Survey of bovine enterovirus in biological and environmental samples by a highly sensitive real-time reverse transcription-PCR
Survey of bovine enterovirus in biological and environmental samples by a highly sensitive real-time reverse transcription-PCR
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DOI：
10.1128/aem.71.7.3536-3543.2005
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发表时间：
2005-07-01
影响因子：
4.4
通讯作者：
Sáiz, JC
中科院分区：
文献类型：
--
作者：
Jiménez-Clavero, MA;Escribano-Romero, E;Sáiz, JC
感染动物粪便中的动物肠道病毒可能是环境污染物，因此可以用作动物粪便污染的指标。先前的工作已经证明，牛粪便中存在的牛肠道病毒（BEV）污染牛群附近的沃茨，BEV样序列也存在于来自同一地理区域的贝类和鹿粪便中。然而，关于BEV野外分离株的流行、分子流行病学和基因组序列变异的信息很少。在这里，我们描述了一种优化的高灵敏度实时逆转录PCR方法来检测生物和环境样品中的BEV RNA。扩增程序与先前描述的使用正电性过滤器的过滤步骤的组合允许我们检测每毫升水高达12个BEV RNA分子。通过对不同来源水样的分析，证实了该方法用于粪便污染高风险地表沃茨中BEV检测的可行性。该方法还用于研究西班牙各地不同牛群中BEV的流行情况，结果显示78%（100个中的78个）的粪便样本为BEV阳性。在绵羊、山羊和马的粪便中也检测到BEV样序列。核苷酸序列分析表明，BEV分离株是相当异质性，并建议存在物种特异性BEV样变异。BEV样序列的检测可能有助于区分和表征动物污染源。
Animal enteroviruses shed in the feces of infected animals are likely environmental contaminants and thus can be used as indicators of animal fecal pollution. Previous work has demonstrated that bovine enterovirus (BEV) present in bovine feces contaminates waters adjacent to cattle herds and that BEV-like sequences are also present in shellfish and in deer feces from the same geographical area. However, little information is available about the prevalence, molecular epidemiology, and genomic sequence variation of BEV field isolates. Here we describe an optimized highly sensitive real-time reverse transcription-PCR method to detect BEV RNA in biological and environmental samples. A combination of the amplification procedure with a previously described filtration step with electropositive filters allowed us to detect up to 12 BEV RNA molecules per ml of water. The feasibility of using the method to detect BEV in surface waters at a high risk of fecal pollution was confirmed after analysis of water samples obtained from different sources. The method was also used to study the prevalence of BEV in different cattle herds around Spain, and the results revealed that 78% (78 of 100) of the fecal samples were BEV positive. BEV-like sequences were also detected in feces from sheep, goats, and horses. Nucleotide sequence analyses showed that BEV isolates are quite heterogeneous and suggested the presence of species-specific BEV-like variants. Detection of BEV-like sequences may help in the differentiation and characterization of animal sources of contamination.