoniae (P1), Chlamydophila pneumoniae (ompA), and Haemophilus influenzae (16S rRNA, with verification PCR for P6). When the protocol was tested on 257 bacterial strains belonging to 37 different species, no false negatives and only one false positive were noted. One Streptococcus mitis out of thirty was positive for lytA. In a pilot application study of 81 sputum samples from different patients with suspected lower respiratory tract infection (LRTI), mPCR identified S. pneumoniae in 25 samples, H. influenzae in 29, M. pneumoniae in 3, and C. pneumoniae in 1. All samples culture positive for S. pneumoniae (n=15) and H. influenzae (n=15) were mPCR positive for the same bacteria. In a pilot control study with nasopharyngeal swabs and aspirates from 10 healthy adults, both culture and mPCR were negative. No PCR inhibition was found in any of the mPCR-negative sputum or nasopharyngeal samples. Whether all samples identified as positive by mPCR are truly positive in an aetiological perspective regarding LRTI remains to be evaluated in a well-defined patient material. In conclusion, the mPCR appears to be a promising tool in the aetiological diagnostics of LRTI.

Adolescent↗

PubMed2000

Development of a single-reaction multiplex PCR toxin typing assay for Staphylococcus aureus strains.

We describe here the development of a single-reaction multiplex PCR assay for the enterotoxin genes from Staphylococcus aureus that utilizes a universal toxin gene primer in combination with toxin-specific primers to amplify characteristic toxin gene products. In combination with a new DNA purification method, the assay can detect enterotoxin genes A to E from a pure culture within 3 to 4 h. The test was used to characterize a diverse set of environmental S. aureus isolates, and a 99% correlation with toxin typing using standard immunological tests was found. The design of the assay allows it to be extended to include both newly characterized and as-yet-unknown toxin genes.

Animals↗

PubMed2000

Multiplex PCR assay for ureC and 16S rRNA genes clearly discriminates between both subspecies of Photobacterium damselae.

A multiplex-PCR approach, employing 2 primer pairs directed to internal regions of the 16S rRNA and ureC genes, was utilized to analyze a collection of Photobacterium damselae strains, including 25 isolates of subspecies piscicida and 15 isolates of subspecies damselae. With this procedure, all the P. damselae subsp. damselae strains yielded 2 amplification products, one of 267 bp and the other of 448 bp, corresponding to internal fragments of the 16S rRNA and ureC genes, respectively. However, P. damselae subsp. piscicida isolates only showed the PCR product of 267 bp (16S rRNA fragment), indicating the absence of the urease gene in its genome. We have constructed a DNA probe directed to an internal region of the ureC gene, and corroborated by dot blot hybridization that the P. damselae subsp. piscicida lacks this gene, whereas it is present in the subspecies damselae. This constitutes the first successful discrimination between both subspecies using a PCR procedure, which could become a useful tool for diagnosis of pasteurellosis in the field. In addition, since these 2 subspecies have been shown to share nearly the same rrn operon sequence, our results provided evidence that one of the steps in the P. damselae speciation proccess included gain/loss events associated with the ure operon.

Animals↗

PubMed2001

Multiplex PCR assay for screening deletions in the low density lipoprotein receptor gene.

BACKGROUND: In different populations of the world, more than 150 genetic alterations of the LDL receptor gene have been identified; each of which can result in hypercholesterolaemia, but no hot spots in the gene were detected so far. Because of the existence of very variable genetic alterations in different ethnic communities, none of the assays developed for screening mutations/deletions in a population defined can be adapted to study the possible genetic defects. The present study was designed to develop a new, multiplex PCR-based, molecular biological method to screen the whole coding region of the LDL receptor gene. METHODS: Using primer pairs completely flanking the promoter and the entire exonal region, in the PCR reactions 83-386-bp long, DNA sequences were synthesised in seven different reaction mixtures. The reaction conditions of the multiplex PCR system were optimised in order to synthesise all exons and the promoter region of the gene using only two annealing temperatures. The products could be visualised separately by agarose gel electrophoresis/ethidium bromide staining. RESULTS: A rapid, effective test enabling the screening of DNA alterations in the entire LDL receptor gene was developed. Using this simple multiplex PCR assay, deletions affecting more than 10 bp in any part of the gene can be easily detected by a single agarose gel electrophoresis. CONCLUSIONS: The simplicity, specificity and versatility of the assay make it suitable system for routine screening of LDL receptor gene mutations in large population samples. This PCR assay can be recommended for screening of LDL-RG deletions in populations or groups at high risk for cardiovascular diseases.

