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Showing papers on "Multiplex polymerase chain reaction published in 1998"


Journal ArticleDOI
TL;DR: Two multiplex PCR assays for the detection and genetic characterization of STEC in cultures of feces or foodstuffs detected STEC of the appropriate genotype in primary fecal cultures from five patients with hemolytic-uremic syndrome and three with bloody diarrhea.
Abstract: Shiga toxigenic Escherichia coli (STEC) comprises a diverse group of organisms capable of causing severe gastrointestinal disease in humans. Within the STEC family, certain strains appear to be of greater virulence for humans, for example, those belonging to serogroups O111 and O157 and those with particular combinations of other putative virulence traits. We have developed two multiplex PCR assays for the detection and genetic characterization of STEC in cultures of feces or foodstuffs. Assay 1 utilizes four PCR primer pairs and detects the presence of stx1, stx2 (including variants of stx2), eaeA, and enterohemorrhagic E. coli hlyA, generating amplification products of 180, 255, 384, and 534 bp, respectively. Assay 2 uses two primer pairs specific for portions of the rfb (O-antigen-encoding) regions of E. coli serotypes O157 and O111, generating PCR products of 259 and 406 bp, respectively. The two assays were validated by testing 52 previously characterized STEC strains and observing 100% agreement with previous results. Moreover, assay 2 did not give a false-positive O157 reaction with enteropathogenic E. coli strains belonging to clonally related serogroup O55. Assays 1 and 2 detected STEC of the appropriate genotype in primary fecal cultures from five patients with hemolytic-uremic syndrome and three with bloody diarrhea. Thirty-one other primary fecal cultures from patients without evidence of STEC infection were negative.

1,102 citations


Journal ArticleDOI
TL;DR: A model multiplex PCR system is demonstrated to have the ability to perform 12-fold multiplex SNP analysis and mass spectra of interleaved genotyping products can be generated with no ambiguity in allele assignment.
Abstract: A primer extension assay is used to perform highly multiplexed genotyping of single nucleotide polymorphisms (SNPs) present in genomic DNA amplified by a multiplex PCR. The assay uses matrix-assisted laser desorption ionization time-of-flight mass spectrometry to accurately measure the masses of short oligonucleotide primers extended by a single dideoxynucleotide. The multiplexed genotyping assays rely on the natural molecular weight differences of DNA bases. By careful analysis of primer composition complementary to the target, or by judicious addition of one or more noncomplementary 5´ bases to the genotyping primers, mass spectra of interleaved genotyping products can be generated with no ambiguity in allele assignment. Using a model multiplex PCR system, we demonstrate the ability to perform 12-fold multiplex SNP analysis.

486 citations


Journal ArticleDOI
TL;DR: Two multiplex PCR enzyme immunoassays (PCR-EIAs) were developed for Staphylococcus aureus exotoxin gene screening as an alternative to the conventional biological assays, which depend on detectable amounts of toxins produced.
Abstract: Two multiplex PCR enzyme immunoassays (PCR-EIAs) were developed for Staphylococcus aureus exotoxin gene screening as an alternative to the conventional biological assays, which depend on detectable amounts of toxins produced. One set of oligonucleotide primers and probes was designed to search for enterotoxin A to E genes (entA, entB, entC, entD, and entE), and the other one was designed to detect the staphylococcal exfoliative toxin genes (eta and etb) and the toxic shock syndrome toxin 1 gene (tst). Oligonucleotide primers were used as published previously, modified or newly developed to meet the requirements of both good size-distinguishable amplification bands of multiplex PCR and the temperature limit of the uracil DNA glycosylase system for carryover protection. Amplification products were visualized by agarose gel electrophoresis, and specificity was controlled with the aid of a DNA EIA system using oligonucleotide probes derived from the sequences of the S. aureus toxin genes. PCR procedures were performed by using template nucleic acids extracted from a panel of S. aureus reference strains and from a collection of 50 clinical strains. The PCR results were compared with those of immunological toxin production assays. This multiplex PCR-EIA system offers an alternative method for the rapid, sensitive, specific, and simultaneous detection of the clinically important exotoxin potency of isolated S. aureus strains for diagnostic purposes as well as research studies.

