Showing papers on "Multiplex polymerase chain reaction published in 2010"

Development of a set of multiplex PCR assays for the detection of genes encoding important β-lactamases in Enterobacteriaceae

Abstract: Objectives To develop a rapid and reliable tool to detect by multiplex PCR assays the most frequently widespread beta-lactamase genes encoding the OXA-1-like broad-spectrum beta-lactamases, extended-spectrum beta-lactamases (ESBLs), plasmid-mediated AmpC beta-lactamases and class A, B and D carbapenemases. Methods Following the design of a specific group of primers and optimization using control strains, a set of six multiplex PCRs and one simplex PCR was created. An evaluation of the set was performed using a collection of 31 Enterobacteriaceae strains isolated from clinical specimens showing a resistance phenotype towards broad-spectrum cephalosporins and/or cephamycins and/or carbapenems. Direct sequencing from PCR products was subsequently carried out to identify beta-lactamase genes. Results Under optimized conditions, all positive controls confirmed the specificity of group-specific PCR primers. Except for the detection of carbapenemase genes, multiplex and simplex PCR assays were carried out using the same PCR conditions, allowing assays to be performed in a single run. Out of 31 isolates selected, 22 strains produced an ESBL, mostly CTX-M-15 but also CTX-M-1 and CTX-M-9, SHV-12, SHV-5, SHV-2, TEM-21, TEM-52 and a VEB-type ESBL, 6 strains produced a plasmid-mediated AmpC beta-lactamase (five DHA-1 and one CMY-2) and 3 strains produced both an ESBL (two SHV-12, one CTX-M-15) and a plasmid-mediated AmpC beta-lactamase (DHA-1). Conclusions We report here the development of a useful method composed of a set of six multiplex PCRs and one simplex PCR for the rapid screening of the most frequently encountered beta-lactamases. This method allowed direct sequencing from the PCR products.

Genotyping of Toxoplasma gondii Isolates with 15 Microsatellite Markers in a Single Multiplex PCR Assay

Abstract: We developed an easy-to-use method for genotyping Toxoplasma gondii isolates in a single multiplex PCR assay with 15 microsatellite markers. This method was validated by testing 26 reference isolates that had been characterized with other sets of markers.

MPprimer: a program for reliable multiplex PCR primer design

Abstract: Background Multiplex PCR, defined as the simultaneous amplification of multiple regions of a DNA template or multiple DNA templates using more than one primer set (comprising a forward primer and a reverse primer) in one tube, has been widely used in diagnostic applications of clinical and environmental microbiology studies. However, primer design for multiplex PCR is still a challenging problem and several factors need to be considered. These problems include mis-priming due to nonspecific binding to non-target DNA templates, primer dimerization, and the inability to separate and purify DNA amplicons with similar electrophoretic mobility.

Abundance of Multiple High-Risk Human Papillomavirus (HPV) Infections Found in Cervical Cells Analyzed by Use of an Ultrasensitive HPV Genotyping Assay

Abstract: PCR methods enable the detection of a large variety of human papillomavirus (HPV) genotypes that infect the anogenital tract. However, PCR with consensus primers, general primers, and, to a lesser extent, broad-spectrum primers may underrepresent the true prevalence of HPV, especially the true prevalence of multiple infections. We compared the rate of HPV positivity determined by a broad-spectrum PCR with primers BSGP5+ and BSGP6+ (BS-PCR) coupled to an established bead-based multiplex HPV genotyping (MPG) assay with the rate of HPV positivity determined by a multiplex PCR with type-specific primers (TS-PCR) coupled to a newly developed MPG assay for 735 selected cervical scraping samples. While the primers used for the BS-PCR are located within the L1 region of the HPV genome, the primers used for the TS-PCR target the E7 gene. The overall rates of positivity for the 19 HPV types included in both assays were 60.9% and 72.2% by the BS-PCR and the TS-PCR, respectively, and the two assays found multiple infections in 34.8% and 58.0% of the specimens, respectively. Both HPV detection assays allowed the semiquantitative detection of HPV types and identified the same dominant HPV type in 66.6% of the multiple infections. In conclusion, the TS-PCR-MPG assay significantly increased the rate of detection of HPV DNA and the number of infections with multiple HPV types detected and demonstrated that the prevalence of low-copy-number HPV infections in the anogenital tract may be strongly underestimated by conventional HPV amplification methods, especially in cases of multiple infections. As a consequence, PCR-TS-MPG appears to be highly suited for analysis of the significance of multiple infections in the development of cervical cancer and for the study the natural history and the latency of HPV.

