切换至 "中华医学电子期刊资源库"

中华临床实验室管理电子杂志 ›› 2025, Vol. 13 ›› Issue (03) : 166 -171. doi: 10.3877/cma.j.issn.2095-5820.2025.03.006

实验研究

基于RAA-CRISPR-Cas12a检测肺炎克雷伯菌KPC型碳青霉烯酶基因方法的建立及评价
胡春财1, 袁伟曦2, 赖少芬1, 魏楚洪1, 余高平1, 肖文玲1, 尹小毛3,()   
  1. 1 528315 广东 佛山,佛山市顺德区乐从医院检验科
    2 528000 广东 佛山,佛山市妇幼保健院新生儿疾病筛查中心
    3 510220 广东 广州,广州市红十字会医院检验科
  • 收稿日期:2024-10-17 出版日期:2025-08-28
  • 通信作者: 尹小毛
  • 基金资助:
    佛山市卫生健康局医学科研课题(20220311); 佛山市科学技术局科技创新项目(2220001004153)

Establishment and evaluation of a RAA-CRISPR-Cas12a method for detecting KPC type carbapenemase genes

Chuncai Hu1, Weixi Yuan2, Shaofen Lai1, Chuhong Wei1, Gaoping Yu1, Wenling Xiao1, Xiaomao Yin3,()   

  1. 1 Department of Clinical Laboratory, Lecong Hospital of Shunde, Foshan Guangdong 528315, China
    2 Newborn Disease Screening Center, Foshan Women and Children Hospital, Foshan Guangdong 528000, China
    3 Department of Clinical Laboratory, Guangzhou Red Cross Hospital, Guangzhou Guangdong 510220, China
  • Received:2024-10-17 Published:2025-08-28
  • Corresponding author: Xiaomao Yin
引用本文:

胡春财, 袁伟曦, 赖少芬, 魏楚洪, 余高平, 肖文玲, 尹小毛. 基于RAA-CRISPR-Cas12a检测肺炎克雷伯菌KPC型碳青霉烯酶基因方法的建立及评价[J/OL]. 中华临床实验室管理电子杂志, 2025, 13(03): 166-171.

Chuncai Hu, Weixi Yuan, Shaofen Lai, Chuhong Wei, Gaoping Yu, Wenling Xiao, Xiaomao Yin. Establishment and evaluation of a RAA-CRISPR-Cas12a method for detecting KPC type carbapenemase genes[J/OL]. Chinese Journal of Clinical Laboratory Management(Electronic Edition), 2025, 13(03): 166-171.

目的

建立一种基于重组酶介导等温扩增技术(RAA)-规律成簇的间隔短回文重复序列及其相关蛋白(CRISPR-Cas12a)快速检测肺炎克雷伯菌碳青霉烯酶(KPC)型碳青霉烯酶(blaKPC)基因的分子检测技术。

方法

根据blaKPC基因序列设计特异性RAA引物和CRISPR RNA(crRNA),构建基于RAA-CRISPR-Cas12a检测blaKPC基因的方法。收集2022至2023年佛山市顺德区乐从医院保存的4株携带blaKPC基因的肺炎克雷伯菌用于方法研究,40株肺炎克雷伯菌临床菌株用于方法评价,同时采用实时荧光定量PCR(qPCR)进行检测,比较两种方法的检出率和一致性。

结果

本研究建立的用以检测blaKPC基因的RAA-CRISPR-Cas12a方法的灵敏度可达到10拷贝/µl。同时采用RAA-CRISPR-Cas12a和qPCR方法检测40株肺炎克雷伯菌临床菌株,两种方法同时检出5株携带blaKPC基因,35株未携带blaKPC基因,以qPCR方法为金标准,本研究建立的方法敏感度为100%(5/5),灵敏度为100%(35/35),两种方法的符合率100%。

结论

建立的RAA-CRISPR-Cas12a方法为精准检测blaKPC基因提供了又一种技术手段,有助于临床快速筛选携带blaKPC基因的菌株。

Objective

To establish a rapid molecular detection method for Klebsiella pneumoniae carbapenemase (KPC) carbapenemase (blaKPC) genes based on recombinase aided amplification (RAA)-clusteredregularly interspaced short palindromic repeats (CRISPR)-CRISPR associated (Cas)12a (CRISPR-Cas12a) technology.

Methods

Specific RAA primers and CRISPR RNA(crRNA) were designed targeting the blaKPC gene sequence to construct a RAA-CRISPR-Cas12a detection assay. 4 strains of Klebsiella pneumoniae carrying the blaKPC gene stored in Lecong Hospital of Shunde, Foshan from 2022 to 2023 were collected for method research, and 40 clinical strains of Klebsiella pneumoniae for method evaluation. At the same time, quantitative real-time polymerase chain reactio (qPCR) method was performed in parallel to compare detection rates and concordance between the two methods.