314 citations


Journal ArticleDOI
TL;DR: The multiplex RT-PCR gave an excellent (100%) correlation with the results of conventional typing and subtyping with specific antisera for the typing andSubtyping of influenza A and B viruses and RSV types A andB.
Abstract: A multiplex reverse transcription (RT)-PCR method that has been developed is capable of detecting and subtyping influenza A (H1N1 and H3N2) and B viruses as well as respiratory syncytial virus (RSV) types A and B in respiratory clinical samples taken as part of a national community-based surveillance program of influenza-like illness in England and Wales. The detection of each different pathogen depended on distinguishing five amplification products of different sizes on agarose gels following RT-PCR with multiple primer sets. The multiplex RT-PCR was tested with 65 nasopharyngeal apirates from which RSV had been isolated and 237 combined nose and throat swabs from which influenza A (H1N1 and H3N2) or B virus had been detected by virus isolation, as well as 40 respiratory samples from which other viruses including cytomegalovirus, herpes simplex virus, enteroviruses, and parainfluenza viruses had been grown. For the typing and subtyping of influenza A and B viruses and RSV types A and B, the multiplex RT-PCR gave an excellent (100%) correlation with the results of conventional typing and subtyping with specific antisera. Multiplex RT-PCR can also be used to accurately detect more than one viral template in the same reaction mixture, allowing viral coinfections to be identified with the same respiratory specimen.

314 citations


Journal ArticleDOI
TL;DR: This dual function microchip provides a means to simplify nucleic acid analyses by integrating in a single device two key steps in the analytical procedure, namely, cell isolation and PCR.

291 citations


Journal ArticleDOI
TL;DR: In this article, a multiplex polymerase chain reaction (MPC) was developed for concurrent detection of rfb sequences specific for the O1 and the O139 serogroups of Vibrio cholerae and for ctxA specific sequences.
Abstract: A multiplex polymerase chain reaction assay was developed for concurrent detection of rfb sequences specific for the O1 and the O139 serogroups of Vibrio cholerae and for ctxA specific sequences. The multiplex PCR assay was found to be highly specific and sensitive and was capable of detecting 65 cfu and 200 cfu per assay of V. cholerae O1 and O139, respectively. Evaluation of the multiplex PCR assay using 121 stool samples from patients admitted to the Infectious Diseases Hospital, Calcutta, showed the assay to be 100% sensitive and 95.2% specific when the culture method was taken as the standard. In addition to the 38 PCR positive stool samples, an additional four samples which were negative by culture method but positive by PCR assay belonged to the O139 serogroup. All the 38 stool samples positive for either O1 or O139 serogroup by PCR assay were also positive for the ctxA amplicon indicating that the O1 and O139 V. cholerae strains have the genetic potential of producing cholera toxin.

271 citations


Journal ArticleDOI
15 Jul 1998-Blood
TL;DR: This multiplex RT-PCR system is an important tool that complements cytogenetic analysis in the up-front screening of acute leukemia patients and should provide a rapid and efficient characterization of leukemia cells, even in situations with sparse patient material.

245 citations


Patent
15 Jun 1998
TL;DR: A method and apparatus for quantitative measurement of gene expression through multiplex competitive reverse transcriptase polymerase chain reaction amplification is described in this article, which is especially useful for analysis of small specimens of cells and tissues.
Abstract: A method and apparatus for quantitative measurement of gene expression through multiplex competitive reverse transcriptase polymerase chain reaction amplification are disclosed. The method and apparatus are especially useful for analysis of small specimens of cells and tissues.

