适用于痰液中结核分枝杆菌RNA提取的实验方法  

Methodological research on extracting total RNA from Mycobacterium tuberculosis of sputum specimen

作　　者：江丽娜[1] 张梅英[1] 穆成[1] 王春花[1] JIANG Li-na;ZHANG Mei-ying;MU Cheng;WANG Chun-hua(Reference Department,Tianjin Center for Tuberculosis Control,Tianjin,300071,China)

机构地区：[1]天津市结核病控制中心参比室

出　　处：《职业与健康》2019年第15期2050-2052,2056,共4页Occupation and Health

基　　金：2017年度结核病控制中心科技基金项目(津疾控科[2013]14号)

摘　　要：目的建立简便有效的确诊肺结核患者痰液中结核分枝杆菌(Mycobacterium tuberculosis,Mtb)总RNA提取的实验方法,以满足后期荧光定量聚合酶链式反应(PCR)的实验要求。方法选择临床上确诊为结核病的10份痰液标本,为使得总RNA纯度和质量更高,本文在传统的Trizol提取结核分枝杆菌RNA的实验方法上,加入DTT使得痰液均质化,加入RNA酶抑制剂抑制内源性和外源性RNA酶的作用,在醋酸钠作用的弱酸性条件下,观察甲醇裂解细胞壁方法对痰液标本中结核分枝杆菌总RNA抽提的实用性,以稳定的核糖体RNA16s为内参基因,通过荧光定量逆转录PCR方法评价痰液中总RNA提取效果的影响。结果甲醇裂解法和传统Trizol法均能成功提取痰液中的结核分枝杆菌总RNA,总RNA的获得率分别为88.5±0.12,85.65±0.18(P>0.01),纯度A260/280的值分别为1.88±0.06,1.80±0.08,差异无统计学意义(P>0.01),荧光定量PCR检测结果显示,两种方法的RNA提取量均能满足后期RT-PCR实验要求,cDNA获得量甲醇裂解细胞壁方法为16.64±0.54,传统的Trizol法为22.53±0.59,前者优于后者(P<0.01)。结论甲醇裂解法与传统Trizol法都能够从确诊肺结核患者痰液中成功提取总RNA,能满足后续荧光定量PCR的实验要求,但前者优于后者,适用于痰液中结核分枝杆菌RND的提取。Objective To establish a simple and effective method for total RNA extraction from Mycobacterium tuberculosis(Mtb) of sputum specimen from the confirmed tuberculosis patients to meet the experimental requirements of fluorescence quantitative PCR.Methods Ten sputum specimen from the confirmed tuberculosis patients were collected.In order to improve the purity and quality of total RNA,DTT was added to homogenize sputum on the basis of the traditional Trizol method for extracting Mtb RNA. RNA enzymes inhibitors were added to suppress the effect endogenous and exogenous RNA enzymes.Under weak acidic condition of sodium acetate,the practicability of methanol cell wall lysis method for extracting total RNA of Mtb in sputum specimen.Using stable ribosomal RNA 16S as internal reference gene,the effect of total RNA extraction from sputum was evaluated by fluorescence quantitative RT-PCR.Results Both methanol pyrolysis method and traditional Trizol method can successfully extract the total RNA of Mtb in sputum specimen,the acquisition rate of total RNA was 88.5±0.12 and 85.65±0.18 respectively(P>0.01), while the value of purity A260/280 was 1.88±0.06 and 1.80±0.08 respectively,and the difference was not statistically significant (P>0.01).The results of fluorescence quantitative RT-PCR showed that the RNA extraction amount of both methods can meet the requirements of RT-PCR.The amount of cDNA was 16.64 ±0.54 in methanol pyrolysis method and 22.53 ±0.59 in traditional Trizol method,and the former was superior to the latter(P<0.01).Conclusion Both methanol pyrolysis method and traditional Trizol method can successfully extract the total RNA of Mtb in sputum specimen,and meet the requirements of RT-PCR,while the former is superior to the latter,which is suitable for the extraction of Mycobacterium tuberculosis RNA from sputum.

关 键 词：痰液 分枝杆菌 甲醇裂解法 逆转录聚合酶链反应 

分 类 号：R117[医药卫生—公共卫生与预防医学]