An Optimized Pentaplex PCR for Detecting DNA Mismatch Repair-Deficient Colorectal Cancers

Abstract: Purpose Microsatellite instability (MSI) is used to screen colorectal cancers (CRC) for Lynch Syndrome, and to predict outcome and response to treatment. The current technique for measuring MSI requires DNA from normal and neoplastic tissues, and fails to identify tumors with specific DNA mismatch repair (MMR) defects. We tested a panel of five quasi-monomorphic mononucleotide repeat markers amplified in a single multiplex PCR reaction (pentaplex PCR) to detect MSI.

The development of a mRNA multiplex RT-PCR assay for the definitive identification of body fluids.

Abstract: With current methodology, DNA profiling can identify an individual from a sample of biological material but it does not reveal what body fluid or tissue source the DNA profile originated from. We have developed a multiplex PCR system using messenger RNA (mRNA) that can identify blood, saliva, semen and menstrual blood in individual stains or in mixtures of body fluids. Messenger RNA transcripts specific to each type of body fluid have been identified and a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) system developed to identify these body fluids along with three housekeeping genes. This multiplex can detect semen and seminal fluid (semen without spermatozoa present). Furthermore, we have targeted the co-isolation of RNA and DNA from the same sample and, with the RT-PCR multiplex, we can determine the type of body fluid present as well as generate a DNA profile(s) from the same stain.

A Multiplex Real-Time Polymerase Chain Reaction for Simultaneous Detection of Salmonella spp., Escherichia coli O157, and Listeria monocytogenes in Meat Products

Abstract: To achieve an effective detection of Salmonella spp., Escherichia coli O157, and Listeria monocytogenes in meat products, a multiplex real-time polymerase chain reaction (PCR) coupled with a multipathogen enrichment strategy was developed in this study. Pathogen-specific DNA sequences in the invA, rfbE, and hlyA genes were employed to design primers and TaqMan probes for identifying Salmonella spp., E. coli O157, and L. monocytogenes, respectively. An internal amplification control (IAC) utilizing a novel DNA sequence from human adenovirus was incorporated into the multiplex PCR assay to indicate false-negative results. Concurrent amplifications of multiple targets and IAC were thoroughly evaluated and optimized to minimize PCR competitions. Combined with a multipathogen enrichment in a selective enrichment broth for Salmonella, Escherichia, and Listeria (SEL), the multiplex real-time PCR assay was able to simultaneously detect all of the three organisms in artificially contaminated ground beef a...

A simplified multiplex PCR assay for fast and easy discrimination of globally distributed staphylococcal cassette chromosome mec types in meticillin-resistant Staphylococcus aureus.

Abstract: A multiplex PCR assay was developed for the identification of major types and subtypes of staphylococcal cassette chromosome mec (SCCmec) in meticillin-resistant Staphylococcus aureus (MRSA) strains. The method uses a novel 9 valent multiplex PCR plus two primer pairs for S. aureus identification and detection of meticillin resistance. All 389 clinical MRSA isolates from Malaysia and 18 European isolates from the Harmony collection harbouring different SCCmec types that we tested were correctly characterized by our PCR assay. SCCmec type III and V were by far the most common types among both hospital- and community-acquired Malaysian MRSA isolates, with an apparent emergence of MRSA harbouring the IVh type.

Molecular detection of invA and spv virulence genes in Salmonella enteritidis isolated from human and animals in Iran

Abstract: It is important to study the genotypic diversity of Salmonella plasmid genes which are responsible for its virulence. In the present study multiplex polymerase chain reaction (multiplex PCR) assay was carried out for detection of Salmonella enteritidisand presence of invA and spv genes. In the first stage of the study, 1001 poultry samples were collected from a slaughterhouse in Kerman province (southern Iran). Biochemical and serological tests were then performed for identification ofSalmonella serovars and 6.79% (68/1001) were positive for Salmonella. Multiplex PCR with three set primers was then applied to confirm serovar enteritidis 51.4% (35/68). Simple-PCR was then applied to detect spvA (Salmonella plasmid virulence), and spvB genes. Finally, multiplex PCR assay was carried out to simultaneously detect and identify invA and spvC genes. The presence of spvA,spvB and spvC in S. enteritidis was 88.6% for each gene. In the second stage of the study, thirty-three bovine (n = 13) and human (n = 20) S. enteritidis strains were isolated from the culture collection in the Department of Microbiology, Faculty of Veterinary Medicine, University of Tehran, Iran. The analyses of the samples revealed that spvA, spvB, and spvC genes were present in 90% of S. enteritidis from human sources as compared to 100% in bovine sources. The study represents the first report in Iran about the genotypic diversity of spvA, spvB and spvC genes of S. enteritidis. Key words: Salmonella enteritidis, multiplex PCR, virulence genes.

