Karim Mohammad R, Fout G Shay, Johnson Clifford H, White Karen M, Parshionikar Sandhya U
Technical Support Center, Office of Ground Water and Drinking Water, United States Environmental Protection Agency, 26 W, Martin Luther King Dr., Cincinnati, OH 45268, USA.
Biohazard Assessment Research Branch, Microbiological and Chemical Exposure Assessment Research Division, National Exposure Research Laboratory, Office of Research and Development, United States Environmental Protection Agency, 26 W, Martin Luther King Dr., Cincinnati, OH 45268, USA.
J Virol Methods. 2015 Jul;219:51-61. doi: 10.1016/j.jviromet.2015.02.020. Epub 2015 Mar 18.
Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the public health significance of positive findings are limited. In this study, PMA RT-PCR and RT-qPCR assays were evaluated for selective detection of infectious poliovirus, murine norovirus (MNV-1), and Norwalk virus. Viruses were inactivated using heat, chlorine, and ultraviolet light (UV). Infectious and non-infectious viruses were treated with PMA before RT-PCR and RT-qPCR. PMA RT-PCR was able to differentiate selectively between infectious and heat and chlorine inactivated poliovirus. PMA RT-PCR was able to differentiate selectively between infectious and noninfectious murine norovirus only when inactivated by chlorine. However, PMA RT-PCR could not differentiate infectious Norwalk virus from virus particles rendered non-infectious by any treatment. PMA RT-PCR assay was not able to differentiate between infectious and UV inactivated viruses suggesting that viral capsid damage may be necessary for PMA to enter and bind to the viral genome. PMA RT-PCR on naked MNV-1 and Norwalk virus RNA suggest that PMA RT-PCR can be used to detect intact, potentially infectious MNV-1 and Norwalk viruses and can be used to exclude the detection of free viral RNA by PCR assay.
目前，尚无成熟的细胞系或小动物模型可用于检测感染性人类诺如病毒。基于逆转录聚合酶链反应（RT-PCR）和实时定量逆转录聚合酶链反应（RT-qPCR）的现有方法可检测感染性和非感染性病毒，因此，关于阳性结果对公共卫生的意义可能得出的结论有限。在本研究中，评估了叠氮溴化丙锭（PMA）RT-PCR和RT-qPCR检测方法对感染性脊髓灰质炎病毒、鼠诺如病毒（MNV-1）和诺沃克病毒的选择性检测能力。使用加热、氯和紫外线（UV）对病毒进行灭活。在进行RT-PCR和RT-qPCR之前，用PMA处理感染性和非感染性病毒。PMA RT-PCR能够选择性地区分感染性脊髓灰质炎病毒与经加热和氯灭活的脊髓灰质炎病毒。仅当鼠诺如病毒被氯灭活时，PMA RT-PCR才能选择性地区分感染性和非感染性鼠诺如病毒。然而，PMA RT-PCR无法区分感染性诺沃克病毒与经任何处理后变为非感染性的病毒颗粒。PMA RT-PCR检测方法无法区分感染性病毒与经紫外线灭活的病毒，这表明病毒衣壳损伤可能是PMA进入并结合病毒基因组所必需的。对裸露的MNV-1和诺沃克病毒RNA进行PMA RT-PCR表明，PMA RT-PCR可用于检测完整的、潜在感染性的MNV-1和诺沃克病毒，并且可用于通过PCR检测排除游离病毒RNA的检测。
