scispace - formally typeset
Search or ask a question

Showing papers on "Multiplex polymerase chain reaction published in 2001"


Journal ArticleDOI
TL;DR: Multiplex PCR should result in significant savings in terms of labour and cost in analysis of a large number of strains when compared with using an individual PCR for targeting each gene.

707 citations


Journal ArticleDOI
TL;DR: A single-tube 5′ nuclease multiplex PCR assay was developed on the ABI 7700 Sequence Detection System (TaqMan) for the detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae from clinical samples of cerebrospinal fluid, plasma, serum, and whole blood.
Abstract: A single-tube 5' nuclease multiplex PCR assay was developed on the ABI 7700 Sequence Detection System (TaqMan) for the detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae from clinical samples of cerebrospinal fluid (CSF), plasma, serum, and whole blood. Capsular transport (ctrA), capsulation (bexA), and pneumolysin (ply) gene targets specific for N. meningitidis, H. influenzae, and S. pneumoniae, respectively, were selected. Using sequence-specific fluorescent-dye-labeled probes and continuous real-time monitoring, accumulation of amplified product was measured. Sensitivity was assessed using clinical samples (CSF, serum, plasma, and whole blood) from culture-confirmed cases for the three organisms. The respective sensitivities (as percentages) for N. meningitidis, H. influenzae, and S. pneumoniae were 88.4, 100, and 91.8. The primer sets were 100% specific for the selected culture isolates. The ctrA primers amplified meningococcal serogroups A, B, C, 29E, W135, X, Y, and Z; the ply primers amplified pneumococcal serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10A, 11A, 12, 14, 15B, 17F, 18C, 19, 20, 22, 23, 24, 31, and 33; and the bexA primers amplified H. influenzae types b and c. Coamplification of two target genes without a loss of sensitivity was demonstrated. The multiplex assay was then used to test a large number (n = 4,113) of culture-negative samples for the three pathogens. Cases of meningococcal, H. influenzae, and pneumococcal disease that had not previously been confirmed by culture were identified with this assay. The ctrA primer set used in the multiplex PCR was found to be more sensitive (P < 0.0001) than the ctrA primers that had been used for meningococcal PCR testing at that time.

590 citations


Journal ArticleDOI
TL;DR: In this paper, the authors combined the Invader assay with multiplex polymerase chain reaction (PCR), carried out in the presence of antibody to Taq polymerase, as well as using a novel 384-well card system that can reduce the required reaction volume.
Abstract: One of the most difficult issues to be solved in genome-wide association studies is to reduce the amount of genomic DNA required for genotyping. Currently available technologies require too large a quantity of genomic DNA to genotype with hundreds or thousands of single-nucleotide polymorphisms (SNPs). To overcome this problem, we combined the Invader assay with multiplex polymerase chain reaction (PCR), carried out in the presence of antibody to Taq polymerase, as well as using a novel 384-well card system that can reduce the required reaction volume. We amplified 100 genomic DNA fragments, each containing one SNP, in a single tube, and analyzed each SNP with the Invader assay. This procedure correctly genotyped 98 of the 100 SNP loci examined in PCR-amplified samples from ten individuals; the genotypes were confirmed by direct sequencing. The reproducibility and universality of the method were confirmed with two additional sets of 100 SNPs. Because we used 40 ng of genomic DNA as a template for multiplex PCR, the amount needed to assay one SNP was only 0.4 ng; therefore, theoretically, more than 200,000 SNPs could be genotyped at once when 100 μg of genomic DNA is available. Our results indicate the feasibility of undertaking genome-wide association studies using blood samples of only 5–10 ml.

425 citations


Journal ArticleDOI
01 Jul 2001-Blood
TL;DR: Of the numerous mutations that have been described, deletions at the α-globin gene locus account for the vast majority of α-thalassemia alleles and the most widely occurring are the -α3.7 and -α4.2.

