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
- 文章访问数: 67
- HTML全文浏览量: 41
- PDF下载量: 4
- 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

下载: 全尺寸图片幻灯片

图 2 RPA扩增^[33]

Figure 2. RPA amplification^[33]

下载: 全尺寸图片幻灯片

图 3 定量RPA ^[33]

Figure 3. Quantitative RPA^[33]

下载: 全尺寸图片幻灯片

图 4 MNAzyme-GNP平台 ^[38]

Figure 4. MNAzyme-GNP platform^[38]

下载: 全尺寸图片幻灯片

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

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

下载: 全尺寸图片幻灯片

图 6 滚环扩增 ^[42]

Figure 6. rolling circle amplification^[42]

下载: 全尺寸图片幻灯片

图 7 i-CoRi平台 ^[43]

Figure 7. i-CoRi platform^[43]

下载: 全尺寸图片幻灯片

图 8 RCA-FLA平台 ^[44]

Figure 8. RCA-FLA platform^[44]

下载: 全尺寸图片幻灯片

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。

下载: 导出CSV

参考文献(44)

[1]	王家琪, 吴佳怡, 林建原, 等. 基于卵黄抗体-金属有机框架复合探针的电化学免疫传感器快速检测金黄色葡萄球菌[J]. 分析化学, 2021, 49(2): 197-206.
[2]	MAHJABEEN F, SAHA U, MOSTAFA MN, et al. An update on treatment options for Methicillin-Resistant Staphylococcus aureus (MRSA) bacteremia: a systematic review[J]. Cureus, 2022, 14(11): e31486.
[3]	SHAHIN IG, MOHAMED KO, TAHER AT, et al. The anti-MRSA activity of phenylthiazoles: a comprehensive review[J]. Curr. Pharm. Des., 2022, 28(43): 3469-3477. doi: 10.2174/1381612829666221124112006
[4]	BASSETTI M, LABATE L, MELCHIO M, et al. Current pharmacotherapy for methicillin-resistant Staphylococcus aureus (MRSA) pneumonia[J]. Expert Opin. Pharmacother., 2022, 23(3): 361-375. doi: 10.1080/14656566.2021.2010706
[5]	BROWN NM, GOODMAN AL, HORNER C, et al. Treatment of methicillin-resistant Staphylococcus aureus (MRSA): updated guidelines from the UK[J]. JAC Antimicrob. Resist., 2021, 3(1): dlaa114. doi: 10.1093/jacamr/dlaa114
[6]	ANDIE SL, HERMÍNIA L, JAVIER G, et al. Methicillin-resistant Staphylococcus aureus[J]. Nat. Rev. Dis. Primers, 2018, 4: 18034. doi: 10.1038/nrdp.2018.34
[7]	NOBLE WC. Antibiotic resistance in the staphylococci[J]. Sci. , Prog., 1997, 80: 5-20.
[8]	HONG J, ENSOM MHH, LAU TTY. What is the evidence for co-trimoxazole, clindamycin, doxycycline, and minocycline in the treatment of Methicillin-Resistant Staphylococcus aureus (MRSA) pneumonia? [J] Ann. Pharmacother. , 2019, 53(11): 1153-1161.
[9]	CONG Y, YANG S, RAO X. Vancomycin resistant Staphylococcus aureus infections: a review of case updating and clinical features[J]. J. Adv. Res., 2019, 21: 169-176.
[10]	MCGUINNESS WA, MALACHOWA N, DELEO FR. Vancomycin resistance in Staphylococcus aureus [J]. Yale J. Biol. Med., 2017, 90(2): 269-281.
[11]	ITO T, KATAYAMA Y, HIRAMATSU K. Cloning and nucleotide sequence determination of the entire mec DNA of pre-methicillin-resistant Staphylococcus aureus N315 [J]. Antimicrob. Agents Chemother., 1999, 43(6): 1449-1458. doi: 10.1128/AAC.43.6.1449
[12]	MLYNARCZYK-BONIKOWSKA B, KOWALEWSKI C, KROLAK-ULINSKA A, et al. Molecular mechanisms of drug resistance in Staphylococcus aureus[J]. Int. J. Mol. Sci., 2022, 23(15): 8088. doi: 10.3390/ijms23158088
