十三种致病菌多重PCR技术检测

Detection of Thirteen-kinds of Pathogenic Bacteria by Multiplex-PCR

目的探讨致病菌多重PCR检测方法。方法利用13种在突发公共事件中可能出现在饮用水水源水体中的致病细菌的特异性序列为靶序列,设计了14对特异性引物并分成3组进行多重PCR反应,建立并优化了多重PCR检测体系。结果通过实验确定适宜的退火温度为66-68℃;3组检测引物的添加比例分别为：第1组引物添加比例为Shi∶Sa∶O157∶Ec∶Sen=1∶1∶1∶2∶3-1∶1∶1∶3∶4,第2组引物添加比例为Lm∶Lp∶Kp∶Hp=1∶1∶4∶1,而第3组引物添加比例为MT∶VC∶BA∶YP∶16S=8∶1∶8∶4∶4;混合引物的用量为0.6~0.8μL。多重PCR的基因组DNA检测灵敏度可达到2×10-2ng/μL;配合浓缩集菌设备,使用研究中建立的多重PCR体系,肠炎沙门菌培养液的检测下限可以达到103cfu/mL。结论该多重PCR检测方法操作简单,速度快,灵敏度高,稳定性和重复性好,具有较广阔的应用前景和较大的经济与社会效益。

Objective To study a rapid detection method based on multiplex PCR technology for 13 kinds of pathogens existed possibly in water.Methods Fourteen pairs of specific primers were designed and three multiplex PCR groups were set up.The annealing temperature,the primers ratios of each group and the mixed primers volume were adjusted to optimize the multiplex PCR system.Results Using optimally designed primers and mPCR system as follows,the genomic DNA could be amplified specifically and sensitively：the annealing temperature was 66-68 ℃,the appropriate ratios of the first group was Shi∶Sa∶O157∶Ec∶Sen=1∶1∶1∶2∶3-1∶1∶1∶3∶4,the second group was Lm∶Lp∶Kp∶Hp=1∶1∶4∶1,and the third group was MT∶VC∶BA∶YP∶16S=8∶1∶8∶4∶4,the appropriate mixed primers amount was 0.6-0.8 μL.The sensitivity of mPCR system was 2×10-2 ng/μL for bacterial genomic DNA,103 cfu/mL for Salmonella cultures.Conclusion The multiplex PCR detection method established in present study is simpler in operation,shorter in detection time,better in sensitivity,stability and repeatability than the traditional method.