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At least 217 records · Page 12Linked to original sources
PubMed2005

Clonorchis sinensis and Opisthorchis viverrini: development of a mitochondrial-based multiplex PCR for their identification and discrimination.

We report a single, one-step PCR approach for detection and discrimination of Clonorchis sinensis and Opisthorchis viverrini in different life-stage forms (adults, metacercariae, and eggs) from fish intermediate hosts and from infected patients. Primers designed for species-specific PCR, amplifying portions of the mitochondrial (mt) genome, were also suitable for a multiplex PCR. The latter was a single, one-step reaction under high stringency conditions, using simultaneously 2 pairs of primers (1 pair for C. sinensis--product size 612 bp, and 1 pair for O. viverrini--product size 1357 bp). Assays using serially diluted templates demonstrated that as little as 0.78 ng of genomic DNA of either species could yield amplicons. Genomic DNA extracted from different life-stage forms including adult worms (of both species), eggs (of O. viverrini), eggs possibly of several trematode species (collected from patients infected with C. sinensis in Vietnam) and mixed metacercariae of common trematodes (collected from fishes in the C. sinensis endemic areas), yielded specific bands of the correct size and their identity was confirmed by sequence analysis. The multiplex PCR approach described here proved to be a species-specific, sensitive and fast tool for accurate diagnosis of clonorchiasis and/or opisthorchiasis, permitting the detection of their metacercariae in infected fishes or adult/eggs from patients in endemic areas.

Animals↗
PubMed1998

Blinded multiplex PCR analyses of middle ear and nasopharyngeal fluids from chinchilla models of single- and mixed-pathogen-induced otitis media.

Multiplex PCR analyses for both bacterial and viral pathogens were conducted in a blinded manner on 33 archival specimens, of known culture status, procured from chinchilla models of both single- and mixed-pathogen-induced otitis media and from a pediatric patient. These specimens had been maintained at -70 degrees C for up to 6 years. Experimental specimens evaluated included middle-ear effusions, nasopharyngeal lavage fluids and middle-ear lavage fluids from animals which were immunologically naive, sham-immunized or actively immunized with nontypeable Haemophilus influenzae antigens. Sampling times used ranged from the day of bacterial or viral challenge to 42 days after challenge. Initial PCR analyses of the 33 specimens matched the traditional culture data in 24 instances (73%), correctly identifying nontypeable H. influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, or adenovirus as the causative agent. A PCR-positive signal for the microbe(s) inoculated was also obtained in four animal model specimens (12%) which were culture negative. One of two culture-negative human effusions was also PCR positive. Thus, overall, results obtained by blinded PCR were 85% concordant with traditional culture methods or correctly indicated the specific pathogen introduced in four specimens that were sterile. In no instance was a false-positive signal obtained for any of the five etiologic agents being evaluated. We conclude that the multiplex PCR analyses are rapid and accurate methodologies when they are used to retrospectively evaluate diverse archival specimens of limited volume from experimental models of otitis media.

Adenoviridae↗
PubMed2000

Multiplex PCR for detection of genes for Staphylococcus aureus enterotoxins, exfoliative toxins, toxic shock syndrome toxin 1, and methicillin resistance.

A multiplex PCR assay for detection of genes for staphylococcal enterotoxins A to E (entA, entB, entC, entD, and entE), toxic shock syndrome toxin 1 (tst), exfoliative toxins A and B (etaA and etaB), and intrinsic methicillin resistance (mecA) was developed. Detection of femA was used as an internal positive control. The multiplex PCR assay combined the primers for sea to see and femA in one set and those for eta, etb, tst, mecA, and femA in the other set. Validation of the assay was performed using 176 human isolates of Staphylococcus aureus. This assay offers a very specific, quick, reliable, and inexpensive alternative to conventional PCR assays used in clinical laboratories to identify various staphylococcal toxin genes.

Bacterial Toxins↗
PubMed2006

Genotyping of Toxoplasma gondii by multiplex PCR and peptide-based serological testing of samples from infants in Poland diagnosed with congenital toxoplasmosis.