[13]	HANSSEN AM, ERICSON SOLLID JU. SCCmec in staphylococci: genes on the move[J]. FEMS Immunol. Med. Microbiol., 2006, 46(1): 8-20. doi: 10.1111/j.1574-695X.2005.00009.x
[14]	PEACOCK SJ, PATERSON GK. Mechanisms of methicillin resistance in Staphylococcus aureus[J]. Annu. Rev. Biochem., 2015, 84: 577-601. doi: 10.1146/annurev-biochem-060614-034516
[15]	LINDEN M, RUTSCHMANN J, MAURER P, et al. PBP2a in β-lactam-resistant laboratory mutants and clinical isolates: disruption versus reduced penicillin affinity[J]. Microb. Drug Resist., 2018, 24(6): 718-731. doi: 10.1089/mdr.2017.0302
[16]	ZAPUN A, CONTRERAS-MARTEL C, VERNET T. Penicillin-binding proteins and beta-lactam resistance[J]. FEMS Microbiol Rev., 2008, 32(2): 361-85. doi: 10.1111/j.1574-6976.2007.00095.x
[17]	MIYACHIRO MM, CONTRERAS-MARTEL C, DESSEN A. Penicillin-binding proteins (PBPs) and bacterial cell wall elongation complexes[J]. Subcell Biochem., 2019, 93: 273-289.
[18]	HASSOUN A, LINDEN PK, FRIEDMAN B. Incidence, prevalence, and management of MRSA bacteremia across patient populations-a review of recent developments in MRSA management and treatment[J]. Crit. Care., 2017, 21(1): 211. doi: 10.1186/s13054-017-1801-3
[19]	PRESTINACI F, PEZZOTTI P, PANTOSTI A. Antimicrobial resistance: a global multifaceted phenomenon[J]. Glob. Health., 2015, 109(7): 309-318.
[20]	DUIN D, PATERSON DL. Multidrug-resistant bacteria in the community: an update[J]. Infect Dis. Clin. North Am., 2020, 34(4): 709-722. doi: 10.1016/j.idc.2020.08.002
[21]	宋晓超, 乔美珍, 丁蔚, 等. 2020-2022年苏州市51所医院区域性医院感染监测平台多药耐药菌数据分析[J]. 中华医院感染学杂志, 2023, 33(21): 3207-3212.
[22]	TRISTAN A, BES M, MEUGNIER H, et al. Global distribution of panton-valentine leukocidin--positive methicillin-resistant Staphylococcus aureus, 2006 [J]. Emerg. Infect. Dis. , 2007, 13(4): 594-600.
[23]	BODULEV OL, SAKHAROV IY. Isothermal nucleic acid amplification techniques and their use in bioanalysis[J]. Biochemistry (Mosc)., 2020, 85(2): 147-166. doi: 10.1134/S0006297920020030
[24]	MAYBORODA O, KATAKIS I, O'SULLIVAN CK. Multiplexed isothermal nucleic acid amplification[J]. Anal. Biochem., 2018, 545: 20-30. doi: 10.1016/j.ab.2018.01.005
[25]	ZHANG C, BELWAL T, LUO Z, et al. Application of nanomaterials in isothermal nucleic acid amplification[J]. Small., 2022, 18(6): e2102711. doi: 10.1002/smll.202102711
[26]	SOROKA M, WASOWICZ B, RYMASZEWSKA A. Loop-mediated isothermal amplification (LAMP): the better sibling of pcr [J]? Cells. , 2021, 10(8): 1931.
[27]	WONG YP, OTHMAN S, LAU YL, et al. Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms[J]. J. Appl. Microbiol., 2018, 124(3): 626-643. doi: 10.1111/jam.13647
[28]	NOTOMI T, OKAYAMA H, MASUBUCHI H, et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Res., 2000, 28(12): E63. doi: 10.1093/nar/28.12.e63
[29]	KOIDE Y, MAEDA H, YAMABE K, et al. Rapid detection of mecA and spa by the loop-mediated isothermal amplification (LAMP) method[J]. Lett. Appl. Microbiol., 2010, 50(4): 386-392. doi: 10.1111/j.1472-765X.2010.02806.x
