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Results for “Multiplex PCR”

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PubMed2002

A PCR assay for rapid detection of vancomycin-resistant enterococci.

Since the first report of a vancomycin-resistant enterococcal clinical isolate, these Gram-positive bacteria have emerged as important nosocomial pathogens. Several glycopeptide resistance phenotypes can be distinguished on the basis of the level and inducibility of resistance to vancomycin and teicoplanin. In the present study, we developed a multiplex PCR, which allows the simultaneous identification of enterococci at the genus level and detection of the most frequent glycopeptide resistance genotypes. Five primer sets targeting the genes vanA, vanB, vanC1, vanC2/C3 and tuf were used in one reaction tube with bacterial DNA extracted from three to five colonies. This PCR method is suitable for the rapid detection of vancomycin-resistant enterococci.

DNA, Bacterial↗

PubMed1999

Rapid simultaneous amplification and detection of the MBR/JH chromosomal translocation by fluorescence melting curve analysis.

Polymerase chain reaction (PCR) amplification and product analysis for the detection of chromosomal translocations, such as the t(14;18), has traditionally been a two-step process. PCR product detection has generally entailed gel electrophoresis and/or hybridization or sequencing for confirmation of assay specificity. Using a microvolume fluorimeter integrated with a thermal cycler and a PCR-compatible double-stranded DNA (dsDNA) binding fluorescent dye (SYBR Green I), we investigated the feasibility of simultaneous thermal amplification and detection of MBR/JH translocation products by fluorescence melting curve analysis. We analyzed DNA from 30 cases of lymphoproliferative disorders comprising 19 cases of previously documented MBR/JH-positive follicle center lymphoma and 11 reactive lymphadenopathies. The samples were coded and analyzed blindly for the presence of MBR/JH translocations by fluorescence melting curve analysis. We also performed dilutional assays using the MBR/JH-positive cell line SUDHL-6. Multiplex PCR for MBR/JH and beta-globin was used to simultaneously assess sample adequacy. All (100%) of the 19 cases previously determined to be MBR/JH positive by conventional PCR analysis showed a characteristic sharp decrease in fluorescence at approximately 90 degrees C by melting curve analysis after amplification. Fluorescence melting peaks obtained by plotting the negative derivative of fluorescence over temperature (-dF/dT) versus temperature (T) showed melting temperatures (Tm) at 88.85+/-1.15 degrees C. In addition, multiplex assays using both MBR/JH and beta-globin primers yielded easily distinguishable fluorescence melting peaks at approximately 90 degrees C and 81.2 degrees C, respectively. Dilutional assays revealed that fluorescence melting curve analysis was more sensitive than conventional PCR and agarose gel electrophoresis with ultraviolet transillumination by as much as 100-fold. Simultaneous amplification and fluorescence melting curve analysis is a simple, reliable, and sensitive method for the detection of MBR/JH translocations. The feasibility of specific PCR product detection without electrophoresis or utilization of expensive fluorescently labeled probes makes this method attractive for routine molecular diagnostics.

Chromosome Mapping↗

PubMed2003

Isolation of Campylobacter jejuni and Campylobacter coli from the gall bladder samples of sheep and identification by polymerase chain reaction.

In this study, 100 gall bladder samples of sheep slaughtered at an abattoir in Elaziğ province were examined for Campylobacter jejuni and Campylobacter coli by culture and polymerase chain reaction (PCR). Preston Campylobacter Agar supplemented with 7% horse blood and Preston Selective Supplement (Oxoid, Hampshire, UK) were used for isolation of the agents. Campylobacter spp. were isolated in 66 samples, and they were identified as 34% C. jejuni and 32% C. coli. A multiplex PCR based upon the use of ceuE gene-specific primers was applied on DNA samples extracted from C. jejuni and C. coli isolates. All C. jejuni and C. coli strains that were positive by culture were also detected to be positive by PCR. This study shows that PCR can be used an alternative, rapid and sensitive test for the identification of C. jejuni and C. coli which threaten human and animal health.

Abattoirs↗

PubMed2003

A single-step, PCR-based method for the detection and differentiation of Plasmodium vivax and P. falciparum.

