Validation of the sensitivities of one-step and two-step reverse-transcription PCR methods for detection of viral hemorrhagic septicemia virus (VHSV) IVa isolates from cultured olive flounder in Korea

Abstract

Viral hemorrhagic septicemia (VHS) is one of the most serious viral diseases of cultured olive flounder in Korea. This study validated the sensitivities of one-step and two-step reverse-transcription PCR (RT-PCR) methods based on the OIE (World Organization for Animal Health) diagnostic manual for the detection of VHSV type IVa isolates from olive flounder in Korea. VHSV type I was used as a positive control for the VHSV VN primer set listed in the OIE manual; the PCR products amplified from VHSV I appeared as strong gel bands of the target size, but the PCR products amplified from the VHSV IVa Korean isolate appeared as faint bands. Sequence comparison revealed that the VHSV VN forward primer was mismatched at 4 out of 24 nucleotide positions within the corresponding VHSV type IVa sequence, but differed only by a single nucleotide in the case of VHSV I. Therefore, the VHSV VN IVa primer set was designed specifically to fit the VHSV IVa sequence, and this was used in RT-PCR. The PCR products amplified from VHSV IVa appeared as strong bands of the target size when the VHSV VN IVa primer set was used, but the PCR products of VHSV I appeared as faint bands. PCR titration results showed that the sensitivity of the VHSV VN IVa primer set for VHSV IVa was 100,000-fold higher than that of the VHSV VN primer set when one-step RT-PCR was used, and that the sensitivity was >10,000-fold higher than that of the VHSV VN primer set when the two-step RT-PCR method was used with oligo dT+random hexamer. The sensitivity of the VHSV VN IVa primer set for VHSV IVa was found to be 10,000-fold higher than that of the VHSV VN primer set when the two-step RT-PCR method was used with the target primer set. Therefore, the RT-PCR methods that are used in pathogen-diagnosis laboratories must be validated according to the specificity of the primer set and the one-step or two-step RT-PCR method employed, using either the target primer set or oligo dT+random hexamer.