AVNV Molecular Epidemiology Survey And The Establishment Of A Loop-mediated Isothermal Amplification Method For Perkinsus Olseni Detection

The scallop Chlamys farreri is one of the main shellfish species cultured innorthern coastal China. Acute Viral Necrosis Virus (AVNV) was proved to be aspherical virus which caused massive mortality in Chlamys farrei. In this paper, theestablished PCR diagnostic technique of AVNV was used to detect Chlamys farreri,fouling organisms and the shellfish from6provinces14sea areas in China, to exploreAVNV distribution throughout the country, the host range, the infection of thevalue-added rule and the transmission route of AVNV. In the course of the study wealso found that the shellfish parasite detection technology lags behind, at the sametime, we established and applied a loop-mediated isothermal amplification (LAMP)method for Perkinsus olseni detection to lay the technical and theoretical foundationfor exploring the shellfish health culture model, proposing and establishing theshellfish mass mortality of the disease epidemic prevention.In this paper, PCR was used to detect the Chlamys farreri from two different seaareas for culturing scallops in2010and2011, to research the pattern of AVNVinfection and explore the shellfish health aquaculture mode. High positive ratio ofscallops was detected in Liuqinghe sea area of Qing Dao during the summer of2010when the Chlamys farreri had a large-scale death.The positive ratio detected of wildseedlings and Penglaihong up to60%and30%respectively. The time that highestpositive ratio detected was in agreement with large-scale death happened. High watertemperature was an important environmental stress factor of Chlamys farreri infectedAVNV. The Sangou Bay is the shellfish and seaweed maricuhur sea area,the scallopscultured in this area carried less AVNV than that in Liuqinghe. August has the highestAVNV positive ratios of the Sanggou Bay, the positive ratio detected of Penglaihong,artificial breeding and the wild seedlings are20%,10%and20%, while the positive ratios were lower during the massive mortality. After the time of massive mortality,we made statistics of the mortality of the three seedlings in two sea areas. Themortalities of the wild seedlings and Penglaihong are37.41%and61.33%inLiuqinghe, while the mortalities of Penglaihong, the artificial breeding and the wildseedlings in Sangou Bay are96.67%,92.5%and94.17%. In conclution, the pattern ofshellfish and seaweed maricuhur may maintain the ecological environment of theChlamys farreri breeding sea in a stable and sustainable development state, inhibiteAVNV infect Chlamys scallops or prevent the spread of AVNV, most important is thatit can also decrease mortality.The fouling organisms which attached scallop farming cages from2010to2011were detected to reseach whether they can carry AVNV or not. The result shows thatMytilus edulis, Skeleton shrimp and Crassostrea gigas can carry AVNV, they are thehosts of AVNV. AVNV was not detected in Hiatella orientalis and Styela clava, soHiatella orientalis and Styela clava may not the host of AVNV.From May2010to December2011,390shellfishes were collected from14seaareas6provinces in china, consist of7bivalvia and2gastropoda. All of the shell,5kinds were detected that can carry AVNV, consist of Patinopecten yessoensis, Mytilusedulis, Perna viridis, Crassostrea gigas, Crassostrea rivularis,;3kinds were notdetected that can carry AVNV, consist of Paphia undulata, Pteria penguin andMeretrix meretrix. But owing to the restriction of regions,time,and numbers ofsamples, further studies is needed to decide whether these species can carry ANNV.Perkinsus olseni(P. olseni) is one of the important pathogenic parasites ofshellfish. With the purpose of building a rapid, accurate, sensitive and easy to usedetection methods for P. olseni, we established a P. olseni loop-mediated isothermalamplification assay (LAMP) based on the internal transcribed spacer (ITS) ofPerkinsus olseni5.8S rDNA sequences. We used the online software Primer ExplorerV4designed a set of4LAMP primers(FIP, BIP, F3and B3) of the P. olseni, then weoptimized the reaction conditions based on the basic reaction system of LAMP,mainly about the reaction temperature, magnesium ion concentration of the reactionsystem and the reaction time. After that, we got the P. olseni25μL LAMP reaction system, the optimal reaction temperature is64℃and the optimal reaction time is60min. In this research, the LAMP products were detected mainly using agarose gelelectrophoresis and visual inspection of a color change due to addition of fluorescentdye. Before confirming the minimum threshold of the LAMP, we constructed P. olsenipositive plasmid also base on the P. olseni5.8S rDNA ITS sequences. The resultshows that the minimum threshold of the LAMP assay is approximately30copies ofplasmid DNA. We proved that the developed LAMP method was highly specific for P.olseni, and no cross-reaction was observed with other pathogens, such as Perkinsusmarinus (P. marinus), Bonamia exitiosa (B. exitiosa), Ichthyobodo sp. and Acute ViralNecrosis Virus (AVNV). A comparative evaluation of the LAMP and PCR assaysusing20Ruditapes philippinarum (R. philippinarum) samples showed that LAMP ismore sensitive and accurate than PCR and the shellfish parasite P. olseni is widelydistributed in farming shellfish of North shellfish farming area. Totally, these resultsindicate that the LAMP method is a kind of simple, sensitive, specific, and reliabletechnique for the detection of P. olseni. The LAMP technique could be used for thedetection of P. olseni in the coastal shellfish farms and laboratiories with simpleequipments.