通过实时PCR快速检测甲型流感病毒并同时进行亚型区分
Rapid detection and simultaneous subtype differentiation of influenza A viruses by real time PCR.
作者信息
Stone Belinda, Burrows Julie, Schepetiuk Sonia, Higgins Geoff, Hampson Alan, Shaw Robert, Kok TuckWeng
机构信息
Infectious Diseases Laboratory, Institute of Medical and Veterinary Science, P.O. Box 14, Frome Road, South Australia, Rundle Mall, S.A. 5000, Australia.
出版信息
J Virol Methods. 2004 May;117(2):103-12. doi: 10.1016/j.jviromet.2003.12.005.
A real time RT-PCR, using the LightCycler, was developed and compared with rapid antigen enzyme immunoassay (AgEIA) and enhanced virus culture for rapid detection of influenza A viruses in stored and prospectively collected respiratory specimens. Specific hybridization probes were used for simultaneous detection and differentiation between H1N1 and H3N2 subtypes. The sensitivity of the RT-PCR for influenza A H1N1 was 120 copies and H3N2 350 copies of in vitro transcribed RNA. A specimen was considered positive for influenza A when it was culture positive or at least two methods yielded a positive test result. Using these criteria, with stored samples, the RT-PCR sensitivity, specificity, positive and negative predictive values were 82.9, 95.5, 98.9 and 52.5%, respectively. In specimens collected prospectively the RT-PCR sensitivity, specificity, positive and negative predictive values were 100, 87.9, 82.8 and 100%, respectively. There was complete concordance with subtype differentiation by hybridization probe melting temperature analysis and haemagglutination inhibition assay.
利用LightCycler开发了一种实时逆转录聚合酶链反应（RT-PCR），并将其与快速抗原酶免疫测定（AgEIA）和增强病毒培养法进行比较，用于快速检测储存和前瞻性收集的呼吸道标本中的甲型流感病毒。使用特异性杂交探针同时检测H1N1和H3N2亚型并进行区分。甲型流感H1N1的RT-PCR敏感性为120拷贝体外转录RNA，H3N2为350拷贝。当培养结果为阳性或至少两种方法检测结果为阳性时，该标本被视为甲型流感阳性。根据这些标准，对于储存样本，RT-PCR的敏感性、特异性、阳性预测值和阴性预测值分别为82.9%、95.5%、98.9%和52.5%。在前瞻性收集的标本中，RT-PCR的敏感性、特异性、阳性预测值和阴性预测值分别为100%、87.9%、82.8%和100%。通过杂交探针熔解温度分析和血凝抑制试验进行的亚型区分结果完全一致。
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