Establishment And Application Of Loop-mediated Isothermal Amplification For Fast Detection Of Lymphocystis Disease Virus And Marine Birnavirus

Lymphocystis disease virus (LCDV) is distributed widely in worldwide freshwater, brackish water and seawater, and can infect more than 140 species of fishes belonging to 9 orders 34 families which is one of the harmful virus diseases to fish. Its infection rate was up to 80 % and the mortality rate could reach 30 %. A specific and sensitive method of a loop-mediated isothermal amplification (LAMP) was developed for rapid detection of lymphocystis disease virus (LCDV). The specific primers were designed according to the highly conservative sequence of major capsid protein (MCP) gene of LCDV using Primer Explorer V3 software. The primers and the reaction condition were optimized to LAMP assy. Moreover, the specificity and sensitivity were estimated and 109 Paralichthys olivaceus samples were tested with this method. It was found that there was high specificity and no cross-reaction with EHNV, TFV, BIV, SGIV, ISKNV, RSIV and WSSV. The detection limit of LAMP assay was 15 fg and similar to real-time quantitative PCR, but 10-fold higher than conventional PCR. The LAMP assay was evaluated using 109 clinical samples and there were the same results between healthy and seriously infected samples, but had 8 differences in unkown samples. The results of 2 detection times indicated real-time quantitative PCR and LAMP were uniform, more sensitive than conventional PCR. Moreover, the results were obtained from extraction of viral DNA using this method only 2 hours. Above all, the LAMP method can effectively detect LCDV at fish farms and in entry-exit inspection and quarantine, and it is credible for specificity, sensitivity and rapidity.Marine birnavirus is existed widely in worldwide seawater and could infect more than 11 species of mollusc, 4 shellfish and more than 10 fishes, which belong to 30 families. It was specialy harmful to young of seriola quinqueradiata and its infection rate was up to 85 %, the mortality rate could reach 62 %. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was developed for marine birnavirus. According to sequence of polyprotein (VP2) gene of MABV from Genbank the specific primers were designed using Primer Explorer V3 software. The RT-LAMP was optimized for its primers and reaction condition. Furthermore, the specificity and sensitinity of RT-LAMP were estimated and Lateolabrax japonicus, Pagrosomus major, Scophthalmus maximus, Paralichthys olivaceus from farms were tested 30 respectively. This assay had high specificity and no cross-reaction among IPNV, IHNV, SVCV, VHSV and VNNV. The detection limit of RT-LAMP was 16.6 fg total RNA and similar to real-time reverse transcription quantitative PCR, but 10-fold higher than conventional reverse transcription PCR. The results of 120 fish samples demonstrated RT-LAMP and real-time quantitative RT-PCR both were good sensitivity. Above results shown RT-LAMP was exact, rapid, specific, sensitive, low-cost assay for MABV, and could be used for fish farms and in entry-exit inspection and quarantine for early diagnosis of MABV.