228 citations


Journal ArticleDOI
TL;DR: Multiplex polymerase chain reaction amplification and genotyping by fluorescent probe melting temperature (Tm) was used to simultaneously detect multiple variants in the hereditary hemochromatosis gene and demonstrates allele multiplexing by Tm for the first time.
Abstract: Multiplex polymerase chain reaction amplification and genotyping by fluorescent probe melting temperature ( T m ) was used to simultaneously detect multiple variants in the hereditary hemochromatosis gene. Homogenous real-time analysis by fluorescent melting curves has previously been used to genotype single base mismatches; however, the current method introduces a new probe design for fluorescence resonance energy transfer and demonstrates allele multiplexing by T m for the first time. The new probe design uses a 3′-fluorescein-labeled probe and a 5′-Cy5-labeled probe that are in fluorescence energy transfer when hybridized to the same strand internal to an unlabeled primer set. Two hundred and fifty samples were genotyped for the C282Y and H63D hemochromatosis causing mutations by fluorescent melting curves. Multiplexing was performed by including two primer sets and two probe sets in a single tube. In clinically defined groups of 117 patients and 56 controls, the C282Y mutation was found in 87% (204/234) of patient chromosomes, and the relative penetrance of the H63D mutation was 2.4% of the homozygous C282Y mutation. Results were confirmed by restriction enzyme digestion and agarose gel electrophoresis. In addition, the probe covering the H63D mutation unexpectedly identified the A193T polymorphism in some samples. This method is amenable to multiplexing and has promise for scanning unknown mutations.

201 citations


Patent
27 Mar 1998
TL;DR: In this article, the authors proposed a method for fast, selective replication of deoxyribonucleic acid (DNA) from biomaterial through the known polymerase chain reaction (PCR), working in individual duplication thermocycles.
Abstract: The invention concerns methods and instruments for fast, selective replication of deoxyribonucleic acid (DNA) from biomaterial through the known polymerase chain reaction (PCR), working in individual duplication thermocycles. The invention consists of extremely brief cycle times of only a few seconds for the PCR reactions, generated, on the one hand, by reaction chambers for the reception of the reaction solution constructed of a pattern of fine capillaries in close proximity to heating and cooling elements in order to optimally accelerate the temperature setting in the reaction solution for the three temperature phases of the PCR duplication cycles and, on the other hand, by keeping the flow rates in the capillaries to a minimum during the amplification phase so that the polymerase reaction is not disturbed. The capillary pattern can be simply produced by means of microsystern technology.

172 citations


Journal ArticleDOI
TL;DR: Multiplex PCR amplification, followed by colorimetric GeneComb™ DNA-DNA hybridization, has been shown to be an effective, sensitive, and rapid method to detect microbial pathogens in shellfish.
Abstract: Multiplex PCR amplification of uidA, cth, invA, ctx, and tl genes was developed enabling simultaneous detection in shellfish of Escherichia coli, an indicator of fecal contamination and microbial pathogens, Salmonella typhimurium, Vibrio vulnificus, V. cholerae, and V. parahaemolyticus, respectively. Each of the five pairs of oligonucleotide primers was found to support PCR amplifications of only its targeted gene. The optimized multiplex PCR reaction utilized a PCR reaction buffer containing 2.5 mM MgCl2 and primer annealing temperature of 55 degrees C. Oyster tissue homogenate seeded with these microbial pathogens was subjected to DNA purification by the Chelextrade mark 100 (BioRad) method. The sensitivity of detection for each of the microbial pathogens was

