目的 利用环介导恒温扩增技术,以空肠弯曲菌的VS1基因设计引物,建立快速检测空肠弯曲菌的方法,以期能应用于急性腹泻和肠炎等疾病的快速检验.方法 ①通过基因比对与引物设计,设计针对空肠弯曲菌的环介导恒温扩增检测(LAMP)方法；②用钙黄绿素、亚甲基蓝、结晶紫、溴化乙锭4种DNA染料对扩增产物进行染色,评价各染料染色效果；③检测该LAMP方法对空肠弯曲菌及其他干扰菌的扩增情况,评价其特异性；④将该LAMP方法与培养法比较,进行统计学分析.结果 ①设计出针对空肠弯曲菌的LAMP检测方法；②钙黄绿素与扩增产物结合后颜色变化明显,是该LAMP方法中较好的DNA染料物质；③本LAMP检测方法只针对空肠弯曲菌有扩增,对其他干扰菌不扩增,显示出良好的特异性；④试验共检测了60株临床和标准菌株,对于菌株的培养物,LAMP检测结果与培养鉴定法比较差异无统计学意义(x2 =0.3333,P＞0.05).结论 本试验设计出了针对空肠弯曲菌LAMP检测方法,该LAMP法较好的DNA染料物质是钙黄绿素,该方法具有良好的特异性,与培养鉴定法比较具有较高的阳性、阴性及总体符合率,能够用于空肠弯曲菌的快速检验.%Objective Basing on the loop-mediated isothermal amplification (LAMP) technology,to design a rapid detection method of Campylobacter jejuni,so as to be applied in clinical inspection of C.jejuni in acute diarrhea and gastroenteritis.Methods By gene alignment and primer design,a set of loop mediated isothermal amplification (LAMP) method for detection of C.jejuni was designed.Four kinds of DNA dye,calcein,methylene blue,crystal violet and EB,were used to stain amplification products,then the dyeing effect could be evaluated.The LAMP method of detecting C.jejuni and other interference bacteria was tested on their specific evaluation.LAMP was Compared with the culture method of identification,and statistics analysis was performed.Results We had designed a LAMP method for detection of C.jejuni.The color changd obviously after combining with amplification products.Calcein was a better DNA dye substancethe of LAMP method.The LAMP method could only amplify C.jejuni,not amplify the other interference bacteria,showing a good specificity.Tests were detected in 60 clinical strains and standard strains.The results of LAMP showed no statistical significance compared with the culture method of identification (x2 =0.3333,P ＞ 0.05).Conclusion The test designed a LAMP detection method of C.jejuni.Better DNA dye of the method is the calcein,and this method has a good specificity.Compared with the culture identification,this method has a higher positive,negative and overall coincidence rate,and can be used as a rapid test for C.jejuni.
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