Detection and Characterization of Human Ureaplasma Species and Serovars by Real-Time PCR

Abstract: We designed primers and probes for the detection and discrimination of Ureaplasma parvum and U. urealyticum and their 14 serovars by real-time PCR. The analytical sensitivity and specificity of the multiplex species-specific PCR were determined by testing corresponding American Type Culture Collection (ATCC) type strains, 47 other microbial species, and human genomic DNA. The limits of the multiplex PCR were 2.8 x 10(-2) CFU/microl PCR mixture for detecting U. parvum and 4.1 x 10(-2) CFU/microl PCR mixture for detecting U. urealyticum. Clinical specificity and sensitivity were proven by comparison with culture and traditional PCR. For the detection of any Ureaplasma species, the clinical sensitivity and specificity of real-time PCR were 96.9% and 79.0%, respectively, using culture as a reference. Multiplex real-time PCR was also more sensitive than traditional PCR in discriminating the two Ureaplasma species in culture-positive subcultures. Each of the 14 monoplex serovar-specific PCR assays was specific for the corresponding ATCC type strain serovar. This new species identification PCR is specific and sensitive in the detection of Ureaplasma species in clinical specimens, and the serovar-specific PCR assays are the first set of complete genotypic assays to differentiate all 14 known Ureaplasma serovars. These assays provide quick and reliable means for investigating the epidemiology and pathogenicity of ureaplasmas at the serovar level.

Multiplex real-time polymerase chain reaction assay for simultaneous detection and quantification of Salmonella species, Listeria monocytogenes, and Escherichia coli O157:H7 in ground pork samples.

Abstract: Salmonella sp., Listeria monocytogenes, and Escherichia coli O157:H7 are foodborne pathogens capable of causing serious gastrointestinal illness. We previously described simultaneous detection of these pathogens by multiplex polymerase chain reaction (PCR) in 44 types of spiked food samples, including meat, produce, fish, and dairy products, targeting genes specific for each pathogen. Based on the previous work, a multiplex real-time PCR assay using fluorescent probes was developed to detect and accurately quantify Salmonella sp., L. monocytogenes, and E. coli O157:H7 in ground pork samples. The detection sensitivity for this method was 2.0 × 102 CFU/mL for each pathogen, and the quantification range was 102–107 CFU/mL with a high correlation coefficient (R2 > 0.99) and high PCR efficiency (84.2% to 99.2%). When this protocol was used for the detection of each of the pathogens in spiked pork samples, one cell per 25 g of inoculated sample after enrichment for 20 h could be detected within 24 h. A...

Rapid diagnostic multiplex PCR (RD-PCR) to discriminate Schistosoma haematobium and S. bovis.

Abstract: Schistosoma haematobium and S. bovis are widespread schistosome species causing human and cattle schistosomiasis, respectively, in Africa. The sympatric occurrence of these two species and their ability to infect the same Bulinus intermediate snail hosts necessitates precise methods of identification of the larval stages. A rapid diagnostic 'mulitplex' one-step polymerase chain reaction protocol (RD-PCR) was developed using cytochrome oxidase subunit 1 (COX1) mitochondrial DNA (mtDNA) to discriminate between S. haematobium and S. bovis. A single forward primer and two species-specific reverse primers were used to produce a polymerase chain reaction (PCR) fragment of 306 bp and 543 bp for S. bovis and S. haematobium, respectively. Serial dilutions were carried out on various lifecycle stages and species combinations to test the sensitivity and specificity of the primers. This RD-PCR proved highly sensitive, detecting a single larval stage and as little as 0.78 ng of genomic DNA (gDNA) from an adult schistosome, providing a cost-effective, rapid and robust molecular tool for high-throughput screening of S. haematobium and S. bovis populations. In areas where human and cattle schistosomiasis overlap and are transmitted in close proximity, this mitochondrial assay will be a valuable identification tool for epidemiological studies, especially when used in conjunction with other nuclear diagnostic markers.

