Abd-Alla Adly, Bossin Hervé, Cousserans François, Parker Andrew, Bergoin Max, Robinson Alan
Entomology Unit, FAO/IAEA Agriculture and Biotechnology Laboratory, A-2444 Seibersdorf, Austria.
J Virol Methods. 2007 Feb;139(2):143-9. doi: 10.1016/j.jviromet.2006.09.018. Epub 2006 Oct 30.
A PCR based diagnostic method to detect salivary gland hypertrophy virus (SGHV) in tsetse flies is described. Two sets of primers GpSGHV1F/GpSGHV1R and GpSGHV2F/GpSGHV2R were selected from a virus-specific sequence. Both primer sets can detect specifically the virus in individual tsetse flies by generating an amplicon of 401 bp. Attempts were made to develop a simple and reliable non-destructive virus detection method in live flies. PCR reactions were performed on either crude or purified tsetse DNA from saliva and legs. While saliva can be an indicator for the presence of the virus in flies, the method is laborious. Crude extract from an excised middle leg resulted in a positive PCR reaction equivalent to crude extract from whole fly. However, sensitivity could be significantly increased when purified DNA was used as the template. In conclusion, PCR using a purified DNA template from a single tsetse leg represents an efficient, non-destructive method for virus diagnosis in live tsetse flies.
本文描述了一种基于聚合酶链反应（PCR）的诊断方法，用于检测采采蝇中的唾液腺肥大病毒（SGHV）。从病毒特异性序列中选择了两组引物GpSGHV1F/GpSGHV1R和GpSGHV2F/GpSGHV2R。这两组引物都能通过产生一个401bp的扩增子，特异性地检测单个采采蝇中的病毒。我们试图开发一种简单可靠的、用于活蝇的非破坏性病毒检测方法。对来自唾液和腿部的粗制或纯化的采采蝇DNA进行PCR反应。虽然唾液可以作为采采蝇中病毒存在的一个指标，但该方法很费力。从切除的中腿获得的粗提物导致的PCR阳性反应与从整个采采蝇获得的粗提物相当。然而，当使用纯化的DNA作为模板时，灵敏度可以显著提高。总之，使用来自单个采采蝇腿部的纯化DNA模板进行PCR，是一种用于活采采蝇病毒诊断的高效、非破坏性方法。
