摘要
目的比较5种痰标本前处理方法,选择适用于基层结核病实验室痰中结核分枝杆菌DNA提取的痰标本前处理方法。方法分别采用DDT、NaOH、NALC-NaOH、Trypsin、Chymotrypsin等5种方法对所收集标本进行前处理,比较提取DNA模板的浓度、纯度和核酸扩增结果。结果经过5种前处理方法处理后,所提取DNA浓度(ng/μl)的最小值依次是27.30、4.10、9.30、18.60和14.80,最大值依次是84.10、40.20、26.10、111.00和159.00,均值依次是(52.84±19.17)、(16.69±12.72)、(14.38±4.57)、(48.65±25.75)和(53.79±46.27);以OD260/OD280的比值衡量DNA纯度,其最小值依次是1.75、1.60、1.64、1.60和1.62,最大值依次是1.86、1.85、1.79、1.88和1.84,均值依次是(1.80±0.29)、(1.66±0.88)、(1.74±0.40)、(1.76±0.86)、(1.73±0.74);16份已知阳性标本采用DDT、NALC-NaOH两种方法处理后扩增结果全部阳性,而采用NaOH、Trypsin、Chymotrypsin处理后分别出现了3、8和5份标本阴性。结论在基层结核病实验室,对拟进行结核分枝杆菌DNA提取的痰标本应首选二硫代苏糖醇进行前处理,其次选择N-乙酰-L-半胱氨酸-氢氧化钠。
Objective To compare the influences of five sputum specimen pre processing methods on the DNA extrac tion of Mycobacterium tuberculosis, so as to explore the feasible method for the tuberculosis research laboratory of basic level. Method The sputum samples were pre - processed by five methods, DL - Dithiothreitol （DDT）, NaOH, N - Acetyl - L - cys- teine（NALC） - NaOH, trypsin and chymotrypsin, and then the concentration, purity and nucleic acid amplification results of the DNA extracted were compared. Results The minimum values of the concentrations （ng/μL） of the DNA extracted after the sputum being pre- processed by five methods were 27.30, 4.10, 9.30, 18.60 and 14.80 in turn. The maximum values were 84.10, 40.20, 26.10, 111.00 and 159.00. The mean values were 52.84 ±19.17, 16.69 ± 12.72, 14.38 ± 4.57, 48.65 ± 25.75 and 53.79 ± 46.27. The minimum values of the DNA purity measured by OD260/OD280 ratio were 1.75, 1.60, 1.64, 1.60 and 1.62 in turn. The maximum values were 1.86, 1.85, 1.79, 1.88 and 1.84. The mean values were 1.80 ±0.29, 1.66 ± 0.88, 1.74 ± 0.40, 1.76 ± 0.86 and 1.73 ± 0.74. When the 16 known - positive sputum samples were pretreated by DDT or NALC- NaOH, all of the samples got positive results after amplification, while when the 16 samples were pretreated by NaOH, trypsin or chymotrypsin, there were respectively 3, 8 and 5 samples with negative results. Conclusions For tubercu- losis research laboratories of basic level, the preferred method to pre - process sputum samples for DNA extraction of Mycobacteri- urn tuberculosis is DL- Dithiothreitolin, followed by NALC - NaOH.
出处
《实用预防医学》
CAS
2013年第9期1056-1059,共4页
Practical Preventive Medicine
基金
长沙市科技局项目(项目编号K1104052-21)
关键词
结核分枝杆菌
DNA提取
痰
前处理方法
Mycobacterium tuberculosis
DNA Extraction
Sputum
Pre- processing method