325 citations


Journal ArticleDOI
TL;DR: The results suggest that microarray analysis of microbial virulence factors might be very useful for automated identification and characterization of bacterial pathogens.
Abstract: Hybridization with oligonucleotide microchips (microarrays) was used for discrimination among strains of Escherichia coli and other pathogenic enteric bacteria harboring various virulence factors. Oligonucleotide microchips are miniature arrays of gene-specific oligonucleotide probes immobilized on a glass surface. The combination of this technique with the amplification of genetic material by PCR is a powerful tool for the detection of and simultaneous discrimination among food-borne human pathogens. The presence of six genes (eaeA, slt-I, slt-II, fliC, rfbE, and ipaH) encoding bacterial antigenic determinants and virulence factors of bacterial strains was monitored by multiplex PCR followed by hybridization of the denatured PCR product to the gene-specific oligonucleotides on the microchip. The assay was able to detect these virulence factors in 15 Salmonella, Shigella, and E. coli strains. The results of the chip analysis were confirmed by hybridization of radiolabeled gene-specific probes to genomic DNA from bacterial colonies. In contrast, gel electrophoretic analysis of the multiplex PCR products used for the microarray analysis produced ambiguous results due to the presence of unexpected and uncharacterized bands. Our results suggest that microarray analysis of microbial virulence factors might be very useful for automated identification and characterization of bacterial pathogens.

266 citations


Journal ArticleDOI
TL;DR: This assay offers a rapid, simple, feasible, specific, sensitive, and accurate identification of mupirocin-resistant MRSA clinical isolates and could be systematically applied as a diagnostic test in clinical microbiology laboratories, facilitating the design and use of antibiotic therapy.
Abstract: In this work, we describe a multiplex PCR assay for the detection of clinically relevant antibiotic resistance genes harbored by some Staphylococcus aureus isolates and for the simultaneous identification of such isolates at the species level. Conditions were optimized for the simultaneous detection of the 310-, 456-, and 651-bp regions of the mecA (encoding high-level methicillin resistance), ileS-2 (encoding high-level mupirocin resistance), and femB (encoding a factor essential for methicillin resistance) genes, respectively, from a single colony in a single reaction tube. The femB PCR fragment allows the specific identification of S. aureus. Validation of the method was performed using 50 human isolates of methicillin-resistant S. aureus (MRSA) and the appropriate control strains. This assay offers a rapid, simple, feasible, specific, sensitive, and accurate identification of mupirocin-resistant MRSA clinical isolates and could be systematically applied as a diagnostic test in clinical microbiology laboratories, facilitating the design and use of antibiotic therapy.

231 citations


Journal ArticleDOI
TL;DR: In conclusion, multiplex PCR followed by electrophoresis seems to be a promising tool for the rapid identification of common and uncommon yeast strains from culture colonies and from yeast-positive blood culture bottles.
Abstract: Multiplex PCR amplification followed by either agarose gel electrophoresis (PCR-AGE) or microchip electrophoresis (PCR-ME) was used to test a total of 120 fungal strains. The internal transcribed spacer 1 (ITS1) and ITS2 regions and the 5.8S ribosomal DNA (rDNA) region of the fungi were amplified by using universal primers ITS1 and ITS4. The ITS2 region was simultaneously amplified by using universal primers ITS3 and ITS4. Since Trichosporon asahi and T. asteroides showed similar lengths for two amplicons, 29 different gel patterns were demonstrated for 30 yeast species tested on the basis of differences in the lengths of one or two amplicons. Of 75 yeast isolates from clinical materials, 5 isolates (6.8%) which were incompletely identified or not identified by the phenotypic method were identified with our PCR-based method (2 isolates as Candida guilliermondii, 2 as C. krusei, and 1 as C. zeylanoides). No differences in discriminating power or sensitivity were observed between the PCR-AGE method and the PCR-ME method. These methods, prospectively applied to 24 yeast-positive blood culture bottles (16 patients), resulted in the correct detection of 24 yeast strains. In conclusion, multiplex PCR followed by electrophoresis seems to be a promising tool for the rapid identification of common and uncommon yeast strains from culture colonies and from yeast-positive blood culture bottles (5.5 h for the PCR-AGE method and 3 h for the PCR-ME method).

