耐甲氧西林金黄色葡萄球菌的核酸等温扩增方法研究进展

赵雨佳; 范培蕾; 李冉; 刘然; 李晶晶; 赵海波; 梁亮

doi:10.12338/j.issn.2096-9015.2024.0039

耐甲氧西林金黄色葡萄球菌的核酸等温扩增方法研究进展

doi: 10.12338/j.issn.2096-9015.2024.0039

北京市计量检测科学研究院，北京 100029

基金项目: 国家重点研发计划项目（2022YFF0606105）；国家市场监督管理总局科技计划项目（2022MK002）；中国博士后面上基金（2023M730312）；北京市计量检测科学研究院自主课题（KJ-2024-16）。

详细信息

作者简介:
赵雨佳，北京市计量检测科学研究院高级工程师，研究方向：生物计量，Zhaoyj@bjjl.cn

通讯作者:
梁亮，北京市计量检测科学研究院高级工程师，研究方向：生物计量，Liangliang@bjjl.cn

计量
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- 被引次数: 0
出版历程
- 收稿日期: 2024-06-30
- 录用日期: 2024-07-25
- 修回日期: 2024-09-26
- 网络出版日期: 2024-10-24

Research Progress on Nucleic Acids Isothermal Amplification Methods of Methicillin-Resistant Staphylococcus Aureus

Beijing Institute of Metrology, Beijing 10024, China

摘要

摘要: 耐甲氧西林金黄色葡萄球菌（MRSA）因高耐药性和高致死率成为医院与社区感染的重要病原。基于PCR检测mecA基因被认为是检测MRSA的“金标准”，然而该方法无法摆脱对精密控温仪器的依赖，难以满足现场检测的需求。与传统的PCR扩增不同，核酸等温扩增能够在恒定温度下进行反应，极大的简化了操作过程，适用于临床快速检测、病房床头诊断、社区基层医疗、现场流行病学调查等多种应用场景。近年来，环介导的等温扩增、重组酶聚合酶扩增和滚环扩增等核酸等温扩增方法被开发用于检测MRSA。对其进行综述，为MRSA的现场检测提供参考。
- 计量学 /
- 耐甲氧西林金黄色葡萄球菌 /
- 核酸等温扩增 /
- 环介导的等温扩增 /
- 重组酶聚合酶扩增 /
- 滚环扩增
Abstract: Methicillin-resistant Staphylococcus aureus (MRSA) has become an important pathogen of nosocomial and community infections due to its high drug resistance and high mortality rate. PCR-based detection of mecA gene is considered to be the "gold standard" for the detection of MRSA, but this method cannot get rid of the dependence on precision temperature control instruments and is difficult to meet the needs of on-site detection. Different from traditional PCR amplification, nucleic acid isothermal amplification can react at a constant temperature, greatly simplifying the operation process, and is suitable for a variety of application scenarios such as clinical rapid detection, bedside diagnosis in wards, community primary care, and on-site epidemiological investigations. In recent years, nucleic acids isothermal amplification methods, such as loop-mediated isothermal amplification, recombinase polymerase amplification and rolling circle amplification have been developed for the detection of MRSA. This article reviews them and provides a reference for the on-site detection of MRSA.
- metrology /
- methicillin-resistant Staphylococcus aureus /
- nucleic acids isothermal amplification /
- loop-mediated isothermal amplification /
- recombinase polymerase amplification /
- rolling circle amplification

HTML全文

图 1 LAMP扩增

Figure 1. LAMP amplification

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图 2 RPA扩增^[33]

Figure 2. RPA amplification^[33]

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图 3 定量RPA ^[33]

Figure 3. Quantitative RPA^[33]

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图 4 MNAzyme-GNP平台 ^[38]

Figure 4. MNAzyme-GNP platform^[38]

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图 5 one-tube RPA-CRISPR/Cas12a平台 ^[39]

Figure 5. one-tube RPA-CRISPR/Cas12a platform^[39]

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图 6 滚环扩增 ^[42]

Figure 6. rolling circle amplification^[42]

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图 7 i-CoRi平台 ^[43]

Figure 7. i-CoRi platform^[43]

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图 8 RCA-FLA平台 ^[44]

Figure 8. RCA-FLA platform^[44]

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表 1 基于等温扩增技术检测MRSA的优缺点

Table 1. Advantages and disadvantages of isothermal amplification technology for the detection of MRSA

技术名称	优点	缺点
LAMP	反应温度：60℃左右。反应时间短：通常在1h内完成扩增。特异性强：通过多个引物与目标序列的特异性结合，减少非特异性扩增的可能。	容易产生气溶胶：操作过程中可能产生气溶胶，增加交叉污染的风险。假阳性：由于引物设计的复杂性，有时可能导致假阳性结果。引物设计要求高：某些序列难以筛选到合适的引物。
RPA	反应温度：(37～42)℃ 反应时间短：通常0.5h内即可完成扩增。	成本较高：虽然不需要昂贵的热循环仪器，但RPA所需试剂盒酶成本较高。
RCA	特异性强：对SNP位点的识别具有极高的选择性。高通量分析：可实现多个目标DNA分子的同时扩增，形成高通量分析技术。	成本高：锁式探针的合成费用较高，尽管可以通过PCR反应来合成探针降低成本，但总体成本仍偏高。操作复杂：相较于LAMP和RPA，RCA的操作更为复杂，需要更高的实验技能。

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