The ability to detect and differentiate between Plasmodium falciparum and P. vivax is of great importance for the routine laboratory diagnosis of malaria, donor-blood screening and epidemiological studies. Most PCR-based methods for the discrimination of these two species require nested protocols or an additional hybridization reaction, leading to high labour costs and long turn-around times. A simple, time-effective and yet sensitive and specific technique, based on a multiplex PCR, has now been developed for the simultaneous detection and differentiation of P. falciparum and P. vivax in blood samples. Compared with the 'gold standard' of microscopy, this method had a sensitivity and specificity of 100%, with a detection limit of just one P. falciparum or three P. vivax parasites/microl blood.

Animals↗

PubMed2006

Virulence-associated gene profiling of Streptococcus suis isolates by PCR.

Definition of virulent Streptococcus suis strains is controversial. One successful approach for identification of virulent European strains is differentiation of capsular serotypes (or the corresponding cps types) and subsequent detection of virulence-associated factors, namely the extracellular factor (EF, epf), the muramidase-released protein (MRP, mrp) and the hemolysin suilysin (SLY, sly). In this work we present a novel multiplex PCR (MP-PCR) and an mrp variant PCR for identification and characterization of virulent S. suis strains. These new methods were used to identify association of disease with particular profiles of virulence-associated genes. The MP-PCR allowed identification of S. suis through detection of the housekeeping gene gdh, differentiation of four cps types (1, 2, 7 and 9), and detection of epf, mrp, sly and arcA (arginine deiminase from S. suis). Furthermore, this study describes the first PCR assay for differentiation of at least six mrp variants. Expression of the corresponding size variants of MRP was shown for four of the six mrp variants, but was undetectable for the two larger mrp variants in the particular strains investigated. The results of this study suggest that cps7 strains are associated with pneumonia and that variation of mrp is very pronounced among these strains. Gene profiles of invasive, pneumonia and carrier S. suis isolates by combination of PCR assays allowed differentiation of 24 different genotypes among cps1, 2, 7 and 9 strains. Forty-five percent of the invasive S. suis diseases investigated in this study were caused by only two of these genotypes, namely cps2/mrp+/epf+/sly+ and cps9/mrp(*)/epf-/sly+. Thus, this study demonstrates for the first time a uniform profile of the particular virulence-associated genes for the vast majority of the investigated invasive cps9 strains.

Animals↗

PubMed2002

The etiology and management of genital ulcers in the Dominican Republic and Peru.

BACKGROUND: Clinical diagnosis of genital ulcers is difficult, and diagnostic tests are least available in settings where rates of disease are highest. The World Health Organization (WHO) has developed protocols for the syndromic management of genital ulcers in resource-poor settings. However, because risk factors, patterns and causes of disease, and antimicrobial susceptibilities differ from region to region and over time, they must be adapted to local situations. GOAL: The goal of this study was to determine etiologic factors, evaluate syndromic management, and compare polymerase chain reaction (PCR) testing with other diagnostic alternatives for genital ulcers among patients attending sexually transmitted disease clinics in the Dominican Republic and Peru. STUDY DESIGN: Eighty-one men with genital ulcers in the Dominican Republic and 63 in Peru underwent identical interviews and identical multiplex PCR (M-PCR) tests of genital lesion specimens for etiologic diagnoses. Algorithms for managing genital ulcers were developed. RESULTS: In the Dominican Republic, 5% were M-PCR-positive for, 26% for, and 43% for herpes simplex virus (HSV); in Peru, 10%, 5%, and 43%, respectively, were positive. The WHO algorithm for treating syphilis and chancroid had a sensitivity of 100%, a positive predictive value (PPV) of 24%, and an overtreatment rate of 76%. A modified algorithm for treating only those without vesicular lesions had 88% sensitivity and a 27% PPV, and the overtreatment rate was reduced to 58%. CONCLUSION: HSV caused 43% of genital ulcers in these populations. The modified algorithm had lower sensitivity but a reduced overtreatment rate. M-PCR testing was more sensitive than standard tests and more specific and sensitive than clinical diagnosis.

Adolescent↗

PubMedDate not supplied

[Species-specific detection of Proteus vulgaris and Proteus mirabilis by the polymerase chain reaction].

