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Results for “Multiplex PCR”

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PubMed1996

Molecular biology of partial D phenotypes.

We have examined all DVI variant phenotypes submitted to the workshop by a combination of RT-PCR, multiplex RHD PCR and immunoblotting with Rh antipeptide sera. Our findings suggest that all DVI phenotypes arise through hybrid RHD-RHCE-RHD genes. Genomic DNA derived from all DVI samples were shown to be RHD intron 4 negative when analysed with an RHD intron 4/exon 10 multiplex assay. We assume therefore that all DVI phenotypes involve gene conversion events involving at least exons 4 and 5 of the RHD gene. Analysis of a novel D and E variant phenotype individual (ISBT49) by RT-PCR has allowed the identification of a hybrid Rh gene composed of exons 1-4 RHD: 5 RHCE/D and 6-10 RHD. We propose that the partial D & E phenotype observed arises through D & E expression on the hybrid RHD-RHCE-RHD protein: as no transcripts encoding Rh E could be found.

DNA↗

PubMed2006

PrimerStation: a highly specific multiplex genomic PCR primer design server for the human genome.

PrimerStation (http://ps.cb.k.u-tokyo.ac.jp) is a web service that calculates primer sets guaranteeing high specificity against the entire human genome. To achieve high accuracy, we used the hybridization ratio of primers in liquid solution. Calculating the status of sequence hybridization in terms of the stringent hybridization ratio is computationally costly, and no web service checks the entire human genome and returns a highly specific primer set calculated using a precise physicochemical model. To shorten the response time, we precomputed candidates for specific primers using a massively parallel computer with 100 CPUs (SunFire 15 K) about 3 months in advance. This enables PrimerStation to search and output qualified primers interactively. PrimerStation can select highly specific primers suitable for multiplex PCR by seeking a wider temperature range that minimizes the possibility of cross-reaction. It also allows users to add heuristic rules to the primer design, e.g. the exclusion of single nucleotide polymorphisms (SNPs) in primers, the avoidance of poly(A) and CA-repeats in the PCR products, and the elimination of defective primers using the secondary structure prediction. We performed several tests to verify the PCR amplification of randomly selected primers for ChrX, and we confirmed that the primers amplify specific PCR products perfectly.

DNA Primers↗

PubMed2004

Identification of Lactobacillus sakei and Lactobacillus curvatus by multiplex PCR-based restriction enzyme analysis.

Two closely related lactic acid bacteria, Lactobacillus sakei and Lactobacillus curvatus, are very difficult to be rapidly differentiated. Here we report multiplex polymerase chain reaction (PCR)-based restriction enzyme analysis that is useful for rapid and reliable identification of these two species. This method employs both polymerase chain reaction (PCR) and restriction enzyme analysis (REA). First, multiplex-PCR using three primers that were designed from 16S rDNA sequence produces two bands, a 433-bp and a 623-bp band. A 433-bp band represents only L. sakei and L. curvatus among lactobacilli and genetically related bacteria, and a 623-bp band is used for further identification by restriction analysis. Second, restriction analysis of 623-bp band using Hind III restriction enzyme discriminates L. sakei from L. curvatus. This method could identify 28 strains as L. sakei or L. curvatus, which were frequently isolated from kimchi, a traditional fermented cabbage product in South Korea. Therefore, these results suggest that this method is simple, rapid, and reliable for the identification of L. sakei and L. curvatus species.

Brassica↗

PubMed2006

Phylogenetic study and multiplex PCR-based detection of Burkholderia plantarii, Burkholderia glumae and Burkholderia gladioli using gyrB and rpoD sequences.

