The Development Of Nested PCR Detection Method And Genotyping System For Ostreid Herpesvirus-1

Since the 1990 s, massive mortality of cultured bivalves outbroke frequently in the summer and spring when the water temperature is high worldwide including China. A kind of herpesvirus is revealed to be closely related to these mortalities by epidemiological investigation and artificial infection experiments,. International Committee on Taxonomy of Virus comes( ICTV) defined the virus as Ostreid herpesvirus 1(OsHV-1) in 2012, the virus is the only member belong to Ostreavirus, Malacoherpesviridae, Herpesviridae. Compared with the reference virus strain, new variants of the virus were discoved by molecular epidemiology investigation with the transformation of time and space. New mutants were found with the transformation of their epidemiological characters sunch as host range, virulence, infection time and the spatial distribution. Our goal is to establish a efficient detection method and the genotyping system for different variants of OsHV-1, and explore the main molecular epidemiological characteristics of the strains. These works will be useful for the prevention and control of the disease. This study also detected the common parasites found frequently in shellfish such as Perkinsus spp., Marteilia refringens and Bonamia spp., sampling test was carried out in order to understand the status of parasitic infection in China. We hope to keep the healthy and steady development of shellfish aquaculture with the implementation of corresponding prevention and treatment measures. The research methods, some results and conclusions of the thesis are listed follows:The first part: In order to establish a detection method suitable for different strains of OsHV-1, primers were designed based on the nuclei acid sequence of the conserved DNA polymerase of OsHV-1. A nested PCR detection method(P-nPCR) was established by optimization of the annealing temperatures of the primers and protocols of PCR. Then, both P-nPCR and C-nPCR were employed to test the infection status of the samples collected from different years and hosts. Our results indicated that the detection limits of the P-nPCR detection method was about 100 copies/μL of recombinant plasmid containing OsHV-1 genes. P-n PCR was more specific than C-nPCR in the detection of different variants of OsHV-1, and resulted in a higher prevalence of OsHV-1 for the same samples. In conclusion, a P-nPCR detection method was developed to detect different variants of OsHV-1 infection. The high specificity of P-n PCR to OsHV-1 ensured that different variants of OsHV-1 could be detected as early as possible, which will provide reliable technical support for the detection and epidemiology studies of OsHV-1.The second part: By using 4 pairs of primers targeted different genomic regions of OsHV-1, C2/C6, Del36-37F2/ Del36-37 R, IA1/IA2 and NC1/NC2, to amplify the DNA extracted from 278 shellfish samples infected with OsHV-1 collected from Liaoning and Shandong provinces in China between 2001 and 2014. The positive PCR products were sended to the sequencing company for sequencing after PCR reaction. Multiple sequence alignment was performed using Mega6.0 with the default settings, to compare the genetic variation between China and the other countries, OsHV-1 sequences identified in previous studies and present in Genbank were also included in the dataset.. According to the results of our analysis, high variability occurred in the genomic region targeted by C2/C6 and NC1/NC2. 11 variants were isolated from 118 sequences according to the sequence variation of C2/C6 region, 26 variants were isolated from 56 samples according to the sequence variation of NC1/NC2 region. Sequence vasriations found in the other genomic regions of OsHV-1 were small compared to the genomic regions of C2/C6 and NC1/NC2.When all of the four genomic regions were considered, the samples selected have a closer relationship with AVNV and OsHV-1-SB than OsHV-1μ Var and OsHV-1, this conclusion consistent with the previous studies. The experimental research takes an example to search the genotyping and evolutionary relationships of OsHV-1.The third part: Using the primers for detecting the common parasites infected bivalves recommended by the world organization for animal health(OIE), moluscs samples collected in Liaoning, Shandong, Guangdong, Guangxi, Fujian and other provinces in China and south Korean coast from 2007 to 2013 were investigated.The host species of samples include mussels, clams, scallops and oysters, the parasites include Perkinsus spp., Marteilia refringens and Bonamia spp..The results show that the infection of Perkinsus spp. mainly occurred in Dalian, Qingdao, Rongcheng and Weifang, no other parasites were found in the samples. The time of infection was mainly in July, August and September when the water temperature was relatively higher. The host species include mussels, Philippines clam and Arca subcrenata.