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周丽娟, 吴艳凌, 周林, 刁叶秋, 王正芳, 王广洲. 检测肺炎支原体新方法的建立及应用[J]. 实用临床医药杂志, 2022, 26(19): 100-104. DOI: 10.7619/jcmp.20221716
引用本文: 周丽娟, 吴艳凌, 周林, 刁叶秋, 王正芳, 王广洲. 检测肺炎支原体新方法的建立及应用[J]. 实用临床医药杂志, 2022, 26(19): 100-104. DOI: 10.7619/jcmp.20221716
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ZHOU Lijuan, WU Yanling, ZHOU Lin, DIAO Yeqiu, WANG Zhengfang, WANG Guangzhou. Establishment and application of a new method for detecting Mycoplasma pneumoniae[J]. Journal of Clinical Medicine in Practice, 2022, 26(19): 100-104. DOI: 10.7619/jcmp.20221716
Citation: ZHOU Lijuan, WU Yanling, ZHOU Lin, DIAO Yeqiu, WANG Zhengfang, WANG Guangzhou. Establishment and application of a new method for detecting Mycoplasma pneumoniae[J]. Journal of Clinical Medicine in Practice, 2022, 26(19): 100-104. DOI: 10.7619/jcmp.20221716
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检测肺炎支原体新方法的建立及应用

Establishment and application of a new method for detecting Mycoplasma pneumoniae

    摘要
  • 摘要:
    目的 建立成簇的规律间隔的短回文重复序列(CRISPR)-Cas12a技术结合环介导等温扩增(LAMP)单管一步法快速检测肺炎支原体(Mp), 并探讨其在临床诊断中的应用价值。
    方法 设计并优化Mp LAMP引物，根据LMAP产物靶标设计Cas12a CrRNA。分析CRISPER-LAMP单管一步法检测Mp DNA的灵敏度; 检测Mp、甲流病毒(FluA)、乙流病毒(FluB)、肺炎克雷伯菌(Kp)和肺炎链球菌(Sp)及50例临床样本，评估CRISPER-LAMP单管一步法的特异性。
    结果 CRISPER-LAMP单管一步法可在1 h内实现Mp DNA的可视化检测，检出限为10 fg/μL, 聚合酶链式反应(PCR)法检出限为100 fg/μL; CRISPER-LAMP单管一步法检测FluA、FluB、Kp和Sp结果均为阴性; 50例临床样本检测结果均与临床诊断结果相符，且CRISPER-LAMP与PCR法一致性较好(Kappa=1)。
    结论 CRISPER-LAMP单管一步法检测Mp简便、快速、灵敏度高，且特异性强，检测不需任何特殊设备，检测过程中无气溶胶污染，其有望成为快速检测Mp的新方法。

     

    Abstract:
    Objective To establish clusters of regularly interspaced short palindromic repeats (CRISPR)-Cas12a technology combined with loop-mediated isothermal amplification (LAMP) single-tube one-step method for rapid detection of Mycoplasma pneumoniae (Mp), and to explore its application value in clinical diagnosis.
    Methods Mp LAMP primers were designed and optimized to design Cas12a CrRNA according to LMAP product targets. The sensitivity of CRISPER-LAMP single-tube one-step method for Mp DNA detection was analyzed; the Mp, influenza virus A (FluA), influenza virus B (FluB), Klebsiella pneumoniae (Kp), Streptococcus pneumoniae (Sp) and 50 clinical samples were detected, and the specificity of CRISPER-LAMP single-tube one-step method was evaluated.
    Results Mp DNA could be visualized within 1 hour by CRISPER-LAMP single-tube one-step method, with a detection limit of 10 fg/μL and a detection limit of 100 fg/μL by polymerase chain reaction (PCR); the FluA, FluB, Kp and Sp were all negative detected by CRISPER-LAMP single-tube one-step method; the detection results of 50 clinical samples were consistent with the clinical diagnosis results, and the consistency between CRISPER-LAMP and PCR method was good (Kappa=1).
    Conclusion CRISPER-LAMP single-tube one-step method for Mp detection is simple, rapid, sensitive and specific. It does not need any special equipment, and there is no aerosol pollution in the detection process. It is expected to become a new method for rapid detection of Mp.

     

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