In order to develop a detection method for the rice pathogens Burkholderia plantarii, Burkholderia glumae and Burkholderia gladioli, the phylogeny of six plant-pathogenic Burkholderia species was analysed using the combined nucleotide sequences of gyrB and rpoD. B. plantarii, B. glumae and B. gladioli formed tight monophyletic branches supported by high bootstrap probabilities. The high sequence similarity revealed a close phylogenetic relationship between B. glumae and B. plantarii. B. plantarii strains were divided into three subclusters comprising rice strains, whereas the single Vanda strain occupied a unique position in the phylogenetic tree. The gyrB and rpoD sequences of all B. glumae strains examined were highly conserved. In contrast, B. gladioli strains demonstrated a far greater sequence diversity, but this diversity did not correlate with pathovar, host plant or geographical origin of the strains. A multiplex-PCR protocol using specific primers from the gyrB sequences was designed that allowed the specific detection and identification of B. plantarii, B. glumae and B. gladioli in rice seeds infected with these pathogenic species.

Bacterial Proteins↗

PubMed1993

Simultaneous amplification of multiple HIV-1 DNA sequences from clinical specimens by using nested-primer polymerase chain reaction.

A sensitive and specific polymerase chain reaction (PCR) protocol with nested primers was developed for simultaneous amplification of three independent human immunodeficiency virus type 1 (HIV-1) DNA sequences from clinical specimens. DNA samples were first amplified with gag, pol, and env outer primer pairs and then with the corresponding three inner primer pairs in the same two-step reaction. Detection of the different amplification products was readily accomplished by simple agarose gel electrophoresis of the reaction product, even when starting with a single copy of HIV-1 DNA. Equivalent amounts of the three PCR products were generated, provided that the relative concentrations of the inner primer pairs were optimized. In addition, a beta-globin control primer pair could be conveniently included in the internal amplification step to verify that the DNA sample was suitable for PCR analysis. One nested multiplex PCR test was sufficient to detect HIV-1 DNA in all of 80 HIV-1-seropositive individuals and none of 50 HIV-1-seronegative healthy blood donors. The nested multiplex PCR procedure provides an attractive means for simple, rapid, and cost-effective direct detection of HIV-1 DNA in patient samples.

Base Sequence↗

PubMed2002

Multiplex quantitative PCR using self-quenched primers labeled with a single fluorophore.

Multiplex quantitative PCR based on novel design of fluorescent primers is described. Fluorogenic primers are labeled with a single fluorophore on a base close to the 3' end with no quencher required. A tail of 5-7 nt is added to the 5' end of the primer to form a blunt-end hairpin when the primer is not incorporated into a PCR product. This design provides a low initial fluorescence of the primers that increases up to 8-fold upon formation of the PCR product. The hairpin oligonucleotides (DeltaG from -1.6 to -5.8 kcal/mol) may be as efficient as linear primers and provide additional specificity to the PCR by preventing primer-dimers and mispriming. Multiple fluorogenic primers were designed by specialized software and used for real-time quantitation of c-myc and IL-4 cDNAs in the presence of reference genes such as beta-actin, GAPDH and 18S rRNA. Targets of 10-10(7) copies were detected with precision in PCR using FAM-labeled primers for variable genes and JOE-labeled primers for the reference genes. This method was also used to detect single nucleotide polymorphism of the human retinal degeneration gene by allele-specific PCR with end-point detection using a fluorescent plate reader or a UV-transilluminator. We conclude that fluorogenic mono-labeled primers are an efficient and cost-effective alternative to FRET-labeled oligonucleotides.

Alleles↗

PubMed2006

Molecular serotype identification of Streptococcus agalactiae of bovine origin by multiplex PCR-based reverse line blot (mPCR/RLB) hybridization assay.

We used a multiplex PCR-based reverse line blot (mPCR/RLB) hybridization assay and sequencing of a variable region of the cps cluster to identify serotypes of 140 Streptococcus agalactiae (group B Streptococcus; GBS) isolates from cattle. Only 71 (51%) isolates were typeable using antisera, but molecular serotypes (MS) were assigned to 133 (95%) and 139 (99%) isolates by partial cpsE-cpsF-cpsG sequencing and mPCR/RLB, respectively. Ninety-four isolates (67%) belonged to MS III and most belonged to a molecular serosubtype (msst) III-3, which is uncommon among GBS isolates from humans. Our results demonstrate that cps clusters of bovine GBS differ significantly from those of GBS isolates from humans.