Toxoplasma gondii has a clonal population genetic structure with three (I, II, and III) lineages that predominate in North America and Europe. Type II strains cause most cases of symptomatic human infections in France and the United States, although few other regions have been adequately sampled. Here we determined the parasite genotype in amniotic fluid and cerebrospinal fluid samples from congenital toxoplasmosis cases in Poland. Nineteen confirmed congenital cases of toxoplasmosis were analyzed, including both severe and asymptomatic cases. The genotype of parasite strains causing congenital infection was determined by direct PCR amplification and restriction fragment length polymorphism analysis. Nested multiplex PCR analysis was used to type four independent polymorphic markers. The sensitivity of multiplex nested PCR was >/=25 parasites/ml in amniotic fluid and cerebral spinal fluid samples. Parasite DNA was successfully amplified in 9 of 19 samples (eight severely affected and one asymptomatic fetus). Only genotype II parasites were identified as the source of T. gondii infection based on restriction fragment length polymorphism analysis. Strains causing congenital infections were also typed indirectly based on detection of antibodies to strain-specific peptides. Serotyping indicated that 12 of 15 cases tested were caused by type II strains and these positives included both symptomatic and asymptomatic infections. Overall, the combined analysis indicated that 14 of the cases were caused by type II strains. Our results are consistent with the hypothesis that parasite burden is associated with severity of congenital toxoplasmosis and indicate that serological testing provides a promising method for genotypic analysis of toxoplasmosis.

Amniotic Fluid↗
PubMed2006

A multiplex PCR for beta-lactamase genes of Haemophilus influenzae and description of a new blaTEM promoter variant.

OBJECTIVES: To establish a multiplex PCR to detect and differentiate the beta-lactamase genes of Haemophilus influenzae. METHODS: A collection of H. influenzae isolates with a range of different beta-lactamase genes were tested in parallel with individual PCRs and the multiplex assay developed in this study. RESULTS: The multiplex method was found to be superior to previous methods and was able to correctly detect and differentiate blaROB-1 and the variants of blaTEM present in the strains tested, including a previously unrecognized variant associated with a 54 bp insertion in the promoter region. CONCLUSIONS: The multiplex PCR will be a useful tool for the surveillance of beta-lactamase genes in H. influenzae.

Anti-Bacterial Agents↗
PubMed2004

Multiplex PCR assay for rapid detection and genotyping of Helicobacter pylori directly from biopsy specimens.

We developed and evaluated a simple, novel multiplex PCR assay for rapid detection of Helicobacter pylori infection and for the determination of vacA and cagA genotypes directly from gastric biopsy specimens. This assay did not require culturing of strains or extraction of DNA from biopsy samples. This multiplex PCR assay would be of particularly great value for laboratories in developing countries.

Antigens, Bacterial↗
PubMed1996

Multiplex PCR provides a low-cost alternative to DNA probe methods for rapid identification of Mycobacterium avium and Mycobacterium intracellulare.

A multiplex PCR designed to differentiate Mycobacterium tuberculosis complex organisms from M. avium and M. intracellulare was used to test 105 isolates identified by DNA probe methods as M. avium, M. intracellulare, or M. avium complex type X. The multiple PCR correctly identified 33 of 34 isolates identified by commercial probe methods as M. avium and all 51 isolates identified as M. intracellulare. The 20 isolates identified as M. avium complex type X by probe were identified as Mycobacterium spp. by the multiplex method. These results confirm that the multiplex PCR, which is simple to perform and cheaper than commercial probe methods, is suitable for routine identification of M. avium and M. intracellulare.

Costs and Cost Analysis↗
PubMed2005

Multiplex-PCR-based recombination as a novel high-fidelity method for directed evolution.

A new and convenient method for the in vitro recombination of single point mutations is presented. This method efficiently reduces the introduction of novel point mutations, which usually occur during recombination processes. A multiplex polymerase chain reaction (multiplex-PCR) generates gene fragments that contain preformed point mutations. These fragments are subsequently assembled into full-length genes by a recombination-PCR step. The process of multiplex-PCR-based recombination (MUPREC) does not require DNase I digestion for gene-fragmentation and is therefore easy to perform, even with small amounts of target DNA. The protocol yields high frequencies of recombination without creating a wild-type background. Furthermore, the low error rate results in high-quality variant libraries of true recombinants, thereby minimizing the screening efforts and saving time and money. The MUPREC method was used in the directed evolution of a Bacillus subtilis lipase that can catalyse the enantioselective hydrolysis of a model meso-compound. Thereby, the method was proved to be useful in producing a reliable second-generation library of true recombinants from which better performing variants were identified by using a high-throughput electrospray ionization mass spectrometry (ESI-MS) screening system.

