Establishment Of Duplex PCR And Real-time PCR Detect Methods For Vibrio Owensii XSBZ03 And V.Alginolyticus XSBZ14

Coral diseases have become one of major threats to coral reef ecosystems,but the technology for the diagnosis of coral diseases and the detection of coral pathogens is absent.In this thesis,we constructed a duplex PCR method and a real-time PCR method for detection of XSBZ03 and XSBZ14 which are the pathogenic strains of Porites andrewsi white syndrome..In this study,according to the whole genome information of strains XSBZ03 and XSBZ14,single-copy genes of XSBZ03 and XSBZ14 were screened by Perl script.The results showed that there were 145 single-copy genes in XSBZ03 and 131 single-copy genes in XSBZ14.Relative Analysis of single-copy genes of XSBZ03 and XSBZ14 were conducted by Blastn of NCBI.The results showed that the single-copy genes S1 and S3 of XSBZ03 and single-copy gene S2 of XSBZ14 had high strain specificity.The primers Z03F1/Z03R1 were designed based on the target sequence S1 and the primers Z14F1/Z14R1 were designed based on the target sequence of S2.The duplex PCR method which was established based on the two primer pairs can specifically detect XSBZ03 and XSBZ14.The detection limits of genomic DNA of XSBZ03 and XSBZ14 are 1.7 pg μL-1 and 2.0 pg μL-1,respectively.In artificial seawater samples,the detection limit for XSBZ03 and XSBZ14 strain concentration is 6×103 CFU mL-1 and 8×103 CFU mL-1,respectively.The real-time PCR primers Z03F2/Z03R2 and Z14F3/Z14R3 for specific detection of XSBZ03 and XSBZ14 were designed based on sequence S3 and S2,respectively.Four different standard curves were constructed to quantify different samples which contained XSBZ03 and XSBZ14.First,the standard curve of DNA concentrations could accurately quantify the DNA concentration of the target strain in the sample.The quantitative limit of XSBZ03 and XSBZ14 DNA are 0.8 pg μL-1 and 0.4 pgμL-1,respectively.Second,the standard curve of strain concentrations could accurately quantify the strain concentration in the sample with a quantitative limit of 1500 CFU mL-1 and 1000 CFU mL-1 for XSBZ03 and XSBZ14.Third,the standard curve of the number of target strains in coral tissue could accurately quantify the number of target strains in coral tissue.The quantitative limit of XSBZ03 in sampling coral is 150 CFU and the quantitative limit of XSBZ14 in sampling coral is 100 CFU.Fourth,the standard curve of the number of target strains in rear seawater could accurately quantify the number of target strains in rear seaawater with a quantitative limit of 1500 CFU for XSBZ03 and 1000 CFU for XSBZ14.Coral pathogens XSBZ03 and XSBZ14 were used to infect Galaxea Fascicularis separately.Then the abundance of XSBZ03 and XSBZ14 in coral tissues and rear seawater was monitored by real-time PCR methods.The results showed that the abundance of the two strains in living coral tissues declined continuously in a week,and the viability of XSBZ03 in rear seawater was better than XSBZ14.The establishment of the duplex PCR method and real-time PCR method for detection of coral pathogens XSBZ03 and XSBZ14 could be applied in epidemiological investigation of XSBZ03 and XSBZ14,diagnosis of coral white syndrome,and the Specific Pathogen Free(SPF)coral transplantation caused by these two pathogens.These results are of great significance for the diagnosis and control of coral diseases.