Animals↗

PubMed2006

Multiplex PCR-based detection of potential fumonisin-producing Fusarium in traditional African vegetables.

Culture-independent methods employed in fungal genetic studies using in vitro amplification (PCR) and analysis of specific genes or gene fragments have proved to be useful for detection, identification, and molecular taxonomy of various plant pathogens including Fusarium spp. This approach may be faster than culture-dependent methods, and could especially be of value for the rapid detection of slow-growing toxin-producing species in food samples. The present study was aimed at the development and evaluation of multiplex PCR-based methods for the detection and identification of potential fumonisin-producing Fusarium spp. in traditional morogo-leafy vegetables supplementing the maize-based staple diet of rural communities in South Africa. In these rural subsistence settings, some morogo plants grow as weeds in maize fields where they might become contaminated with potential fumonisin-producing Fusarium strains before being collected for consumption. Substances released by senescent vegetables could induce toxin production during storage. Using fumonisin-positive MRC Fusarium verticillioides strains as reference cultures, the following primer sets for the detection of specific gene fragments in fumonisin-positive Fusarium spp. were evaluated: (i) the conserved transcription elongation factor gene (EF-1), (ii) the FUM1 gene encoding polyketide synthase for fumonisin B1 production, and (iii) 18S rRNA gene. Preliminary results indicated that, these DNA fragments were amplified from MRC reference strains as well as Fusarium spp. isolated from morogo. The annealing temperature for the multiplex PCR was 55 degrees C and each reaction contained 25 pmol of each of the primer sets EF and FUM1 and 12.5 pmol of the 18S primer set. The detection limit of the individual primers was up to 1 ng and for the multiplex up to 10 ng. This demonstrates the potential of this method for the detection of potential fumonisin-positive strains.

Fumonisins↗

PubMed2004

Rapid detection and identification of human adenovirus species by adenoplex, a multiplex PCR-enzyme hybridization assay.

Human adenoviruses (AdV) have been implicated in a wide variety of diseases and are ubiquitous in populations worldwide. These agents are of concern particularly in immunocompromised patients, children, and military recruits, resulting in severe disease or death. Clinical diagnosis of AdV is usually achieved through routine viral cell culture, which can take weeks for results. Immunofluorescence and enzyme-linked immunosorbent assay-based techniques are more timely but lack sensitivity. The ability to distinguish between the six different AdV species (A to F) is diagnostically relevant, as infections with specific AdV species are often associated with unique clinical outcomes and epidemiological features. Therefore, we developed a multiplex PCR-enzyme hybridization assay, the Adenoplex, using primers to the fiber gene that can simultaneously detect all six AdV species A through F in a single test. The limit of detection (LOD) based on the viral 50% tissue culture infective dose/ml for AdV A, B, C, D, E, and F was 10(-2), 10(-1), 10(-1), 10(-2), 10(-1), and 10(-2), respectively. Similarly, the LOD for the six DNA controls ranged from 10(2) to 10(3) copies/ml. Twelve common respiratory pathogens were tested with the Adenoplex, and no cross-reactivity was observed. We also validated our assay using clinical specimens spiked with different concentrations of AdV strains of each species type and tested by multiplex PCR and culture. The results demonstrated an overall sensitivity and specificity of Adenoplex of 100%. This assay can be completed in as few as 5 h and provides a rapid, specific, and sensitive method to detect and subtype AdV species A through F.

Adenoviridae Infections↗

PubMed2006

Multiplex primer extension analysis for rapid detection of major European mitochondrial haplogroups.

