啮齿类实验动物唐菖蒲伯克霍尔德菌荧光定量PCR方法的建立及应用

Establishment and Application of Fluorescent Quantitative PCR method for Burkholderia Gladioli in Rodent Laboratory Animal

目的建立用于检测实验动物小鼠中唐菖蒲伯克霍尔德菌的荧光定量PCR方法。方法参考团标公布的序列合成唐菖蒲伯克霍尔德氏菌引物、探针和质粒,建立荧光PCR方法,并对方法的线性、灵敏度、重复性和特异性等性能进行验证。同时应用建立的荧光PCR方法和细菌培养法对110份小鼠送检样品进行比对检测。结果建立的唐菖蒲伯克霍尔德菌荧光PCR方法,相关系数R2可达0.998,线性良好;方法最低可检测至1×10^(2) copies/5μL,灵敏度高;方法检测培养的唐菖蒲伯克霍尔德菌为阳性,检测金黄色葡萄球菌、肺炎克雷伯杆菌、产酸克雷伯杆菌、绿脓杆菌和嗜肺巴斯德杆菌均为阴性,特异性良好。采集110只安乐死小鼠样品,分别进行细菌培养和荧光PCR检测,结果均为阴性,2种方法结果完全符合。结论建立的荧光PCR方法性能良好,可用于实验动物小鼠唐菖蒲伯克霍尔德菌的检测。

Objective To establish a fluorescent quantitative PCR(q-PCR)method for detection of Burkholderia gladioli(B.gladioli)in laboratory mouse.Method the q-PCR method was established with the primers,probe and plasmid of B.gladioli which were synthesized according to the sequence published by Group Standard.And the linearity,sensitivity,repeatability and specificity of the method were verified.110 samples of mice were tested for B.gladioli by the q-PCR method and bacterial culture method.Result The established q-PCR method for B.gladioli showed good linearity with correlation coefficient R20.998,and high sensitivity with a detection limit of 1×10^(2) copies/5μL.The assay could test specifically for B.gladioli with no cross reaction for Staphylococcus aureus,Klebsiella pneumoniae,Klebsiella oxytoca,Pseudomonas aeruginosa and Pasteurella pneumotropica.110 samples were collected from euthanized mice and tested by bacterial culture and the q-PCR method respectively.The result with the two method were all negative,which were completely consistent.Conclusion The established q-PCR method had good performance and could be used for the detection of B.gladioli in laboratory mouse.