228 citations


Journal ArticleDOI
TL;DR: The development of a multiplex PCR protocol for the diagnosis of staphylococcal infection was reported, which identified two isolates that were mecA positive but were oxacillin sensitive according to conventional methods.
Abstract: We report the development of a multiplex PCR protocol for the diagnosis of staphylococcal infection The protocol was designed to (i) detect any staphylococcal species to the exclusion of other bacterial pathogens (based on primers corresponding to Staphylococcus-specific regions of the 16S rRNA genes), (ii) distinguish between S aureus and the coagulase-negative staphylococci (CNS) (based on amplification of the S aureus-specific clfA gene), and (iii) provide an indication of the likelihood that the staphylococci present in the specimen are resistant to oxacillin (based on amplification of the mecA gene) The expected fragments were amplified from each of 60 staphylococcal isolates (13 oxacillin-resistant S aureus isolates, 23 oxacillin-sensitive S aureus isolates, 17 oxacillin-resistant CNS, and 7 oxacillin-sensitive CNS) No amplification products were observed with template DNA from nonstaphylococcal species, and the efficiency of amplification of staphylococcal targets was not adversely affected by the presence of DNA from other bacterial species in the same sample The utility of the protocol for the analysis of clinical samples was verified by analysis of aliquots taken directly from BacT/Alert blood culture bottles Of 77 blood cultures tested, only 7 yielded results inconsistent with those of conventional methods of diagnosis and susceptibility testing Of those, one was identified as a CNS species by PCR and S aureus by conventional methods We also identified two isolates that were mecA positive but were oxacillin sensitive according to conventional methods The other four samples failed to yield any amplification product even with a control set of primers corresponding to a conserved region of the eubacterial rRNA genes

209 citations


Journal ArticleDOI
TL;DR: The results show that DNA microarray technology provides a useful supplement to PCR-based diagnostic methods and is used to type and subtype several previously unsequenced influenza virus isolates.
Abstract: A model DNA microarray has been prepared and shown to facilitate typing and subtyping of human influenza A and B viruses. Reverse transcriptase PCR was used to prepare cDNAs encoding ∼500-bp influenza virus gene fragments, which were then cloned, sequenced, reamplified, and spotted to form a glass-bound microarray. These target DNAs included multiple fragments of the hemagglutinin, neuraminidase, and matrix protein genes. Cy3- or Cy5-labeled fluorescent probes were then hybridized to these target DNAs, and the arrays were scanned to determine the probe binding site(s). The hybridization pattern agreed perfectly with the known grid location of each target, and the signal-to-background ratio varied from 5 to 30. No cross-hybridization could be detected beyond that expected from the limited degree of sequence overlap between different probes and targets. At least 100 to 150 bp of homology was required for hybridization under the conditions used in this study. Combinations of Cy3- and Cy5-labeled DNAs can also be hybridized to the same chip, permitting further differentiation of amplified molecules in complex mixtures. In a more realistic test of the technology, several sets of multiplex PCR primers that collectively target influenza A and B virus strains were identified and were used to type and subtype several previously unsequenced influenza virus isolates. The results show that DNA microarray technology provides a useful supplement to PCR-based diagnostic methods.

183 citations


Journal ArticleDOI
TL;DR: Two opposing rRNA gene-targeted primers have been developed for specific detection of this microorganism by PCR and the specificity and sensitivity of this detection with a pure culture of B. lactis were evaluated and verified.
Abstract: The species Bifidobacterium lactis, with its main representative strain Bb12 (DSM 10140), is a yoghurt isolate used as a probiotic strain and is commercially applied in different types of yoghurts and infant formulas In order to ensure the genetic identity and safety of this bacterial isolate, species- and strain-specific molecular tools for genetic fingerprinting must be available to identify isolated bifidobacteria or lactic acid bacteria from, eg, various clinical environments of relevance in medical microbiology Two opposing rRNA gene-targeted primers have been developed for specific detection of this microorganism by PCR The specificity of this approach was evaluated and verified with DNA samples isolated from single and mixed cultures of bifidobacteria and lactobacilli (48 isolates, including the type strains of 29 Bifidobacterium and 9 Lactobacillus species) Furthermore, we performed a Multiplex-PCR using oligonucleotide primers targeting a specific region of the 16S rRNA gene for the genus Bifidobacterium and a conserved eubacterial 16S rDNA sequence The specificity and sensitivity of this detection with a pure culture of B lactis were, respectively, 100 bacteria/ml after 25 cycles of PCR and 1 to 10 bacteria/ml after a 50-cycle nested-PCR approach

