Institut Pasteur in Cambodia, Réseau International des Instituts Pasteur, Virology Unit, 5 Monivong Blvd, PO Box 983, Phnom Penh, Cambodia.
J Virol Methods. 2011 Sep;176(1-2):69-73. doi: 10.1016/j.jviromet.2011.06.002. Epub 2011 Jun 12.
Contaminated mud and soil may play roles as reservoirs and sources of transmission for avian influenza A virus. However, the persistence of highly pathogenic avian influenza (HPAI) H5N1 virus in soil or mud has not been well documented, and specific methods of H5N1 virus detection in mud and soil specimens have not been described. The aim of this work was to evaluate the capacities of five different commercial kits and one elution-concentration technique to extract nucleic acids from H5N1 virus and to detect infectious viral particles in experimentally infected mud specimens. The viral RNA detection thresholds for the QIAamp kit, Trizol LS and the MagNA Pure LC kit were 5 × 10(2)RNA copies per gram of mud. Trizol reagent and the RNA PowerSoil™ kit were unsuccessful in recovering any viral RNA from mud. When the elution-concentration technique was performed prior to nucleic acid extraction, the performance of the MagNA Pure kit increased to a level that allowed the detection of H5N1 nucleic acids in naturally contaminated environmental samples that had previously tested negative after direct extraction using commercial kits. The levels of detection of infectious virus after inoculation into embryonated eggs were higher in concentrates than in eluates.
受污染的泥土可能是禽流感病毒的储存库和传播源。然而，高致病性禽流感（HPAI）H5N1 病毒在土壤或泥浆中的持久性尚未得到很好的记录，也没有描述在泥浆和土壤样本中检测 H5N1 病毒的具体方法。本研究旨在评估五种不同商业试剂盒和一种洗脱浓缩技术从 H5N1 病毒中提取核酸并检测实验感染泥浆样本中传染性病毒颗粒的能力。QIAamp 试剂盒、Trizol LS 和 MagNA Pure LC 试剂盒的病毒 RNA 检测阈值为每克泥浆 5×10(2)RNA 拷贝。Trizol 试剂和 RNA PowerSoil™试剂盒均无法从泥浆中回收任何病毒 RNA。当在核酸提取之前进行洗脱浓缩时，MagNA Pure 试剂盒的性能提高到能够检测先前使用商业试剂盒直接提取后呈阴性的自然污染环境样本中的 H5N1 核酸。接种到鸡胚中的传染性病毒的检测水平在浓缩物中高于洗脱液。
