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At least 325 records · Page 18Linked to original sources
PubMed2003

Use of multienzyme multiplex PCR amplified fragment length polymorphism typing in analysis of outbreaks of multiresistant Klebsiella pneumoniae in an intensive care unit.

We developed and optimized a new modified amplified fragment length polymorphism (AFLP) typing method to obtain a multibanding fingerprint that can be separated by agarose gel electrophoresis. Both to maximize the discriminatory power and to facilitate the computer-assisted analysis, bacterial DNA was digested with four different restriction enzymes. After ligation of adaptors to the DNA fragments, PCR testing of various single primers was performed. Two single primers that gave optimal results with regard to band resolution and discriminatory power were selected and combined. The computer-assisted analysis of fingerprint patterns was performed with Pearson's product-moment correlation values of densitometric curves, without assigning bands to peaks. Thus, the analysis is not subject to human interpretation errors. With this method, we investigated two outbreaks of multiresistant Klebsiella pneumoniae in an intensive care unit and various sporadic isolates of K. pneumoniae and Klebsiella oxytoca. Cluster analysis of isolates analyzed in different experiments and on different gels showed that fingerprint patterns clustered correctly according to subspecies or to the outbreaks. Multienzyme multiplex PCR AFLP revealed that the first outbreak was caused by two different types of strains. Outbreak two was caused by yet another strain of K. pneumoniae. In conclusion, the typing method used here is easy to perform and highly reproducible, and due to generation of complex banding patterns, it has a higher discriminatory power. Furthermore, the multienzyme multiplex PCR fingerprints are easy to analyze, and a reliable database can be stored in the computer to facilitate comparison of future isolates of Klebsiella spp. The method can be performed in every clinical microbiology laboratory.

Cross Infection↗
PubMed2003

[Diagnosis of imported malaria with multiplex PCR on LighCycler apparatus].

For the diagnosis of imported malaria, a competitive multiplex PCR using LightCycler was developed and compared for 3 months with a traditional PCR method. Hundred eighty three patients were tested by these 2 techniques of molecular diagnosis: 60 were positive for P. falciparum, 9 were positive for other Plasmodium species and 114 were negative. The LightCycler method was found in total agreement with the traditional PCR.

Animals↗
PubMed1994

Comparison of multiplex PCR and standard bacteriological methods of detecting Salmonella on chicken skin.

A multiplex PCR technique was compared with standard bacteriological techniques for the detection of Salmonella, and Salm. enteritidis in particular, in samples of chicken skin obtained from retail outlets. Five of 68 samples were positive by both systems (7.4%), 58 of 68 were negative by both systems (85%), five were positive by PCR only (7.4%) but none were positive by culture only. The PCR was quicker and more sensitive than the bacteriological methods, finding 15% of samples positive for Salmonella compared to 7.4%, and taking less than 24 h to reach a result as opposed to 2-3 d.

Animals↗
PubMedDate not supplied

[Clarification of a break-in theft crime by multiplex PCR analysis of cigarette butts].

This paper describes the first use of multiplex PCR amplification kits for the analysis of DNA extracted from cigarette butts in a criminal case. Two suspects could be excluded as potential contributors to the samples, whereas the multi locus PCR-based DNa profile derived from the cigarette butts was consistent with a DNA profile derived from a third suspect. For identity testing in criminal cases where cigarette butts are involved, commercially available PCR amplification kits provide currently the most powerful tool. Furthermore this PCR-based analysis can be implemented into most application orientated laboratories.

DNA↗
PubMed1999

Simultaneous detection of erythromycin-resistant methylase genes ermA and ermC from Staphylococcus spp. by multiplex-PCR.

A comparative analysis of the two most dominant erythromycin-resistance determinant genes in Staphylococcus sppnamely, the ermA and ermC genes, was carried out. Sixty erythromycin-resistant strains of Staphylococcus spp. were tested, of which 24 were avian and 36 were clinical isolates. Our results indicated the prevalence of ermA over the ermC gene as opposed to the widely held opinion of the ermC gene being the most dominant resistance determinant gene. A multiplex-PCR assay was developed to detect the presence of ermA and ermC genes. Two pairs of primers, specific for the detection of ermA and ermC genes, were used in a multiplex-polymerase chain reaction (PCR) assay to yield amplified DNA products of 610 and 520 bp, respectively. Their digestion with restriction enzyme FokI that yielded a 477 bp and a 132 bp digestion product for ermA and a 333 bp and a 187 bp digestion product for ermC confirmed the authenticity of PCR products. The method could be used to amplify the ermA and ermC genes with as little as 5 pg of template DNA. The use of excess primers or the template DNA resulted in gene-specific amplification and no non-specific amplification was observed by changing the primer to primer or template to primer ratios. Furthermore, no amplification from erythromycin-sensitive S. aureus strain was observed. Using this assay, the poultry strains were found to contain either ermA alone (50%) or a combination of ermA (100%) and ermC (50%) both. The clinical strains contained either ermA (94.5%) or ermC (5.5%) but never both. The gene-specific internal probes were also used to verify the above findings and a high degree of correlation between the multiplex PCR and Southern hybridization data was observed.