155 citations


Journal ArticleDOI
TL;DR: The use of matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF) is incorporated in the laboratory as a tool for differentiating genotypes based on the mass of the variant DNA sequence, and the MS technique provides a cost‐effective and efficient method for high‐throughput genotyping.
Abstract: Single nucleotide polymorphisms (SNPs) are currently being identified and mapped at a remarkable pace, providing a rich genetic resource with vast potential for disease gene discovery, pharmacogenetics, and understanding the origins of modern humans. High-throughput, cost effective genotyping methods are essential in order to make the most advantageous and immediate use of these SNP data. We have incorporated the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) in our laboratory as a tool for differentiating genotypes based on the mass of the variant DNA sequence, and have utilized this method for production scale SNP genotyping. We have combined a 4 μl PCR amplification reaction using 3 ng of genomic DNA with a secondary enzymatic reaction (mini-sequencing) containing oligonucleotide primers that anneal immediately upstream of the polymorphic site, dideoxynucleotides, and a thermostable polymerase used to extend the PCR product by a single base pair. Mass spectrometry (MS) analysis of mini-sequencing reactions was performed using a MALDI-TOF instrument (Voyager-DE, Perseptive Biosystems, Framingham, MA). We performed both single and multiplex PCR and mini-sequencing reactions, and genotyped seven different variant sites in a random sample of 989 individuals. Genotypes generated with MS methods were compared with genotypes produced using a 5′ exonuclease fluorescence-based assay (Taqman, Applied Biosystems, Foster City, CA) and a gel-based genotyping protocol. Because multiple polymorphisms can be detected in a single reaction, the MS technique provides a cost-effective and efficient method for high-throughput genotyping. Hum Mutat 17:296–304, 2001. © 2001 Wiley-Liss, Inc.

Journal ArticleDOI
TL;DR: This multiplex PCR assay was developed for the simultaneous detection of the four major bacterial causes of bovine mastitis, Staphylococcus aureus, Streptococcus agalactiae, StrePTococcus dysgalactia, and Streptitis uberis and suggested that it could be used as an alternative method in routine diagnosis for rapid, sensitive, and specific simultaneous detection in milk samples.

Journal ArticleDOI
TL;DR: A multiplex polymerase chain reaction (PCR) assay was developed for the detection and differentiation of enterotoxigenic Staphylococcus aureus in dairy products and allows presumptive identification and differentiation in less than 6 h.

Journal ArticleDOI
TL;DR: Improved a multiplex PCR method described in the previous report in order to distinguish the five lines of genetically modified (GM) maize, and re-designed new primer pairs to increase the specificity of PCR to distinguishFive lines of GM maize by multipleX PCR.
Abstract: 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.

Journal ArticleDOI
TL;DR: A multiplex PCR method was developed to rapidly identify clinically important yeasts that cause fungemia and is simpler than any previously reported molecular method for the identification of blood yeasts.
Abstract: Yeasts are emerging as important etiological agents of nosocomial bloodstream infections. A multiplex PCR method was developed to rapidly identify clinically important yeasts that cause fungemia. The method amplified the internal transcribed spacer 1 (ITS1) region between the 18S and 5.8S rRNA genes and a specific DNA fragment within the ITS2 region of Candida albicans. With this method, C. albicans produced two amplicons, whereas other species produced only one. Through sequence analysis, the precise lengths of the PCR products were found to be as follows: C. glabrata (482 or 483 bp), C. guilliermondii (248 bp), C. parapsilosis (229 bp), C. albicans (218 or 219 and 110 bp), C. tropicalis (218 bp), Cryptococcus neoformans (201 bp), and C. krusei (182 bp). The PCR products could be effectively separated by disk polyacrylamide gel electrophoresis. The method was used to test 249 positive blood cultures (255 isolates), from which the following species (strain number) were isolated: C. albicans (128), C. tropicalis (51), C. glabrata (28), C. parapsilosis (23), C. neoformans (9), C. krusei (5), C. guilliermondii (3), and other, minor species (8). The test sensitivity of the method was 96.9% (247 of 255 isolates). The eight minor species were either misidentified (one strain) or not identified (seven strains). From the time at which a positive bottle was found, the multiplex PCR could be completed within 8 h; the present method is simpler than any previously reported molecular method for the identification of blood yeasts.

