中国组织工程研究

中国组织工程研究 ›› 2025, Vol. 29 ›› Issue (5): 1040-1049.doi: 10.12307/2025.279

• 口腔组织构建 oral tissue construction • 上一篇    下一篇

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

王玥晖1，尚  进1，杨  晨1，符冬格1，曹  灿1，张晓东1，王敬夫1，2   

  1. 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


  • 收稿日期:2024-01-27 接受日期:2024-03-09 出版日期:2025-02-18 发布日期:2024-06-04
  • 通讯作者: 张晓东，博士，主任医师，北部战区总医院口腔科，辽宁省沈阳市 110016 共同通讯作者：王敬夫，博士，主治医师，北部战区总医院口腔科，辽宁省沈阳市 110016；空军军医大学第三附属医院颌面外科，陕西省西安市 710004
  • 作者简介:王玥晖，女，1998年生，湖南省娄底市人，汉族，大连医科大学在读硕士，医师，主要从事口腔正畸学与基础的研究。
  • 基金资助:
    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

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

Wang Yuehui1, Shang Jin1, Yang Chen1, Fu Dongge1, Cao Can1, Zhang Xiaodong1, Wang Jingfu1, 2   

  1. 1Department of Stomatology, General Hospital of Northern Theater Command, Shenyang 110016, Liaoning Province, China; 2Department of Maxillofacial Surgery, The Third Affiliated Hospital of Air Force Military Medical University, Xi’an 710004, Shaanxi Province, China
  • Received:2024-01-27 Accepted:2024-03-09 Online:2025-02-18 Published:2024-06-04
  • Contact: Zhang Xiaodong, MD, Chief physician, Department of Stomatology, General Hospital of Northern Theater Command, Shenyang 110016, Liaoning Province, China Co-corresponding author: Wang Jingfu, MD, Attending physician, 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
  • About author:Wang Yuehui, Master’s candidate, Physician, Department of Stomatology, General Hospital of Northern Theater Command, Shenyang 110016, Liaoning Province, China
  • Supported by:
    Liaoning Provincial Science and Technology Plan (General Program), No. 2022-MS-043 (to CS)

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摘要：




文题释义：
FTA卡：是一种经过特殊试剂处理后的棉纤维卡片，可以直接在室温下采集样品的基因，裂解细胞膜后释放的核酸可被FTA卡的纤维缠绕固定，随后经特殊的纯化试剂洗脱干燥即可用于后续的扩增检测。
LAMP：环介导等温扩增技术主要特征是采用4或6条能够识别目的基因上的6个特异区域的引物，依赖于Bst DNA聚合酶的强链置换活性，可使反应在30-60 min内DNA扩增达109-1010倍。因其具有种高敏、高特异性、结果可视化等特点，设备仅需一个恒温箱即可快速扩增目的基因，现被应用于临床检测中。

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(4.2×107，4.2×106，4.2×105，4.2×104，4.2×103，4.2×102，4.2×10 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4.2×103 CFU/mL，FTA卡提取法最低检测限为4.2×104 CFU/mL，试剂盒提取法和常规煮沸法最低检测限分别为4.2×
106 CFU/mL和4.2×107 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。

https://orcid.org/0009-0000-4927-7359(王玥晖)

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: 变异链球菌, 环介导等温扩增, FTA卡, DNA, 可视化

Abstract: 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 (4.2×107, 4.2×106, 4.2×105, 4.2×104, 4.2×103, 4.2×102, 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 4.2×103 CFU/mL for the lysate method, 4.2×104 CFU/mL for the FTA Elute card extraction method, 4.2×106 CFU/mL for the kit extraction method, and 4.2×107 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 FTA Elute card method, the lysate extraction method was superior in terms of time, but it had a high single cost and required more equipment. To conclude, the FTA-LAMP technology established in this study has the advantages of ease of operation, high specificity, high sensitivity, and visualization, which is expected to be a new way for efficient extraction and detection of Streptococcus mutans.  

Key words: Streptococcus mutans, Loop-mediated isothermal amplification, FTA Elute card, DNA, visualization

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