Development and Evaluation of a Fluorogenic Real-Time RT-PCR for the Detection of Dengue 3 Virus

作者：Tan Irene L; Dimamay Mark Pierre S; Buerano Corazon C; Alfon Jhoe Anthony R; Tanig Carol Z; Matias Ronald R; Natividad Filipinas F^*

来源：Journal of Medical Virology, 2010, 82(12): 2053-2063.

DOI：10.1002/jmv.21853

• light upon extension
• nonstructural 1 gene
• specificity
• sensitivity
• dengue virus

A dengue-3-specific real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was developed using the novel Light Upon eXtension (LUX(TM)) fluorogenic technology. A labeled forward primer and a standard reverse primer that target a conserved region within the non-structural 1 (NS1) gene of dengue 3 strains were designed. The dengue-3-specific assay did not recognize other dengue serotypes and related flaviviruses. Using a tenfold serial dilution of plasmid DNA containing the dengue 3 NS1 gene as standards, the range of dengue virus detection was determined to be from 10(3) to 10(9) copies/ml or from 80 to 8 x 10(7) copies/reaction with an average correlation coefficient of >= 0.99. The mean intra-assay coefficient of variation (CV) at 2.01% and the mean inter-assay CV at 2.68% suggest the repeatability of the procedure. Moreover, the fluorogenic assay was evaluated by using clinical specimens and comparing test results with historical data obtained from conventional RT-PCR, which served as the criterion standard. Using patient sera as test samples, the assay demonstrated 95.45% sensitivity and 100% specificity, respectively. These results reveal that the real-time RT-PCR assay may be utilized as a rapid, convenient, and sensitive tool for the detection of dengue 3 in clinical and laboratory specimens. J. Med. Virol. 82:2053-2063,2010.

• 出版日期2010-12