Electrophoresis, Agar Gel↗

PubMed2006

Development of a sensitive and specific assay combining multiplex PCR and DNA microarray primer extension to detect high-risk mucosal human papillomavirus types.

The importance of assays for the detection and typing of human papillomaviruses (HPVs) in clinical and epidemiological studies has been well demonstrated. Several accurate methods for HPV detection and typing have been developed. However, comparative studies showed that several assays have different sensitivities for the detection of specific HPV types, particularly in the case of multiple infections. Here, we describe a novel one-shot method for the detection and typing of 19 mucosal high-risk (HR) HPV types (types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, and 82). This assay combines two different techniques: multiplex PCR with HPV type-specific primers for amplification of viral DNA and array primer extension (APEX) for typing. This novel method has been validated with artificial mixtures of HPV DNAs and clinical samples that were already analyzed for the presence of mucosal HPV types by a different consensus PCR method, i.e., GP5+/GP6+. Our data showed a very good agreement between the results from the multiplex PCR/APEX assay and those from the GP5+/GP6+ PCR (overall rates of HPV positivity, 63.0 and 60.9%, respectively). Whereas the GP5+/GP6+ PCR was slightly more sensitive for the detection of HPV type 16 (HPV-16), multiplex PCR-APEX found a higher number of infections with HPV-33, HPV-53, and multiple HPV types. These favorable features and the high-throughput potential make our present novel assay ideal for large-scale clinical and epidemiological studies aimed at determining the spectrum of mucosal HR HPV types in cervical specimens.

DNA Primers↗

PubMed1997

Multiplex PCR detection of vanA, vanB, vanC-1, and vanC-2/3 genes in enterococci.

Vancomycin-resistant enterococci (VRE) are increasingly isolated from clinical specimens. One hundred clinical isolates of enterococci (E. casseliflavus/E. flavescens [n = 10], E. faecalis [n = 34], E. faecium [n = 43], E. avium [n = 1], E. gallinarum [n = 11], and E. raffinosus [n = 1]) were examined for the presence of vanA, vanB, vanC-1, and vanC-2/3 genes by a single multiplex PCR performed directly with colonies from blood agar plates. Six previously characterized VRE strains which carry either vanA, vanB, vanC-1, or vanC-2 genes were used as controls. To discriminate among van genes, the PCR amplicons were digested with MspI and were electrophoresed on agarose gels. Because of significant sequence homology between vanC-2 and vanC-3 genes, this assay is unable to discriminate these genes from each other; therefore, these are referred to as vanC-2/3 genes. PCR products were detected in 63 of the 100 clinical isolates. The restriction fragment length patterns were consistent with vanA for 10 strains, vanB for 30 strains, vanC-1 for 12 strains, vanC-2 for 6 strains, and vanA and vanC-1 for 1 strain. The vancomycin MICs for the isolates with restriction fragment length patterns consistent with vanA and vanB were all > and = 64 micrograms/ml. The vancomycin MICs for the isolates with restriction fragment length patterns consistent with vanC-1 or vanC-2 were 4 to 8 micrograms/ml. The vancomycin MICs for the isolates from which no PCR amplicons were produced were 2 to 4 micrograms/ml. A PCR product was produced in four isolates (vancomycin MICs, 4 to > 256 micrograms/ml) with restriction fragment length patterns differing from those for the control vanA, vanB, vanC-1, and vanC-2 isolates. DNA sequencing of these amplicons revealed that two of the four isolates had nucleic acid sequences which were closely related to the published sequence for the vanB gene and two had nucleic acid sequences which were closely related to the published sequence for the vanC-2 and vanC-3 genes. Multiplex PCR-restriction fragment length polymorphism appears to be a useful and convenient method for rapidly detecting and discriminating genotypes for vancomycin-resistant Enterococcus spp. in the clinical laboratory. In instances in which unusual restriction fragment patterns of PCR amplicons occur, DNA sequencing can be performed to discriminate van genotypes.

Amino Acid Sequence↗