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[目的]建立阪崎肠杆菌环介导等温扩增(loop-mediated isothermal amplification,LAMP)快速检测技术,并与PCR检测方法进行比较,为阪岐肠杆菌感染的快速诊断提供实验依据。[方法]根据阪崎肠杆菌外膜蛋白OmpA基因,设计特异性引物,建立LAMP和PCR检测技术体系,并对2种方法检测的灵敏度、特异性和实际样品的检测结果进行比较。[结果]建立了优化的LAMP和PCR检测体系,灵敏度检测结果显示,LAMP检测限为101cfu/ml,PCR检测限为102cfu/ml,LAMP检测灵敏度高于PCR;对36株近源菌进行特异性检测,结果显示,LAMP仅8株阪崎肠杆菌得到阳性结果,PCR产物则出现非特异性条带,LAMP特异性比PCR高。应用于46份奶粉样品的检测,LAMP和PCR均有清晰、特异的预期条带和结果产生,并与常规检测结果一致。[结论]LAMP检测技术作为一种快速检测阪崎肠杆菌的方法具有简便、灵敏快速,与PCR方法比较,特异性强、操作简便、检测成本低,耗时短,更有望发展成为快速检测阪岐肠杆菌的有效手段。
[Objective] To establish rapid detection loop of loop-mediated isothermal amplification (LAMP) of Enterobacter sakazakii and compare with PCR detection method to provide experimental basis for rapid diagnosis of Enterobacter sakazakii infection. [Method] According to the OmpA gene of Enterobacter sakazakii outer membrane protein, specific primers were designed and LAMP and PCR detection systems were established. The sensitivity, specificity and actual results of two methods were compared. [Result] The optimized detection system of LAMP and PCR was established. The results of sensitivity test showed that the detection limit of LAMP was 101 cfu / ml, the detection limit of PCR was 102 cfu / ml, and the detection sensitivity of LAMP was higher than that of PCR. The specificity of 36 strains The results showed that there were only 8 positive strains of Enterobacter sakazakii in LAMP, non-specific PCR products appeared, and LAMP specificity was higher than that of PCR. For 46 samples of milk powder, both LAMP and PCR have clear, specific expected bands and results, consistent with routine test results. [Conclusion] As a rapid and sensitive method for rapid detection of Enterobacter sakazakii, LAMP detection technique is more sensitive and rapid than the PCR method. It has the advantages of high specificity, simple operation, low detection cost and short time-consuming. Effective means of Enterobacter.