FTA-环介导等温扩增技术直接提取变异链球菌DNA的效果评价  

Evaluation of FTA-LAMP direct extraction method for extracting DNA from Streptococcus mutans

作　　者：王玥晖 尚进 杨晨 符冬格 曹灿 张晓东 王敬夫 Wang Yuehui;Shang Jin;Yang Chen;Fu Dongge;Cao Can;Zhang Xiaodong;Wang Jingfu(Department of Stomatology,General Hospital of Northern Theater Command,Shenyang 110016,Liaoning Province,China;Department of Maxillofacial Surgery,The Third Affiliated Hospital of Air Force Military Medical University,Xi’an 710004,Shaanxi Province,China)

机构地区：[1]北部战区总医院口腔科,辽宁省沈阳市110016 [2]空军军医大学第三附属医院颌面外科,陕西省西安市710004

出　　处：《中国组织工程研究》2025年第5期1043-1049,共7页Chinese Journal of Tissue Engineering Research

基　　金：辽宁省科学技术计划项目面上项目(2022-MS-043),项目负责人:曹灿。

摘　　要：背景:变异链球菌是龋病的重要病原菌,及时检测变异链球菌水平对龋病的早发现、早治疗有重要意义。目的:建立并评价FTA-环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术直接提取变异链球菌DNA的应用效果。方法:①制备含有ATCC标准菌株变异链球菌的菌悬液,接种于脑心浸出液培养基,充分混匀后按10倍梯度稀释成7种浓度(4.2×10^(7),4.2×10^(6),4.2×10^(5),4.2×10^(4),4.2×10^(3),4.2×10^(2),4.2×10 CFU/mL),每个稀释级做2个平行对照,并增加无菌水作为空白对照;②分别采用FTA卡、常规煮沸法、试剂盒提取及裂解液提取4种方法直接提取菌株DNA,通过LAMP技术进行扩增,并进行特异性试验,比较4种提取方法的差异。结果与结论:①4种方法提取的DNA均满足LAMP扩增的要求;②特异性试验结果显示,只有变异链球菌才可特异扩增出靶基因;③裂解液提取法最低检测限为4.2×10^(3) CFU/mL,FTA卡提取法最低检测限为4.2×10^(4) CFU/mL,试剂盒提取法和常规煮沸法最低检测限分别为4.2×10^(6) CFU/mL和4.2×10^(7) CFU/mL;④4种提取方法其他方面的比较显示,试剂盒提取法的实验成本、步骤数和时间都是最高;其他3种方法步骤数一致,其中FTA卡所需仪器设备最少,常规煮沸法单次成本最低,裂解液提取法所需时间最少;FTA卡和裂解液提取法仅需少量菌即可提取成功,后者在时间方面优于FTA卡,但相较于FTA卡其单次成本高,所需设备多;⑤结果说明,该研究建立的FTA-LAMP技术具有操作简便、特异性强、灵敏度高、结果可视化等优势,有望为高效提取检测变异链球菌提供新途径。BACKGROUND:Streptococcus mutans is an important pathogen of dental caries,and timely detection of its levels is of great significance for early detection and treatment of dental caries.OBJECTIVE:To evaluate the effect of loop-mediated isothermal amplification(FTA-LAMP)direct extraction of Streptococcus mutans DNA.METHODS:(1)Bacterial suspensions containing ATCC standard strains(Streptococcus mutans)were prepared and inoculated into the brain-heart leachate medium.After mixed thoroughly,the mixture was then diluted in a 10-fold gradient into seven concentrations(4.2×10^(7),4.2×10^(6),4.2×10^(5),4.2×10^(4),4.2×10^(3),4.2×10^(2),4.2×10 CFU/mL),two parallel controls were made for each dilution level,and sterile water was used as a blank control.(2)The DNA of Streptococcus mutans was extracted using FTA Elute card,boiling method,kit extraction and lysate extraction methods separately and then amplified using LAMP technology was amplified.A specificity test was also performed to compare the differences between the four DNA extraction methods.RESULTS AND CONCLUSION:The DNA extracted by all four methods met the requirements for LAMP amplification.Specificity test results showed that only Streptococcus mutans could specifically amplify the target gene.The detection limit value of the DNA concentration was 4.2×10^(3) CFU/mL for the lysate method,4.2×10^(4) CFU/mL for the FTA Elute card extraction method,4.2×10^(6) CFU/mL for the kit extraction method,and 4.2×10^(7) CFU/mL for the boiling method.In the other aspects of the four extraction methods,the kit extraction method had the highest experimental cost,number of steps and time;the other three methods had the same number of steps,with the FTA Elute card method requiring the least amount of instruments,the boiling method having the lowest single cost,and the lysate extraction method taking the least amount of time.Only a small amount of bacteria were needed for successful extraction using both the FTA Elute card and lysate extraction methods.Compared with the FT

关 键 词：变异链球菌 环介导等温扩增 FTA卡 DNA 可视化 

分 类 号：R496[医药卫生—康复医学] R318[医药卫生—临床医学] R782