The evolution of the human mitochondrial genome is reflected in the existence of ethnically distinct lineages or haplogroups. Alterations of mitochondrial DNA (mtDNA) have been instrumental in studies of human phylogeny, in population genetics, and in molecular medicine to link pathological mutations to a variety of human diseases of complex etiology. For each of these applications, rapid and cost effective assays for mtDNA haplogrouping are invaluable. Here we describe a hierarchical system for mtDNA haplogrouping that combines multiplex PCR amplifications, multiplex single-base primer extensions, and CE for analyzing ten haplogroup-diagnostic mitochondrial single nucleotide polymorphisms. Using this rapid and cost-effective mtDNA genotyping method, we were able to show that within a large, randomly selected cohort of healthy Austrians (n = 1172), mtDNAs could be assigned to all nine major European haplogroups. Forty-four percent belonged to haplogroup H, the most frequent haplogroup in European Caucasian populations. The other major haplogroups identified were U (15.4%), J (11.8%), T (8.2%) and K (5.1%). The frequencies of haplogroups in Austria is within the range observed for other European countries. Our method may be suitable for mitochondrial genotyping of samples from large-scale epidemiology studies and for identifying markers of genetic susceptibility.

Austria↗

PubMed2004

Characterization of Hprt mutations in cDNA and genomic DNA of T-cell mutants from control and 1,3-butadiene-exposed male B6C3F1 mice and F344 rats.

A multiplex PCR procedure for analysis of genomic DNA mutations in the mouse hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene was developed and then used with other established methods for the coincident identification of large- and small-scale genetic alterations in the Hprt gene of mutant T-cell isolates propagated from sham- and 1,3-butadiene (BD)-exposed mice and rats. The spectra data for RT-PCR/cDNA analysis and multiplex PCR of genomic DNA from Hprt mutants were combined, and statistical analyses of the mutant fractions for the classes of mutations identified in control versus exposed animals were conducted. Under the assumption that the mutant fractions are distributed as Poisson variates, BD exposure of mice significantly increased the frequencies of (1) nearly all types of base substitutions; (2) single-base deletions and insertions; and (3) all subcategories of deletions. Significantly elevated fractions of G:C-->C:G and A:T-->T:A transversions in the Hprt gene of BD-exposed mice were consistent with the occurrence of these substitutions as the predominant ras gene mutations in multiple tumor types increased in incidence in carcinogenicity studies of BD in mice. BD exposure of rats produced significant increases in (1) base substitutions only at A:T base pairs; (2) single-base insertions; (3) complex mutations; and (4) deletions (mainly 5' partial and complete gene deletions). Future coincident analyses of large- and small-scale mutations in rodents exposed to specific BD metabolites should help identify species differences in the sources of deletion mutations and other types of mutations induced by BD exposures in mice versus rats.

Animals↗

PubMed2005

Characteristics of cytotoxic necrotizing factor and cytolethal distending toxin producing Escherichia coli strains isolated from meat samples in Northern Ireland.

Swabs collected from pig, lamb and beef carcasses and samples of pork, lamb and beef mince were cultured for Escherichia coli strains. Strains harbouring cytotoxic necrotizing factors (CNF1 and 2) and cytolethal distending toxins (CDT-I,-II,-III and -IV) were identified in plate cultures of the isolates by colony hybridization with labelled probes and multiplex PCR assays. Simplex and multiplex PCR assays were used to further characterize the isolates to determine the presence of P, S and F17 fimbriae as well as afimbrial adhesins and haemolysin. The serotype was also determined where possible. Thirty strains with the capacity to code for CNF (4), CDT (24) or both (2) were isolated and characterized, and a wide range of associated factor patterns was observed. The methods utilized were successful in demonstrating the detection of viable strains with potentially significant pathogenic factors from human food sources.

Adhesins, Bacterial↗

PubMed2006

Isolation of bacteriocinogenic Lactobacillus plantarum strains from ben saalga, a traditional fermented gruel from Burkina Faso.

