Gene Detection And Analysis Of Vibrio Parahaemolyticus And Vibrio Vulnificus In Aquatic Seashells

Techniques of quick detection and isolation for pathogenic microorganisms have been concentrated on recent years. Polymerase chain reaction (PCR) is an effective detection technology, which can be used to quickly detect pathogenic microorganisms. Probe in situ hybridization is a valid approach as well. In this paper, PCR and tlh and tdh probe were used to isolate Vibrio parahaemolyticus and Vibrio vulnificus strains in seashells.Six Vibrio parahaemolyticus strains have been isolated from seashells by using tlh and tdh probe. Two results of probe tlh hybridization were positive and all results of probe tdh hybridization were negative in about one month. It was demonstrated that all the six isolated tlh positive strains were tlh positive indeed by the PCR assay. The six Vibrio parahaemolyticus strains obtained from the Disease Control Center of Qingdao were examined by PCR assay and probe hybridization. All of the six strains were tlh positive and five of them were tdh positive, and non-new O3 :K6 Vibrio parahaemolyticus strain was found. The Vibrio vulnificus strains have been detected for three times in seashells(mainly in clams) in 4 months by PCR assay and two Vibrio vulnificus strains have been isolated with WAP probe labled with alkaline phosphatase(AP).The gene type of the six positive tdh strains were analyzed by random amplified polymorphic DNA (RAPD) technology. The results showed that although the six strains were all tdh positive, they were different in genome. The strain of VPS remarkably differed from the other five Vibrio parahaemoliticus isolated from Qingdao. The maximum polymorphic strips of nine were got in primer oligol?.Now oligo nucleotide can't be labeled with alkaline phosphatase in China. So probe labeled with biotin was used instead of AP, and similar cross bred effect was obtained by numerous investigations.The tdh gene of VPS isolated from Qingdao was cloned and sequence analysis showed a homology of 100% between the VP3's tdh gene and the reference strain WPl's tdh gene available in Genbank. The homology between VPS's tdh gene and VPS's tdh gene was 99.5%. The vvhA genes of two isolated Vibrio vulnificus were cloned and analyzed with BLASTn. The data showed that the whA gene sequences of these two strains have the highest homology with L-180. The homology between VV1 and L-180 was 99.52%, and the homology between VV2 and L-180 was 99.72%.