Mohamed Nahla, Belák Sándor, Hedlund Kjell-Olof, Blomberg Jonas
Section of Clinical Virology, Department of Medical Sciences, Academic Hospital, Uppsala University, Sweden.
J Virol Methods. 2006 Mar;132(1-2):69-76. doi: 10.1016/j.jviromet.2005.09.006. Epub 2005 Nov 9.
Detection of caliciviruses requires high mutation tolerance and throughput. The development of a rational simple, single tube reverse transcription-real-time quantitative PCR (QPCR) technique for human noroviruses (NV) is reported here. A dual-probe, triple-primer system (NM system) was used for simultaneous detection and preliminary differentiation of NV genogroups in fecal samples. The design was based on a comprehensive analysis of all 1140 NV sequences available in GenBank. A touch-down amplification protocol improved the frequency of detection. The final QPCR was evaluated with 71 fecal samples from outbreak and sporadic cases in Sweden (1997-2004), all calicivirus-positive by electron microscopy. Up to 56 (79 %) were positive. The method is more rational than NV detection methods described previously, and should be a developmental basis for large-scale routine methods for detection of NV.
杯状病毒的检测需要高突变耐受性和高通量。本文报道了一种用于人类诺如病毒（NV）的合理、简单的单管逆转录实时定量PCR（QPCR）技术的开发。采用双探针、三引物系统（NM系统）同时检测粪便样本中的NV基因组群并进行初步鉴别。该设计基于对GenBank中所有1140条NV序列的综合分析。一种降落式扩增方案提高了检测频率。最终的QPCR用来自瑞典（1997 - 2004年）暴发和散发病例的71份粪便样本进行评估，所有样本经电子显微镜检测均为杯状病毒阳性。多达56份（79%）样本呈阳性。该方法比先前描述的NV检测方法更合理，应为大规模常规NV检测方法的发展奠定基础。
