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

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.