Single-tube heminested PCR coupled with ‘touchdown’ PCR for the analysis of the walleye dermal sarcoma virus env gene

Abstract

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.