食品中空肠弯曲菌荧光重组酶聚合酶扩增检测方法的建立

Development of a Detection Method for Food-borne Campylobacter jejuni Based on Fluorescence Probe-based Recombinase Polymerase Amplification

摘要:

本研究为了实现空肠弯曲菌的快速和便捷检测，建立了一种基于荧光重组酶聚合酶扩增技术（exo-RPA）快速检测空肠弯曲菌的方法。通过对空肠弯曲菌和对照菌株的exo-RPA检测来判断该方法的特异性。用梯度稀释的空肠弯曲菌作为模板进行检测来分析exo-RPA方法的灵敏度。通过对模拟污染样品检测来分析exo-RPA的应用效果。分别以exo-RPA和荧光PCR检测实际食品样品来分析二者的检测效果。空肠弯曲菌exo-RPA方法可特异检出空肠弯曲菌，检测灵敏度达到6.0×102 CFU/mL。在模拟污染试验中，含2.5×101 CFU/mL空肠弯曲菌的增菌液在培养24 h后可以被exo-RPA检测出阳性信号。exo-RPA和荧光PCR对于40份样品的检测结果相同。本研究建立的空肠弯曲菌exo-RPA具有特异、灵敏和抗干扰性强的特点，方法操作简便快速，具有较好的应用前景。

Abstract:

In order to achieve rapid and convenient detection of Campylobacter jejuni, a rapid detection method of Campylobacter jejuni based on fluorescence probe-based recombinase polymerase amplification (exo-RPA) was developed. The specificity of the method was determined by exo-PA detection of Campylobacter jejuni and control strains. The gradient-diluted Campylobacter jejuni was used as a template to analyze the sensitivity of the exo-RPA method. The application effect of the exo-RPA method was analyzed through detecting the simulated contaminated samples.The exo-RPA and fluorescence PCR were used to test real food samples respectively, in order to analyze their detection effects. The Campylobacter jejuni exo-RPA method can specifically detect Campylobacter jejuni with a detection sensitivity of 6.0×102 CFU/mL. In the simulated contamination tests, the enrichment broth medium containing 2.5×101 CFU/mL Campylobacter jejuni was tested positive after 24 hours of culture by the exo-RPA assay. The exo-RPA and fluorescence PCR methods led to the same detection results for the 40 samples. The Campylobacter jejuni exo-RPA assay developed in this study has high specificity, sensitivity and anti-interference ability. The method is simple, convenient and rapid, with a good application prospect.