Sets of primers for the species-specific detection of P. mirabilis and P. vulgaris by the polymerase chain reaction (PCR) were developed. As targets for these primers beta-lactamase and 16S rRNA gene fragments were chosen on the basis of the multiple leveling of the sequences of the DNA of all known P. mirabilis and P. vulgaris isolates. For differential detection oligonucleotides were selected in such a way that primers, specific for P. vulgaris, contained the non-paired nucleotide for P. mirabilis isolate at the 3'-end, and all other nucleotides were complementary to the beta-lactamase gene fragment. Primers, specific for gene 16S rRNA of P. mirabilis, contained the non-paired nucleotide for P. vulgaris isolates at the 3'-end. Standard PCR was carried out for 6 P. mirabilis and P. vulgaris strains. The use of PCR species-specific primers to P. vulgaris DNA made it possible to amplify the DNA fragment of the expected length only for P. vulgaris isolates, while the result of PCR for P. mirabilis was negative. PCR with primers specific to P. mirabilis permitted the detection of amplicon sized 101 nucleotides pairs only for P. mirabilis strains. These primers were optimized so as to use them in the specific differentiation of closely related P. mirabilis and P. vulgaris species by multiplex PCR. Genus-specific primers permitted the detection of bacterial gyrB gene of the genus Proteus were developed also.

Bacteriological Techniques↗

PubMed1999

Clinical impact of rapid oxacillin susceptibility testing using a PCR assay in Staphylococcus aureus bactaeremia.

OBJECTIVES: The aim of this work was to establish the clinical impact of rapid oxacillin susceptibility testing in nosocomial Staphylococcus aureus bacteraemia. METHODS: This study was performed in 145 critically ill patients infected by S. aureus. Patients were randomly assigned to one of two groups: patients for whom susceptibility testing was performed using a rapid same day multiplex PCR assay for detection of the staphylococcal mecA (mean delay of response: 6 h) and those for whom testing was accomplished using traditional overnight techniques (21 h). RESULTS: The results of this study showed no significant difference between the two groups in terms of age, Simplified Acute Physiologic Score, severity of infection, severity of underlying disease and clinical outcome (control vs. PCR): unfavourable outcome of infection, 12.32 vs. 12.5%; 95% CI for the difference = -11.49 to 11.09 (P = 0.975); unfavourable general outcome, 16.43 vs. 20.83%; 95% CI for the difference = -17.35 to 8.50 (P = 0.497). For the oxacillin-susceptible S. aureus bactaeraemia, results were: unfavourable outcome of infection = 13.04 vs. 11.11%; 95% CI for the difference = -11.38 to 16.18 (P = 0.767); unfavourable general outcome = 13.04 vs. 20.37%; 95% CI for the difference = -22.12 to 8.07 (P = 0.331). CONCLUSION: This study seemed to demonstrate that rapid oxacillin susceptibility testing using a PCR assay did not have a major impact on the care and outcome of patients with S. aureus bactaeremia.

Adult↗

PubMed2004

Simultaneous PCR detection of ica cluster and methicillin and mupirocin resistance genes in catheter-isolated Staphylococcus.

Recent data show that more than 50% of catheter-associated bloodstream infections are caused by staphylococci. Staphylococcal infections produced by intercellular-adhesion cluster (ica) carriers can be even more problematic due to the presence of methicillin and mupirocin resistance genes. In the present study, a multiplex PCR protocol that allows the simultaneous identification of staphylococci and detection of both the ica and methicillin and/or mupirocin resistance genes was developed. Furthermore, the method allows differential detection of the ica locus from Staphylococcus aureus and Staphylococcus epidermidis.

Base Sequence↗

PubMed1999

Simultaneous detection of IFN-gamma and IL-4 mRNAs using RT-PCR and time-resolved fluorometry.

