Regional Virus Laboratory, Royal Victoria Hospital, Belfast, Northern Ireland, United Kingdom.
J Med Virol. 2011 Sep;83(9):1650-6. doi: 10.1002/jmv.22162.
There is a need to provide rapid, sensitive, and often high throughput detection of pathogens in diagnostic virology. Viral gastroenteritis is a serious health issue often leading to hospitalization in the young, the immunocompromised and the elderly. The common causes of viral gastroenteritis include rotavirus, norovirus (genogroups I and II), astrovirus, and group F adenoviruses (serotypes 40 and 41). This article describes the work-up of two internally controlled multiplex, probe-based PCR assays and reports on the clinical validation over a 3-year period, March 2007 to February 2010. Multiplex assays were developed using a combination of TaqMan™ and minor groove binder (MGB™) hydrolysis probes. The assays were validated using a panel of 137 specimens, previously positive via a nested gel-based assay. The assays had improved sensitivity for adenovirus, rotavirus, and norovirus (97.3% vs. 86.1%, 100% vs. 87.8%, and 95.1% vs. 79.5%, respectively) and also more specific for targets adenovirus, rotavirus, and norovirus (99% vs. 95.2%, 100% vs. 93.6%, and 97.9% vs. 92.3%, respectively). For the specimens tested, both assays had equal sensitivity and specificity for astrovirus (100%). Overall the probe-based assays detected 16 more positive specimens than the nested gel-based assay. Post-introduction to the routine diagnostic service, a total of 9,846 specimens were processed with multiplex 1 and 2 (7,053 pediatric, 2,793 adult) over the 3-year study period. This clinically validated, probe-based multiplex testing algorithm allows highly sensitive and timely diagnosis of the four most prominent causes of viral gastroenteritis.
在诊断病毒学中，需要快速、灵敏且通常具有高通量的病原体检测方法。病毒性肠胃炎是一个严重的健康问题，常导致婴幼儿、免疫功能低下者和老年人住院治疗。病毒性肠胃炎的常见病因包括轮状病毒、诺如病毒（I 型和 II 型）、星状病毒和 F 组腺病毒（血清型 40 和 41）。本文描述了两种内部对照多重、探针基 PCR 检测方法的工作流程，并报告了 3 年（2007 年 3 月至 2010 年 2 月）的临床验证结果。多重检测方法使用 TaqMan™和小沟结合（MGB™）水解探针的组合开发。使用一组 137 个经嵌套凝胶基检测方法证实为阳性的标本对检测方法进行验证。该检测方法对腺病毒、轮状病毒和诺如病毒的检测灵敏度提高（分别为 97.3%比 86.1%、100%比 87.8%和 95.1%比 79.5%），对腺病毒、轮状病毒和诺如病毒的检测特异性也更高（分别为 99%比 95.2%、100%比 93.6%和 97.9%比 92.3%）。对于测试的标本，两种检测方法对星状病毒的检测灵敏度和特异性均为 100%。总体而言，探针基检测方法比嵌套凝胶基检测方法多检测出 16 个阳性标本。在引入常规诊断服务后，3 年研究期间，共对 9846 个标本（7053 个儿科，2793 个成人）使用多重 1 和 2 进行了处理。这种经临床验证的探针基多重检测算法可高度灵敏且及时地诊断出四种最主要的病毒性肠胃炎病因。
