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3种检测布鲁氏菌荧光定量PCR方法的比较
董浩,原霖,赵明海,许中衎,刘巍,徐阳,陈亚娜,梁春南
0
(中国食品药品检定研究院,北京102629; 中国动物疫病预防控制中心,北京 102618)
摘要:
为了筛选适用于家畜布鲁氏菌病检测的荧光定量PCR方法,合成目前报道最多的布鲁氏菌IS711插入序列、per基因和bcsp31基因的引物和探针,并分别对这3种荧光定量PCR方法的敏感性、特异性、重复性以及63份临床样品检测效果进行比较。结果显示:3种荧光定量PCR方法均具有较好的敏感性且均不与其他常见细菌发生交叉反应;3种方法的批内变异系数和批间变异系数均小于5%。在检测临床样品时,3种方法的敏感性均高于套式PCR方法,其中IS711荧光定量PCR方法与套式PCR方法符合率为95.24%,其他2种方法与套式PCR方法的符合率为98.41%。比较而言,IS711荧光定量PCR敏感性最高,更适合于布鲁氏菌核酸含量低的样品的检测。
关键词:  布鲁氏菌  荧光定量PCR  方法比对  IS711插入序列
DOI:10.13610/j.cnki.1672-352x.20220106.022
基金项目:国家自然科学基金(31602055)资助。
Comparison of three real time PCR assays for Brucella
DONG Hao,YUAN Lin,ZHAO Minghai,XU Zhongkan,LIU Wei,XU Yang,CHEN Yana,LIANG Chunnan
(National Institutes for Food and Drug Control, Beijing 102629; China Animal Disease Control Center, Beijing 102618)
Abstract:
To select a real time PCR method for the detection of animal brucellosis, the primers and probes of three real time PCR methods (IS711 insertion sequence, per gene and bcsp31 gene), which were most reported, were synthesized. The sensitivity, specificity and repeatability of the three methods were tested. In addition, 63 clinical samples were also tested using the three real time PCR methods. The results indicated that the three real time PCR methods had good sensitivity and did not cross-react with the DNA samples from other common bacteria. Both intra-batch coefficient of variation and inter-batch coefficient of variation of the three methods were less than 5%. When testing clinical samples, the sensitivity of the three methods were higher than the nested PCR method. The coincidence rate between the IS711-based real time PCR method and the nested PCR method was 95.24%, and which of the other two real time PCR methods and the nested PCR method were 98.41%. In comparison, the IS711-based real time PCR has the highest sensitivity, and it is more suitable for the detection of samples with low content of Brucella DNA.
Key words:  Brucella  real time PCR  comparison of methods  IS711 insertion sequence

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