[30]	WANG XR, WU LF, WANG Y, et al. Rapid detection of Staphylococcus aureus by loop-mediated isothermal amplification [J]. Appl. Biochem. Biotechnol. 2015, 175(2): 882-891.
[31]	WU C, ZENG Y, HE Y. Rapid visualization and detection of Staphylococcus aureus based on loop-mediated isothermal amplification[J]. World J. Microbiol Biotechnol., 2021, 37(12): 209. doi: 10.1007/s11274-021-03178-0
[32]	MENG X, ZHANG G, SUN B, et al. Rapid detection of mecA and femA genes by loop-mediated isothermal amplification in a microfluidic system for discrimination of different Staphylococcal species and prediction of methicillin resistance [J]. Front Microbiol. 2020, 11: 1487.
[33]	TAN M, LIAO C, LIANG L, et al. Recent advances in recombinase polymerase amplification: principle, advantages, disadvantages and applications [J]. Front Cell Infect Microbiol. 2022, 12: 1019071.
[34]	LOBATO IM, O'SULLIVAN CK. Recombinase polymerase amplification: Basics, applications and recent advances[J]. Trends Analyt Chem., 2018, 98: 19-35. doi: 10.1016/j.trac.2017.10.015
[35]	MUNAWAR MA. Critical insight into recombinase polymerase amplification technology [J]. Expert Rev. Mol. Diagn. 2022, 22(7): 725-737.
[36]	PIEPENBURG O, WILLIAMS CH, STEMPLE DL, et al. DNA detection using recombination proteins[J]. PLoS Biol., 2006, 4(7): e204. doi: 10.1371/journal.pbio.0040204
[37]	LUTZ S, WEBER P, FOCKE M, et al. Microfluidic lab-on-a-foil for nucleic acid analysis based on isothermal recombinase polymerase amplification (RPA) [J]. Lab Chip. 2010, 10(7): 887-93.
[38]	ABDOU MOHAMED MA, KOZLOWSKI HN, KIM J, et al. Diagnosing antibiotic resistance using nucleic acid enzymes and gold nanoparticles[J]. ACS Nano., 2021, 15(6): 9379-9390. doi: 10.1021/acsnano.0c09902
[39]	LI Y, SHI Z, HU A, et al. Rapid one-tube RPA-CRISPR/Cas12 detection platform for methicillin-resistant Staphylococcus aureus. Ddiagnostics (basel) [J]. 2022, 12(4): 829.
[40]	YAO C, ZHANG R, TANG J, et al. Rolling circle amplification (RCA)-based DNA hydrogel[J]. Nat. Protoc., 2021, 16(12): 5460-5483. doi: 10.1038/s41596-021-00621-2
[41]	XU L, DUAN J, CHEN J, et al. Recent advances in rolling circle amplification-based biosensing strategies-a review[J]. Anal. Chim. Acta., 2021, 1148: 238187. doi: 10.1016/j.aca.2020.12.062
[42]	GOO NI, KIM DE. Rolling circle amplification as isothermal gene amplification in molecular diagnostics [J]. Biochip J. 2016, 10(4): 262-271.
[43]	KANG YK, IM SH, RYU JS, et al. Simple visualized readout of suppressed coffee ring patterns for rapid and isothermal genetic testing of antibacterial resistance [J]. Biosens. Bioelectron. 2020, 168: 112566.
[44]	LEE HN, LEE J, KANG YK, et al. A lateral flow assay for nucleic acid detection based on rolling circle amplification using capture ligand-modified oligonucleotides [J]. Biochip J. 2022, 16(4): 441-450.