Identification and evaluation of Vibrio vulnificus-specific target genes

Vibrio vulnificus is a marine seafood-borne pathogen that will cause death in susceptible individuals after consumption of raw or uncooked contaminated seafood around the world.So,early detection and identification of V.vulnificus strains in food and clinical samples is essential for diagnosis and reducing the incidence of food-borne disease.PCR assay has been one of the most important and extensive method to detect pathogenic bacteria.Previous study depended on vvhA as the target gene to detect this bacterium.In this study,we constructed a local BLAST database and identified 34 candidates as V.vulnificus-specific target genes distributed in 3 isolates(CMCP6,MO6-24/O,and YJ016)with published completed genome.Among these candidate-specific targets,VV1_2692,VV2_0075,and VV2_0939 genes are known for their functions,while the rests encode hypothetical protein of unknown function.To evaluate the specificity of above 3 genes,PCR amplification of genomic DNA from a V.vulnificus strain resulted in a product with predicted length,whereas no products were detected from 14 non-V.vulnificus bacterial strains.The minimum detectable limits of VV1_2692,VV2_0075,and VV2_0939 genes were 103,101,and 102 cfu/mL,respectively.A total of 137 seafood samples(e.g.,fish,prawn,crab,shell)from Hangzhou city were detected by both PCR assay and biochemical method.Among them,39 V.vulnificus isolates were detected using biochemical method,while 38,39,39,and 39 V.vulnificus isolates were detected using PCR assay of VV1_2692,VV2_0075,VV2_0939,and vvhA genes,respectively.The negative result of VV1_2692 probably resulted from its relative high detectable limit,and both VV2_0075 and VV2_0939 genes might be suitable species-specific targets to detect V.vulnificus in seafood.Moreover,28.5% of 137 seafood samples contained V.vulnificus,and oyster had the highest ratio(68%),suggesting status of V.vulnificus pollution was extremely serious in coastal seafood of Hangzhou city,especially oyster.