One Step Extraction of Mycoplasma Pneumoniae Genomic DNA in Application Research of the Visuable LAMP System / 现代检验医学杂志

ABSTRACT

Objective The modified rapid one-step extraction (ROSE) method was employed in the Mycoplasma pneumoniae (Mp)genomic DNA extraction,in order to establish an easy DNA extraction method,which is used in Mp Loop Mediated Isothermal Amplification(LAMP) and suitable for high throughput without any expensive instruments.Methods In line with the character of Mp,a one-step Mp acid extraction method was established on the basis of ROSE.The studies had been carried out on comparing the modified ROSE DNA extraction with the classical methods of Boiling,CTAB,Alkali solution and reagent kit in extraction quality,quantity,reagent kinds,operation steps and extraction time.The extracted DNA of the Mp culture solution by these five methods had been used in LAMP to test the extraction efficiency.20 cases of Throat swab specimens from children with mycoplasma pneumonia were selected from January to March of 2016,and the throat swab specimens were extracted by the improved ROSE method then applied to LAMP detection system.Results Purity,comparison of ROSE method and other four methods were P＜0.05,the difference was statistically significant and ROSE was slightly better than the alkaline lysis method and boiling method,but lower than the CTAB method and kit method;Yield,results were compared with ROSE method and other four methods:there was no significant difference in q=0.95 (P＞0.05) compared with ROSE method and boiling method.The ROSE method was significantly different from the other three methods (P＜0.05),so the yield of ROSE method was higher than that of alkaline lysis method,CTAB method and kit method.Instrument and equipment,extracting procedure,extracting time and quantity ROSE,Boiling and Alkali solution were obvious ly better than CTAB and reagent kit.The extracted DNA by these five methods could be used in LAMP reaction,and the results showed no significant difference.Conclusion This study established an original one-step DNA genomic extraction method of Mp,which had been successfully applied in the Mp LAMP withoutany expensive instruments.Offer a groundbreaking method detecting Mp for the hospitals at all levels including the primary medical organizations.