Animals↗
PubMed1998

Advantages of a new Taq DNA polymerase in multiplex PCR and time-release PCR.

Extensive diagnostic and scientific investigations are often restricted by limited availability of material. Therefore, methods like multiplex PCR strategies are needed to conserve as much sample as possible. Unfortunately, the establishment of such procedures poses several difficulties. Here we describe the advantages of a new enzyme, AmpliTaq Gold DNA Polymerase, in multiplex and time-release PCR. The application of this thermostable recombinant Taq DNA polymerase allows the specific amplification of DNA/cDNA targets with very high sensitivity. With our protocol, the specific amplification of 13 different cDNAs of cytokines and cytokine receptors can be realized in three multiplex PCRs (IL-2R alpha, IL-2/15R beta, gamma c-chain, IL-4 and IL-4R alpha; IL-10, IL-15 and IL-15R alpha; and IL-2, IFN gamma, IL-7, IL-7R alpha and IL-9R alpha). The novel application of AmpliTaq Gold DNA Polymerase in a time-release PCR protocol allows specific amplification of target DNA/cDNA when only limited amounts of material are available or only low-copy-number DNA/cDNA is suspected. No IL-9 cDNA can be detected in peripheral blood mononuclear cells (PBMC) in the absence of any stimulation, thus it was difficult to amplify this target with routine PCR protocols. Here we demonstrate the reliable and reproducible amplification of IL-9 cDNA in the Hodgkin's lymphoma cell line KM-H2, in PBMC and in stimulated PBMC. Results with AmpliTaq Gold DNA Polymerase were more sensitive and specific compared with AmpliTaq DNA Polymerase, with and without manual hot-start procedure.

DNA, Complementary↗
PubMed2002

Rapid detection of methicillin-resistant staphylococci from blood culture bottles by using a multiplex PCR assay.

Rapid detection and accurate identification of methicillin-resistant staphylococci are critical for the effective management of infections caused by these organisms. We describe a multiplex PCR-based assay for the direct detection of methicillin-resistant staphylococci from blood culture bottles (BacT/Alert; Organon-Teknika, Durham, N.C.). A simple lysis method followed by a multiplex PCR assay designed to detect the nuc, mecA, and bacterial 16S rRNA genes was performed. A total of 306 blood culture specimens were collected over a period of 10 months from June 1998 to April 1999, consisting of 236 blood cultures growing staphylococci (including 124 methicillin-resistant Staphylococcus spp.), 50 positive blood cultures which grew organisms other than staphylococci, and 20 blood cultures that were negative for bacterial and fungal pathogens after 5 days of incubation and terminal subculture. DNA extraction, PCR, and detection could be completed in 2.5 h. Of the positive blood cultures with staphylococci, the multiplex PCR assay had a sensitivity and specificity of 99.2% and 100%, respectively. Our results show that rapid, direct detection of methicillin-resistant staphylococci is possible, allowing clinicians to make prompt and effective decisions for the management of patients with staphylococcal bacteremia.

Bacteremia↗
PubMed1998

Multiple genetic diagnoses from single cells using multiplex PCR: reliability and allele dropout.

We used a multiplex fluorescent PCR system containing seven primer sets on single cells from three different cell types (buccal, corneal and blastomere cells) and more than 3500 heterozygous alleles to investigate reliability and extent of allele dropout in multiplex PCRs at the single cell level. All three cell types gave similarly high reliability, accuracy and allele dropout rates, with similar reliability between singleplex and multiplex PCRs. Allele dropout was also consistent between the three cell types and did not significantly increase as allele size increased. These results indicate that multiplex fluorescent PCR is a reliable and accurate method of obtaining multiple diagnosis (eight chromosomes simultaneously) from single cells and maximizes the information available from single cell analysis.

Alleles↗
PubMed1996

Identification of root rot fungi in nursery seedlings by nested multiplex PCR.

