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目的通过对常规聚合酶链反应(PCR)与多重PCR在检测6种食物中毒致病菌中的比较研究,建立一个快速、准确、灵敏、有效的针对该6种致病菌进行PCR检测的平台。方法利用6种导致患者腹泻的食物中毒致病菌的特异性序列为靶序列,设计了6对特异性引物进行PCR反应,优化并建立了PCR检测体系。结果通过实验确定常规PCR适宜的退火温度为58℃,而多重PCR适宜的退火温度为62℃;常规PCR的基因组DNA检测灵敏度可达到2×10-7ng/μl,而多重PCR的基因组DNA检测灵敏度只有2×10-6ng/μl;常规PCR比多重PCR更容易从食物中毒样品中检测出致病菌。结论 6种导致患者腹泻的食物中毒微生物完全可以在同批次中完成检测,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 establish a rapid, accurate, sensitive and effective platform for PCR detection of six kinds of pathogenic bacteria by comparing PCR and multiplex PCR in the detection of six food poisoning pathogenic bacteria . Methods Six specific pathogen-causing pathogens causing diarrhea in patients were used as target sequences. Six pairs of specific primers were designed for PCR reaction, and the PCR detection system was optimized. The results showed that the suitable annealing temperature of conventional PCR was 58 ℃, while the optimum annealing temperature of multiplex PCR was 62 ℃. The detection sensitivity of genomic DNA by conventional PCR was 2 × 10-7ng / μl, while the sensitivity of multiplex PCR Only 2 × 10-6ng / μl; conventional PCR is more likely to detect pathogens from food poisoning samples than multiplex PCR. Conclusion Six kinds of food poisoning microorganisms that cause diarrhea in patients can be completely detected in the same batch. PCR detection method is simple, rapid, sensitive, stable and reproducible, with broad application prospects and large economy And social benefits.