Development And Application Of Loop-Mediated Isothermal Amplification For Detection Of Chinese Giant Salamander Ranavirus

Chinese Giant salamander (Andrias davidianus) belongs to Amphibia, Anura, cryptobr--achidae, it’s the largest remaining precious amphibian species and endemic in China. It is defined as Chinese class II national key protected wild animals. In the 1970s, China began the breeding trials of Chinese giant salamander with the establishment and implementation of the aquatic wildlife policy and huge success have been made in artificial breeding of giant salamander in the provinces of Sichuan, Shanxi, Hunan, Chognqing and Gansu. Chinese Giant Salamander Ranavirus was a new disease of the giant salamander. The major symptoms include surface bleeding spots, ulcers, head and swelling of the limbs, which was called the canker or "Canker or Bigfoot disease".Once occurred, the disease often leads to high mortality rates which could reach 100% to the farmers as it called "salamander cancer". LAMP possesses better specificity and sensitivety, so as to provide a new way to prediction and dignosis of the infection of RanavirusAccording to the sequence of MCP (major capsid protein) of CGSRV (HQ684746)on the GenBank, primers F3:5’-GAGGGCGTACTTTTGGGC-3’; B3:5’-ATGCCACCTCCATCCCA-3’;FIP: 5’-TAACGTCACCCTGTCCGCTGA-CAGAGTTGTCACCTCCGC-3’; BIP 5’-TCTGCCGTAATTGGTGGATCCG-GGCACCACCTCTACTCCTA-3’were designed. In the following step, several important parameters of LAMP were optimized including Mg2+ concentration、pirmer concentration、dNTPs concentration and Bst DNA polymerase concentration,the temperature and reaetion time of the reaction (reaction system:1μL F3(10μM), (B3(10μM), (FIP(50μM), (BIP(50μM), 3.5μL dNTPs (25mM),4μL 10×Thermopol buffer,8U Bst DNA polymerase,3μL MgCl2(25mM),2μL DNA and7.5μL ddH2O; reaction condition:62℃ 40min. Results showed that LAMP was highly sensitive to Chinese Giant Salamander Ranavirus with a detection limit of 5 copies/μL, which was 10-fold and 103-fold higher than that of nested-PCR and traditional PCR for CGSRV. Moreover, the assay was specific for the detection of CGSRV and was not susceptible to cross reaction with other viruses, including koi herpesvirus (KHV)、Goldfish Herpes Virus (CyHV-2)、Marbled sleepy goby iridovirus (MSGIV)、Infectious Spleen And Kidney Necrosis Virus(ISKNV)、Edwardsiella tarda、Aeromonashydrophila、Aeromonas veronii. Using LAMP、Nested-PCR and conventional PCR detection 55 suspected samples and 8 health of Chinese giant salamanders, same result was obtained which indicated the accuracy of the LAMP assay. In 32 unkown samples, the detection rate of LAMP was 93.75%,which was much higher than the Nested-PCR, which was 87.5% and conventional PCR,which was 68.75%. In conclution, the detection of LAMP method was time-saving, specificity which could increase the detection speed and accuracy of CGSRV.it is suitable for the clinical diagnosis and epidemiological investigation CGSRV.