Amino Acid Sequence↗
PubMed2005

Multiplex PCR for identification of seven Streptococcus pneumoniae serotypes targeted by a 7-valent conjugate vaccine.

Here we describe a Streptococcus pneumoniae one-step multiplex PCR assay which identifies by amplicon size the seven capsular polysaccharide serotypes targeted by the 7-valent conjugate vaccine. The multiplex PCR assay was used to blindly assay clinical isolates recovered during 1999 in the Republic of Ireland from cases of invasive disease whose serotypes were previously determined by classical methods.

Adolescent↗
PubMed2004

Multiplex PCR assay for detection of Streptococcus suis species and serotypes 2 and 1/2 in tonsils of live and dead pigs.

A PCR assay was developed for the detection of Streptococcus suis serotypes 2 and 1/2. This multiplex PCR is based on the amplification of the gene coding for 16S rRNA of S. suis and on the amplification of the cps2J gene coding for the capsule of S. suis serotypes 2 and 1/2. An internal control was constructed and added in this test to monitor the efficiency of amplification in each reaction. To evaluate the specificity of the test, 31 strains of other bacterial species related to S. suis or isolated from pigs and 42 strains of S. suis serotypes 1 and 3 to 34 were analyzed. The detection threshold of the test was 28 S. suis CFU/ml. The specificity and the sensitivity of the multiplex PCR test and the presence of an internal control allowed the analysis of biological samples without a culture step. The PCR assay was then applied to the detection of 14 S. suis serotype 1/2 strains, 88 S. suis serotype 2 strains isolated from pigs, and 25 S. suis serotype 2 strains isolated from humans. This test was also applied to analyze tonsil samples of pigs experimentally infected and carrier pigs without any symptoms.

Animals↗
PubMed2003

Detection of norovirus (GI, GII), Sapovirus and astrovirus in fecal samples using reverse transcription single-round multiplex PCR.

A reverse transcription (RT) single-round multiplex polymerase chain reaction (smPCR) assay was developed to detect simultaneously Norovirus genogroup I and II, Sapovirus and astrovirus. A total of 377 diarrhea stool samples (screened for rotavirus- and adenorivus-negative) from four regions in Japan during July 2000 to June 2001 were examined by RT-smPCR. The positive rate was 16.4% (62 out of 377 stool samples). Norovirus, Sapovirus and astrovirus were detected in 42, 16, 4 of 60 positive samples, respectively. Coinfection was not found in these samples. Infections occurred mainly in November, December and January. The key elements of the RT-smPCR are (i) the cDNA synthesis with the Superscript RTII and random primer at 42 degrees C for 1 h, at 99 degrees C for 5 min, and (ii) single-round multiplex PCR by using Taq polymerase mixed together with a mixture of four different primer pairs (G1-SKF/G1-SKR for Norovirus genogroup I, COG2F/G2-SKR for Norovirus genogroup II, SLV5317/SLV5749 for Sapovirus, PreCAP1/82b for astrovirus). All of the four primer pairs amplify the capsid region of target viral genome, produce four size-specific amplicons of 330, 387, 434, 719 bp for Norovirus genogroup I and II, Sapovirus and astrovirus, respectively. This assay provides a more rapid and efficient way to detect these viruses from fecal samples in a single test, and also offers the potential for their molecular detection in food and environmental samples.

Adolescent↗
PubMed1999

Simultaneous detection of Bacteroides forsythus and Prevotella intermedia by 16S rRNA gene-directed multiplex PCR.

In a 16S rRNA gene-directed multiplex PCR, Prevotella intermedia- and Bacteroides forsythus-specific reverse primers were combined with a single conserved forward primer. A 660-bp fragment and an 840-bp fragment that were specific for both species could be amplified simultaneously. A total of 152 clinical samples, subgingival plaque and swabs of three different oral mucosae, from 38 periodontitis patients were used for the evaluation.

Adult↗
PubMed2005

Multiplex PCR for typing strains of Toxoplasma gondii.

