Partial Cloning Of The Genome Of The Acute Viral Necrobiotic Virus From Chlamys Farreri And The Nucleic Acid Diagnosing Methods

It is the acute viral necrobiotic virus (AVNV) that causes the massive death of the cultured scallop, Chlamys farreri in the coast of Shandong since 1997. AVNV is isolated and purified from moribund scallop (Chlamys farreri) through differential centrifugation and discontinuous sucrose gradient centrifugation. The virual nucleic acid is extracted from AVNV by proteinase K digesting. The AVNV genome is a single-stranded and non-sectional RNA molecule. The total RNA is extracted from AVNV with Trizol reagent. The cDNA is synthesized with random hexamers and Oligo(dT) method. After ligating of Size-Fractionated cDNA to the plasmid vector pUC118 and introducing into E. coli DH5a. The recombinant plasmids are screened by blue and white colonies, checked by PCR amplification and analyzed by restricted enzyme. The partial cDNA clones are sequenced and analyzed. According to the results of nucleotide sequence analysis, none of the 23# and 52# AVNV clones shows significant homology with those of other known sequences in GenBank.A rapid, sensitive and highly specific detection method for AVNV based on reverse transcription-polymerase chain reaction (RT-PCR) is developed. Two pairs of PCR primers are synthesized according to the 23# and 52# cloned cDNA sequences. For each primer combination only one specific major product is obtained when amplification is performed by using AVNV RNA. The lengths of their expected products are 406bp and 412bp, respectively. No products are obtained when the genomic DNA is extracted from the mantle of the scallop Chlamys farreri other thanAVNV RNA is used as RT-PCR templates. To assess the sensitivity of the method, dilutions of purified AVNV RNA (0.1pg-1000pg) are amplified and quantities of as little as 1pg and 100pg of purified AVNV RNA are respectively detected by using 23F, 23R and 52F, 52R primers when the amplification products are analyzed by 1% agarose gel electrophoresis.The 23# and 52# AVNV digoxigenin (DIG) labeled cDNA probes are produced by PCR. The sizes of the 23# and 52# DIG-labeled probes are respectively 406bp and 412bp, and their production is respectively 60ng/HL and 40ng/PL. Using dot blot hybridization the sensitivity and specificity are tested with dilutions of purified AVNV RNA (0.061pg-6100pg), DNA extracted from the mantle of the scallop Chlamys farreri. The results show the 23# and 52# probes have high sensitivity and strong specificity that the sensitivity of the two probes is respectively 6.1pg and 610pg to AVNV RNA. No hybridization signal is observed using DNA extracted from the mantle of the scallop Chlamys farreri. The two probes are used to detect the different tissues of the cultured scallop Chlamys farreri. The detecting results show the positive hybridization signals are in the mantle, gill, kidney, and the digestive gland of the cultured scallop Chlamys farreri, but no hybridization signal is observed in the gonad and muscle of the cultured scallop Chlamys farreri.All the results show that both the RT-PCR amplification method and the dot blot hybridization by the DIG labeled probe produced by PCR method are rapid, sensitive and highly specific methods for detection of AVNV, and are of significant importance for early diagnosis, prophylaxis and treatment of the acute viral necrobiotic disease (AVND) and AVNV resistant breeding.