| The 16S-23S rDNA IGSs of V.splendidus were amplified and cloned with pMD19-T Vector,then sequ- enced. Based on the sequences analysis results,species-specific PCR primers were successfully designed a- nd the PCR methods were developed. A 177 base pair (bp) region from the purified Vibrio splendidus DNA and infected tissue DNA of Apostichopus japonicus was amplified successfully. the sensitivity and specifi- city of PCR method were studied. The sensitivity test illustrated that the detection limit of PCR was 0.5pg V. splendidus DNA,and it was only positive for V. splendidus DNA, while it was all negative for DNAs of V. fluvialis, V. anguillarum, V. alginolyticus, V. parahaemolyticus, V. harveyi, V. vulnificus and Aeromonas hydrophila.The PCR amplification Vibrio splendidus described above was labeled with digoxigenin( DIG) as a p- robe used for dot blot hybridization and in situ hybridization test. The sensitivity and specificity was tested by dot blot bridization.And the sensitivity was 6.25 pg to Vibrio splendidus DNA. The results showed high sensitivity and strong specificity of the DNA probe. The method of PCR and dot-blot hybridization was c- onsidered to be accurate in the detection of V. splendidus as the positive ratio was as high as 100% for the tissue samples of clinically healthy Apostichopus japonicus and the samples from Qingdao, Weihai and Y- antai areas.Using the probe described above to detect Vibrio splendidus by in situ hybridization,the results show- ed that positive siginals could be detected in the connective tissue,muscle tissue,epithelium of intestines and water-vaacular.The three developed methods were evaluated to detecte the pathogenic bacteria from the infection test. The under-test samples were detected directly without extracting DNA carefully. Results showed approximately96.9%,90.6% and 87.5%of the samples were found to be positive by PCR, dot-blot hybridization and in situ hybridization respectively.
|
PDF Full Text Request