A collection of lactic acid bacteria isolated from ben saalga, a traditional fermented gruel from Burkina Faso, was screened for bacteriocin production. Seven isolates were selected for their broad antimicrobial spectra, which overall included strains of Bacillus cereus, Bacillus licheniformis, Enterococcus faecalis, Listeria innocua, Listeria monocytogenes, Staphylococcus aureus, Escherichia coli and Salmonella enterica. Cluster analysis of RAPD-PCR patterns revealed that six of the isolates represent different strains. The six selected strains were identified as Lactobacillus plantarum by 16S rDNA sequencing, species-specific PCR and multiplex PCR of the recA gene. PCR amplification revealed the presence of genes of the plantaricin cluster described in L. plantarum C11. Among them, strain 5.2.2 carried the largest number of genes from this cluster.

Bacteriocins↗

PubMed2003

Relationship between HPV typing and abnormality of G1 cell cycle regulators in cervical neoplasm.

OBJECTIVE: In tumorigenesis, loss of function of the G1 pathway (p16-CDK4/cyclinD1-pRB pathway (RB pathway) and p14-MDM2-p53 pathway (p53 pathway)) is a theoretically essential event. The simultaneous analysis of all components of the RB and p53 pathway may be able to explain cervical tumorigenesis. However, there are no reports in which all components of the G1 pathway and HPV typing were examined simultaneously in cervical cancer. METHODS: We examined HPV typing and the status of the G1 pathways simultaneously by PCR-SSCP, multiplex PCR, methylation-specific PCR, and immunohistochemical techniques in cervical neoplasia. A total of 105 samples (normal, 10; cervical intraepithelial neoplasm (CIN), 42; invasive cancer (IC), 53) were included. RESULTS: Abnormality of the RB pathway tended to be more frequent in ICs (60.4%) than in CINs (31.0%) (P = 0.069). The primary target was p16 (CIN, 14.3%; IC, 43.4%; P = 0.032). Abnormality of the p53 pathway was detected in ICs (56.6%) and in CINs (40.5%) (P = 0.1494). In particular, strong expression of MDM2 was higher in ICs (32.1%) than in CINs (7.1%) (P = 0.0045). Abnormalities of the RB and p53 pathways were higher in low-risk and negative HPV than in high-risk HPV (81.3% vs 51.4%, P = 0.0657; 81.3% vs 45.9%, P = 0.0328). Seven HPV-negative cases had abnormalities in the RB or p53 pathways. CONCLUSION: In conclusion, abnormality of the G1 pathway may be one of the important mechanisms for carcinogenesis of low-risk and negative HPV cases.

Cyclin D1↗

PubMed2001

Polymorphisms of the CYP1A1 and GSTM1 genes in relation to individual susceptibility to lung carcinoma in Chinese population.

Cytochrome P450 1A1 (CYP1A1) and glutathione S-transferase M1 (GSTM1) metabolize tobacco-related carcinogens. To investigate the prevalence of CYP1A1 and GSTM1, and their association with increased risk of lung carcinoma in Chinese, allele-specific PCR and multiplex PCR technique were employed to identify the genotypes of CYP1A1 and GSTM1 in a case-control study of 106 lung carcinoma patients with histopathological diagnosis and 106 matched controls free of malignancy in Jiangsu Province, China. Logistic regression analysis was performed to calculate the odds ratio (OR) and 95% confidence intervals (CI). The results showed that individuals with GSTM1 null, and the combined GSTM1 null/CYP1A1 Ile/Val or GSTM1 null/CYP1A1 Val/Val had an elevated risk of lung carcinoma, with the OR, 1.92 (P=0.02; CI, 1.07-3.46), 3.27 (P=0.01; CI, 1.23-8.84) and 9.33 (P=0.04; CI, 1.01-217.42), respectively. Light smokers (<30 pack-years) carrying GSTM1 null genotype were shown to have the increased risk to lung carcinoma (OR=3.47; CI, 1.13-7.57). Our study suggested that the null GSTM1 genotype, independently or in combined with at least one Val allele of CYP1A1, might affect the genetic susceptibility for lung carcinoma in Chinese population.

Adult↗

PubMed2005

Facile, comprehensive, high-throughput genotyping of human genital papillomaviruses using spectrally addressable liquid bead microarrays.