Journal ArticleDOI
TL;DR: The present multiplex PCR assay detects simultaneously five different herpesviruses and sample suitability for PCR in a single amplification round of 40 cycles with an excellent sensitivity can provide an early, rapid, reliable noninvasive diagnostic tool allowing the application of antiviral therapy on the basis of a specific viral diagnosis.
Abstract: A sensitive multiplex PCR assay for single-tube amplification that detects simultaneous herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella-zoster virus (VZV), human cytomegalovirus (CMV), and Epstein-Barr virus (EBV) is reported with particular emphasis on how the method was optimized and carried out and its sensitivity was compared to previously described assays. The assay has been used on a limited number of clinical samples and must be thoroughly evaluated in the clinical context. A total of 86 cerebrospinal fluid (CSF) specimens from patients which had the clinical symptoms of encephalitis, meningitis or meningoencephalitis were included in this study. The sensitivity of the multiplex PCR was determined to be 0.01 and 0.03 50% tissue culture infective doses/the reciprocal of the highest dilution positive by PCR for HSV-1 and HSV-2 respectively, whereas for VZV, CMV and EBV, 14, 18, and 160 ag of genomic DNA were detected corresponding to 48, 66, and 840 genome copies respectively. Overall, 9 (10.3%) of the CSF samples tested were positive in the multiplex PCR. HSV-1 was detected in three patients (3.5%) with encephalitis, VZV was detected in four patients (4.6%) with meningitis, HSV-2 was detected in one neonate (1.16%), and CMV was also detected in one neonate (1.16%). None of the samples tested was positive for the EBV genome. None of the nine positive CSF samples presented herpesvirus coinfection in the central nervous system. Failure of DNA extraction or failure to remove any inhibitors of DNA amplification from CSF samples was avoided by the inclusion in the present multiplex PCR assay of α-tubulin primers. The present multiplex PCR assay detects simultaneously five different herpesviruses and sample suitability for PCR in a single amplification round of 40 cycles with an excellent sensitivity and can, therefore, provide an early, rapid, reliable noninvasive diagnostic tool allowing the application of antiviral therapy on the basis of a specific viral diagnosis. The results of this preliminary study should prompt a more exhaustive analysis of the clinical value of the present multiplex PCR assay.

Journal ArticleDOI
TL;DR: Microsatellite marker analysis with multiplex PCR and automated procedures has a high throughput and should be suitable for large epidemiologic studies of C. albicans, confirming that candidemia usually originates from the colonizing isolate.
Abstract: To obtain a rapid genotyping method of Candida albicans, three polymorphic microsatellite markers were investigated by multiplex PCR. The three loci, called CDC3, EF3, and HIS3, were chosen because they are on different chromosomes so as to improve the chances of finding polymorphisms. One set of primers was designed for each locus, and one primer of each set was dye-labeled to read PCR signals by using an automatic sequencer. Amplifications were performed directly from the colonies harvested on the agar plate without a sophisticated DNA extraction step. At total of 27 reference strains and 73 clinical independent isolates were tested. The numbers of allelic associations were 10, 22, and 25 for the loci CDC3, EF3, and HIS3, respectively. The combined discriminatory power of the three microsatellites markers was 0.97. The markers were stable after 25 subcultures, and the amplifications were specific for C. albicans. An initial study of 17 clinical isolate pairs, including blood culture and peripheral sites, showed a similar genotype for 15 of them, confirming that candidemia usually originates from the colonizing isolate. Therefore, microsatellite marker analysis with multiplex PCR and automated procedures has a high throughput and should be suitable for large epidemiologic studies of C. albicans.

Journal ArticleDOI
TL;DR: A PCR protocol for rapid identification of enterohemorrhagic Escherichia coli on a LightCycler instrument and the genes encoding Shiga toxin 1 and Shiga toxins 2 are detected in a single reaction capillary.
Abstract: In this report, we present a PCR protocol for rapid identification of enterohemorrhagic Escherichia coli on a LightCycler instrument. In a multiplex assay, the genes encoding Shiga toxin 1 and Shiga toxin 2 are detected in a single reaction capillary. A complete analysis of up to 32 samples takes about 45 min.

