景明.逆转录定量PCR选择性检测环境样品中的活性分枝杆菌[J].环境科学学报,2015,35(2):484-490
逆转录定量PCR选择性检测环境样品中的活性分枝杆菌
- Selective detection of viable Mycobacterium spp. in environmental samples using reverse transcription quantitative PCR
- 基金项目：国家水体污染控制与治理科技重大专项(No.2011ZX07506-003-005)
- 景明
- 1. 苏州环境监测中心, 苏州 215000;2. 同济大学环境科学与工程学院, 上海 200092
- 摘要：以分枝杆菌(Mycobacterium spp.)为研究对象,建立了一种逆转录定量PCR(Reverse transcription quantitative PCR,RT-qPCR)方法,选择性检测环境样品中的活性分枝杆菌.研究结果表明,稳定期(48~144 h)分枝杆菌细胞内hsp65基因的RNA转录稳定,单位细胞内hsp65的cDNA含量约为1拷贝,以此作为活性分枝杆菌的定量依据,达到快速确定环境样品中活性分枝杆菌浓度的目的.相比qPCR,RT-qPCR方法能够区分3个数量级的非活性分枝杆菌;样品的底物基质对RT-qPCR方法的影响较小;对于实际样品,与qPCR方法相比,RT-qPCR方法检测实际样品中活性分枝杆菌的结果与培养法更接近,线性关系更加显著(R2=0.9390).本研究表明,作为一种新的检测技术,RT-qPCR可以快速、准确地检测环境样品中的活性分枝杆菌.
- Abstract：A detection method based on reverse transcription quantitative PCR (RT-qPCR) was developed to quantify the concentration of viable Mycobacterium spp. in environmental samples. The results showed that the transcription of hsp65 gene of Mycobacterium spp. was stable during the stationary grow phase (48~144 h), and the concentration of cDNA targeting on hsp65 was about 1 copy per cell. Based on the above results, the established method can quantify viable Mycobacterium spp. in environmental samples. Compared to qPCR, RT-qPCR could distinguish inactivated Mycobacterium spp. only with 3 lg level. Besides, the substrate of environmental sample had non-significant effect on RT-qPCR. Finally, RT-qPCR results were similar and highly linearly correlated to those obtained by culture assay for environmental samples, and the linear correlation (R2) between RT-qPCR and culture-based method was 0.9390. In summary, RT-qPCR shows to be a promising method that can detect viable Mycobacterium spp. in environmental samples rapidly and accurately.
摘要点击次数： 1443 全文下载次数： 2866
