[1]严春霞,闻人庆,陆伟宏,等.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[J].现代检验医学杂志,2017,32(02):82-85.[doi:10.3969/j.issn.1671-7414.2017.02.022]
　YAN Chun-xia,WEN Ren-qing,LU Wei-hong,et al.One Step Extraction of Mycoplasma Pneumoniae Genomic DNA in Application Research of the Visuable LAMP System[J].Journal of Modern Laboratory Medicine,2017,32(02):82-85.[doi:10.3969/j.issn.1671-7414.2017.02.022]
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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()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第32卷
期数:
2017年02期
页码:
82-85
栏目:
论著
出版日期:
2017-03-25

文章信息/Info

Title:
One Step Extraction of Mycoplasma Pneumoniae Genomic DNA in Application Research of the Visuable LAMP System
文章编号:
1671-7414(2017)02-082-04
作者:
严春霞; 闻人庆; 陆伟宏; 何国产
金华职业技术学院,浙江金华 321007
Author(s):
YAN Chun-xia; WEN Ren-qing; LU Wei-hong; HE Guo-chan
Jinhua College of Profession and Technology,Zhejiang Jinhua 321007,China
关键词:
一步法;  DNA提取;  肺炎支原体
分类号:
R375.2; Q781
DOI:
10.3969/j.issn.1671-7414.2017.02.022
文献标志码:
A
摘要:
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。
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 inMp Loop Mediated Isothermal Amplification(LAMP)and suitable for high throughputwithout any expensive instruments.Methods In line withthe 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 ROSEDNA extraction with the classical methods of Boiling,CTAB,Alkali solution andreagent kit in extraction quality,quantity,reagent kinds,operation steps andextraction 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 Throatswab 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 obviously 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 establishedan 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.

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备注/Memo

备注/Memo:
基金项目:金华市科技计划项目(2014-3-034),浙江省科技计划项目(2017C37085)。
作者简介:严春霞(1977-),女,硕士,主管技师,主要从事病原微生物检测的研究,Tel:13757990496,0579-82265099。
更新日期/Last Update: 2017-04-10