Journal ArticleDOI
R L Hu1, G Huang1, W Qiu1, Z H Zhong1, X Z Xia1, Z Yin1 
TL;DR: One pair of common primers was designed and synthesized according to the sequences of the E3 and flanking regions and a polymerase chain reaction (PCR) assay was established using these two primers to amplify the virus-specific DNA fragment from clinical specimens as well as from cell cultures, making it applicable in the rapid confirmation of diagnosis and differentiation of the two types of canine adenoviruses.
Abstract: Canine adenovirus type 1 (CAV-1) and type 2 (CAV-2) can be categorized in the laboratory by haemagglutination and neutralization tests, but they are difficult to differentiate from each other in specimens, especially when infection occurs in the digestive tract. The object of this study was to develop a simple method of detecting and differentiating them. One pair of common primers was designed and synthesized according to the sequences of the E3 and flanking regions and a polymerase chain reaction (PCR) assay was established using these two primers to amplify the virus-specific DNA fragment from clinical specimens as well as from cell cultures. After elecctrophoresis, under the same amplification conditions, 508 bp and 1030 bp PCR products were observed for CAV-1 and CAV-2, respectively. These were further shown to be adenovirus specific by dot hybridization and sequencing. As only one pair of primers was involved in the PCR procedure, it was faster and easier to perform than any of the other assays used for detecting canine adenovirus, making it applicable in the rapid confirmation of diagnosis and differentiation of the two types of canine adenoviruses.

Journal ArticleDOI
TL;DR: Two new approaches for the molecular determination of RhD zygosity are explored, based on the double Amplification Refractory Mutation System and multiplex real-time quantitative PCR, which accurately distinguished homozygous from heterozygous RhD-positive samples.
Abstract: Background: Rh isoimmunization and hemolytic disease of the newborn still occur despite the availability of Rh immunoglobulin. For the prenatal investigation of sensitized RhD-negative pregnant women, determination of the zygosity of the RhD-positive father has important implications. The currently available molecular methods for RhD zygosity assessment, in general, are technically demanding and labor-intensive. Therefore, at present, rhesus genotype assessment is most commonly inferred from results of serological tests. The recent elucidation of the genetic structure of the prevalent RHD deletion in Caucasians, as well as the development of real-time PCR, allowed us to explore two new approaches for the molecular determination of RhD zygosity. Methods: Two methods for RhD zygosity determination were developed. The first was based on the double Amplification Refractory Mutation System (double ARMS). The second was based on multiplex real-time quantitative PCR. For the double ARMS assay, allele-specific primers were designed to directly amplify the most prevalent RHD deletion found in RhD-negative individuals in the Caucasian population. The multiplex real-time quantitative PCR assay, on the other hand, involved coamplification and quantification of RHD -specific sequences in relation to a reference gene, albumin , in a single PCR reaction. A ratio, ΔCt, based on the threshold cycle, was then determined and reflects the RHD gene dosage. Results: The allele-specific primers of the double ARMS assay reliably amplified the RHD -deleted allele and therefore accurately distinguished homozygous from heterozygous RhD-positive samples. The results were in complete concordance with serological testing. For the multiplex real-time quantitative PCR assay, the ΔCt values clearly segregated into two distinct populations according to the RHD gene dosage, with mean values of 1.70 (SD, 0.17) and 2.62 (SD, 0.29) for the homozygous and heterozygous samples, respectively ( P <0.001, t -test). The results were in complete concordance with the results of serological testing as well as with the double ARMS assay. Conclusion: Double ARMS and real-time quantitative PCR are alternative robust assays for the determination of RhD zygosity.

Journal ArticleDOI
TL;DR: A novel allele-specific primer elongation protocol using a DNA polymerase on oligonucleotide chips greatly simplifies preamplification of SNP-containing regions, thereby providing a framework for typing hundreds of mtDNA polymorphisms.
Abstract: We have developed a novel allele-specific primer elongation protocol using a DNA polymerase on oligonucleotide chips. Oligonucleotide primers carrying polymorphic sites at their free 3'end were covalently bound to glass slides. The generation of single-stranded targets of genomic DNA containing single nuclotide polymorphisms (SNPs) to be typed was achieved by an asymmetric PCR reaction or exonuclease treatment of phosphothioate (PTO)-modified PCR products. In the presence of DNA polymerase and all four dNTPs, with Cy3-dUTP replacing dTTP, allele-specific extension of the immobilized primers took place along a stretch of target DNA sequence. The yield of elongated products was increased by repeated reaction cycles. We performed multiplexed assays with many small DNA targets, or used single targets of up to 4.4 kb mitochondrial DNA (mtDNA) sequence to detect multiple SNPs in one reaction. The latter approach greatly simplifies preamplification of SNP-containing regions, thereby providing a framework for typing hundreds of mtDNA polymorphisms.

