海产品中溶藻弧菌的分离及多种方法鉴定效果的比较

Comparison of the identification methods of VITEK,RT-PCR and rpoB sequencing on Vibrio alginolyticus isolated from seafood

目的了解北京市市售海产品中溶藻弧菌的污染情况,并对使用不同方法鉴定溶藻弧菌的效果进行比较。方法样品经碱性蛋白胨水增菌后,分别于硫代硫酸盐柠檬酸盐胆盐蔗糖(TCBS)琼脂培养基和科玛嘉弧菌显色培养基上划线培养,实时荧光聚合酶链式反应(PCR)法鉴定可疑菌落。以rpoB基因测序方法为参考,比较了实时荧光PCR和VITEK两种方法的鉴定效果。结果对北京市水产品市场随机采集的116份海产品进行了检测,实时荧光PCR法鉴定出溶藻弧菌阳性样品95份,检出率高达82%。使用科玛嘉弧菌显色培养基的检出率高于TCBS培养基,分别为82%(95/116)和72%(83/116)。经rpoB基因核苷酸序列测定确定95株疑似菌株为溶藻弧菌。采用VITEK 2 COMPACT GN鉴定卡对这95株菌株进行鉴定,31株鉴定为溶藻弧菌,其余未能得到准确的鉴定结果。结论北京市市售海产品中溶藻弧菌检出率高。科玛嘉弧菌显色培养基比TCBS琼脂培养基更适于溶藻弧菌的分离,实时荧光PCR法的鉴定效果优于VITEK法。

Objective To investigate the contamination of Vibrio alginolyticus in seafood sold in Beijing and compare the different identification method. Methods After enrichment with alkaline peptone water（ APW）,a loopful from APW culture was streaked on thiosulfate citrate bile salts sucrose agar（ TCBS） and CHROMagar Vibrio simultaneously. Typical colonies of Vibrio alginolyticus were identified with reverse transcription polymerase chain reaction（ RT-PCR）. The isolated 95 Vibrio alginolyticus strains were also identified with VITEK 2 COMPACT GN card and rpo B gene sequencing.Results Vibrio alginolyticus strains were isolated from 116 seafood which were collected randomly from markets in Beijing.The result indicated that the detection of Vibrio alginolyticus in seafood was high in Beijing,about 82 %（ 95/116）.CHROMagar Vibrio media got higher detection rate than TCBS media,which were 82 %（ 95/116） and 72 %（ 83/116）respectively. RT-PCR result were in good accordance with that of rpo B gene sequencing,while VITEK 2 COMPACT GN card could only identify 32. 6 %（ 31/95） of the isolated strains as Vibrio alginolyticus. Conclusion The contamination of Vibrio alginolyticus in seafood was serious in Beijing. CHROMagar Vibrio media was better than TCBS media for isolation.RT-PCR could give a more precise result than VITEK.