Multiplex polymerase chain reaction

About: Multiplex polymerase chain reaction is a research topic. Over the lifetime, 6409 publications have been published within this topic receiving 221244 citations.

Specific information concerning taxonomy, pathogenicity and methicillin resistance of staphylococci obtained by a multiplex PCR.

Abstract: The use of DNA amplification techniques such as the polymerase chain reaction (PCR) in modern diagnostic microbiology not only allows the sensitive and specific identification of micro-organisms but also the detection of specific antibiotic resistance genes. This study describes a multiplex PCR on bacterial colonies picked directly from agar plates without preceding DNA preparation. Eubacteria and staphylococci were identified by 16S rRNA specific PCR products. In parallel, specific primers were used for the detection of staphylococcal coa and mecA genes. This 4-h multiplex PCR, consisting of four sets of primers, was evaluated for rapid and specific differential diagnosis of methicillin-resistant and methicillin-susceptible strains of Staphylococcus aureus and coagulase-negative staphylococci. To analyse specificity of the amplification products, 100 non-staphylococcal, eubacterial isolates and 20 Candida albicans strains were tested. In a first step, specificity of all four single sets of primers was evaluated before the co-amplification within the multiplex PCR procedure was performed. The results were compared with those of conventional susceptibility and typing methods. The specific 16S rRNA PCR product for eubacterial isolates (n = 786) and staphylococci (686) was found in all strains tested. The coa gene was detected only in S. aureus (488) strains with a specificity of 100%, and was not detected in any of the coagulase-negative staphylococci (198). The mecA gene was detected in 98% of methicillin-resistant staphylococci (393) and in 2% of all methicillin-susceptible staphylococci (293). The multiplex PCR with co-amplification of different determinants provides rapid reliable information on staphylococcal identification and methicillin susceptibility supporting the diagnosis, treatment and control of staphylococcal infections.

Enhanced Polymerase Chain Reaction Methods for Detecting and Quantifying Phytophthora infestans in Plants

Abstract: Sensitive and specific primer sets for polymerase chain reaction (PCR) for Phytophthora infestans, the oomycete that causes late blight of potato and tomato, were developed based on families of highly repeated DNA. The performance of these primers was compared to those developed previously for the internal transcribed spacer (ITS) of ribosomal DNA. The detection limit using the new primers is 10 fg of P. infestans DNA, or 0.02 nuclei. This is about 100 times more sensitive than ITS-directed primers. Nested polymerase chain reaction (PCR) allows the measurement of down to 0.1 fg of DNA using the new primers. To enhance the reliability of diagnostic assays, an internal positive control was developed using an amplification mimic. The mimic also served as a competitor for quantitative PCR, which was used to assess the growth of P. infestans in resistant and susceptible tomato. A key dimension of this study was that two laboratories independently checked the specificity and sensitivity of each primer ...

Molecular Methods for Virus Detection

Abstract: D.L. Wiedbrauk and A.M. Drevon, Nucleic Acid Detection Methods. D.L. Wiedbrauk and J. Stoerker, Quality Assurance in the Molecular Biology Laboratory. D.L. Stoler and N.L. Michael, Nucleic Acid Blotting Techniques for Virus Detection. J.Carr, In Situ Hybridization. R.L. Hodinka, Antiviral Susceptibility Testing Using DNA-DNA Hybridization. J.C. Wilbur and M.S. Urdea, Quantitation of Viral Nucleic Acids Using Branched DNA (bDNA) Signal Amplification. I. Bronstein and C.E.M. Olesen, Chemiluminescence Methods. B.J. McCreedy, Polymerase Chain Reaction. F. Ferre, P. Pezzoli, E. Buxton, C. Duffy, A. Marchese, and A. Diagle, Quantitation of RNA Targets Using the Polymerase Chain Reaction. J.B. Mahony and M.A. Chernesky, Multiplex Polymerase Chain Reaction (M-PCR). G.J. Nuovo, PCR in Situ Hybridization. R. Sooknanan, B. van Gemen, and L.T. Malek, Nucleic Acid Sequence Based Amplification (NASBA(tm)). S.S.Ghosh, E. Fahy, and T.R. Gingeras, The Self-Sustained Sequence Replication (3SR) Reaction and its Application in Clinical Diagnostics and Molecular Biology. J.D. Burczak, S. Ching, H.-Y. Hu, and H.H. Lee, Ligase Chain Reaction (LCR) for the Detection of Infectious Agents. G. T. Walker, et al., A Chemiluminescent DNA Probe Test Based upon Strand Displacement Amplification (SDA). D.M. Schuster, M.S. Berninger, and A. Rashtchian, Ligation Activated Transcription Amplification: Amplification and Detection of Human Papillomaviruses. Subject Index.

Identification of root rot fungi in nursery seedlings by nested multiplex PCR.

Abstract: The internal transcribed spacer (ITS) of the ribosomal DNA (rDNA) subunit repeat was sequenced in 12 isolates of Cylindrocladium floridanum and 11 isolates of Cylindrocarpon destructans. Sequences were aligned and compared with ITS sequences of other fungi in GenBank. Some intraspecific variability was present within our collections of C. destructans but not in C. floridanum. Three ITS variants were identified within C. destructans, but there was no apparent association between ITS variants and host or geographic origin. Two internal primers were synthesized for the specific amplification of portions of the ITS for C. floridanum, and two primers were designed to amplify all three variants of C. destructans. The species-specific primers amplified PCR products of the expected length when tested with cultures of C, destructans and C. floridanum from white spruce, black spruce, Norway spruce, red spruce, jack pine, red pine, and black walnut from eight nurseries and three plantations in Quebec. No amplification resulted from PCR reactions on fungal DNA from 26 common contaminants of conifer roots. For amplifications directly from infected tissues, a nested primer PCR using two rounds of amplification was combined with multiplex PCR approach resulting in the amplification of two different species-specific PCR fragments in the same reaction. First, the entire ITS was amplified with one universal primer and a second primer specific to fungi; a second round of amplification was carried out with species-specific primers that amplified a 400-bp PCR product from C. destructans and a 328-bp product from C. floridanum. The species-specific fragments were amplified directly from infected roots from which one or the two fungi had been isolated.