Time-resolved fluorometry was applied in the detection of RT-PCR amplified mRNAs for the Th1 and Th2 cell-derived cytokines interferon gamma (IFN-gamma) and interleukin (IL-)4, respectively. RNA stimulated cells was reverse transcribed and the cDNAs for the cytokine mRNAs and the constantly expressed beta-actin (beta-ACT) mRNA were simultaneously amplified in one multiplex PCR reaction. The PCR conditions were optimized to minimize mutual inhibition of individual amplifications. One of the PCR primers in each primer pair was biotinylated, and the PCR products were captured onto streptavidin-coated microtitre plates. The three PCR products were detected with three different lanthanide labelled target-specific probes in solution hybridization. IFN-gamma, IL-4 and beta-ACT were detected with europium (Eu), terbium (Tb) and samarium (Sm) labelled probes, respectively, using time-resolved fluorometry. Small cell numbers used in microtitre plate cultures were sufficient to detect cytokine messages after mitogen stimulation. This sequence-based method provides a sensitive, specific, fast and nonisotopic alternative to conventional blotting and hybridisation with radioactive probes. In addition, the multiplex fluorogenic dye detection facilitates relative quantification of target mRNAs.

Actins↗

PubMed2002

Evaluation of a single-step multiplex RT-PCR for influenza virus type and subtype detection in respiratory samples.

A single-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) for the simultaneous detection of influenza virus type and subtypes was evaluated for its use in clinical specimens. One part of each specimen was tested using an established standard culture and/or monoclonal antibody-based immunofluorescence assay method, and the other part was tested by the multiplex RT-PCR method for the presence or absence of the influenza virus, and its type and subtype. Sixty-seven specimens were examined. The results revealed a strong agreement between the data obtained by the established method and those obtained by the multiplex RT-PCR.

Antigens, Viral↗

PubMed1998

Evaluation of the BeTha gene 1 kit for the qualitative detection of the eight most common Mediterranean beta-thalassemia mutations.

We describe the evaluation of the Bio-Rad BeTha Gene 1 kit (Bio-Rad Laboratories, Hercules, CA), a DNA-probe assay designed for the qualitative determination of the eight most common Mediterranean beta-thalassemia mutations. The kit utilizes the principle of allele-specific oligonucleotide (ASO) hybridization. Following sample preparation and in vitro DNA amplification by the polymerase chain reaction (PCR), an allele-specific detection of the amplified products by a nonradioactive enzymatic assay is performed. Genomic DNA is prepared from an individual's whole blood with a DNA purification matrix. In a second step, the beta-globin gene is amplified in a multiplex PCR reaction containing four 5' biotinylated oligonucleotide primers. In a final step, an aliquot of the PCR reaction is first chemically denatured and then captured in two eight-well strips of a 96-well enzyme-linked immunosorbent assay (ELISA) plate by hybridization to an immobilized ASO probe. Each DNA sequence at each of the eight mutation sites is represented by one normal and one mutant ASO. During this capture/hybridization step, which is performed at 37 degrees C, only perfectly matched PCR products will be captured by an ASO. Subsequently, the allele-specific captured biotin-labeled PCR products are detected by a colorimetric enzymatic reaction. The system permits the detection of 16 beta-thalassemia alleles using a high-throughput format that can be automated easily. A clinical feasibility study was performed to evaluate the functionality (method comparison study, assay validity using samples previously collected and stored at various temperatures for different periods of time, interference on kit performance, and assay validity for prenatal diagnosis) and the usability (ease of use, sample throughput) of the kit. The analysis of 110 samples previously studied with reference methods showed 100% clinical sensitivity and specificity. We demonstrate here that the procedure not only increases the throughput of beta-thalassemia allele genotyping but also provides an accurate, rapid, reliable, and nonisotopic diagnostic tool.

Alleles↗

PubMed2005

Mutation rates in the dystrophin gene: a hotspot of mutation at a CpG dinucleotide.

An analysis of mutations was performed in 141 Duchenne muscular dystrophy (DMD) patients previously found to be negative for large deletions by standard multiplex PCR assays. Comprehensive mutation scanning of all coding exons, adjacent intronic splice regions, and promoter sequences was performed by DOVAM-S, a robotically enhanced, high throughput method that detects essentially all point mutations. Samples negative for point mutations were further analyzed for duplications by multiplex amplifiable probe hybridization (MAPH). Presumptive causative mutations were detected in 90% of the patients (70% protein truncating point mutations, 13% duplications, and 7% deletions not detected by the standard multiplex screening method). A total of 40 of the mutations are putatively novel. Most duplications involve multiple exons with an average and median size of about 160 and 153 kb, respectively. This is the first analysis of the absolute and relative rates of point mutations in the dystrophin gene. Relative to microdeletions (0.68 x 10(-9) per bp per generation), transitions at CpG dinucleotides are enhanced 150-fold while complex indels, the least common mutation type, are less frequent than microdeletions by a factor of five. The frequency of microdeletions and microinsertions at mononucleotide repeats increases exponentially with length. When compared to the well-studied human factor IX gene (F9), the results are similar, with two exceptions: a hotspot of mutation in the dystrophin gene (c.8713C>T/p.R2905X) at a CpG dinucleotide and an altered size distribution of microdeletions. The hotspot reflects a difference in the underlying pattern of mutation, while the altered size distribution of microdeletions reflects certain abundant sequence motifs within the dystrophin coding sequence (relative to factor IX).

