牛乳中蜡样芽孢杆菌荧光定量PCR检测方法的建立及验证

Establishment and verification of fluorescence quantitative PCR assay for Bacillus cereus in milk

目的建立一种快速检测牛乳中蜡样芽孢杆菌的荧光定量PCR方法,并进行验证。方法根据GenBank中登录的蜡样芽孢杆菌gyrB基因序列及荧光定量PCR要求,设计合适的特异性引物,提取菌体DNA,对目的基因进行扩增及克隆;以重组质粒为模板进行荧光定量PCR扩增,绘制标准曲线;以蜡样芽孢杆菌DNA为模板进行荧光定量PCR扩增,确定其扩增条件。对建立的方法进行特异性及灵敏性验证。结果建立的方法检测其他3种牛乳中常见致病菌(大肠埃希菌、志贺菌、沙门菌)未见特异性扩增,即无交叉反应;最低检出限为1×10^(-7) ng/μL。以掺入蜡样芽孢杆菌的牛乳为样品,可检测到牛乳中10. 4 CFU/mL的蜡样芽孢杆菌,优于国标方法(GB 4789. 14-2014)检测含有相同菌落数的蜡样芽孢杆菌(仅能检测到1. 04×10~2 CFU/mL)。结论建立的方法特异性强,灵敏性高,适用于快速、准确检测牛乳中的蜡样芽孢杆菌,为实验室快速检测致病菌提供了参考。

Objective To establish and verify a fluorescence quantitative PCR assay for rapid detection of Bacillus cereus in milk. Methods Specific primers were designed according to the gyr B gene sequence of B. cereus in GenBank and the requirement of fluorescence quantitative PCR,based on which the bacterial DNA was extracted,and the target gene was cloned. Fluorescence quantitative PCR assay was performed using recombinant plasmid as a template,and a standard curve was plotted. Meanwhile,the fluorescence quantitative PCR assay was performed using DNA template of B. cereus,and the condition for amplification was determined. The established method was verified for specificity and sensitivity.Results The established fluorescence quantitative PCR assay showed no cross reactions with three common pathogenic bacteria in B. cereus,E. coli,Shigella shigae and salmonella. The minimum detection limit of the developed method was1 × 10-7 ng/L. The B. cereus at a concentration of 10. 4 CFU/mL in milk was detected by the method. However,by the method in GB 4789. 14-2014,only the B. cereus at a concentration of 1. 04 × 102 CFU/mL was detected.Conclusion The developed fluorescence quantitative PCR assay showed high specificity and sensitivity,which was suitable for rapid and accurate detection of B. cereus in milk. It provided a reference for rapid detection of pathogenic bacteria in laboratory.