Wallace L A, McAulay K A, Douglas J D, Elder A G, Stott D J, Carman W F
Institute of Virology, University of Glasgow, Scotland.
J Infect. 1999 Nov;39(3):221-6. doi: 10.1016/s0163-4453(99)90053-1.
To compare the conventional virus isolation method for diagnosis of influenza infection with reverse-transcription polymerase chain reaction (RT-PCR) in prospectively collected nose and throat swabs from elderly patients during the winter influenza season. The use of a denaturing buffer as an alternative to viral transport medium (VTM) for submission of combined nose and throat swabs to the laboratory for PCR was then investigated in a second study.
Virus was cultured in microtitre plates using two different cell lines and detected using monoclonal antibody staining. A multiplex, matrix gene PCR assay was optimized to increase the sensitivity and specificity of detection of influenza A (H3 and H1) and B nucleic acid.
The multiplex assay detected all viruses with equal sensitivity to individual assays. In a large, multicentre field study PCR detected twice as many influenza infections compared with virus isolation. No positive culture was missed. PCR has a rapid turn around time (< 36 h) vs. a minimum of 7 days for virus isolation. Greater sensitivity and specificity in the PCR were achieved using a 'hot-start' method. Although the numbers were small, the detection rate using PCR was greater for swabs submitted in denaturing buffer than in VTM.
PCR significantly increased the sensitivity and clinical utility of influenza A (H3 and H1) and B diagnosis. There were a number of advantages in using denaturing buffer for submission of samples, including high sensitivity, rapidity, ease of use and no requirement for the virus to be viable on arrival at the laboratory. Therefore, PCR is a rapid, sensitive and user-friendly alternative for influenza diagnosis. Virus isolation technology should be limited to referral centres for further epidemiological characterization.
比较传统病毒分离方法与逆转录聚合酶链反应（RT-PCR）在冬季流感季节前瞻性收集的老年患者鼻拭子和咽拭子中诊断流感感染的效果。在第二项研究中，研究了使用变性缓冲液替代病毒运输培养基（VTM）将联合鼻拭子和咽拭子送检至实验室进行PCR检测的情况。
使用两种不同的细胞系在微量滴定板中培养病毒，并使用单克隆抗体染色进行检测。优化了多重基质基因PCR检测方法，以提高甲型流感（H3和H1）和乙型流感核酸检测的灵敏度和特异性。
多重检测对所有病毒的检测灵敏度与单独检测相同。在一项大型多中心现场研究中，PCR检测出的流感感染病例数是病毒分离法的两倍。没有漏检阳性培养物。PCR的周转时间很快（<36小时），而病毒分离至少需要7天。使用“热启动”方法可提高PCR的灵敏度和特异性。虽然数量较少，但使用变性缓冲液送检的拭子PCR检测率高于使用VTM送检的拭子。
PCR显著提高了甲型流感（H3和H1）和乙型流感诊断的灵敏度和临床实用性。使用变性缓冲液送检样本有许多优点，包括高灵敏度、快速、易用且无需病毒在到达实验室时仍保持活性。因此，PCR是一种快速、灵敏且用户友好的流感诊断替代方法。病毒分离技术应仅限于转诊中心用于进一步的流行病学特征分析。
