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Results for “Multiplex PCR”

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PubMed2001

Fully integrated PCR-capillary electrophoresis microsystem for DNA analysis.

A fully integrated genomic analysis microsystem including microfabricated heaters, temperature sensors, and PCR chambers directly connected to capillary electrophoretic separation channels has been constructed. Valves and hydrophobic vents provide controlled and sensorless sample positioning and immobilization into 200 nL PCR chambers. The use of microfabricated heating and temperature sensing elements improves the heating and cooling rates for the PCR reaction to 20 degree C s(-1). The amplified PCR product, labeled on-column with an intercalating fluorescent dye, is injected into the gel-filled capillary for electrophoretic analysis. Successful sex determination using a multiplex PCR reaction from human genomic DNA is demonstrated in less than 15 min. This device is an important step toward a microfabricated genomic microprocessor for use in forensics and point-of-care molecular medical diagnostics.

DNA↗

PubMed1999

High-resolution analysis of gene rearrangements in lymphoid malignancies.

As T-cell receptor and immunoglobulin gene rearrangements provide specific clonal markers for lymphoid cell proliferations, analysis of these genes is useful for distinguishing between reactive and malignant disease. We have developed an automated, high-resolution analysis of PCR fragments to identify clonally rearranged TCR-gamma (TCR gamma) genes and IgH genes. Consensus primers are used to detect the majority of possible rearrangements in multiplex PCR assays, and the PCR products are fluorescently labelled for visualisation with ABI Genescan software. Polyclonal populations of lymphoid cells are represented by a spectrum of fragments, whereas a monoclonal population of cells is represented by one or two discrete bands, indicating rearrangement of one or both alleles. For TCR-gamma PCR, the rearranged DNA fragment from a monoclonal population of T-cells diluted to 0.1% in DNA from a polyclonal population of cells is still readily distinguishable from the polyclonal background. Similarly, for IgH PCR, the gene rearrangement from a monoclonal population of B-cells is still distinguishable to 0.5% in a polyclonal background. As this technique allows semi-quantitative resolution of fragments one base different in size, it is ideal for detecting monoclonal and oligoclonal populations of B- and T-cells. The accurate size determination of PCR fragments also minimises the risk of false positives resulting from contamination, as individual monoclonal rearrangements are frequently patient-specific on the basis of size alone.

Clone Cells↗

PubMed1991

OSP: a computer program for choosing PCR and DNA sequencing primers.

OSP (Oligonucleotide Selection Program) selects oligonucleotide primers for DNA sequencing and the polymerase chain reaction (PCR). The user can specify (or use default) constraints for primer and amplified product lengths, %(G+C), (absolute or relative) melting temperatures, and primer 3' nucleotides. To help minimize nonspecific priming and primer secondary structure, OSP screens candidate primer sequences, using user-specifiable cutoffs, against potential base-pairing with a variety of sequences present in the reaction, including the primer itself, the other primer (for PCR), the amplified product, and any other sequences desired (e.g., repetitive element sequences in genomic templates, vector sequence in cloned templates, or other primer pair sequences in multiplexed PCR reactions). Base-pairing involving the primer 3' end is considered separately from base-pairing involving internal sequences. Primers meeting all constraints are ranked by a "combined score," a user-definable weighted sum of any of the above parameters. OSP is being routinely and extensively used to select sequencing primers for the Caenorhabditis elegans genome sequencing project and human genomic PCR primer pairs for the Washington University Genome Center mapping project, with success rates exceeding 96% and 81%, respectively. It is available for research purposes from the authors, at no cost, in both text output and interactive graphics (X windows) versions.

Animals↗

PubMed1992

Analysis of quantitative PCR for the diagnosis of deletion and duplication carriers in the dystrophin gene.

A direct, non-radioactive method of quantitative PCR amplification has been investigated for the diagnosis of deletion and duplication carriers in the dystrophin gene. The simultaneous amplification of two loci, or several loci using multiplex PCR, allows for the direct comparison of relative amounts of products from normal homozygous loci and potentially heterozygous deleted/duplicated loci. Sufficient cycles of PCR are performed to enable visual analysis or densitometric quantification of products on ethidium bromide stained gels. The method has been verified in blind trials performed on known genotypes and by showing that under the conditions used the assay remains within the exponential phase of amplification.

Chromosome Deletion↗

PubMed2000

Rapid combined assay for Salmonella detection in food samples.

A rapid method was developed to detect salmonellae in food samples. The method gave a possibility to obtain results after 28 h 30 min. The preenrichment in buffered peptone water lasted for 6 h, the enrichment in Rappaport-Vassiliadis medium was applied for 18 h followed by PCR with INVA1-INVA2 primer pair, adapting Chiu and Ou's method. This procedure was suitable to demonstrate salmonella contamination at min. 10 cfu/25 g sample. Out of 18 samples there was a good agreement between the results of the conventional and rapid methods in case of 17 samples. PCR with SPVC1-SPVC2 primer pair informing about the presence of virulence plasmid was performed in separate tubes, because decreased sensitivity was observed in case of multiplex PCR.