Human papillomavirus (HPV) is the worldwide cause of carcinoma of the uterine cervix, a cancer that is the second most common neoplasm in women, resulting in nearly 250,000 deaths a year. The magnitude of the risk of cancer after HPV infection, however, is virus type-specific. Over 40 HPV types can infect the genital tract. Comprehensive, high-throughput typing assays for HPV, however, are not currently available. Blending multiplex PCR and multiplex hybridization using spectrally addressable liquid bead microarrays we have developed a high-throughput, fast, single-tube-typing assay capable of simultaneously typing 45 HPV. The overall incidence of HPV in 429 women tested using this new assay was 72.2% for those with squamous intraepithelial lesions, 51.5% for those with atypical squamous cells of undetermined significance and 15.4% for women with normal cytology, respectively. This compared well with the incidence of HPV detected by a parallel non-typing generic high-risk assay. The new assay detected a wide spectrum of HPV types and a high incidence of mixed infections. We believe our assay may find widespread applications in areas requiring virus type-specific information, such as in epidemiological studies, cancer screening programs, monitoring therapeutic interventions, and evaluating the efficacy of HPV vaccine trials.

DNA, Viral↗

PubMed2002

Toward efficient analysis of mutations in single cells from ethanol-fixed, paraffin-embedded, and immunohistochemically stained tissues.

Only a few studies have demonstrated successful molecular analysis after whole genome amplification using single cells dissected from paraffin-embedded tissues. The results in these studies were limited by low-amplification efficiency and high rates of allele dropout. In the present study, the amplification rate using a thoroughly modified primer extension and preamplification-PCR protocol was improved significantly for single cells microdissected from paraffin-embedded and immunohistochemically stained tissues. Tissue fixation with ethanol (85%) and the addition of 0.2 mmol/L EDTA helped to achieve an amplification rate between 67% (segments 200 to 400 bp) and 72% (segments <200 bp). Normal tissue sections were immunohistochemically double stained for overabundance of p53 protein and proliferating cell nuclear antigen. Microdissection of single cells was performed with a manual micromanipulator equipped with a Tungsten needle. Sequence analysis of the TP53 gene was performed after improved primer extension preamplification-PCR and multiplex PCR from single microdissected cells. The rate of allele dropout was at least 68%. These technical advances facilitate routine mutation analysis using a single cell or a few cells microdissected from routinely processed paraffin-embedded normal and tumor tissues. Allele dropout still represents a serious problem in single-cell mutation analysis, especially in samples with limited template DNA and prone to DNA damage.

Breast↗

PubMed2003

Laboratory diagnosis of Mycoplasma pneumoniae infection.

Diagnosis of Mycoplasma pneumoniae infection is challenging due to the fastidious nature of the pathogen, the considerable seroprevalence, and the possibility of transient asymptomatic carriage. During recent years, various new techniques have been adapted for the diagnosis of M. pneumoniae infection, notably in the field of molecular biology. Standard polymerase chain reaction (PCR) is currently the method of choice for direct pathogen detection, but several PCR-related methods provide enhanced sensitivity or more convenient handling procedures, and have been successfully applied for research purposes. Among these techniques are real-time PCR, nested PCR, reverse transcriptase PCR (RT-PCR) and multiplex PCR. Generally, amplification-based methods have replaced hybridization assays and direct antigen detection. Serology, which is the basic strategy for mycoplasma diagnosis in routine clinical practice, has been improved by the widespread availability of sensitive assays for separate detection of different antibody classes. For the diagnosis of mycoplasma pneumonia, serology and direct pathogen detection should be combined. Extrapulmonary diseases may be diagnosed by direct pathogen detection alone, but the value of this diagnostic approach is limited by the probably immunologically mediated pathogenesis of some manifestations. This review summarizes the current state of Mycoplasma pneumoniae diagnosis, with special reference to molecular techniques. The value of different methods for routine diagnosis and research purposes is discussed.

Culture Media↗