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Position: Home > Articles > Development and Application of Real-time PCR Assay for Quantitative Detection of Campylobacter spp China Animal Health Inspection 2014 (12) 70-74

弯曲菌荧光定量PCR检测方法的建立及初步应用

作  者:
朱佳琪;张小燕;黄金林;焦新安
单  位:
江苏省动物重要疫病与人兽共患病防控协同创新中心;扬州大学;江苏省人兽共患病学重点实验室
关键词:
弯曲菌;16S r RNA基因;荧光定量PCR
摘  要:
根据弯曲菌16S r RNA基因的靶序列设计特异性引物和探针,对反应条件和试剂浓度进行优化,建立了快速检测鸡肉中弯曲菌的荧光定量PCR方法,并对其特异性、敏感性和重复性进行评价。该方法除了对弯曲菌有扩增曲线外,对其他常见食源性病原菌均未有扩增曲线,具有较好的特异性;弯曲菌的最低检测限为10 CFU/m L;重复性实验获得标准曲线相关系数为R2=0.999,批内可重复性变异系数在0.12%-2.1%之间,批间可重复性变异系数为1.7%。应用该方法对68份鸡肉样品检测弯曲菌阳性率为95.6%(65/68),传统分离方法阳性率为89.5%(60/68),两种方法的阳性符合率为89.7%。本研究建立的荧光定量PCR方法灵敏度高、特异性好、操作简单,大大缩短检测周期,可作为检测鸡肉样品中弯曲菌的手段,为鸡肉样品弯曲菌的流行病学调查研究提供新的工具。
译  名:
Development and Application of Real-time PCR Assay for Quantitative Detection of Campylobacter spp
作  者:
Zhu Jiaqi;Zhang Xiaoyan;Huang Jinlin;Jiao Xinan;Jiangsu Key Lab of Zoonosis/Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou University;
关键词:
Campylobacter spp.;;16S r RNA gene;;real-time PCR
摘  要:
To establish a rapid assay for quantitative detection of Campylobacter spp.,a pair of primers and fl uorescent probe were designed according to 16 S r RNA sequence of Campylobacter spp.,and then a real-time PCR method was successfully established to detect Campylobacter spp. Results demonstrated that the method was specifi c for amplifi cation of Campylobacter spp.,but no amplifi cation for other related bacteria. The detection limit of the assay was 10CFU/m L and the correlation coeffi cient of standard curve(R2)was 0.999. The detection of 68 chicken meat samples showed that the positive rate was 95.6%,while the positive rate was 89.5% by traditional culture method. The method was simple,rapid,specifi c and sensitive for rapid detection of Campylobacter spp. and a new tool for epidemiological investigation of Campylobacter spp. in chicken meats.

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