Culture Media↗

PubMed1998

Strategies for preimplantation genetic diagnosis of single gene disorders by DNA amplification.

Preimplantation genetic diagnosis (PGD) is an alternative to prenatal diagnosis for ensuring the genetic health of offspring born to families affected by inherited disease. This paper sets out to review current protocols for the diagnosis of single gene defects in human preimplantation embryos. These methods, which depend on DNA amplification using PCR, are subject to a variety of pitfalls, such as allele dropout (ADO), contamination and reduced amplification efficiency. Advances in single cell DNA amplification, such as improved multiplex PCR protocols, fluorescent-PCR and whole genome amplification (WGA), can be applied to address some of these problems. Different PGD strategies are discussed in the context of their clinical application.

DNA Mutational Analysis↗

PubMed2006

Detection of fetal DNA in maternal plasma by microarray coupled with emulsions PCR.

BACKGROUND: The presence of fetal DNA in maternal plasma made non-invasive prenatal diagnosis possible. Although fetal DNA has been used in several genetic disease diagnoses, many challenges remained in the detection methods. We attempted to develop a sensitive and reliable microarray coupled with emulsions PCR method to detect the fetal DNA in the plasma of pregnant women. METHOD: Fetal DNAs extracted from the plasma of pregnant women were amplified in emulsions, and fluorescence was labeled at the same time. The labeled target DNAs were hybridized and detected by the capturing DNA probes on a modified slide. Six Y chromosome special sequences in gene of SRY, DYS and DYZ as the marker of fetal DNAs were detected simultaneously in this study, and the beta-globin gene was detected as marker of total DNA from maternal plasma. An unrelated sequence was also detected as negative control in this study. 76 pregnant women in the first trimester of gestation joined in this study. Conventional PCR and real time PCR were also carried out for comparison. RESULTS: We could detect the fetal DNAs reliably with this method in early stage of gestation. The 6Y chromosome sequences were detected in 40 of the 42 male fetus carrier samples, and no Y special sequence was detected in the female fetus carrier samples. The earliest plasma sample which we could detect in this study was collected on the 31st day after pregnancy. CONCLUSIONS: The results suggest that the microarray coupled with emulsions PCR method could be used for fetal DNA detections, and emulsions were useful in DNA amplification as reaction media to overcome the primer incompatibility which frequently encounters in multiplex PCR amplification. Our methods have the potential in high-throughput assays and could be widely used in clinical researches and diagnosis.

DNA↗

PubMed2004

Four-color multiplex 5' nuclease assay for the simultaneous detection of the factor V Leiden and the prothrombin G20210A mutations.

We developed a real-time multiplex four-color assay for the simultaneous detection of the factor V Leiden (FVL) and prothrombin (PT) G20210A mutations in one closed tube using a single thermocycling protocol. The assay combines the power of multiplex PCR with the specificity provided by allele-specific oligonucleotide (ASO) hybridization using the 5' nuclease assay format. Human genomic DNA is prepared from whole blood with standard procedures. A 97-bp DNA sequence of the coagulation factor V gene is co-amplified with a 111-bp DNA sequence of the coagulation factor II (PT) gene using four PCR primers. In addition, the reactions included four differentially labeled ASO probes for the specific detection of the different FVL/PT G20210A genotypes. To evaluate the assay's performance characteristics, we performed a comparison of two methods. We analyzed DNA samples from 52 individuals with known FVL/PT G20210A genotypes that were previously genotyped with an assay that combined PCR with the use of restriction fragment length polymorphisms. We found a 100% concordance between the results generated by both methodologies. We conclude that the four-color multiplex assay is specific and reproducible for the detection of the FVL/PT G20210A mutations, and it can be easily adapted for the detection of other SNPs.

Alleles↗

PubMed1999

Detection of clinically relevant genotypes of vancomycin-resistant enterococci in nosocomial surveillance specimens by PCR.

This study evaluated a PCR method for the rapid detection of clinically significant genotypes of vancomycin-resistant enterococci (VRE) in nosocomial surveillance specimens. Detection of the vanA and vanB genes by multiplex PCR using 657 specimens that showed presumptive growth of VRE on bile esculin azide agar containing 6 mg of vancomycin/liter was compared to the conventional method. The diagnostic values for the PCR compared to the phenotypic method were as follows: 99.8% specificity, 95.4% sensitivity, 98.8% positive predictive value, and 99.3% negative predictive value. The average cost per test for PCR is $8.26, compared to $9.45 for the phenotypic method. The average turnaround time for detecting a VRE is 48 h for PCR, compared to 96 h for the conventional method.