Journal ArticleDOI
TL;DR: Multiplex RT-PCR assays that detect and differentiate HPIV serotypes 1, 2, and 3 in a combined reaction are described, providing sensitive, specific, and simplified tools for the rapid diagnosis of HPIV infections.
Abstract: Reverse transcription (RT)-PCR assays have been widely described for use in the diagnosis of human parainfluenza viruses (HPIVs) and other respiratory virus pathogens. However, these assays are mostly monospecific, requiring separate amplifications for each HPIV type. In the present work, we describe multiplex RT-PCR assays that detect and differentiate HPIV serotypes 1, 2, and 3 in a combined reaction. Specifically, a mixture of three pairs of primers to conserved regions of the hemagglutinin-neuraminidase gene of each HPIV serotype was used for primary amplification, yielding amplicons with similar sizes. For typing, a second amplification was performed with a mixture of nested primers, yielding amplicons with sizes easily differentiated by agarose gel electrophoresis. A modified single-amplification RT-PCR assay with fluorescence-labeled nested primers, followed by analysis of the labeled products on an automated sequencing gel, was also evaluated. Fifteen temporally and geographically diverse HPIV isolates from the Centers for Disease Control and Prevention archives and 26 of 30 (87%) previously positive nasopharyngeal specimens (8 of 10 positive for HPIV serotype 1 [HPIV1], 9 of 10 positive for HPIV2, and 9 of 10 positive for HPIV3) were positive and were correctly typed by both assays. Negative results were obtained with naso- or oropharyngeal specimens and/or culture isolates of 33 unrelated respiratory tract pathogens, including HPIV4, enterovirus, rhinovirus, respiratory syncytial virus, adenovirus, influenza virus, and Streptococcus pneumoniae. Our multiplex RT-PCR assays provide sensitive, specific, and simplified tools for the rapid diagnosis of HPIV infections.

Journal ArticleDOI
TL;DR: Random amplification of the human genome using the degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) proved to be a suitable template for multiplex PCR as long as amplicon size was < 250 bp.

Journal ArticleDOI
TL;DR: The results exemplified by PCR-amplified 217-bp and 389-bp target DNA sequences demonstrate that the analysis based on two-color fluorescence cross-correlation is a powerful method for simplifying the identification of targets in PCR for medical use.

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

Journal ArticleDOI
TL;DR: Ref refined polymerase chain reaction (PCR) assays for the detection of sickle cell anaemia, the delta F 508 deletion causing cystic fibrosis, and the IVS1–110 mutation leading to beta‐thalassaemia, allowing them to be successfully performed upon single cells using fluorescent primers.
Abstract: We have refined polymerase chain reaction (PCR) assays for the detection of sickle cell anaemia, the delta F 508 deletion causing cystic fibrosis, and the IVS1-110 mutation leading to beta thalassaemia, allowing them to be successfully performed upon single cells using fluorescent primers. We have also assessed the possibility of detecting aneuploidies of chromosomes 13, 18 and 21 using a quantitative fluorescent polymerase chain reaction (QF-PCR) with primers flanking polymorphic short tandem repeat (STR) markers. Trisomies were readily diagnosed by the detection of tri-allelic patterns. However some heterozygote normal and trisomic diallelic patterns did not produce the expected ratios of amplified PCR products due to preferential DNA sequence amplification. Total allelic drop out (ADO) did not occur with any of the cells tested. Multiplex QF-PCR assays can be performed on a single cell in under 6 h and simultaneously provide diagnosis of single gene defects, sex determination and an indication of selected chromosome aneuploidy.