Journal Article
TL;DR: The data suggest that such assays provide a good basis for the clinical examination of multiple fetal loci, in particular Rhesus D status or fetal sex, and can be performed effectively using real-time multiplex PCR assays.
Abstract: Questions under study Pregnancies with a Rhesus constellation still present a considerable obstetric problem. In addition, pregnancies with male Rhesus D fetuses are more severely affected by haemolytic disease of the newborn, requiring more transfusions in utero and having a three fold higher mortality than female Rhesus D fetuses. Furthermore, almost 150 X-linked genetic deficiencies have now been characterised, increasing the need for prenatal sex determination in pregnancies at risk for such a disorder. In order examine these two important fetal loci in a risk free manner, we have developed a novel multiplex real-time PCR assay for the analysis of extracellular fetal DNA in maternal plasma. Methods Cell free DNA was isolated from 34 maternal plasma samples and examined by a multiplex real-time PCR assay for the Rhesus D gene and the SRY locus on the Y chromosome. Results Our study showed that we were able to genotype 12/13 Rhesus D males correctly. All 5 Rhesus d males were correctly identified. In addition a 100% concordance was found in the 16 samples obtained from pregnancies with female Rhesus D or Rhesus d fetuses. Conclusions By developing a novel multiplex real-time PCR assay we present the first report describing the determination of multiple fetal loci from cell free DNA in maternal plasma by these means. As this assay is suitable for automation, our data, therefore, suggest that such assays provide a good basis for the clinical examination of multiple fetal loci, in particular Rhesus D status or fetal sex, and can be performed effectively using real-time multiplex PCR assays.

Journal ArticleDOI
TL;DR: The results show that multiplex PCR and quantitative real-time PCR assays can be used in resistance screening to measure the interaction between different pathogens and their hosts at different growth stages, and in specific tissues.
Abstract: A multiplex Polymerase Chain Reaction (PCR) assay was developed to detect and quantify four fungal foliar pathogens in wheat. For Septoria tritici (leaf blotch) and Stagonospora nodorum (leaf and glume blotch), the β-tubulin gene was used as the target region. Diagnostic targets for Puccinia striiformis (stripe or yellow rust) and P. recondita (brown rust) were obtained from PCR products amplified with β-tubulin primer sequences. Final primer sets were designed and selected after being tested against several fungi, and against DNA of infected and healthy wheat leaves. For detection of the four pathogens, PCR products of different sizes were amplified simultaneously, whereas no products were generated from wheat DNA or other non-target fungi tested. The presence of each of the diseases was wheat tissue- and cultivar specific. Using real-time PCR measurements with the fluorescent dye SYBR Green I, PCR-amplified products could be quantified individually, by reference to a standard curve generated by adding known amounts of target DNA. Infection levels for each of the diseases were measured in the flag leaf of 19 cultivars at Growth Stage (GS) 60–64 in both 1998 and 1999. The infection levels for the cultivars were ranked, and showed, with a few exceptions, a good correlation with the NIAB Recommended List for winter wheat, which is based on visual assessment of symptoms. With PCR, the presence of the different pathogens was accurately diagnosed and quantification of pre-symptomatic infection levels was possible. Although sampling and DNA detection methods need further optimisation, the results show that multiplex PCR and quantitative real-time PCR assays can be used in resistance screening to measure the interaction between different pathogens and their hosts at different growth stages, and in specific tissues. This should enable an earlier identification of specific resistance mechanisms in both early-stage breeding material and field trials.

Journal ArticleDOI
TL;DR: A 24‐h system for the detection of Listeria monocytogenes in ham is developed to develop a 24-h system to detect the bacterium in ham.
Abstract: Aims: To develop a 24-h system for the detection of Listeria monocytogenes in ham. Methods and Results: An immunomagnetic separation (IMS) of bacteria directly from ham followed by extraction of DNA and detection using a new multiplex polymerase chain reaction (PCR) was used. The PCR method used one primer pair targeted at the listeriolysin O gene of L. monocytogenes and the other pair for a region of the 23S rRNA genes of Listeria, giving products of 706 and 239 bp, respectively. The combined IMS/PCR was calculated to be capable of detecting as few as 1·1 L. monocytogenes cells g–1 in a 25-g ham sample. Conclusions: The process produced acceptable results, but the IMS step is the main barrier to further improvement of sensitivity. The DNA isolation was the most time-consuming step in the process. Significance and Impact of the Study: A 24-h test for the presence of L. monocytogenes will be useful to the food industry and significantly assist in the timely investigation of outbreaks.

