Eltahir Y M, Dovas C I, Papanastassopoulou M, Koumbati M, Giadinis N, Verghese-Nikolakaki S, Koptopoulos G
Laboratory of Microbiology and Infectious Diseases, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece.
J Virol Methods. 2006 Aug;135(2):240-6. doi: 10.1016/j.jviromet.2006.03.010. Epub 2006 May 2.
A PCR assay was developed for the reliable detection of small ruminant lentivirus (SRLV) proviral DNA. The method involved the use of degenerate deoxyinosine-substituted primers and a second semi-nested PCR step that increased the polyvalency and sensitivity of the detection, respectively. Primers were designed from the pol gene conserved motifs of 85 SRLV isolates and were evaluated using different SRLV isolates together with Maedi-Visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV) reference strains. The method successfully detected SRLV proviral DNA in total DNA extracts originating from whole blood samples, separated peripheral blood mononuclear cells (PBMCs) and tissue cultures. The semi-nested PCR was compared with the agar gel immunodiffusion test and proved to be highly sensitive, specific and capable of detecting many SRLV variants in infected or suspect animals. Therefore, it would be useful in the diagnosis of natural SRLV infections, in eradication programs and epidemiological studies. Whole blood samples can be used directly, thus alleviating the need for PBMC separation, and thereby enables a simple, fast and cost-effective analysis of a large number of samples.
开发了一种用于可靠检测小反刍兽慢病毒（SRLV）前病毒DNA的聚合酶链反应（PCR）检测方法。该方法使用了简并脱氧肌苷取代引物以及第二个半巢式PCR步骤，分别提高了检测的通用性和灵敏度。引物是根据85株SRLV分离株的pol基因保守基序设计的，并使用不同的SRLV分离株以及梅迪-维斯纳病毒（MVV）和山羊关节炎-脑炎病毒（CAEV）参考毒株进行了评估。该方法成功地在源自全血样本、分离的外周血单核细胞（PBMC）和组织培养物的总DNA提取物中检测到了SRLV前病毒DNA。将半巢式PCR与琼脂凝胶免疫扩散试验进行了比较，结果证明其具有高度敏感性、特异性，并且能够检测感染或疑似动物中的许多SRLV变体。因此，它在自然SRLV感染的诊断、根除计划和流行病学研究中将会很有用。全血样本可直接使用，从而无需分离PBMC，进而能够对大量样本进行简单、快速且经济高效的分析。
