Bhattacharya Siddhartha S, Kulka Michael, Lampel Keith A, Cebula Thomas A, Goswami Biswendu B
Division of Molecular Biology, HFS-025, Office of Applied Research and Safety Assessment, Food and Drug Administration, 8301 Muirkirk Road, Laurel, MD 20708, USA.
J Virol Methods. 2004 Mar 15;116(2):181-7. doi: 10.1016/j.jviromet.2003.11.008.
Hepatitis A virus (HAV) is a major cause of infectious hepatitis worldwide. Detection of HAV in contaminated food or water is a priority research area in laboratories worldwide. Our laboratory has reported previously the development of reverse transcription-polymerase chain reaction (RT-PCR) based detection and typing methods for HAV in contaminated shellfish and produce. It is commonly held that RT-PCR can detect viral genome, but cannot distinguish between infectious and inactivated virus. Therefore, signals obtained after PCR should be considered as false positives unless it can be shown that the sample contains virus capable of infecting a suitable host cell line in culture. We present data to show that this general assumption is not valid. Evidence is provided that demonstrate that signals generated after RT-PCR amplification of viral genome correlated well with the presence of infectious virus in the sample. Viral samples inactivated by heat or UV treatment produced significantly lower signal strength that paralleled infectivity of the sample in cultured cells. The loss of signal strength is most likely the result of damage to the viral RNA that renders it unsuitable for RT-PCR. The correlation between PCR signal and infectivity was better following UV inactivation than heat treatment. The procedure may be adapted to other viruses and inactivating agents.
甲型肝炎病毒（HAV）是全球范围内传染性肝炎的主要病因。在受污染的食物或水中检测HAV是全球实验室的重点研究领域。我们实验室此前曾报道过基于逆转录-聚合酶链反应（RT-PCR）的方法，用于检测受污染贝类和农产品中的HAV并进行分型。人们普遍认为RT-PCR可以检测病毒基因组，但无法区分感染性病毒和灭活病毒。因此，除非能证明样品中含有能够感染合适培养宿主细胞系的病毒，否则PCR后获得的信号应被视为假阳性。我们提供的数据表明这一普遍假设并不成立。有证据表明，病毒基因组RT-PCR扩增后产生的信号与样品中感染性病毒的存在密切相关。经加热或紫外线处理灭活的病毒样品产生的信号强度明显较低，这与样品在培养细胞中的感染性平行。信号强度的损失很可能是病毒RNA受损导致其不适用于RT-PCR的结果。紫外线灭活后PCR信号与感染性之间的相关性比热处理更好。该方法可适用于其他病毒和灭活剂。