Adolescent↗

PubMed2001

Development of a PCR-based method for specific identification of genotypic markers of shiga toxin-producing Escherichia coli strains.

A simple, rapid and specific PCR-based method for identification of shiga toxin-producing Escherichia coli (STEC) was developed. The procedure involves amplification of the E. coli-specific universal stress protein A (uspA) gene (uspa-PCR), with the primer pair described by other authors, which allows differentiation of E. coli (STEC and non-STEC) from other gram-negative bacteria followed by identification of the main genetic virulence traits of the uspA-positive isolates. For this purpose, two multiplex PCR assays, based on previously published primer sequences, were established. Assay 1 (mPCR-1) uses three primer pairs and detects the genes encoding O157 (rfb), enterohemolysin (ebly) and shiga toxin (stx), generating amplification products of 420, 534 and 230 bp, respectively. Assay 2 (mPCR-2) uses four primer pairs specific for rfb (E. coli O157), eaeA (intimin), stx1 and stx2 (shiga toxin 1 and 2, respectively), generating PCR amplicons of 420, 840, 348 and 584 bp, respectively. These two assays were validated by testing several E. coli reference strains and 202 previously characterized E. coli isolates originating from calves and from children, and 100% agreement with previous results was obtained. The method developed can be used for specific identification of STEC bacteria including those of the O157 serogroup.

Animals↗

PubMed2000

Current diagnostic techniques in genital herpes: their role in controlling the epidemic.

Genital herpes continues to be a public health problem in both developed and developing countries. Laboratory confirmation of clinical diagnosis is important, particularly as there are other conditions which present similarly to genital herpes, while atypical presentations of genital herpes also occur. Herpes simplex virus type 2 (HSV-2) is the most common cause of genital herpes globally, although HSV-1 genital herpes infections occur also. Type-specific serology has overcome the technical problems posed by earlier cross-reactive HSV serological assays, hence HSV-2 specific antibodies can be identified using both in-house and commercial assays. All HSV-2 serological assays require an appropriately thorough validation, and here an understanding of the natural history of genital HSV infection is important. Validated HSV-2 specific antibody assays have featured in seroepidemiological studies which have emphasised the largely asymptomatic nature of this infection. Subsequent seroepidemiological studies have included a sexual lifestyle questionnaire to identify risk factors for genital HSV-2 infection. This has given rise to serological screening proposals which, it is argued, may arrest the spread of genital herpes in the general population. However, counter arguments to such proposals are important to consider. As regards diagnosis and management of genital herpes in individual patients, situations have been identified where type-specific serology may be of benefit. PCR has become the method of laboratory diagnosis for HSV encephalitis over the past decade, but its role in the diagnosis of genital herpes has been addressed only in recent years. Evaluation of HSV PCR on specimens from genital herpes cases has shown PCR to be more sensitive than virus culture, the traditional "gold standard" of HSV identification. However, questions remain regarding the acceptance of HSV into routine diagnostic settings, particularly concerning sample preparation, although automation and the ability to include diagnosis of other genital infections in a multiplex PCR is an advantage. Such developments should enhance the role of PCR in genital herpes diagnosis and ultimately reduce costs relative to traditional methods such as culture and HSV antigen detection. Finally, the use of type-specific serology and HSV PCR in genital herpes research is noted.

Antibodies, Viral↗

PubMed2005

Projector 2: contig mapping for efficient gap-closure of prokaryotic genome sequence assemblies.

