Application Of Real-time Quantitative PCR Assay For IHHNV And LCDV

Infectious hypodermal and hematopoietic necrosis virus(IHHNV) can infect worldwide cultured shrimp and cause serious economic loss to the shrimp culture industry. The experiment surveyed 84 Litopenaeus vannamei samples from Guangxi Province with real-time quantitative PCR (rt-qPCR) for the first time, with the conventional PCR as a contrast. The positive rate of rt-qPCR was 79.8% and that of the conventional PCR was 40.5%, and each sample positive by conventional PCR was also positive by rt-qPCR, indicating that the infection rate of Litopenaeus vannamei with IHHNV in Guangxi Province was high. Nucleotide sequencing was performed on 30 samples that were IHHNV positive. The results indicated that they were IHHNV sequence when analysed with the soft package of DNA STAR and compared with the GenBank using the NCBI Blast program. The sequences of the 30 samples were conservative with variation on two positions and classified to 4 types. rt-qPCR can be an effective diagnostic tool for detecting IHHNV infections on shrimp.LCDV was widely distributed in worldwide freshwater, brackish water and seawater and could infect more than 140 species of fishes belonging to 9 orders, 34 families. LCD was one of the harmful virus diseases. Its infection rate was up to 80% and the mortality rate could reach 30%. The research established sensitive and specific Taqman,SYBR GreenⅠreal-time PCR assay for the detection of lymphocystis disease virus(LCDV). Selected the conserved sequences of major capsid protein of LCDV and applied Primer Express2.0 to design primer and probe.In Taqman real-time quantitative PCR the gradient diluted plasmids containing LCDV objective amplification fragment were used as standards for sensitivity detection. The result indicated that there were amplification curves when the plasmid concentration was between 3×107 and 30, and the detection limit was 30 copy numbers. According to the relationship between virus copies(X)and Ct values the standard curve was prepared, the relationship formula was Ct=-3.63lgX+41.86 with the correlation coefficient R2=0.9915. The specificity analysis revealed that there weren't amplified reactions for EHNV, BIV, SGIV and RSIV. 12 batches of fish samples were found to be positive using established method in LCDV detection and the quantitative analysis was also performed. Conventional PCR products of positive samples were purified and then sequenced and the results were confirmed to be LCDV sequences. Taqman real-time quantitative PCR has the characteristics of high sensitivity,high specificity and performing quantitative analysis in LCDV detection, and it can be used for early diagnosis of LCDV infection.In SYBR GreenⅠreal-time PCR the optimal conditions was established and the optimal primer concentration was 0.5μM for the established system. In the specificity test LCDV plasmid was positive, while EHNV,TFV,BIV,SGIV,ISKNV and RSIV were negative. For the 12 diseased Paralichthys olivaceus samples the results were positive. In the repetition test SYBR GreenⅠreal-time PCR showed good inner and inter repetition in LCDV plasmid DNA detection. SYBR GreenⅠreal-time PCR established in this study is a low-cost, specific, sensitive and repetitive method for the detection of LCDV, and it can also be used for early diagnosis of LCDV infection.