纳米金PCR技术检测副溶血弧菌方法的建立与初步应用

Establishment and primary application of nanoparticle-based assisted PCR for Detection of Vibrio parahaemolytious

纳米金PCR是在普通PCR基础上发展起来的新技术,它能够明显提高普通PCR的灵敏度和特异性。根据副溶血弧菌(VP)的toxR基因序列,应用primer6.0设计一对特异性引物,建立了纳米金PCR方法,并对该方法的最佳反应条件、循环数、特异性和灵敏度进行测定。采用2.0%琼脂糖电泳进行检测,结果表明能扩增得到与试验设计相符的208bp(VP)的特异性条带;与溶藻弧菌、霍乱弧菌、麦氏弧菌、沙门氏菌、枸橼酸杆菌没有交叉反应;纯培养细菌检测灵敏度为3cfu/反应体系。与普通PCR法进行比较,该方法检测灵敏度比普通PCR高10倍。经临床检测,从150份带鱼、鱿鱼和鱼粉中检出5份阳性样品,与常规细菌分离鉴定方法符合率100%。该试验方法的建立对于加强进出口水产品中VP的检验检疫具有十分重要的意义。

Nanogold-assisted PCR（NP-PCR） is a new technology developed on the basis of ordinary PCR, it can obviously improve the sensitivity and specificity of ordinary PCR.Nanogold-assisted PCR was established and optimized to detect Vibrio parahaemolytictts （VP）.One set of specific primers were designed according to the toxR sequence of VP in the GenBank by primer 6.0.The reaction conditions, cycle numbers, sensitivity and specialty of the method was optimized and assessed. It was showed that all samples which contained VP could be amplified by the nanogold-assisted PCR using this set primers. One specific band of VP 208 bp was detected using 2.0% agarose gel electrophoresis in this nanogold -assisted PCR and accorded with designed result, other bacterial （such as Vibrio alginolyticus, V.cholera, Vibrio metschnikovii,salmonella, Citro Bacter）controls gave negative results. The nanogold-assisted PCR was able to detect as little as 3 cfu/reaction system for VP from pure culture samples and more sensitive than polymerase chain reaction. The experiment proved that NP-PCR is 10 times than ordinary PCR in sensitivity.The method provides a fast and reliable way of identifying ~P.5 from 150 samples were positive by two methods and their coincidence rate was 100% .The method could prove very useful for identification and inspection of VP from fishery product in import and export.