The Establishment Of CPA Techniques In Detecting OsHV-1-SB And Epidemiological Survey Of Perkinsus Sp. In Shellfish

SB strain of Ostreid herpesvirus-1(Os HV-1-SB) was detected from Scapharca broughtonii samples, these samples were from different aquaculture farm, such as Chang Dao, Lai Zhou, Ri Zhao and Ji Mo of Shan Dong and so on, acquired by our laboratory in June of 2012. Healthy Scapharca broughtonii after infected with Os HV-1-SB, its’ s main behaviori are slow response, double shell is not closed or closed slowly after touched it, mantle indentation and the color of internal organs present Grey white or Black grey. The individual general appeared above symptoms would die in 1 day, the mortality rate could be up to 75%, the effective detection method on the virus has not yet been established at present. According to the whole genome sequence of Os HV-1-SB, which have been sequenced by our laboratory. Using cross priming amplification(CPA) technology, through the optimization of reaction conditions, the enzyme identification of CPA product, CPA sensitivity experiment, CPA specific experiment and practical testing of the samples, this paper aimed to establish a simple, rapid, high sensitive CPA-based field detecting method for Os HV-1-SB. Meanwhile, we used Polymerase chain reaction(PCR) technique for epidemiological investigation of Perkinsus olseni, the samples of shellfish were collected by our laboratory in 2010, in order to provide a certain theoretical basis for effective prevention and control of Perkinsus olseni disease. Research method and some results are as follows:Part 1: According to the whole genome sequences of the SB strain of Os HV-1, which have been sequenced by our laboratory(the article not published), select its conservative areas as CPA target sequence, use Primer Explorer 4.0 primer design software, design a set of primers CPA responsea. Then the reaction temperature, time and concentrations of d NTPs and Mg2+ were optimized. Results showed the optimum temperature and time of the assay were 63℃ and 60 min, and the optimum concentration of d NTP and Mg2+ were 1.4 mmol/L and 6 mmol/L, respectively. The CPA assay was highly sensitive with detection limit approximately 30 copies recombinant plasmid DNA per μl. The assay was also highly specific for Os HV-1-SB detection. No cross-reaction was found when detecting Acute viral necrosis virus, Abalone herpes viruses, Perkinsus sp., Bonamia sp., Martelia sp., White Spot Syndrome Virus and Vibrio parahaemolyticus. Furthermore, we used experimental CPA detection method to detect a total of 22 Os HV-1-SB infection status unknown samples, those were collected from Gyeongsangnam-do of Korea, Changdao of Shandong province, Dalian of Liaoning province. The results showed that the CPA assay is a simple, rapid, sensitive, specific and reliable technique. Additionally, since the test results can be simply by centrifugal observation white precipitate or to join in the reaction tube nucleic acid fluorescent Gene FinderTM masculine judgment through the color change, this assay can be used in shellfish farms, local laboratories with poor conditions and has good application prospect.Part 2: Perkinsus olseni is a major parasite in shellfish aquaculture. In this study, I extracted DNA from a total of 568 shellfish samples, which were collected from Qingdao, Rongcheng, Changdao of Shandong province, Huian of Guangzhou province and Xiapu of Fujian province. After extracted DNA, using OIE recommendation’s PCR method to detected Perkinsus olseni. The results showed that the positive rate of Perkinsus olseni reached up to 21.46% in all samples. Among, the positive rate of Chlamys farreri(Penglaihong and wild seedlings) samples was 22.00%, Perna sp. was 55.00%, which was collected from Liuqinghe of Qingdao. the positive rate of Chlamys farreri(Penglaihong, wild seedlings and artificial breeding) samples was 16.30%, Perna sp. was 18.52%, which was collected from Sanggou Bay of Rongcheng.The shellfish samples’ s positive rate was low collected from Yantai Changdao town, only Haliotis discus hannai was detected positive, the Perkinsus olseni was not detected in Chlamys farreri wild seedlings and Patinopecten yessoensis. Detecting Perkinsus olseni of Haliotis discus hannai, the positive rate in Xiapu of Fujian province reached 70%, but Huian of Guangzhou province was not detected. Meanwhile, this study analysised overall positive ratio of Perkinsus olseni’s of each month shellfish samples. The results indicated Perkinsus olseni infection most occurred in Summer high temperature season, especially reached a peak in July. However, In January, February and March Low temperature period, Perkinsus olseni infection positive samples were not detected.Part 3: Acute Viral Necrosis Virus(AVNV) was proved to be a spherical virus which caused massive mortality in Chlamys farrei. In this paper, based on the completed AVNV whole genome sequence, is obtained by PCR technology coding AVNV multiple ORF110 across membrane protein gene, and TA cloning p MD18-T-110 plasmid, the restriction enzymes Sna B Ⅰ and Not Ⅰ after double enzyme and eukaryotic expression vector p PIC9 K connection, transformation into the top 10 E.coli cells, get used to build the protein expression of recombinant plasmid p PIC9K-ORF110. After electricity transformation, the recombinant plasmid into pichia GS115, using methanol has carried on the induction expression, at the same time in the process of the expression of yeast form using the optical microscope observation, in order to confirm the express no bacterial pollution in the process.