Plague Research Today is a free monthly online journal that collates and summarizes the latest research about Plague, including details on bubonic plague, yersinia pestis, infection, types, treatment. | ||||||||
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Cost effective real time RT-PCR to screen for Dengue followed by rapid single-tube multiplex RT-PCR for serotyping of the virus.Lai YL, Chung YK, Tan HC, Yap HF, Yap G, Ooi EE, Ng LC National Environment Agency, Environmental Health Institute, Singapore; National Environment Agency, Environmental Health Department, Singapore; and DSO National Laboratories, Singapore. Virus detection methodology provides detection of Dengue in the early phase of the disease. Polymerase chain reaction (PCR), targeting at complementary deoxyribonucleic acid (cDNA) derived from viral ribonucleic acid (RNA), has been used as a laboratory-based molecular tool for the detection of Dengue. We report the development of three real-time one step reverse transcriptase (RT)-PCR assays and their employment to detect Dengue cases, and serotype the virus involved. The first RT-PCR assay uses SYBR green I as the reporting dye for the purpose of cost effective screening for Dengue virus. The detection limit of the SYBR green I assay was 10 plague forming unit (PFU)/ml (0.01 equivalent PFU per assay) for all four Dengue serotypes. The second RT-PCR assay is a duplex fluorogenic probe-based real-time RT-PCR for serotyping clinical samples for Dengue viruses. The detection threshold of the probe-based RT-PCR format was 0.1 PFU for Dengue-1 and Dengue-2, 1 PFU for Dengue-3 and 0.01 PFU for Dengue-4. The third is a fourplex assay that detects any of the 4 serotypes in a single closed tube with comparable sensitivity. Validation of the assays with local clinical samples collected from 2004 to 2006 revealed that there was an 88% positive correlation between virus isolation and RT-PCR with regards to Dengue detection and 100% correlation with seroconversion in subsequent samples. The serotyping results derived from duplex and fourplex assays agree fully with each other and with that derived from immunofluorescence assays (IFA). Published 11 January 2007 in J Clin Microbiol.
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