With genome sequencing efforts increasing exponentially, valuable information accumulates on genomic content of the various organisms sequenced. Projector 2 uses (un)finished genomic sequences of an organism as a template to infer linkage information for a genome sequence assembly of a related organism being sequenced. The remaining gaps between contigs for which no linkage information is present can subsequently be closed with direct PCR strategies. Compared with other implementations, Projector 2 has several distinctive features: a user-friendly web interface, automatic removal of repetitive elements (repeat-masking) and automated primer design for gap-closure purposes. Moreover, when using multiple fragments of a template genome, primers for multiplex PCR strategies can also be designed. Primer design takes into account that, in many cases, contig ends contain unreliable DNA sequences and repetitive sequences. Closing the remaining gaps in prokaryotic genome sequence assemblies is thereby made very efficient and virtually effortless. We demonstrate that the use of single or multiple fragments of a template genome (i.e. unfinished genome sequences) in combination with repeat-masking results in mapping success rates close to 100%. The web interface is freely accessible at http://molgen.biol.rug.nl/websoftware/projector2.

Algorithms↗

PubMed2003

Molecular immunoglobulin/T- cell receptor clonality analysis in cutaneous lymphoproliferations. Experience with the BIOMED-2 standardized polymerase chain reaction protocol.

BACKGROUND AND OBJECTIVES: Molecular clonality analysis of immunoglobulin (Ig) and T-cell receptor (TCR) genes is a widely used diagnostic tool for discrimination between polyclonal, oligoclonal, and monoclonal lymphoproliferative skin lesions. We studied Ig/TCR clonality in a series of 60 patients with an initial suspicion of (primary) cutaneous B- or T-cell lymphoma (CBCL/CTCL). Clonality of Ig/TCR gene rearrangements was assessed by Southern blot (SB) and polymerase chain reaction (PCR) analysis using standardized PCR primers and protocols of the BIOMED-2 Concerted Action BMH4-CT98-3936. The obtained PCR products were subjected to heteroduplex (HD) and GeneScan (GS) analysis. We compared the data of 154 samples with the histopathologic diagnosis, based on the EORTC classification of skin lymphomas. DESIGN AND METHODS: Molecular results were largely concordant with histopathology. In 12 CBCL patients PCR analysis of Ig gene rearrangements detected clonality in 83% of cases whereas SB did so in 92%. Clonal TCR gene rearrangements were detected by SB in 68% of CTCL patients, whereas TCRG and TCRB PCR analysis detected clonality in 76% and 66% of cases, respectively. PCR GS analysis of TCR rearrangements appeared to be slightly more informative than HD analysis. Clonality assessment was particularly informative for studying involvement of extracutaneous sites, such as regional lymph nodes, peripheral blood, and bone marrow. INTERPRETATION AND CONCLUSIONS: Our study shows that the BIOMED-2 multiplex PCR analysis strategy is a reliable and useful technique in the diagnostic process of patients with an initial suspicion of (primary) CBCL/CTCL and for assessment of extracutaneous dissemination, provided that the results are interpreted in the context of clinical, histologic and immunophenotypic data.

Clone Cells↗

PubMed1999

PCR detection of aminoglycoside resistance genes: a rapid molecular typing method for Acinetobacter baumannii.

Aminoglycoside resistance is common among strains of Acinetobacter baumannii responsible for nosocomial infections, and inactivation of these antibiotics by enzymatic modification is the main mechanism. Different types of aminoglycoside acetyltransferases (AAC), nucleotidyltransferases (ANT), and phosphotransferases (APH) are synthesized by clinical isolates, and several enzymes can be produced by a single strain. Using a multiplex PCR procedure carried out on bacterial thermolysates, we analyzed the aminoglycoside resistance gene content of strains belonging to eight clusters identified by pulsed-field gel electrophoresis. In a single reaction were combined three primer pairs in order to amplify the genes coding for AAC(6')-Ih, AAC(3)-I, and AAC(3)-II, three primer pairs for the genes coding for ANT(2'')-I, APH(3')-VI, and rRNA 16S as internal control, and finally two primer pairs for the genes coding for AAC(6')-Ib and APH(3')-I. According to the aminoglycoside resistance gene patterns, the strains of the eight clusters were distributed into seven classes. This simple and rapid (< 8 h) fingerprinting technique could be a useful tool for the epidemiological investigation of A. baumannii nosocomial infections.

Acetyltransferases↗