张军鸽, 赵飞, 刘立雍, 马闪珊, 单小云. 多种一步法快速提取DNA对肺炎支原体PCR检测的影响[J]. 疾病监测, 2018, 33(6): 503-509. DOI: 10.3784/j.issn.1003-9961.2018.06.014
引用本文: 张军鸽, 赵飞, 刘立雍, 马闪珊, 单小云. 多种一步法快速提取DNA对肺炎支原体PCR检测的影响[J]. 疾病监测, 2018, 33(6): 503-509. DOI: 10.3784/j.issn.1003-9961.2018.06.014
Zhang Junge, Zhao Fei, Liu Liyong, Ma Shanshan, Shan Xiaoyun. Influence of different rapid one-step DNA extraction on PCR detection of Mycoplasma pneumoniae[J]. Disease Surveillance, 2018, 33(6): 503-509. DOI: 10.3784/j.issn.1003-9961.2018.06.014
Citation: Zhang Junge, Zhao Fei, Liu Liyong, Ma Shanshan, Shan Xiaoyun. Influence of different rapid one-step DNA extraction on PCR detection of Mycoplasma pneumoniae[J]. Disease Surveillance, 2018, 33(6): 503-509. DOI: 10.3784/j.issn.1003-9961.2018.06.014

多种一步法快速提取DNA对肺炎支原体PCR检测的影响

Influence of different rapid one-step DNA extraction on PCR detection of Mycoplasma pneumoniae

  • 摘要: 目的 对比不同一步法提取DNA在肺炎支原体PCR检测中的效果及应用范围,为临床标本中肺炎支原体的检测提供依据。方法 以两种经典的DNA提取一步法(ROSE和Chelex-100法)及其优化方案和商品化DNA提取试剂盒,提取肺炎支原体纯培养菌株及30份肺炎支原体阳性临床咽拭子标本DNA,分别进行普通PCR和荧光PCR检测,对比不同方法提取的DNA对PCR检测结果的影响。结果 ROSE法和Chelex-100一步法中的SDS严重影响Taq酶活性,提取物稀释100倍方可进行荧光PCR扩增,稀释10倍可用于普通PCR扩增。改良的Chelex-100一步法提取肺炎支原体纯菌DNA产量最高,且PCR扩增效果最好。对于30份临床标本来说,试剂盒法、改良Chelex-100法和水煮法的阳性率分别为100.00%(30/30)、80.00%(24/30)和43.33%(13/30),且对相同肺炎支原体阳性标本提取的DNA,试剂盒法检测的循环阈值(Ct值)比上述两种方法分别小1.95和2.38。结论 经典一步法提取的DNA必须经稀释后方可作为扩增模板。改良的Chelex-100法操作简便,可用于纯菌培养的DNA提取。临床标本中肺炎支原体DNA提取以试剂盒法最优,改良的Chelex-100法提取的DNA也可用于临床标本中肺炎支原体的检测,但不可用于定量分析。

     

    Abstract: Objective To understand the effect of one-step nucleic acid extraction method and its optimization scheme in the detection of Mycoplasma pneumoniae (MP). Methods The DNA of strains and eight positive clinical throat swab specimens of MP were extracted with two classical DNA extraction one-step methods (ROSE method and Chelex-100 method)and their optimized programs and commercial nucleic acid kit (QIAGEN). PCR and real time PCR were applied to compare different DNA extraction methods. Results The SDS widely used in DNA extraction could influence Taq enzyme activity seriously. The DNA extracted by the classical DNA extraction one-step methods could not be applied to PCR detection unless it was diluted more than 10 times,even 100 times. There was the most yield of DNA in improved Chelex-100 one-step extraction. For 30 clinical specimens,the positive detection rate of commercial nucleic acid kit,modified Chelex-100 method and boiling method were 100.00% (30/30),80.00% (24/30)and 43.33% (13/30),respectively,and the detection CT values of the commercial nucleic acid was lower than the other two methods. Conclusion The DNA extracted by the classical one-step method must be diluted before being used for PCR amplification. The improved Chelex-100 method was simple and effective,which can be used for MP strains. The commercial kit (Qiagen)was best in DNA extraction for clinical specimens. The improved Chelex-100 method could be also used for clinical specimens,but it was not suitable for the determination of pathogen load.

     

/

返回文章
返回