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Molecular Methods for the Detection of Infectious Enteroviruses in Source or Finished Waters

机译：检测源水或成品水中感染性肠病毒的分子方法

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Environmental or source water samples and even finished drinking watersamples may contain virus particles detectable by molecular methods such asPCR and RT-PCR. These methods in conjunction with confirmatory analyses(e.g., nucleic acid hybridization or nucleotide sequencing) are useful indetermining the potential presence or absence of viruses in waters, even at verylow levels. However, these tests do not conclusively determine whether theviruses are infectious or non-infectious. The current standard method fordetection of infectious viruses in water samples or concentrates is cell culturewith two passages for confirmation of cytopathic effect (CPE). Viruses that donot cause CPE or have limited replication are not detected. Furthermore, theprocedure requires weeks and is costly. Evidence of replication in cell culturewithout repeated passage may be obtained by evaluation of infected cells for thepresence of viral nucleic acid; however, testing of dilutions is necessary todemonstrate replication. We have developed molecular methods to directlyconfirm replication or viability of positive-sense, single-stranded RNA viruses incell culture, regardless of their ability to produce CPE. Key enteric key viralpathogens with such nucleic acid include enteroviruses (coxsackieviruses andechoviruses and polioviruses), caliciviruses, hepatitis A and E viruses andastroviruses. During replication of these viruses, a complimentary negativestrand is made that is present primarily paired with the positive strands asreplicative intermediate RNAs in infected cells. Detection of the negative strandsor the double stranded replicative form RNA complexes containing positive andnegative strands is proof of viral replication. We have developed RT-PCRmethods to detect the negative strands or double-stranded forms of viral RNA inthe infected cultures using Coxsackievirus B3 as a model virus. Specific primerpairs for CVB3 were used for detection of the negative strand. With the use ofpanenterovirus primers for detection of the double-stranded forms, we havedemonstrated that as low as 4 infectious units can be detected within 24 hrs afterinfection. The methods we have developed offer great promise as rapid,sensitive, and specific approaches to the molecular detection of infectious humanenteric viruses in source water and drinking water supplies.
机译：环境或原水样本，甚至是饮用水 样本中可能包含可通过分子方法（例如 PCR和RT-PCR。这些方法与验证性分析相结合 （例如核酸杂交或核苷酸测序）可用于 确定水中是否存在病毒，即使在非常高的温度下 低水平。但是，这些测试并不能最终确定是否 病毒是传染性的或非传染性的。当前的标准方法 细胞培养检测水样或浓缩物中的传染性病毒 两次确认细胞病变作用（CPE）。可以的病毒 不会导致CPE或复制受到限制。此外， 该过程需要数周时间，而且成本很高。细胞培养中复制的证据 无需重复传代即可通过评估感染细胞的 病毒核酸的存在;但是，稀释测试对于 演示复制。我们已经开发出分子方法可以直接 确认正链单链RNA病毒在以下细胞中的复制或生存能力 细胞培养，无论其产生CPE的能力如何。关键肠溶病毒 具有此类核酸的病原体包括肠病毒（柯萨奇病毒和 回声病毒和脊髓灰质炎病毒），杯状病毒，甲型和戊型肝炎病毒以及 星状病毒。在这些病毒的复制过程中，互补阴性 产生的链主要与正链成对存在，例如 感染细胞中的复制中间RNA。负链的检测 或含有正向和反向双链复制形式的RNA复合体 负链是病毒复制的证据。我们开发了RT-PCR 检测病毒RNA负链或双链形式的方法 使用柯萨奇病毒B3作为模型病毒感染被感染的培养物。特异引物 CVB3对被用于检测负链。随着使用 用于检测双链形式的全肠病毒引物，我们有 证明在感染后24小时内可检测到4个感染单位 感染。我们开发的方法可带来快速发展的巨大希望， 传染性人类分子检测的灵敏，特异方法 饮用水和饮用水中的肠道病毒。

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