Zhang Z, Martineau D
Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Canada.
J Virol Methods. 1996 Jun;60(1):29-37. doi: 10.1016/0166-0934(96)02024-1.
The nucleotide sequences of geographically distant isolates of a newly characterized retrovirus of fish, the walleye dermal sarcoma virus (WDSV) were compared. Primers were designed based on the nucleotide sequence of a single molecular clone isolated from a tumor collected from a fish in New York state, USA. Conventional polymerase chain reaction (PCR) did not allow the satisfactory amplification of WDSV isolates from a different geographic region (Quebec, Canada), probably owing to the high genetic variability of retroviruses. To allow the successful amplification of Quebec WDSV isolates, heminested PCR and 'touchdown' PCR were combined in a highly sensitive, short and simple protocol taking place in a single tube, thus minimizing cross contamination. This technique allowed the first unambiguous detection of a fish retrovirus in Canada and the phylogenetic analysis of various geographic isolates of this virus. Other potential applications for this technique include PCR amplification using degenerate primers, and amplification of templates with some expected degree of genetic variability.
对一种新鉴定的鱼类逆转录病毒——大眼狮鲈皮肤肉瘤病毒（WDSV）在地理上相距遥远的分离株的核苷酸序列进行了比较。根据从美国纽约州一条鱼身上采集的肿瘤中分离出的单个分子克隆的核苷酸序列设计了引物。常规聚合酶链反应（PCR）无法令人满意地扩增来自不同地理区域（加拿大魁北克）的WDSV分离株，这可能是由于逆转录病毒的高遗传变异性所致。为了成功扩增魁北克WDSV分离株，将半巢式PCR和“降落”PCR在一个高度灵敏、简短且简单的单管方案中结合起来，从而将交叉污染降至最低。这项技术首次明确检测到了加拿大的一种鱼类逆转录病毒，并对该病毒的各种地理分离株进行了系统发育分析。该技术的其他潜在应用包括使用简并引物进行PCR扩增，以及扩增具有一定预期遗传变异性的模板。
