Amplicon Rescue Multiplex PCR (Arm-PCR): a Novel Tool for Simultaneous Detection of Seven Types of Fish Viruses

Abstract:

Major fish viruses that are severely harmful in aquaculture industry include Lymphocystis disease virus (LCDV), Megalocytivirus (Mega), red-spotted grouper nervous necrosis virus (RGNNV), infectious haematopoietic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV), viral hemorrhagic septicemia virus (VHSV) and infectious salmon anaemia virus (ISAV). Here we developed a specific amplicon rescue multiplex PCR (Arm-PCR) combined with gene microarray technique for the simultaneous detection of the seven types of fish viruses. First we optimized the conditions of Arm-PCR such as the annealing temperature and the concentrations of Taq DNA polymerase, Mg2+, dNTP and Primer Mix shown as follows. Reaction mixture (50 μl) consisted of 1.0 μl Taq DNA polymerase (2.5 U/μl), 5 μl 10×PCR Buffer (20 mmol/L Mg2+), 5 μl dNTP (2.5 mmol/L each), 9 μl 10×Primer Mix (2 μmol/L), and 1 μl template. The annealing temperature was 56℃. This method could simultaneously produce specific amplicons in one tube. The detection sensitivity of the Arm-PCR was 101 copies/μl for RGNNV, VHSV, non-structural protein of ISAV (ISAV-NS), and matrix protein of ISAV(ISAV-MA), 102 copies/μl for LCDV, Mega, IHNV, and IPNV, and 103 copies/μl for TRBIV (Turbot reddish body iridovirus). The Arm-PCR did not cause cross reactions with genomic DNA from healthy fish such as half smooth tongue sole, grouper, turbot and flounder.