Cost-effective real-time reverse transcriptase PCR (RT-PCR) to screen for dengue virus followed by rapid single-tube multiplex RT-PCR for serotyping of the virus
Autor(es): Lai, Yee-Ling; Chung, Youne-Kow; Tan, Hwee-Cheng; Yap, Hoon-Fang; Yap, Grace; Ooi, Eng-Eong; Ng, Lee-Ching
Resumo: Virus detection methodology provides detection of dengue virus in the early phase of the disease. PCR, targeting cDNA derived from viral RNA, has been used as a laboratory-based molecular tool for the detection of Dengue virus. We report the development and use of three real-time one-step reverse transcriptase PCR (RT-PCR) assays 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 PFU/ml (0.01 equivalent PFU per assay) for all four dengue virus 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 serotypes Dengue-1 and Dengue-2, 1 PFU for serotype Dengue-3, and 0.01 PFU for serotype Dengue-4. The third is a fourplex assay that detects any of the four 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 regard to dengue virus detection and a 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.
Palavras-Chave: Aedes - Albopictus skuse; Immunoglobulin-M Igm; Hemorrhagic - Fever; Singapore -City; Aegypti L.; Diagnosis; Assay; Infection; System; Challenges
Imprenta: Journal of Clinical Microbiology, v. 45, n. 3, p. 935-941, 2007
Identificador do objeto digital: 10.1128/JCM.01258-06
Descritores: Aedes aegypti - RNA ; Aedes aegypti - PCR detection ; Aedes aegypti - RT-PCR ; Aedes aegypti - Real Time PCR ; Aedes aegypti - Molecular screening
Data de publicação: 2007