A multiplex PCR assay was designed for multilocus strain typing of Toxoplasma gondii based on length polymorphism of five microsatellite markers. Eight T. gondii strains already sequenced at these five markers were used as control isolates. This method is simple, rapid, reproducible, and adapted to a large set of isolates.

Animals↗
PubMed2001

A multiplex PCR method of detecting recombinant DNAs from five lines of genetically modified maize.

Seven lines of genetically modified (GM) maize have been authorized in Japan as foods and feeds imported from the USA. We improved a multiplex PCR method described in the previous report in order to distinguish the five lines of GM maize. Genomic DNA was extracted from GM maize with a silica spin column kit, which could reduce experimental time and improve safety in the laboratory and potentially in the environment. We sequenced recombinant DNA (r-DNA) introduced into GM maize, and re-designed new primer pairs to increase the specificity of PCR to distinguish five lines of GM maize by multiplex PCR. A primer pair for the maize intrinsic zein gene (Ze1) was also designed to confirm the presence of amplifiable maize DNA. The lengths of PCR products using these six primer pairs were different. The Ze1 and the r-DNAs from the five lines of GM maize were qualitatively detected in one tube. The specific PCR bands were distinguishable from each other on the basis of the expected length. The r-DNA could be detected from maize samples containing 0.5% of each of the five lines of GM maize. The sensitivity would be acceptable to secure the verification of non-GMO materials and to monitor the reliability of the labeling system.

DNA Primers↗
PubMed1997

Selecting optimal oligonucleotide primers for multiplex PCR.

We investigate the problem of designing efficient multiplex PCR for medical applications. We show that the problem is NP-complete by transformation to the Multiple Choice Matching problem and give an efficient approximation algorithm. We developed this algorithm in a computer program that predicts which genomic regions may be simultaneously amplified by PCR. Practical use of the software shows that the method can treat 250 non-polymorphic loci with less than 5 simultaneous experiments.

Algorithms↗
PubMed1999

Optimized multiplex PCR: efficiently closing a whole-genome shotgun sequencing project.

A new method has been developed for rapidly closing a large number of gaps in a whole-genome shotgun sequencing project. The method employs multiplex PCR and a novel pooling strategy to minimize the number of laboratory procedures required to sequence the unknown DNA that falls in between contiguous sequences. Multiplex sequencing, a novel procedure in which multiple PCR primers are used in a single sequencing reaction, is used to interpret the multiplex PCR results. Two protocols are presented, one that minimizes pipetting and another that minimizes the number of reactions. The pipette optimized multiplex PCR method has been employed in the final phases of closing the Streptococcus pneumoniae genome sequence, with excellent results.

Algorithms↗
PubMed2005

Identification of Mytilus spp. and Pecten maximus in Irish waters by standard PCR of the 18S rDNA gene and multiplex PCR of the 16S rDNA gene.

Two molecular protocols for the identification of mussel and scallop have been developed using specific primers targeting the mitochondrial 16S ribosomal DNA gene and the nuclear 18S ribosomal DNA gene. Primers for the mitochondrial 16S ribosomal DNA gene in multiplex polymerase chain reaction (PCR) protocols yielded diagnostic DNA fragments for the mussels Mytilus edulis, Mytilus galloprovincialis, and the hybrid Mytilus edulis/galloprovincialis (335 bp), the king scallop Pecten maximus (382 bp) and the black scallop Mimachlamys varia (398 bp). DNA from the queen scallop Aequipecten opercularis showed no consistent PCR amplification of the 16S rDNA gene. Primers for the nuclear 18S rDNA gene in standard PCR protocols yielded similar-sized, diagnostic DNA fragments (approx. 190 bp) for the mussels Mytilus edulis, Mytilus galloprovincialis, and the hybrid Mytilus edulis/galloprovincialis, the king scallop Pecten maximus, the black scallop Mimachlamys varia, and the queen scallop Aequipecten opercularis. Both protocols have been tested with Mytilus spp., P. maximus, and 6 other bivalve species from a wide range of locations in Irish and European waters. Cross reaction of the specific primers with DNA template from any of the 6 other bivalve species was not observed. Rapid DNA extraction using FTA Card technology and the16S rDNA primers allowed for the detection of at least 10 mussel larvae in a subsample of natural plankton.

Animals↗