The internal transcribed spacer (ITS) of the ribosomal DNA (rDNA) subunit repeat was sequenced in 12 isolates of Cylindrocladium floridanum and 11 isolates of Cylindrocarpon destructans. Sequences were aligned and compared with ITS sequences of other fungi in GenBank. Some intraspecific variability was present within our collections of C. destructans but not in C. floridanum. Three ITS variants were identified within C. destructans, but there was no apparent association between ITS variants and host or geographic origin. Two internal primers were synthesized for the specific amplification of portions of the ITS for C. floridanum, and two primers were designed to amplify all three variants of C. destructans. The species-specific primers amplified PCR products of the expected length when tested with cultures of C, destructans and C. floridanum from white spruce, black spruce, Norway spruce, red spruce, jack pine, red pine, and black walnut from eight nurseries and three plantations in Quebec. No amplification resulted from PCR reactions on fungal DNA from 26 common contaminants of conifer roots. For amplifications directly from infected tissues, a nested primer PCR using two rounds of amplification was combined with multiplex PCR approach resulting in the amplification of two different species-specific PCR fragments in the same reaction. First, the entire ITS was amplified with one universal primer and a second primer specific to fungi; a second round of amplification was carried out with species-specific primers that amplified a 400-bp PCR product from C. destructans and a 328-bp product from C. floridanum. The species-specific fragments were amplified directly from infected roots from which one or the two fungi had been isolated.

Base Sequence↗
PubMed2002

A multiplex-PCR for the differentiation of Mycobacterium bovis and Mycobacterium tuberculosis.

A multiplex-polymerase chain reaction (PCR) assay based on one-step amplification and detection of two different mycobacterial genomic fragments was designed for differentiation of Mycobacterium bovis and Mycobacterium tuberculosis. The oligonucleotide primers were chosen from a 500-bp genomic fragment which is well conserved in M. bovis and the pncA gene (based on M. tuberculosis-specific nucleotide polymorphism, a cytosine residue at position 169), specific for M. tuberculosis. The multiplex-PCR allowed detection of a single product of 500 bp in M. bovis isolates while M. tuberculosis isolates generated a single product of 185 bp, with or without an additional product of 500 bp. None of the atypical mycobacterial isolates revealed any amplification products. The method was found to be highly specific and could detect as little as 20 pg of pure DNA. This multiplex-PCR assay, based on the 500-bp fragment and the pncA gene, may be very useful for the rapid and specific differentiation of these two closely related mycobacteria and easy to use in medical and veterinary microbiological laboratories.

Amidohydrolases↗
PubMed2005

Typing of Y chromosome SNPs with multiplex PCR methods.

We describe a method for the simultaneous typing of Y-chromosome single nucleotide polymorphism (SNP) markers by means of multiplex polymerase chain reaction (PCR) strategies that allow the detection of 35 Y chromosome SNPs on 25 amplicons from 100 to 200 pg of chromosomal deoxyribonucleic acid (DNA). Multiplex PCR amplification of the DNA was performed with slight modifications of standard PCR conditions. Single-base extension (SBE) was performed using the SNaPshot kit containing fluorescently labeled ddNTPs. The extended primers were detected on an ABI 3100 sequencer. The most important factors for the creation of larger SNP typing PCR multiplexes include careful selection of primers for the primary amplification and the SBE reaction, use of DNA primers with homogenous composition, and balancing the primer concentrations for both the amplification and the SBE reactions.

Base Sequence↗
PubMed2004

Multiplex PCR for rapid detection of pseudorabies virus, porcine parvovirus and porcine circoviruses.

A multiplex PCR (mPCR) assay was developed and subsequently evaluated for its effectiveness as a means to simultaneously detect multiple viral infections of swine. Specific primers for each of four common DNA viruses, namely, pseudorabies virus (PRV), porcine circovirus type I (PCV1), porcine circovirus type II (PCV2), and porcine parvovirus (PPV), were used for testing procedure. The assay was shown to be highly sensitive in that as little as 10(-4) ng of each of the respective amplicons (approximately equal to 10,000 molecules) was detected when a composite of all four viruses (including both field and gene-deleted permutations of PRV) was tested as a single sample. It was also effective for detecting one or more of these same viruses in various combinations in specimens including lymph nodes, lungs, spleens, and tonsils collected from clinically ill pigs, and in specimens in spleen collected from aborted fetuses. The relative efficiency (compared to performing separate assays for each virus) and apparent sensitivity of mPCR suggest its potential application for routine molecular diagnostic purposes.

