Establishment And Application Of Amplicon Rescue Multiplex PCR(Arm- PCR) For The Simultaneous Detection Of Seven Kinds Of Fish Viruses

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) were major fish viruses in aquaculture. In this report, a specific amplicon rescue multiplex polymerase chain reaction(Arm-PCR) combined with a gene microarray for the simultaneous detection of above seven kinds of fish viruses were established. The concentration of Taq DNA polymerase, Mg2+, d NTP, Primer Mix and annealing temperature in the first step of Arm-PCR were optimized in this study. In 50 μL of reaction volume, the optimized parameters were Taq DNA polymerase(2.5 U/μL) 1.0 μL, 10 × PCR Buffer(20 mmol/L Mg2+) 5 μL, d NTP(2.5 mmol/L each) 5 μL, 10 × Primer Mix(2 μmol/L) 9 μL, template 1 μL. The annealing temperature was 56 °C.The result showed that the Arm-PCR reported here could produce specific amplicons in one tube simultaneously. The sensitivities of the Arm-PCR were 101 copies/μL(RGNNV, VHSV, ISAV-NS, ISAV-MA), 102 copies/μL(LCDV, Mega, IHNV, IPNV) and 103 copies/μL(TRBIV), respectively. The established gene microarray showed good specificity and reliability by preliminary application. There were no cross reactions with genomic DNA from healthy fish, such as half smooth tongue sole, grouper, turbot and flounder accorrding to the Arm-PCR.This study established an amplicon rescue multiplex polymerase chain reaction(Arm-PCR) method which could detect seven fish viruses simultaneously. With the advantages of high flux, high sensitivity and high accuracy, the method could improve work efficiency. It will be used in the field of fish virus screening and epidemiological investigations.