Cross Infection↗

PubMed2006

A universal microsatellite multiplex kit for genetic analysis of great apes.

DNA profiling with microsatellite markers is a commonly used genetic method of studying the great apes. An efficient method of generating the genetic data is amplification of multiple microsatellites in a single PCR reaction. Here we describe a PCR multiplex in which 9 genetic markers can be amplified simultaneously, thereby saving time, expenses and DNA. This marker system can discriminate between all the great ape species except bonobos and chimpanzees. Furthermore, the cumulative probability of identity values were low for all 4 species tested.

Animals↗

PubMed2003

Molecular approaches to the identification and treatment monitoring of periodontal pathogens.

Two different PCR-based molecular approaches, a commercial kit for detection of A. actinomycetemcomitans, P. gingivalis, P. intermedia, B. forsythus and T. denticola (Amplimedical "Paradonthosis") and a home-made multiplex PCR for A. actinomycetemcomitans, P. gingivalis and B. forsythus were compared for monitoring the efficacy of different dental treatments on localized persistent periodontal pockets. 44 sites were randomized in two treatment groups: mechanical treatment (22 control sites) and in conjunction with the application of tetracycline fibres (22 experimental sites). 40/44 sites were found positive with both tests for A. actinomycetemcomitans, P. gingivalis and B. forsythus pretheraphy. P. intermedia was detected alone in only three sites during the follow-up, while T. denticola. was always associated with the other pathogens. 20 sites were positive in conventional cultures for one to three of the pathogens. PCR-based approaches provided a sensitive and reliable method for identification and monitoring treatment of periodontal pathogens.

Actinobacillus Infections↗

PubMed2004

Application of extended single-reaction multiplex polymerase chain reaction for toxin typing of Staphylococcus aureus isolates in South Korea.

The extended single-reaction multiplex PCR (esr-mPCR) developed in this study to detect staphylococcal enterotoxins (SEs), including SEA, SEB, SEC, SED, SEE, SEH, SEI, and SEJ, requires fewer sets of primers than other conventional multiplex PCRs and can be used to detect newly identified staphylococcal enterotoxins SEs more readily. Esr-mPCR analysis of 141 isolates of Staphylococcus aureus obtained from abattoir and livestock product samples revealed that 27 of the S. aureus isolates were toxigenic, and two were 2 multitoxigenic isolates. The most prevalent SE type was SEI followed by SEA and SEH. In addition, we investigated the clonal relatedness of toxigenic S. aureus isolates by arbitrarily primed PCR (AP-PCR). AP-PCR analysis of toxigenic S. aureus isolates revealed that the discriminatory power of AP-PCR was 9 (D=0.81), 8 (D=0.77), and 10 types (D=0.83) with primers AP1, ERIC2, and AP7, respectively. The combination of three each AP-PCR result could rearrange toxigenic S. aureus isolates into 10 types and five subtypes, with the D-value of 0.92. Interestingly, our data showed that toxigenic S. aureus isolates from different sources had different fingerprinting patterns although some of them carried the same types of SE genes. These data suggest that combinations of esr-mPCR and AP-PCR can provide a powerful approach for epidemiological investigation of toxigenic S. aureus isolates.

Abattoirs↗

PubMed2004

Forensic DNA-typing of dog hair: DNA-extraction and PCR amplification.

The forensic application of DNA-typing for the identification of dog hair provides objective evidence in the characterisation of traces found at crime scenes. During the past few years forensic dog identity testing has been improved considerably using multiplex PCR systems. However, DNA-typing from samples of one up to 10 dog hairs is often problematic in forensic science. A single dog hair contains very small quantities of DNA or the hair sample consists of hairs with roots of bad quality or even of broken hairshafts without roots. Here we describe an experimental study about dog hairs by means of a Ca(2+) improved DNA-extraction method, quantification and amplification.

Animals↗

PubMed1999

Swiss Caucasian population data for 13 STR loci using AmpFISTR profiler plus and cofiler PCR amplification kits.

Allele and genotype frequencies for the 13 core STR loci (D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, THO1, TPOX, CSF1PO, and D16S539) were determined in a Swiss Caucasian population sample (n = 206) using two commercially available multiplex PCR kits (AmpFISTR Profiler Plus and AmpFISTR Cofiler) and subsequent electrophoresis on an ABI PRISM CE 310 Genetic Analyzer instrument. All loci meet Hardy-Weinberg expectations. In addition, there is little evidence for association of alleles among the 13 loci. The allelic frequency data can be used in forensic analyses and paternity tests to estimate the frequency of a multiple STR locus DNA profile in the Swiss population.