Animals↗
PubMed2004

Automation of a fluorescence-based multiplex PCR for the laboratory confirmation of common bacterial pathogens.

A fluorescence-based multiplex PCR was automated for the simultaneous detection of Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae in clinical samples from patients with suspected meningitis. Sensitivity of one to two genome copies per 100 microl sample and specificity of 100% for each organism were shown. Automation of DNA extraction, liquid handling, PCR and analysis are achieved on a single platform, which enables a high throughput and rapid turnaround of clinical samples that, in turn, leads to faster diagnosis. This is ultimately beneficial to the treatment of the patient and for public health management.

Adolescent↗
PubMed2004

Modified multiplex PCR method for detection of pyrogenic exotoxin genes in staphylococcal isolates.

A modified multiplex PCR method for detection of nine Staphylococcus aureus enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, and sej) and one form of immunoreactive toxic shock syndrome toxin based on a previously published method (S. R. Monday and G. A. Bohach, J. Clin. Microbiol. 37:3411-3414, 1999) has been developed. The modified PCR protocol seems robust and gives reliable results.

Bacterial Toxins↗
PubMed2005

A collagenase-targeted multiplex PCR assay for identification of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus.

A multiplex PCR assay using three collagenase-targeted primer pairs for the species-specific detection of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus was developed. The results highlight the species specificity of the three primer sets designed. Because of the increasing importance of Vibrio spp. in human foodborne diseases, molecular approaches for routine microbial screening and monitoring of clinical, environmental, and food samples also have become more important. The results of this study indicate that the gene coding for collagenase should be used as an alternative molecular target to discriminate among the three Vibrio species.

DNA Primers↗
PubMed1990

Multiplex PCR amplification and immobilized capture probes for detection of bacterial pathogens and indicators in water.

Detection of pathogens (Legionella species) and indicator bacteria (coliform bacteria) was achieved by multiplex (simultaneous) PCR amplification of diagnostic gene sequences and by hybridization to immobilized poly-dT-tailed capture probes using a dot- or slot-blot approach. Complex manipulations of primer concentrations and staggered additions of primers were required in order to achieve equal amplification of multiple genes. Multiplex PCR amplification of two different Legionella genes, one specific for L. pneumophila (mip) and the other for the genus Legionella (5S rRNA), was achieved by staggered amplification. Multiplex PCR amplification using differing amounts of primers specific for lacZ and lamB genes permitted the detection of coliform bacteria and those associated with human faecal contamination, including the indicator bacterial species E. coli and enteric pathogens Salmonella and Shigella. Hybridization of biotin-labelled amplified DNA, in which the biotin was incorporated during PCR amplification from biotinylated-dUTP, to immobilized 400-dT-tailed capture probes permitted specific and sensitive detection of target gene sequences. The sensitivity of colorimetric detection achieved by PCR amplification of target DNA was at a level equivalent to 1-2 bacterial cells, which is the same level of sensitivity obtained with radioactive detection. The simultaneous amplification of several genes and hybridization to immobilized capture probes with colorimetric detection is an effective, efficient and rapid detection method for various human bacterial pathogens.

Bacteriological Techniques↗
PubMed2003

Rapid identification of the species of the Bacteroides fragilis group by multiplex PCR assays using group- and species-specific primers.

We report a rapid and reliable two-step multiplex polymerase chain reaction (PCR) assay to identify the 10 Bacteroides fragilis group species - Bacteroides caccae, B. distasonis, B. eggerthii, B. fragilis, B. merdae, B. ovatus, B. stercoris, B. thetaiotaomicron, B. uniformis and B. vulgatus. These 10 species were first divided into three subgroups by multiplex PCR-G, followed by three multiplex PCR assays with three species-specific primer mixtures for identification to the species level. The primers were designed from nucleotide sequences of the 16S rRNA, the 16S-23S rRNA intergenic spacer region and part of the 23S rRNA gene. The established two-step multiplex PCR identification scheme was applied to the identification of 155 clinical isolates of the B. fragilis group that were previously identified to the species level by phenotypic tests. The new scheme was more accurate than phenotypic identification, which was accurate only 84.5% of the time. The multiplex PCR scheme established in this study is a simple, rapid and reliable method for the identification of the B. fragilis group species. This will permit more accurate assessment of the role of various B. fragilis group members in infections and of the degree of antimicrobial resistance in each of the group members.

Bacteriological Techniques↗