肺炎支原体基因组DNA一步法提取在可视化环介导等温扩增体系中的应用研究

One Step Extraction of Mycoplasma Pneumoniae Genomic DNA in Application Research of the Visuable LAMP System

目的探究建立肺炎支原体(Mp)基因组DNA一步提取方法,应用于显色肺炎支原体环介导等温扩增(LAMP)体系,以期研制出高灵敏度和高特异度肺炎支原体的检测方法。方法分别用5种方法提取肺炎支原体培养液基因组DNA,并分别从Mp DNA的纯度、产量、操作步骤、仪器设备和提取时间方面进行比较;最后将5种方法提取的基因组DNA同时应用于已建立的可视化LAMP体系检验提取效果;选取2016年1月~3月20例支原体肺炎住院患儿的咽拭子标本,并用改良后的ROSE法提取咽拭子标本,并鉴定提取效果。结果提取纯度上,ROSE法和其他四种方法进行两两比较,差异均具有统计学意义(P均<0.05),即ROSE法略好于碱裂解法和煮沸法,但低于CTAB法和试剂盒法;产量上,ROSE法和其他四种方法进行两两比较的结果是:和煮沸法比差异无统计学意义(q=0.95,P>0.05),和其他三种方法比差异有统计学意义(P均<0.05),因此ROSE法提取的产量要高于碱裂解法、CTAB法和试剂盒法;在仪器设备、操作步骤和提取时间上ROSE法、碱裂解法和煮沸法优于CTAB法和试剂盒法;5种方法提取的DNA均能应用于LAMP反应,除煮沸法外,其余四种方法结果无明显差异。结论该研究建立了肺炎支原体一步法基因组DNA提取方法,成功应用于显色Mp LAMP体系中,真正实现不需要任何贵重仪器即可检测肺炎支原体。

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 I- sothermal 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 car- ried 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,compari- son of ROSE method and other four methods were P〈0.05 ,the difference was statistically significant and ROSE was slight- ly 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） com- pared 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. In- strument 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 re- sults 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 ground- breaking method detecting Mp for the hospitals at all levels including the primary medical organizations.