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王春晖, 赵素慧, 韦耀, 周莹, 万成松. A型肉毒梭菌atx基因TaqMan探针荧光定量PCR检测[J]. 中国公共卫生, 2012, 28(6): 863-865. DOI: 10.11847/zgggws-2012-28-06-59
引用本文: 王春晖, 赵素慧, 韦耀, 周莹, 万成松. A型肉毒梭菌atx基因TaqMan探针荧光定量PCR检测[J]. 中国公共卫生, 2012, 28(6): 863-865. DOI: 10.11847/zgggws-2012-28-06-59
WANG Chun-hui, ZHAO Su-hui, WEI Yao, . Detection of atx gene in Clostridium botulinum using TaqMan probe FQ-PCR[J]. Chinese Journal of Public Health, 2012, 28(6): 863-865. DOI: 10.11847/zgggws-2012-28-06-59
Citation: WANG Chun-hui, ZHAO Su-hui, WEI Yao, . Detection of atx gene in Clostridium botulinum using TaqMan probe FQ-PCR[J]. Chinese Journal of Public Health, 2012, 28(6): 863-865. DOI: 10.11847/zgggws-2012-28-06-59

A型肉毒梭菌atx基因TaqMan探针荧光定量PCR检测

Detection of atx gene in Clostridium botulinum using TaqMan probe FQ-PCR

  • 摘要: 目的采用TaqMan探针荧光定量PCR方法对A型肉毒梭菌atx基因进行检测。方法以atx基因为靶基因,设计引物和TaqMan探针,优化反应条件,制作定量标准曲线,进行灵敏度、特异性和重复性验证,建立A型肉毒梭菌TaqMan荧光定量PCR检测方法。结果构建质粒pUC57-△atx,标准曲线在103~103拷贝数之间有较好的线性关系,相关系数为0.997,灵敏度达到22个拷贝数,比普通PCR提高约100倍;能选择性检测A型肉毒梭菌,与其他5种食源性病原菌无交叉反应,结果与普通PCR一致;重复性试验表明,同一浓度的15个平行样品的变异系数为1.0%。结论 A型肉毒梭菌TaqMan探针荧光定量PCR方法具有灵敏度高、特异性强、重复性好等特点,可以快速、准确、定量地检测A型肉毒梭菌。

     

    Abstract: ObjectiveTo detect the atx gene of Clostridium botulinum(C.botulinum)type A neurotoxin(bont/A) using TaqMan probe fluorescence quantitative polymerase chain reaction(FQ-PCR).MethodsBy targeting the gene atx of bont/A,designing primer and TaqMan probe,optimizing the reaction conditions,making quantitative standard curve,and verifying sensitivity,specificity and repeatability of the method,we established a TaqMan FQ-PCR method to detect bont/A.ResultsPlasmid pUC57-△atx was constructed successfully.The standard curve established had good linearity when gene quantity was between 103-107 copies,and the coefficient of correlation was 0.997.The limit of detection for FQ-PCR was 22 copies.The sensitivity was about 100 times higher than that of ordinary PCR.The FQ-PCR method could selectively detect C.botulinum and there were no cross reactions with other five food-borne pathogen samples,consisting with the results of ordinary PCR.Repeatability tests showed that the coefficient of variation of 15 parallel samples in same concentration was only 1.0%.ConclusionA TaqMan probe FQ-PCR method with high sensitivity and specificity,and good repeatability was established for rapid,accurate and quantitative detectoin of C.botulinum type A.

     

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