Importance of the 3'-Terminal Nucleotide of the Forward Primer for Nucleoprotein Gene Detection of Viral Hemorrhagic Septicemia Virus by Conventional Reverse-Transcription PCR

Viral hemorrhagic septicemia virus (VHSV), causing severe diseases in farmed fish, is detected and genotyped using conventional reverse-transcription PCR (cRT-PCR) targeting the nucleoprotein gene with corresponding VN F (forward) and VN R (reverse) primers. However, these primers have low sensitivity to VHSV subtype IVa; I investigated the cause for the poor cRT-PCR performance using various primer combinations. The results demonstrated that a 3'-end mismatch in the VN F primer reduced sensitivity and plays a critical role in VHSV detection by cRT-PCR.