CAJ | 학술논문

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。
위건립쾌속감별진단결핵활/사균적방법，본연구응용PMA염료여실시정량PCR기술결합(PMA-qPCR)，이결핵표준주16 sRNA기인위파기인，PMA대양품기인조제취물진행처리，PMA능구여사세포DNA분자공개교련병억제DNA분자확증。결과현시：PMA농도재3μg/mL，폭광시간대우15 min시，PMA기불영향활균적적확증，우능유효억제사균적확증；당PMA농도대우20μg/mL시，PMA영향활균DNA적확증；PMA-qPCR기술능구정량불동비례적결핵활/사균현액중적활균。결론：PMA-qPCR기술능구쾌속준학검측출결핵간균중적활세포。
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.