Journal ArticleDOI
TL;DR: Overall, the PCR technique was more accurate and sensitive than conventional methodology in detecting MRSA, and results were available within 24 h of screening swabs arriving in the laboratory, compared with a minimum of 48-72 h by conventional techniques.
Abstract: A multiplex polymerase chain reaction (PCR), involving detection of the mecA and femB genes, was combined with a novel immunoassay system capable of detecting specific PCR products. The resulting PCR-immunoassay was evaluated in comparison with conventional microbiological techniques used in the routine diagnostic laboratory for the rapid identification of methicillin-resistant Staphylococcus aureus (MRSA), either in pure culture or in overnight broth cultures obtained following enrichment of patient screening swabs. Among the 480 purified isolates of staphylococci and 246 enrichment broths examined, only one 'false-negative' result was obtained by PCR, compared with 18 'false-negative' results obtained by conventional methodology and demonstrated by further conventional examination. Five demonstrable 'false-positive' results were obtained by conventional methodology, compared with a possible 10 by the PCR-immunoassay, although it was not certain that these 10 PCR results were true 'false positives' as, by definition, MRSA could not be isolated by conventional methodology. The results indicated that the routine diagnostic laboratory was encountering difficulties in identifying MRSA correctly, and that the conventional microbiological techniques lacked sensitivity. Overall, the PCR technique was more accurate and sensitive than conventional methodology in detecting MRSA, and results were available within 24 h of screening swabs arriving in the laboratory, compared with a minimum of 48-72 h by conventional techniques. The immunoassay system added to the usefulness of the method by allowing the detection of specific PCR products within 5 min of completing the PCR, without the normal additional step of agarose gel electrophoresis.

Journal ArticleDOI
TL;DR: Genomic polymorphisms among nine strains of Blastocystis hominis were examined by random amplified polymorphic DNA (RAPD) using four different arbitrary polymerase chain reaction (PCR) primers to classify several subtypes among B. hominis groups based on the difference of genomic DNA.

Journal ArticleDOI
P.C Choppa, A Vojdani1, C Tagle, R Andrin, L. Magtoto 
TL;DR: This assay provides a rapid and cost efficient procedure to screen either cell cultures or clinical samples for the presence of three potentially pathogenic species of mycoplasma with a high level of sensitivity and specificity.

Journal ArticleDOI
TL;DR: High resolution agarose gel electrophoresis was unable to satisfactorily resolve 1–2 repeat unit differences in the common size range for SSR loci, and was therefore unsuitable for mass screening of allelic variants.
Abstract: Microsatellite (SSR) profiles from 65 wheats and 135 barleys have been generated, involving 14 and 22 loci, respectively The wheat and barley varieties were chosen to represent the bulk of the area sown to these crops in the UK over the past 70 years The profiling identified genotypic mixtures in some seed samples Null alleles were common in wheat, but rare in barley We describe attempts to increase the efficiency of data acquisition High resolution agarose gel electrophoresis was unable to satisfactorily resolve 1–2 repeat unit differences in the common size range for SSR loci, and was therefore unsuitable for mass screening of allelic variants Multiplex PCR was very dependent on the choice of primer combinations and seldom produced amplifications as consistently as when primer pairs were used individually Background (non-specific) amplification was common to many primer pairs, and this hindered the use of both multiplex PCR and multiple sample loading Sequential sample loading was the most effective strategy, although this was the least time-efficient of the measures used

Journal ArticleDOI
TL;DR: The multiplex polymerase chain reaction-allele specific oligonucleotides (PCR/ASO) dot blot hybridization method was used to detect 44 mitochondrial DNA point mutations and indicated that analysis of 12 recurrent disease-causing point mutations in category I identified 5.4% of the patients suspected as having mitochondrial DNA disorders.
Abstract: The multiplex polymerase chain reaction–allele specific oligonucleotides (PCR/ASO) dot blot hybridization method was used to detect 44 mitochondrial DNA point mutations in 2,000 patients suspected as having mitochondrial DNA disorders. These point mutations are classified into four categories. Category I consists of primary disease-causing, heteroplasmic point mutations. Homoplasmic nucleotide substitutions that have been reported to be possibly disease associated are in Category II. Homoplasmic nucleotide substitutions that are thought to be benign polymorphism are included in category III. The novel nucleotide substitutions recently discovered in our laboratory by single strand conformation polymorphism analysis are in category IV. Frequencies of these 44 nucleotide substitutions in 2,000 patients and 262 control individuals were studied. The results indicated that analysis of 12 recurrent disease-causing point mutations in category I identified 5.4 % of the patients suspected as having mitochondrial DNA disorders. Since the mitochondrial disorders are a group of complex, heterogeneous, and multisystemic diseases, it is often difficult to confirm clinical diagnosis without molecular studies. Thus, the multiplex PCR/ASO method is an effective approach for initial screening of mtDNA mutations in patients suspected as having mitochondrial DNA disorders. Am. J. Med. Genet. 77:395–400, 1998. © 1998 Wiley-Liss, Inc.

