高通量荧光PCR组合方法鉴定16种金黄色葡萄球菌肠毒素的方法建立及评估

Establishment and evaluation of high-throughput fluorescent PCR assay for identifying 16 kinds of Staphylococcus aureus enterotoxins

目的建立一种高效、特异的荧光探针熔解曲线方法,用于鉴定16种金黄色葡萄球菌肠毒素。方法根据金黄色葡萄球菌肠毒素SEA～SEQ特异基因序列设计不同杂交连接探针,建立金黄色葡萄球菌肠毒素检测体系,评估其最低检出限、特异度、可重复性等指标。与普通PCR方法比较,采用荧光探针熔解曲线方法对本实验室的158株食物中毒金黄色葡萄球菌进行检测,评估新方法的灵敏度和特异度。结果荧光探针熔解曲线方法最低检出限为0.80～2.15 ng/μl,特异度为100.0%,无交叉荧光信号产生,变异系数<1%。与普通PCR方法比较,新方法的灵敏度为95.4%,特异度为100.0%,Kappa值为0.88。结论荧光探针熔解曲线技术检测金黄色葡萄球菌肠毒素的方法可覆盖金黄色葡萄球菌16种肠毒素,具有快速、准确、特异性高的特点。

Objective To establish a high-throughput fluorescent PCR assay（fluorescent probe melting curve method） for the identification of 16 kinds of Staphylococcus aureus enterotoxins. Methods Primers were designed according to S. aureus enterotoxin SEA-SEQ gene sequences with the different annealing temperature,and the PCR assay was established, then the limit detection, sensitivity, specificity and repeatability of the assay were evaluated by detecting 158 strains of food borne S. aureus in comparison with conventional PCR. Results The limit detection of the established PCR assay ranged from0.80 ng/μl to 2.15 ng/μl. The specificity was 100.0%, no cross fluorescence signal was produced, and the coefficient of variation was lower than 1%. Compared with conventional PCR, the sensitivity and specificity of new PCR assay were 95.4%and 100.0%, the Kappa was 0.88. Conclusion The established PCR assay is rapid, accurate and specific and can be used to identify all the 16 kinds of enterotoxins of S. aureus.