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gaoshandian1金虫 (小有名气)
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[交流]
Real-time PCR in virology
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Ian M. Mackay1,2,3,*, Katherine E. Arden4 and Andreas Nitsche5 1Clinical Virology Research Unit, Sir Albert Sakzewski Virus Research Centre, Royal Children’s Hospital, Brisbane, Australia, 2Department of Paediatrics and 3Clinical Medical Virology Centre, University of Queensland, Queensland, Australia, 4Membrane Transport Laboratory, Division of Cancer and Cell Biology, Queensland Institute of Medical Research, Queensland, Australia and 5TIB MOLBIOL, Tempelhofer Weg 11–12, D-10829 Berlin, Germany The use of the polymerase chain reaction (PCR) in molecular diagnostics has increased to the point where it is now accepted as the gold standard for detecting nucleic acids from a number of origins and it has become an essential tool in the research laboratory. Real-time PCR has engendered wider acceptance of the PCR due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination. There are currently five main chemistries used for the detection of PCR product during real-time PCR. These are the DNA binding fluorophores, the 5' endonuclease, adjacent linear and hairpin oligoprobes and the self-fluorescing amplicons, which are described in detail. We also discuss factors that have restricted the development of multiplex real-time PCR as well as the role of real-time PCR in quantitating nucleic acids. Both amplification hardware and the fluorogenic detection chemistries have evolved rapidly as the understanding of real-time PCR has developed and this review aims to update the scientist on the current state of the art. We describe the background, advantages and limitations of real-time PCR and we review the literature as it applies to virus detection in the routine and research laboratory in order to focus on one of the many areas in which the application of real-time PCR has provided significant methodological benefits and improved patient outcomes. However, the technology discussed has been applied to other areas of microbiology as well as studies of gene expression and genetic disease. * To whom correspondence should be addressed at: CVRU, SASVRC, Royal Children’s Hospital, Herston Road, Herston, Queensland 4029, Australia. Tel: +61 3636 8716; Fax: +61 3636 1401; Email: i.mackay@mailbox.uq.edu.au |
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