Efficient differentiation of Mycobacterium avium complex species and subspecies by use of five-target multiplex PCR.

Abstract: Infections caused by the Mycobacterium avium complex (MAC) are on the rise in both human and veterinary medicine. A means of effectively discriminating among closely related yet pathogenetically diverse members of the MAC would enable better diagnosis and treatment as well as further our understanding of the epidemiology of these pathogens. In this study, a five-target multiplex PCR designed to discriminate MAC organisms isolated from liquid culture media was developed. This MAC multiplex was designed to amplify a 16S rRNA gene target common to all Mycobacterium species, a chromosomal target called DT1 that is unique to M. avium subsp. avium serotypes 2 and 3, to M. avium subsp. silvaticum, and to M. intracellulare, and three insertion sequences, IS900, IS901, and IS1311. The pattern of amplification results allowed determination of whether isolates were mycobacteria, whether they were members of the MAC, and whether they belonged to one of three major MAC subspecies, M. avium subsp. paratuberculosis, M. avium subsp. avium, and M. avium subsp. hominissuis. Analytical sensitivity was 10 fg of M. avium subsp. paratuberculosis genomic DNA, 5 to 10 fg of M. avium subsp. avium genomic DNA, and 2 to 5 fg of DNA from other mycobacterial species. Identification accuracy of the MAC multiplex was evaluated by testing 53 bacterial reference strains consisting of 28 different mycobacterial species and 12 nonmycobacterial species. Identification accuracy in a clinical setting was evaluated for 223 clinical MAC isolates independently identified by other methods. Isolate identification agreement between the MAC multiplex and these comparison assays was 100%. The novel MAC multiplex is a rapid, reliable, and simple assay for discrimination of MAC species and subspecies in liquid culture media.

A highly sensitive and specific multiplex PCR assay for simultaneous detection of Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus

Abstract: Aims: To develop an effective multiplex PCR for simultaneous and rapid detection of Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus, the three most important Vibrio species that can cause devastating health hazards among human. Methods and Results: Species-specific PCR primers were designed based on toxR gene for V. cholerae and V. parahaemolyticus, and vvhA gene for V. vulnificus. The multiplex PCR was validated with 488 Vibrio strains including 322 V. cholerae, 12 V. vulnificus, and 82 V. parahaemolyticus, 20 other Vibrio species and 17 other bacterial species associated with human diseases. It could detect the three target bacteria without any ambiguity even among closely related species. It showed good efficiency in detection of co-existing target species in the same sample. The detection limit of all the target species was ten cells per PCR tube. Conclusions: Specificity and sensitivity of the multiplex PCR is 100% each and sufficient for simultaneous detection of these potentially pathogenic Vibrio species in clinical and environmental samples. Significance and Impact of the Study: This simple, rapid and cost-effective method can be applicable in a prediction system to prevent disease outbreak by these Vibrio species and can be considered as an effective tool for both epidemiologist and ecologist.

Development of a qualitative, multiplex real-time PCR kit for screening of genetically modified organisms (GMOs)

Abstract: The number of commercially available genetically modified organisms (GMOs) and therefore the diversity of possible target sequences for molecular detection techniques are constantly increasing. As a result, GMO laboratories and the food production industry currently are forced to apply many different methods to reliably test raw material and complex processed food products. Screening methods have become more and more relevant to minimize the analytical effort and to make a preselection for further analysis (e.g., specific identification or quantification of the GMO). A multiplex real-time PCR kit was developed to detect the 35S promoter of the cauliflower mosaic virus, the terminator of the nopaline synthase gene of Agrobacterium tumefaciens, the 35S promoter from the figwort mosaic virus, and the bar gene of the soil bacterium Streptomyces hygroscopicus as the most widely used sequences in GMOs. The kit contains a second assay for the detection of plant-derived DNA to control the quality of the often processed and refined sample material. Additionally, the plant-specific assay comprises a homologous internal amplification control for inhibition control. The determined limits of detection for the five assays were 10 target copies/reaction. No amplification products were observed with DNAs of 26 bacterial species, 25 yeasts, 13 molds, and 41 not genetically modified plants. The specificity of the assays was further demonstrated to be 100% by the specific amplification of DNA derived from reference material from 22 genetically modified crops. The applicability of the kit in routine laboratory use was verified by testing of 50 spiked and unspiked food products. The herein described kit represents a simple and sensitive GMO screening method for the reliable detection of multiple GMO-specific target sequences in a multiplex real-time PCR reaction.