基于热对流PCR技术对乳品中布鲁杆菌的检测技术研究  

Detection for Brucella in dairy products based on thermal convection PCR technology

作　　者：杨洪彩[1] 赵国玉[1] 陈欣乐 艾依热提·买买提 木合塔尔·艾山[1] 马玉文豪 张杰[3] YANG Hong-cai;ZHAO Guo-yu;CHEN Xin-le;Aiyireti MAIMAITI;Muhetaer AISHAN;MAYUWen-hao;ZHANG Jie(The Center for Disease Control and Prevention of Xinjiang Uygur Autonomous Region,Urumqi,Xinjiang 830002,China;不详)

机构地区：[1]新疆维吾尔自治区疾病预防控制中心,乌鲁木齐830002 [2]北京华安瑞基生物科技有限公司,北京102488 [3]新疆医科大学公共卫生学院,乌鲁木齐830011

出　　处：《疾病预防控制通报》2021年第3期6-9,共4页Bulletin of Disease Control & Prevention(China)

基　　金：新疆维吾尔自治区自然科学基金资助项目(2018D01C085)。

摘　　要：目的运用热对流PCR技术检测乳品中布鲁杆菌。方法根据布鲁杆菌保守基因序列,设计合成引物和探针,通过优化反应体系,配合商品化核酸提取试剂盒,建立检测乳品中布鲁杆菌的热对流PCR方法,并与常规荧光定量PCR方法的敏感性与特异性比较,以验证该检测方法的可靠性。结果热对流PCR检测敏感性与常规荧光定量PCR检测敏感性基本一致,差异无统计学意义(t=85.500,P>0.05),且该布鲁杆菌热对流PCR方法能检测出的乳品样本中布鲁杆菌含量最低浓度为10 cfu/ml;含有大肠杆菌、溶血性链球菌、克雷伯杆菌、金黄色葡萄球菌、绿脓杆菌的乳液经扩增,均为阴性结果;只有含布鲁杆菌乳液的阳性对照,经热对流PCR仪扩增后结果显示为阳性;热对流PCR方法(22.3%)与常规荧光定量PCR方法(24.1%)阳性检出率差异无统计学意义(χ^(2)=0.250,P>0.05)。结论热对流PCR方法检测乳品中布鲁杆菌可靠性高、扩增反应时间短、方便快捷、仪器成本低廉,适用于基层现场检测病原。Objective To detect Brucella in dairy products by thermal convection PCR.Methods According to the conserved gene sequence of Brucella,primers and probes were designed and synthesized.By optimizing the reaction system and combining with commercial nucleic acid extraction kit,a thermal convection PCR method was established to detect Brucella in dairy products.The sensitivity and specificity of the method were compared with that of conventional fluorescent quantitative PCR method to verify the reliability of the method.Results The sensitivity of thermal convection PCR was essentially consistent with that of conventional fluorescent quantitative PCR,without statistical difference(t=85.500,P>0.05).The minimum concentration of Brucella detected by thermal convection PCR was 10 cfu/ml.After amplification,the dairy samples containing Escherichia coli,Hemolytic streptococcus,Klebsiella,Staphylococcus aureus and Pseudomonas aeruginosa showed negative results.Only the positive control with Brucella displayed positive after amplification by thermal convection PCR.There was no significant difference in the positive rate between thermal convection PCR and conventional fluorescent quantitative PCR(χ^(2)=0.250,P>0.05).Conclusions The thermal convection PCR method is of the advantages of high reliability,short amplification time,convenience and low cost.It is suitable for rapid field detection for Brucella.

关 键 词：布鲁杆菌 乳品 热对流PCR 现场检测 

分 类 号：R378.5[医药卫生—病原生物学] R446.5[医药卫生—基础医学]