创伤弧菌物种特异性检测靶标基因的发掘及评价

实验根据GenBank公布的细菌全基因组数据,采用比较基因组学分析创伤弧菌与其它细菌基因组间的差异,筛选出34个潜在的创伤弧菌物种特异性检测靶标基因,其中VV1_2692、VV2_0075和VV2_0939这3个基因已有功能注释.常规PCR扩增结果显示这3个基因均具有良好的特异性和灵敏度.进而以这3个基因和传统靶基因vvhA作为检测靶标,采用常规PCR技术检测来自杭州市农贸市场的137份海产品中创伤弧菌,发现VV2_0075、VV2_0939和vvhA基因的检测结果与传统生化鉴定方法一致,表明新发掘的VV2_0075和VV2_0939基因在对海产品中创伤弧菌检测时具有很好的稳定性和很强的抗干扰能力.在137份海产品中创伤弧菌的检出率为28.5％,其中牡蛎的检出率最高(68％),表明杭州市农贸市场售卖的海产品,尤其是牡蛎中的创伤弧菌污染现状严重,应加强对杭州市海产品中创伤弧菌污染状况的监测.

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 andVV2_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.