| 引用本文: | 李珏,王银环,王庭璋,张林爽,陈欢,李钧,郑小玲,王知坚.洋葱伯克霍尔德菌群3种检测方法的比较[J].中国现代应用药学,2024,41(8):1091-1098. |
| LI Jue,WANG Yinhuan,WANG Tingzhang,ZHANG Linshuang,CHEN Huan,LI Jun,ZHENG Xiaoling,WANG Zhijian.Comparison of Three Detection Methods for Burkholderia Cepacia Complex[J].Chin J Mod Appl Pharm(中国现代应用药学),2024,41(8):1091-1098. |
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| 洋葱伯克霍尔德菌群3种检测方法的比较 |
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李珏1, 王银环1, 王庭璋2, 张林爽1, 陈欢3, 李钧4, 郑小玲1, 王知坚1
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1.浙江省食品药品检验研究院, 国家药品监督管理局药品微生物检测与预警重点实验室, 药品接触材料质量控制研究重点实验室, 杭州 310052;2.浙江省微生物技术与生物信息学研究重点实验室, 杭州 310012;3.杭州微数生物科技有限公司, 杭州 311202;4.杭州市疾病预防控制中心, 杭州 310021
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| 摘要: |
| 目的 通过对环介导等温扩增(loop-mediated isothermal amplification，LAMP)、基于聚合酶链式扩增(polymerase chain reaction，PCR)的SYTO 9染料法及TaqMan探针实时荧光定量PCR法(简称TaqMan探针法)3种检测方法的比较，旨在建立一种能够快速、准确检测24株洋葱伯克霍尔德菌群的PCR方法。方法 根据 NCBI 数据库中24株洋葱伯克霍尔德菌的分子生物学信息，筛选出洋葱伯克霍尔德菌所特有的多个候选序列片段，设计能同时检出24株洋葱伯克霍尔德菌的特异性引物和探针，同时探索了LAMP法、基于PCR的SYTO 9染料法及Taqman探针法3种检测方法，优化筛选出最佳退火温度，并采用39株实验菌株对洋葱伯克霍尔德菌群检测方法开展了验证研究。结果 采用LAMP法无法实现对洋葱伯克霍尔德菌群的有效检出，采用SYTO 9染料法和TaqMan探针法可以实现对20多株洋葱伯克霍尔德菌群的有效检出，而TaqMan探针法扩增效率更高，检测灵敏度、重复性、稳定性更好，能够满足本研究的要求。结论 本研究建立了洋葱伯克霍尔德菌群的TaqMan探针快速检测方法，相较于LAMP法和SYTO 9染料法，该方法具有快速、简便和敏感性高等优点，为洋葱伯克霍尔德菌群的快速检测提供了技术支持。 |
| 关键词: 洋葱伯克霍尔德菌群 TaqMan探针 qPCR法 |
| DOI：10.13748/j.cnki.issn1007-7693.20232897 |
| 分类号:R917 |
| 基金项目:浙江省药品监管系统科技计划项目(2022035)；浙江省市场监督管理局科技计划项目(20210128)；内蒙古自治区药品监督管理局药品安全监管科研项目(NMYJ-KJ-202303) |
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| Comparison of Three Detection Methods for Burkholderia Cepacia Complex |
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LI Jue1, WANG Yinhuan1, WANG Tingzhang2, ZHANG Linshuang1, CHEN Huan3, LI Jun4, ZHENG Xiaoling1, WANG Zhijian1
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1.Zhejiang Institute for Food and Drug Control, NMPA Key Laboratory for Testing and Risk Warning of Pharmaceutical Microbiology, Key Laboratory of Drug Contacting Materials Quality Control of Zhejiang Province Research on Physical Safety & Chemical Compatibility, Hangzhou 310052, China;2.Key Laboratory of Microbial Technology and Bioinformatics of Zhejiang Province, Hangzhou 310012, China;3.Hangzhou Digital-Micro Biotech Co., Ltd., Hangzhou 311202, China;4.Hangzhou Center for Disease Control and Prevention, Hangzhou 310021, China
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| Abstract: |
| OBJECTIVE To establish a rapid and accurate PCR method for detecting 24 strains of Burkholderia cepacia complex(Bcc) by comparing three detection methods of loop-mediated isothermal amplification(LAMP), SYTO 9 dye method based on polymerase chain reaction(PCR) and TaqMan probe real-time fluorescent quantitative PCR method( TaqMan probe method). METHODS According to the molecular biological information of 24 strains of Bcc in the NCBI database, multiple candidate sequence fragments unique to Bcc were screened out, and specific primer and probe that could simultaneously detect 24 strains of Bcc were designed. At the same time, the detection methods of LAMP, SYTO 9 dye method based on PCR and Taqman probe were explored, and the optimal annealing temperature was optimized and screened. The 39 experimental strains were used to verify the Bcc detection method. RESULTS LAMP method could not effectively detect Bcc, SYTO 9 dye method and TaqMan probe method could effectively detect more than 20 strains of Bcc, while TaqMan probe method had higher amplification effect, better detection sensitivity, repeatability and stability, which could meet the requirements of this study. CONCLUSION In this study, a TaqMan probe method for rapid detection of Bcc was established. Compared with LAMP method and SYTO 9 dye method, this method has the advantages of fast, simple and high sensitivity, and provides technical support for the rapid detectionof Bcc. |
| Key words: Burkholderia cepacia Complex TaqMan probe qPCR method |
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