不同破壁方法提取结核分枝杆菌总RNA的比较

The comparison of different wall-breaking method for extracting total RNA of Mycobacterium tuberculosis

目的建立简便有效的结核分枝杆菌(Mycobacterium tuberculosis,Mtb)总RNA提取方法。方法采用5种方法,分别为160、240和400W超声波联合Trizol法(简称"160W超声法"、"240W超声法"和"400W超声法")、玻璃珠(简称"玻珠")联合Trizol法(简称"玻珠法")和Trizol法(简称"不加玻珠法"),对结核分枝杆菌标准株H37Rv进行破壁,提取总RNA。通过荧光定量逆转录PCR评价不同破壁方法对Mtb总RNA提取效果的影响,以灭菌超纯水代替模板为阴性对照。每种方法分别提取3份样本进行检测。最后结果为3份标本的平均值。结果玻珠法检测组Ct值为20.67,△Rn值为0.644;400W超声法检测组Ct值为22.09,△Rn值为0.571;240W超声法检测组Ct值为21.86,△Rn值为0.503;160W超声法检测组Ct值为25.21,△Rn值为0.411;不加玻珠法检测组Ct值为28.40,△Rn值为0.299;阴性对照Ct值为40.00,△Rn值为0.000。结论直观地从试验结果来看,玻珠法对结核分枝杆菌破壁效果好于超声法。

Objective To establish a simple and effective total RNA extraction method of Mycobacterium tu- berculosis. Methods Cell wall of Mycobacterium tuberculosis were treated by ultrasonic and glass-bead associated with Trizol method respectively, the total RNA was extracted and the influence of different total RNA extraction methods of Mtb were evaluated through fluorescent quantitative RT-PCR. The negative control is ultrapure water instead of template. Each method were extracted from 3 samples for testing. The final result is the average of 3 specimen. Results The Ct and △Rn value of glass-bead group is 20.67 and 0. 644, 400 W ultrasonic group is 22.09 and 0. 571, 240 W ultrasonic group is 21.86 and 0. 503, 160 W ultrasonic group is 25.21 and 0. 411, bead absent group is 28.40 and 0. 299, negative control is 40. 00 and 0. 000. Conclusion The wall-breaking effection of glass-bead method is better than that of ultrasonic method.