食品中幽门螺杆菌荧光PCR快速检测方法的建立及评价

Establishment and evaluation of real time PCR for detection of Helicobacter pylori in food

目的:建立一种快速、特异、灵敏的幽门螺杆菌检测方法,并对方法进行评价,为快速、准确、有效检测幽门螺杆菌方法的建立提供依据。方法:以尿素酶基因序列为靶位点,用Primer Express 3.0软件设计引物及探针,经Blast软件对相似序列搜索后,筛选出一套最优与常见致病菌无交叉反应的引物;分别用食品中常见致病菌大肠杆菌、金黄色葡萄球菌、单增李斯特菌、沙门菌和空肠弯曲菌DNA进行特异性实验;将计数过的幽门螺杆菌菌悬液与牛奶样品混合液,梯度稀释后分别提取DNA进行荧光PCR扩增,确定检测方法的灵敏度;对人工污染幽门螺杆菌的生奶及生肉样品进行检测验证方法的可行性。结果:利用尿素酶基因设计的引物及探针仅对幽门螺杆菌有扩增曲线,而其他对照以及阴性对照均未有明显扩增曲线;能够对生奶及生肉中污染的幽门螺杆菌进行有效扩增;设计的引物对幽门螺杆菌的检测敏感性可达到3CFU.mL-1,检测可以在4d内完成。结论:荧光PCR方法特异性强,灵敏度高,可以快速、准确检测食品中污染的幽门螺杆菌。

Objective To establish a rapid,specific and sensitive method for the detection of Helicobacter pylori,and provide an effective detecting evidence for Helicobacter pylori.Methods A pair of primers and probe corresponding to the urease gene for real time PCR were designed according to Primer Express 3.0 software,similar sequences were searched by Blast method,and the excellent primers and probe were selected.DNA from common bacterial pathogens such as Escherichia coli,Staphylococcus,Listeria monocytogenes,Campylobacter and Salmonella in food was used for specific test;the counted Helicobacter pylori in bacterium suspension and the bacterium and milk mixture were serially diluted,the DNA was extracted for real time PCR amplification,the sensitivity of method was tested.The practicability of the method was demonstrated through the detection of the artificial contaminative samples by real time PCR.Results The primers and probe according to urease gene could only amplify Helicobacter pylori DNA,but not other reference bacterium DNA.Helicobacter pylori from the artificial contaminative samples could be amplified by the method.It can detect 3 CFU·mL-1 of Helicobacter pylori.Its sensitivity was sufficient to detect 3 CFU·mL-1 of Helicobacter pylori.The method was enough rapid to finish detection in 4 d.Conclusion Real time PCR can rapidly and accurately detect the Helicobacter pylori contamination in food with high specificity and sensitivity.