Tang Y W, Heimgartner P J, Tollefson S J, Berg T J, Rys P N, Li H, Smith T F, Persing D H, Wright P F
Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2605, USA.
Diagn Microbiol Infect Dis. 1999 Aug;34(4):333-7. doi: 10.1016/s0732-8893(99)00049-8.
We developed a colorimetric microtiter plate (MTP) PCR system for specific detection of the respiratory syncytial virus (RSV) nucleocapsid gene and differentiation of viral subtypes A and B. Of 47 pediatric nasal aspirate specimens, the sensitivity and specificity were 94.4% (17 of 18) and 100% (15 of 15), respectively, when compared with RSV cell culture isolation in HEp-2 cells. An additional 14 specimens positive for adenoviruses, rhinoviruses, influenza, or parainfluenza viruses did not give positive reactions. PCR testing detected a mean of 0.15 (0.01 to 7.00) plaque-forming units of RSV virions. Inhibition of PCR amplification was detected in 33.3% (6/18) of undiluted specimens and could be avoided by a dilution (1:10) of extracted RNA without decreasing test sensitivity. RSV subtype, as determined by allele-specific probes, was identical to that determined by an immunofluorescence assay. These results indicate that the MTP PCR system provides a sensitive and specific test for clinical laboratory diagnosis and simultaneous subgroup classification of RSV infection.
我们开发了一种比色微量滴定板（MTP）PCR系统，用于特异性检测呼吸道合胞病毒（RSV）核衣壳基因并区分病毒A和B亚型。在47份儿科鼻吸出物标本中，与在HEp-2细胞中进行RSV细胞培养分离相比，其灵敏度和特异性分别为94.4%（18份中的17份）和100%（15份中的15份）。另外14份腺病毒、鼻病毒、流感病毒或副流感病毒阳性的标本未出现阳性反应。PCR检测平均检测到0.15（0.01至7.00）个RSV病毒体空斑形成单位。在33.3%（6/18）的未稀释标本中检测到PCR扩增受到抑制，通过将提取的RNA稀释（1:10）可以避免这种情况，且不会降低检测灵敏度。通过等位基因特异性探针确定的RSV亚型与通过免疫荧光测定法确定的亚型相同。这些结果表明，MTP PCR系统为临床实验室诊断和RSV感染的同时亚组分类提供了一种灵敏且特异的检测方法。
