Technical Support Center, U.S. Environmental Protection Agency, Mailstop 140, 26 W. Martin Luther King Dr., Cincinnati, OH 45268, USA.
Appl Environ Microbiol. 2010 Jul;76(13):4318-26. doi: 10.1128/AEM.02800-09. Epub 2010 May 14.
Human enteric viruses can be present in untreated and inadequately treated drinking water. Molecular methods, such as the reverse transcriptase PCR (RT-PCR), can detect viral genomes in a few hours, but they cannot distinguish between infectious and noninfectious viruses. Since only infectious viruses are a public health concern, methods that not only are rapid but also provide information on the infectivity of viruses are of interest. The intercalating dye propidium monoazide (PMA) has been used for distinguishing between viable and nonviable bacteria with DNA genomes, but it has not been used to distinguish between infectious and noninfectious enteric viruses with RNA genomes. In this study, PMA in conjunction with RT-PCR (PMA-RT-PCR) was used to determine the infectivity of enteric RNA viruses in water. Coxsackievirus, poliovirus, echovirus, and Norwalk virus were rendered noninfectious or inactivated by treatment with heat (72 degrees C, 37 degrees C, and 19 degrees C) or hypochlorite. Infectious or native and noninfectious or inactivated viruses were treated with PMA. This was followed by RNA extraction and RT-PCR or quantitative RT-PCR (qRT-PCR) analysis. The PMA-RT-PCR results indicated that PMA treatment did not interfere with detection of infectious or native viruses but prevented detection of noninfectious or inactivated viruses that were rendered noninfectious or inactivated by treatment at 72 degrees C and 37 degrees C and by hypochlorite treatment. However, PMA-RT-PCR was unable to prevent detection of enteroviruses that were rendered noninfectious by treatment at 19 degrees C. After PMA treatment poliovirus that was rendered noninfectious by treatment at 37 degrees C was undetectable by qRT-PCR, but PMA treatment did not affect detection of Norwalk virus. PMA-RT-PCR was also shown to be effective for detecting infectious poliovirus in the presence of noninfectious virus and in an environmental matrix. We concluded that PMA can be used to differentiate between potentially infectious and noninfectious viruses under the conditions defined above.
人类肠道病毒可能存在于未经处理和处理不当的饮用水中。分子方法，如逆转录 PCR（RT-PCR），可以在几个小时内检测到病毒基因组，但它们无法区分传染性和非传染性病毒。由于只有传染性病毒才是公共卫生关注的问题，因此，不仅快速而且提供有关病毒感染力的信息的方法是人们感兴趣的。嵌入染料吖啶橙单叠氮化物（PMA）已用于区分具有 DNA 基因组的有活力和无活力的细菌，但尚未用于区分具有 RNA 基因组的传染性和非传染性肠道病毒。在这项研究中，PMA 与 RT-PCR（PMA-RT-PCR）结合使用，以确定水中肠道 RNA 病毒的感染力。柯萨奇病毒、脊髓灰质炎病毒、埃可病毒和诺如病毒通过加热（72°C、37°C 和 19°C）或次氯酸盐处理而失去传染性或失活。将传染性或天然的和非传染性或失活的病毒用 PMA 处理。然后进行 RNA 提取和 RT-PCR 或定量 RT-PCR（qRT-PCR）分析。PMA-RT-PCR 结果表明，PMA 处理不会干扰对传染性或天然病毒的检测，但会阻止通过在 72°C 和 37°C 以及次氯酸盐处理而失去传染性或失活的非传染性或失活病毒的检测。然而，PMA-RT-PCR 无法防止通过在 19°C 处理而失去传染性的肠病毒的检测。在 37°C 处理后，PMA 使脊髓灰质炎病毒失去传染性，但通过 qRT-PCR 无法检测到，而 PMA 处理不会影响诺如病毒的检测。还证明 PMA-RT-PCR 可用于在存在非传染性病毒和环境基质的情况下检测有感染力的脊髓灰质炎病毒。我们得出结论，在上述定义的条件下，PMA 可用于区分潜在传染性和非传染性病毒。
