摘要
目的将叠氮溴化丙锭(propidium monoazide,PMA)与实时定量聚合酶链式反应(RTi-PCR)技术相结合,建立以一种快速准确的创伤弧菌检测方法。方法以vvh基因作为检测创伤弧菌的靶基因,用PMA对样品基因组提取进行前处理,抑制该DNA分子的RTi-PCR扩增。结果终浓度为3.0mg/L的PMA可有效抑制死细胞(1×108 CFU/mL)DNA的扩增;当PMA浓度小于或等于5.0mg/L时,对创伤弧菌DNA的扩增没有显著的影响;含有不同比例死活细菌的创伤弧菌混合液RTi-PCR结果表明,PMA RTi-PCR能选择性定量创伤弧菌中的活菌。纯培养创伤弧菌活细胞的检测灵敏度检测极限为2.5×101 CFU/mL,并且Ct值与细胞数的对数呈良好的线性关系,相关系数R2值为0.9982。结论 PMA RTi-PCR是一种快速灵敏且能有效鉴别并定量检测病原活细胞的新方法。
A rapid and accuyate detection method of Vibrio vutnificus was developed by adding propidium monoazide （PMA） in the process of RTi-PCR. The results showed that amplification of DNA derived from 1 × 10^8 CFU/mL Vibrio vulnificus dead cells could be inhibited by PMA with a concentration of 3.0 mg/L, while the use of 5.0 mg/L or less of PMA did not inhibit the RTi PCR amplification of DNA derived from viable cells, and for that PMA treatment, the number of viable Vibrio vulnificus cells in varying ratios of viable to dead cells could be selectively quantified by RTi-PCR. The detection limit of the RTi-PCR assay was 2.5 × 10^1CFU/mL in pure cultures, and the copy number of vaccine gene and the magnitude of cycle threshold （Ct） presented the satisfactory linear dependability and the coefficient of correlation R2 approached 0. 9982. It may be a fast and sensitive method to identify and quantify pathogenic viable cells effectively.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2013年第1期54-58,共5页
Chinese Journal of Zoonoses
基金
广州市医药卫生科技项目(201102A213240)资助~~
