LAMP法检测牛奶中金黄色葡萄球菌、鼠伤寒沙门菌和大肠杆菌O517:H

Detection of Staphylococcus Aureus,Salmonella Typhimurium and Escherichia Coli O517:H7 in Milk by LAMP

目的建立一种能快速准确检测牛奶中金黄色葡萄球菌(Staphylococcus aureus)、鼠伤寒沙门菌(Salmonella typhimurium)和大肠杆菌O517:H7(Escherichia coli O517:H7)的环介导恒温扩增方法(loop mediated isothermal amplification,LAMP)。方法以牛奶为研究对象,分别利用金黄色葡萄球菌nuc,鼠伤寒沙门菌invA和大肠杆菌O517:H7的rfbE保守基因设计LAMP特异引物,通过优化反应温度、时间等条件,建立检测牛奶中金黄色葡萄球菌、鼠伤寒沙门菌和大肠杆菌O517:H7的LAMP反应体系。并验证了本方法的特异性和灵敏度。结果 LAMP法对牛奶中金黄色葡萄球菌、鼠伤寒沙门菌和大肠杆菌O517:H7的灵敏度均为1.0×10~2拷贝/μL,比常规PCR灵敏度提高了10倍。金黄色葡萄球菌和大肠杆菌O517:H7的LAMP反应条件为60℃、30 min,鼠伤寒沙门菌为60℃、60 min。利用SYBR Green I染料在紫外光下可直接观察检测结果,金黄色葡萄球菌、鼠伤寒沙门菌和大肠杆菌O517:H7阳性样品均呈绿色;进一步利用荧光定量PCR仪可检测到对应的扩增曲线。通过牛奶样品模拟掺杂实验,验证了方法的实用性和可靠性。结论本研究建立的检测牛奶中金黄色葡萄球菌、鼠伤寒沙门菌和大肠杆菌O517:H7的LAMP方法具有特异性强、灵敏度高、操作简便等特点,可用于牛奶中相关致病菌的检测,对预防和控制3种致病菌的传染有重要意义。

Objective Todevelopa loop-mediated isothermal amplification（LAMP） method for rapid diagnosis of Staphylococcus aureus, Salmonellatyphimurium, and Escherichia coliO517：H7in milk.Methods Using milk asresearch object, specific primers were designed based on conserved region of nuc of Staphylococcus aureus, invAof SalmonellatyphimuriumandrfbE of Escherichia coliO517：H7. By optimization of reaction time and temperature,the LAMP method was established, and the reaction specificity and sensitivity were verified.Results The detectionsensitivity of the LAMP method for Staphylococcus aureus, Salmonellatyphimuriumand Escherichia coliO517：H7was1.0×102copys/μL, which was as 10 times high as general PCR. The rapid detection for Staphylococcus aureusand Escherichia coliO517：H7could be completed in 30 min at 60℃, and for Salmonellatyphimurium could becompleted in 60 min at 60℃.After addition of SYBR Green I, three kinds of pathogenic bacteriacould be detectedunder ultraviolet light. The corresponding amplification curvesof three pathogenic bacteria could also be detected byfluorogenic real-time PCR. The practicability and reliability of LAMP method for detection of Staphylococcus aureus,Salmonellatyphimurium and Escherichia coliO517：H7in milk were verified by doping test. Conclusion The practiceproves that the LAMP method is of high specificity and sensitivity, and is easy to operate, thus can be widely used forthe detection of pathogens in milk.This method has important significance for the prevention and control of infectionof three kinds of pathogenic bacteria.