Journal ArticleDOI
TL;DR: A new multiplex-PCR method was used that allowed single bacterial colonies grown on agar plates to be used directly in the PCR assay without preceding preparation and rapidly generated reliable information concerning the toxin-producing capacity of staphylococcal strains.
Abstract: As well as conventional methods such as immunodiffusion, ELISA, or agglutination for the detection of toxin production in Staphylococcus aureus, amplification techniques like PCR allow a very sensitive and specific identification of the genes responsible for enterotoxin B and C, and TSST-1 production. These toxins might be a cause of the toxic shock syndrome (TSS). For that reason an easy and quick test system for determining the toxin production pattern of S. aureus isolates is desirable so that strains suspected to be toxin producers may be identified much faster and easier. In the present investigation, a new multiplex-PCR method was used that allowed single bacterial colonies grown on agar plates to be used directly in the PCR assay without preceding preparation. This procedure generated information concerning the presence of seb, sec-1 and tst genes within 4 h in a single test. To analyse the sensitivity and the specificity of this procedure, 100 methicillin-resistant S. aureus (MRSA), 50 coagulase-negative staphylococci and 50 other eubacterial isolates were tested initially with sets of single primer pairs followed by a combined multiplex-PCR. Results of this amplification technique were compared to a conventional and widely used method for toxin detection, reversed passive latex agglutination (RPLA). With the RPLA assay results as the basis, sensitivity and specificity of the seb and tst primer sets were 100%, whereas sensitivity and specificity of the sec-1 primer set were 100% and 82%, respectively. With the sec-1 primer set, two isolates were identified as carrying the corresponding toxin gene although the RPLA test did not show any detectable toxin. The multiplex-PCR rapidly generated reliable information concerning the toxin-producing capacity of staphylococcal strains and could be easily integrated into a multiplex procedure described previously. The latter enabled the identification of specific PCR products for eubacteria and staphylococci as well as the detection of the coa and mecA genes.


Journal ArticleDOI
TL;DR: This work describes the development and evaluation of a multiplex polymerase chain reaction (PCR) for the detection of Bacillus anthracis strains harbouring plasmid pX02, which incorporated an internal control (IC) to avoid false negative reactions.

Journal ArticleDOI
TL;DR: The use of PCR to amplify a specific virA gene fragment serves as a highly specific and sensitive method to detect virulent bacteria of the genus Shigella and enteroinvasiveEscherichia coli.
Abstract: The use of PCR to amplify a specific virA gene fragment serves as a highly specific and sensitive method to detect virulent bacteria of the genus Shigella and enteroinvasive Escherichia coli. Amplification of a 215-bp DNA band was obtained by using isolated genomic DNA of Shigella, individual cells of Shigella dysenteriae, and mayonnaise contaminated with S. dysenteriae. Moreover, a multiplex PCR with specific (virA) and bacterium-restricted (16S ribosomal DNA) primers generated an amplification product of approximately 755 bp for all bacteria tested and an additional 215-bp product for Shigella and enteroinvasive E. coli.


