PMA-qPCR快速检测结核活菌方法的建立

Establisment of Rapid Detection for Live/Dead Mycobacterium tuberculosis with PMA-qPCR

为建立快速鉴别诊断结核活/死菌的方法,本研究应用PMA染料与实时定量PCR技术结合(PMA-qPCR),以结核标准株16 sRNA基因为靶基因,PMA对样品基因组提取物进行处理,PMA能够与死细胞DNA分子共价交联并抑制DNA分子扩增。结果显示:PMA浓度在3μg/mL,曝光时间大于15 min时,PMA既不影响活菌的的扩增,又能有效抑制死菌的扩增;当PMA浓度大于20μg/mL时,PMA影响活菌DNA的扩增;PMA-qPCR技术能够定量不同比例的结核活/死菌悬液中的活菌。结论:PMA-qPCR技术能够快速准确检测出结核杆菌中的活细胞。

To establish a rapid method for differential diagnosis of live or dead Mycobacterium tuberculosis,PMA dye technology combined with quantitative real-time PCR （PMA-qPCR） was utilized based on 16 sRNA genes of M.tuberculosis strain H37Rv. As a DNA-banding dye,PMA could penetrate damaged cells and inhibit DNA amplification prior to DNA extraction.Our results showed that concentration of 3 μg/mL PMA could effectively inhibit the amplification of dead cells without affecting the amplification of living cells when exposed to 650W halogen lamps for more than 15 min.Concentration of 20 μg/mL PMA significantly inhibited PCR amplification of living cells,indicating that higher concentration of PMA were toxic to living cells.As a rapid diagnostic method,PMA-qPCR can be effectively utilized to identify living cells and may be widely used in the detection of other bacteria.