Alleles↗

PubMed2001

Validation and casework application of a Y chromosome specific STR multiplex.

A series of validation experiments was performed for a Y chromosome specific STR multiplex system following the suggestions made by the Technical Working Group DNA Analysis Methods (TWGDAM). The multiplex PCR products were detected on Perkin-Elmer 373 and 377 automated sequencers using two labeling colors. No problems regarding the stability, robustness and sensitivity of the Y STR multiplex were observed. Mixture studies revealed a cut off rate similar to autosomal STRs for mixtures of male DNAs and no interference of any female admixture. The comparison of the Y STR results to the autosomal typing results for 56 nonprobative semen stains and swabs, showed a slightly higher success rate in detecting the semen donor's alleles for the Y STR multiplex. Two examples are shown to illustrate the usefulness of Y STR typing for DNA mixtures. In one case the Y STR results confirmed an isolated exclusion; in the other case, the interpretation of a mixture was clarified since the Y STR results proved the presence of DNA from at least two semen donors. Y STR typing is a valuable addition to the forensic DNA testing panel.

Alleles↗

PubMed2006

Detection of Salmonella enterica serovar Typhi (S. Typhi) by selective amplification of invA, viaB, fliC-d and prt genes by polymerase chain reaction in mutiplex format.

AIMS: Development of a PCR assay that can target multiple genes for rapid detection of Salmonella enterica serovar Typhi (S. Typhi) from water and food samples. METHODS AND RESULTS: PCR primers for invasion, O, H and Vi antigen genes, invA, prt, fliC-d and viaB were designed and used for the rapid detection of S. Typhi by multiplex PCR. Internal amplification control, which co-amplified with prt primers, was also included in the assay. The results showed that all cultures of Salmonella were accurately identified by the assay with no nonspecific amplification in other cultures. The assay had 100% detection probability when a cell suspension of 10(4) CFU ml(-1) (500 CFU per reaction) was used. Salmonella Typhi bacteria were artificially inoculated in the water and food (milk and meat rinse) samples and detected by mPCR after overnight pre-enrichment in buffered peptone water. No Salmonella bacteria could be detected from water samples collected from the field by mPCR or standard culture method. CONCLUSIONS: The developed mPCR assay provides specific detection of S. Typhi. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid methods for detection of S. Typhi from complex environmental matrices are almost nonexistent. The mPCR assay reported in this study can be useful to identify S. Typhi bacteria in field environmental samples.

Antigens, Bacterial↗

PubMed2005

Use of pyrosequencing to differentiate Streptococcus pneumoniae serotypes 6A and 6B.

Accurate serotyping of Streptococcus pneumoniae remains important to monitor the changes in seroepidemiology of the organism over time. Though several PCR-based systems have been developed for this purpose, the cross-reactivity within serogroups often limits discrimination between types. All serogroup 6 isolates can be identified using a multiplex PCR system; however, due to the high sequence homology between the cps-6B and cps-6A loci, serotypes 6A and 6B cannot be differentiated by this method. We describe the use of pyrosequencing to reliably differentiate between serotypes 6A and 6B using a previously described single nucleotide polymorphism at codon 195 of the cps locus wciP gene. We observed complete concordance between capsular serotyping results and wciP pyrosequencing among 210 isolates examined, indicating that pyrosequencing is a rapid and accurate technique for deducing serotypes 6A and 6B.

Bacterial Capsules↗

PubMed1998

Simplified polymerase chain reaction detection of new world Leishmania in clinical specimens of cutaneous leishmaniasis.

Polymerase chain reaction (PCR)-based detection of New World Leishmania from different types of clinical specimens has been further streamlined for field use by simplifying sample preparation and modifying published protocols. A multiplex PCR reaction was developed that allows simultaneous detection of the Leishmania genus and identification of the L. braziliensis complex. For typing isolates in culture, we found that simply boiling diluted cultured strains was sufficient preparation for the PCR. We have demonstrated that Leishmania parasites can be reliably detected from boiled dermal scrapings, instead of the more invasive skin biopsies often used as PCR specimens. The PCR of dermal scrapings yielded a sensitivity of 100% and a specificity of 100%, as compared with microscopic examination. In a study population, PCR was more sensitive than classic diagnostic techniques. The PCR detection of Leishmania in biopsies and peripheral blood mononuclear cells (PBMCs) was investigated. Diluting crude extracts of skin biopsies was sufficient to eliminate sample inhibition yet maintain required sensitivity. Leishmania braziliensis was also detected by PCR in PBMCs of individuals with active cutaneous leishmaniasis. The simplifications described here significantly improve the feasibility of using the PCR in endemic countries as the primary method for detection and preliminary characterization of Leishmania in clinical specimens of cutaneous leishmaniasis.

Animals↗