Gregory Jason B, Litaker R Wayne, Noble Rachel T
University of North Carolina, Institute of Marine Sciences, 3431 Arendell St., Morehead City, NC 28557, USA.
Appl Environ Microbiol. 2006 Jun;72(6):3960-7. doi: 10.1128/AEM.02291-05.
Human enteroviruses can serve as a more accurate indicator of human fecal contamination than conventional bacteriological fecal indicators. We describe here a quantitative reverse transcriptase PCR (qRT-PCR) assay specifically tailored to detect these viruses in environmental waters. The assay included a competitive internal positive control (CIPC) that allowed the inhibition of qRT-PCRs to be quantitatively assessed. Coamplification of the CIPC with enteroviral genetic material did not affect the sensitivity, specificity, or reproducibility of the enteroviral qRT-PCR assay. The assay is rapid (less than 5 h from sample to result), has a wide dynamic range (>3 logs), and is capable of detecting as few as 25 enteroviral genomes with an average amplification efficiency of 0.91. In samples with low or moderate inhibition, the delay in CIPC amplification was used to adjust enterovirus qRT-PCR concentrations to account for losses due to inhibition. Samples exhibiting significant inhibition were not corrected but instead diluted twofold and immediately assayed again. Using significantly inhibited samples, it was found that dilution relieved inhibition in 93% (25 of 27) of the samples. In addition, 15% (4 of 27) of these previously negative samples contained enteroviral genomes. The high-throughput format of the assay compared to conventional culture-based methods offers a fast, reliable, and specific method for detecting enteroviruses in environmental water samples. The ability of the assay to identify false negatives and provide improved quantitative assessments of enterovirus concentrations will facilitate the tracking of human fecal contamination and the assessment of potential public health risk due to enteroviruses in recreational and shellfish harvesting waters.
与传统的粪便细菌学指标相比，人类肠道病毒可作为人类粪便污染更准确的指标。我们在此描述一种专门设计用于检测环境水体中这些病毒的定量逆转录聚合酶链反应（qRT-PCR）检测方法。该检测方法包括一个竞争性内部阳性对照（CIPC），可对qRT-PCR的抑制作用进行定量评估。CIPC与肠道病毒遗传物质的共扩增不影响肠道病毒qRT-PCR检测方法的灵敏度、特异性或重现性。该检测方法快速（从样品到结果少于5小时），具有宽动态范围（>3个对数），能够检测低至25个肠道病毒基因组，平均扩增效率为0.91。在抑制作用低或中等的样品中，利用CIPC扩增的延迟来调整肠道病毒qRT-PCR浓度，以补偿抑制造成的损失。对表现出显著抑制作用的样品不进行校正，而是将其稀释两倍并立即再次检测。使用显著抑制的样品发现，93%（27个样品中的25个）的样品稀释后可解除抑制。此外，这些先前为阴性的样品中有15%（27个中的4个）含有肠道病毒基因组。与传统的基于培养的方法相比，该检测方法的高通量形式为检测环境水样中的肠道病毒提供了一种快速、可靠且特异的方法。该检测方法识别假阴性的能力以及对肠道病毒浓度进行改进的定量评估，将有助于追踪人类粪便污染，并评估娱乐用水和贝类捕捞水域中肠道病毒导致的潜在公共卫生风险。