Journal ArticleDOI
TL;DR: The novel application of AmpliTaq Gold DNA Polymerase in a time-release PCR protocol allows specific amplification of target DNA/ cDNA when only limited amounts of material are available or only low-copy-number DNA/cDNA is suspected.
Abstract: Extensive diagnostic and scientific investigations are often restricted by limited availability of material. Therefore, methods like multiplex PCR strategies are needed to conserve as much sample as possible. Unfortunately, the establishment of such procedures poses several difficulties. Here we describe the advantages of a new enzyme, AmpliTaq Gold DNA Polymerase, in multiplex and time-release PCR. The application of this thermostable recombinant Taq DNA polymerase allows the specific amplification of DNA/cDNA targets with very high sensitivity. With our protocol, the specific amplification of 13 different cDNAs of cytokines and cytokine receptors can be realized in three multiplex PCRs (IL-2R alpha, IL-2/15R beta, gamma c-chain, IL-4 and IL-4R alpha; IL-10, IL-15 and IL-15R alpha; and IL-2, IFN gamma, IL-7, IL-7R alpha and IL-9R alpha). The novel application of AmpliTaq Gold DNA Polymerase in a time-release PCR protocol allows specific amplification of target DNA/cDNA when only limited amounts of material are available or only low-copy-number DNA/cDNA is suspected. No IL-9 cDNA can be detected in peripheral blood mononuclear cells (PBMC) in the absence of any stimulation, thus it was difficult to amplify this target with routine PCR protocols. Here we demonstrate the reliable and reproducible amplification of IL-9 cDNA in the Hodgkin's lymphoma cell line KM-H2, in PBMC and in stimulated PBMC. Results with AmpliTaq Gold DNA Polymerase were more sensitive and specific compared with AmpliTaq DNA Polymerase, with and without manual hot-start procedure.

Journal ArticleDOI
TL;DR: The multiplex PCR is a potentially powerful technique for rapid and sensitive co-detection of both pathogens in foods and other types of samples.
Abstract: Contamination of foods with pathogens such as Escherichia coli O157:H7 and Salmonella is a major concern worldwide and rapid, sensitive, and reliable methods are needed for detection of these organisms. Since these pathogens can contaminate similar foods and other types of samples, a multiplex polymerase chain reduction (PCR) was designed to allow simultaneous detection of both E. coli O157:H7 and Salmonella spp directly from enrichment cultures. Samples of apple cider, beef carcass wash water, ground beef, and bovine feces were inoculated with both E. coli O157:H7 and S. typhimurium at various bacterial levels. Following enrichment culturing for 20–24 h at 37°C in modified EC broth or buffered peptone water both containing novobiocin, the samples were subjected to a DNA extraction technique or to immunomagnetic separation then tested by the multiplex PCR assay. Four pairs of primers were employed in the PCR: primers for amplification of E. coli O157:H7 eaeA, stx 1/2 and plasmid sequences and for amplification of a portion of the Salmonella invA gene. Four fragments of the expected sizes were amplified in a single reaction and visualized following agarose gel electrophoresis in all the samples inoculated with ≤ 1 CFU g−1 or ml−1. Results can be obtained in approximately 30 h. The multiplex PCR is a potentially powerful technique for rapid and sensitive co-detection of both pathogens in foods and other types of samples.

Journal ArticleDOI
TL;DR: PCR is a useful assay for rapid detection of Clostridium perfringens in feed, and for confirmation of the identity of isolates presumed to be C. perfringen from feces and intestinal contents of pigs and from feed samples from pig farms in Iowa.

Journal ArticleDOI
TL;DR: Species-specific internal ITS primers that amplify polymerase chain reaction (PCR) products of different lengths were selected to distinguish the morphologically similar ectomycorrhizal fungi T. melanosporum, T. brumale and T. indicum by aligning their internal transcribed spacer sequences and taking into account any incidence of intraspecific variability.
Abstract: Species-specific internal ITS primers that amplify polymerase chain reaction (PCR) products of different lengths were selected to distinguish the morphologically similar ectomycorrhizal fungi T. melanosporum, T. brumale and T. indicum by aligning their internal transcribed spacer sequences and taking into account any incidence of intraspecific variability. In multiplex PCR experiments, the species-specific primers yielded the expected amplicons on template DNA isolated from the above mentioned species, while there was no amplification in PCR reactions carried out on fungal DNA from competing truffle species and host plants.