Journal ArticleDOI
TL;DR: A multiplex polymerase chain reaction (PCR) test was developed for identifying gastrointestinal (GI) nematodes that commonly infect cattle and a closely migrating doublet was generated suggesting size heterogeneity in the ETS which is consistent with multiple rDNA repeat units within this species.

Journal ArticleDOI
Nao Suzuki1, Yoshio Nakano1, Yasuo Yoshida1, Daisuke Ikeda1, Toshihiko Koga1 
TL;DR: Oligonucleotide primers specific for gene clusters involved in the biosynthesis of serotype-specific polysaccharide antigens were designed to identify Actinobacillus actinomycetemcomitans serotypes using the multiplex PCR, and may be useful for serotypes-specific genotyping rapidly and directly from clinical samples containing various organisms.
Abstract: Oligonucleotide primers specific for gene clusters involved in the biosynthesis of serotype-specific polysaccharide antigens were designed to identify Actinobacillus actinomycetemcomitans serotypes a to e using the multiplex PCR. This method may be useful for serotype-specific genotyping rapidly and directly from clinical samples containing various organisms.

Journal ArticleDOI
TL;DR: A multiplex PCR assay for type‐specific identification of Ad3, Ad7, and Ad21 isolates will provide a rapid and convenient tool for the epidemiologic investigation of Ad‐associated respiratory illness.
Abstract: Human adenovirus (Ad) serotypes 3, 7, and 21 of DNA cluster B:1 are often associated with severe respiratory illness, particularly in infants and young children and, in addition to Ad4, are among the most important causes of acute respiratory disease syndrome in new military recruits. To address the inherent problems associated with classic typing methods, we developed a multiplex PCR assay for the rapid, specific identification of Ad3, Ad7, and Ad21 field isolates. To design type-specific primers for our assay, we sequenced the Ad21 hexon gene and compared this sequence with previously published sequences of Ad3, Ad7, and Ad16. The overall nucleotide (nt) and amino acid (aa) identities between Ad21 and Ad3, Ad7, and Ad16 were similar (ranges 78.3-80.8% nt; 84.1-86.2% aa), with significantly greater variability in the regions of the hexon that encode surface loops 1 and 2. Type-specific primers designed to the hypervariable regions correctly identified Ad3, Ad7, and Ad21 prototype strains and 53 previously typed Ad field isolates. No cross-reactions with other Ad serotypes were identified. Our multiplex PCR assay for type-specific identification of Ad3, Ad7, and Ad21 isolates will provide a rapid and convenient tool for the epidemiologic investigation of Ad-associated respiratory illness.

Journal ArticleDOI
TL;DR: Specific attention is given to advances provided by multiplex PCR, fluorescence-based "real-time" PCR, and the utilization of PCR as a quantitative technique in the application of PCR methodology to parasite detection and differentiation and the diagnosis of disease.

Journal ArticleDOI
TL;DR: This new procedure, called two primers (TP)‐RAPD fingerprinting, is rapid, sensitive, reliable, highly reproducible and suitable for experiments with a large number of microorganisms, and can be applied to bacterial taxonomy, ecological studies and for the detection of new bacterial species.
Abstract: Polymerase chain reation (PCR) fingerprints are used to characterize and recognize bacteria and are generally obtained using universal primers that generate an array of DNA amplicons, which can be separated by electrophoresis Universal primers 8F and 1491 R have been used to amplify specifically 16S rDNA We have used these primers at an annealing temperature of 50 degrees C Agarose gel electrophoresis of PCR products revealed several bands The band pattern of each bacterial species was different and the strains belonging to the same species shared an identical pattern The patterns obtained did not show variations with plasmid DNA content or the growth stage of the bacteria The peculiarity of the randomly amplified polymorphic DNA (RAPD) described in this work lies in the use of two large primers (proximately 20 nt) to obtain the pattern, since normally a only smaller primer is used, and in the new application for the primers used to amplify 16S rDNA This new procedure, called two primers (TP)-RAPD fingerprinting, is thus rapid, sensitive, reliable, highly reproducible and suitable for experiments with a large number of microorganisms, and can be applied to bacterial taxonomy, ecological studies and for the detection of new bacterial species

Journal ArticleDOI
TL;DR: DNA fingerprinting by randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) and DNA fingerprinting after restriction with XbaI showed that all 28 E. coli O157:H7 strains were resistant to two or more antibiotics tested.