布鲁氏菌、弓形虫及巴尔通体多重TaqMan荧光PCR检测方法的建立及应用
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S855.9

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Establishment and Application of Multiplex TaqMan Real-time PCR for Brucella,Toxoplasma gondii and Bartonella
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    摘要:

    为建立一种应用于宠物布鲁氏菌、弓形虫及巴尔通体的高通量、高灵敏、高特异的检测方法,根据各自保守序列合成了特异性引物及TaqMan探针,通过优化反应体系,建立了多重TaqMan荧光PCR检测方法,并利用该方法对2021年厦门市抽检的304份犬猫全血样品进行了检测。结果显示:该方法特异性好,可特异性扩增布鲁氏菌、弓形虫和巴尔通体阳性核酸,其他对照菌株核酸均无扩增信号;抗干扰性强,检测混合模板时,不同成分间无交叉干扰反应;灵敏度高,最低检出限均为3×100 copies/μL;重复性好,批内及批间重复试验变异系数均小于2%。应用该方法在4 h内即可完成304份临床样品的快速检测,此次临床抽检样品中未检出布鲁氏菌、弓形虫及巴尔通体阳性核酸。该方法大大提高了检测效率,为多种动物布鲁氏菌、弓形虫及巴尔通体感染的早期诊断及筛查提供了技术支持。

    Abstract:

    In order to establish a method with high-throughput,sensitivity and specificity detecting Brucella,Toxoplasma gondii and Bartonella in pets,specific primers and TaqMan probes were synthesized according to their respective conservative sequences. By optimizing the reaction system,a triple fluorescent PCR assay was established to detect 304 whole blood samples collected from dogs and cats in Xiamen in 2021. The results showed that the method,with good specificity,could specifically amplify the positive nucleic acids of Brucella,Bartonella and Toxoplasma gondii,but failed for other control strains;with strong anti-interference performance,could make different components fail to react with each other when a mixed template was tested;with strong sensitivity,could reach the lowest detection limit of 3×100 copies/μL;with good repeatability,the variable coefficient of intra and inter batch repeated tests were less than 2%. The method could be used to rapidly detect 304 clinical samples within 4 hours,but no positive nucleic acids of Brucella,Toxoplasma gondii and Bartonella were detected. In conclusion,the detection efficiency could be greatly improved by the method,which technically supported early diagnosis and screening of Brucella,Toxoplasma gondii and Bartonella prevalent in various animals.

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赵冉,蔡振鸿,陈琼,徐晔,陈永锋,连玉华,李谓娟,温亚萍,吕菊香.布鲁氏菌、弓形虫及巴尔通体多重TaqMan荧光PCR检测方法的建立及应用[J].《中国动物检疫》编辑部,2023,40(1):109-114.

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  • 在线发布日期: 2023-01-10
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