Unité de Sécurité Microbiologique, Institut Pasteur de Lille, 1 rue du Prof. Calmette, BP 245, 59019 Lille, France.
J Virol Methods. 2012 Jan;179(1):148-53. doi: 10.1016/j.jviromet.2011.10.013. Epub 2011 Oct 20.
The role of environmental reservoirs in avian influenza virus (AIV) transmission has been investigated during AIV-associated outbreaks. To date, no method has been defined for detection of AIV from mud samples. A procedure using elution and polyethylene glycol (PEG) concentration steps was designed to detect AIV by RT-PCR from 42g of raw mud, corresponding to 30g of the solid fraction of mud. RNA was recovered with MagMAX AI/ND Viral RNA Isolation kit (Ambion, Austin, TX). Three elution buffers were studied and viral recoveries higher than 29% were yielded by elution with a 10% beef extract solution (pH 7). The overall method showed that, under some conditions, virus was not detectable in PEG samples, whereas viruses were detected in the elution fractions. PCR curves were improved significantly by running the amplification reaction with a mixture containing a PCR additive for inhibitor removal, such as T4 gene 32 protein (Gp32), although PCR inhibitors from mud were removed partially from PEG samples. A theoretical detection threshold of 5×10(5) RNA copies of H5N1 virus per 30g of solid mud could be obtained by elution. The overall method has proved successful for detecting H5N1 virus contamination of mud specimens collected during outbreak investigations of avian influenza in Cambodia.
环境储层在禽流感病毒（AIV）传播中的作用已在与 AIV 相关的暴发期间进行了研究。迄今为止，尚无从泥浆样本中检测 AIV 的方法。设计了一种使用洗脱和聚乙二醇（PEG）浓缩步骤的程序，通过 RT-PCR 从 42g 原始泥浆（相当于泥浆固相的 30g）中检测 AIV。使用 MagMAX AI/ND 病毒 RNA 分离试剂盒（Ambion，奥斯汀，TX）回收 RNA。研究了三种洗脱缓冲液，通过用 10%牛肉提取物溶液（pH 7）洗脱，可获得高于 29%的病毒回收率。总体方法表明，在某些条件下，PEG 样品中无法检测到病毒，但在洗脱部分检测到病毒。通过在扩增反应中使用包含用于去除抑制剂的 PCR 添加剂（如 T4 基因 32 蛋白（Gp32））的混合物运行，PCR 曲线得到了显著改善，尽管从 PEG 样品中部分去除了泥浆中的 PCR 抑制剂。通过洗脱，可以获得每 30g 固相泥浆中 5×10(5)个 RNA 拷贝 H5N1 病毒的理论检测下限。该综合方法已成功用于检测柬埔寨禽流感暴发调查中采集的泥浆样本中 H5N1 病毒的污染。