Results

The RAA-CRISPR-Cas12a method achieved a sensitivity of 10 copies/µl for blaKPC gene detection. At the same time, RAA-CRISPR-Cas12a and qPCR methods were used to detect 40 clinical strains of Klebsiella pneumoniae. Both methods simultaneously detected 5 blaKPC-positive and 35 blaKPC-negative isolates. Using qPCR as the gold standard, the sensitivity of the method established in this study was 100% (5/5), and the sensitivity was 100% (35/35), with a 100% agreement rate between the two methods.

Conclusion

The established RAA-CRISPR-Cas12a method provides a reliable and efficient tool for blaKPC gene detection, facilitating rapid clinical screening of blaKPC gene.

表1 RAA引物、crRNA、qPCR和探针
图1 不同RAA引物在RAA-CRISPR-Cas12a方法中的检测
图2 RAA-CRISPR-Cas12a检测不同稀释浓度的blaKPC DNA模板 注:RAA-5~RAA-0分别表示105、104、103、102、10、1拷贝/µl的核酸浓度。
图3 RAA-CRISPR-Cas12a方法检测blaKPCblaNDMblaVIMblaIPMblaOXA-48
表2 2种方法检测40株肺炎克雷伯菌携带blaKPC基因/株
1
CHOBY J E, HOWARD-ANDERSON J, WEISS D S. Hypervirulent Klebsiella pneumoniae-clinical and molecular perspectives[J]. Journal of internal medicine, 2020, 287(3): 283-300.
2
CHANG D, SHARMA L, DELA CRUZ C S, et al. Clinical epidemiology, risk factors, and control strategies of Klebsiella pneumoniae infection[J]. Frontiers in microbiology, 2021, 12: 750662.
3
QIN X, DING L, HAO M, et al. Antimicrobial resistance of clinical bacterial isolates in China: Current status and trends[J]. JAC-antimicrobial resistance, 2024, 6(2): dlae052.
4
LUO Q, LU P, CHEN Y, et al. ESKAPE in China: Epidemiology and characteristics of antibiotic resistance[J]. Emerging microbes & infections, 2024, 13(1): 2317915.
5
LUTGRING J D. Carbapenem-resistantEnterobacteriaceae: An emerging bacterial threat[J]. Seminars in diagnostic pathology, 2019, 36(3): 182-186.
6
KARAISKOS I, GALANI I, PAPOUTSAKI V, et al. Carbapenemase producing Klebsiella pneumoniae: Implication on future therapeutic strategies[J]. Expert review of anti-infective therapy, 2022, 20(1): 53-69.
7
CANDAN E D, AKSÖZ N. Klebsiella pneumoniae: Characteristics of carbapenem resistance and virulence factors[J]. Acta biochimica polonica, 2015, 62(4): 867-874.
8
GAO H, LIU Y, WANG R, et al. The transferability and evolution of NDM-1 and KPC-2 co-producing Klebsiella pneumoniae from clinical settings[J]. EBioMedicine, 2020, 51: 102599.
9
KARAMPATAKIS T, ZARRAS C, PAPPA S, et al. Emergence of ST39 carbapenem-resistant Klebsiella pneumoniae producing VIM-1 and KPC-2[J]. Microbial pathogenesis, 2022, 162: 105373.
10
LIU J, YU J, CHEN F, et al. Emergence and establishment of KPC-2-producing ST11 Klebsiella pneumoniae in a general hospital in Shanghai, China[J]. European journal of clinical microbiology & infectious diseases: Official publication of the European society of Clinical Microbiology, 2018, 37(2): 293-299.
11
CHENG J, ZHAO D, MA X, et al. Molecular epidemiology, risk factors, and outcomes of carbapenem-resistant Klebsiella pneumoniae infection in a tertiary hospital in eastern China: For a retrospective study conducted over 4 years[J]. Frontiers in microbiology, 2023, 14: 1223138.
12
JIANG W, BIKARD D, COX D, et al. RNA-guided editing of bacterial genomes using CRISPR-Cas systems[J]. Nature biotechnology, 2013, 31(3): 233-239.
13
周红蕾, 程淑琴, 李佳鹏, 等. 猫杯状病毒RAA-CRISPR/Cas12a-LFS检测方法的建立及初步应用[J]. 中国预防兽医学报, 2023, 45(5): 494-501.
14
徐蛟, 王英丽, 王莹, 等. 基于RAA-CRISPR/Cas12a快速检测尼帕病毒方法的建立[J]. 中国动物检疫, 2023, 40(10): 95-99, 111.
15
林冬媛, 谢龙飞, 李芙蓉, 等. 基于CRISPR-Cas12a系统的鼠伤寒沙门氏菌可视化检测方法的建立[J]. 黑龙江畜牧兽医, 2024(8): 68-76.
16
苏璇, 葛以跃, 张倩, 等. CRISPR-Cas13a辅助RAA快速检测金黄色葡萄球菌的研究[J]. 中国病原生物学杂志, 2020, 15(3): 253-258.
17
李婷, 刘燕红, 赵松, 等. 重组酶介导的核酸等温扩增荧光法快速检测日本血吸虫感染性钉螺[J]. 中国血吸虫病防治杂志, 2019, 31(2): 109-114, 120.
18
章太婵, 车玉传, 梁雪雁, 等. RT-RAA联合CRISPR/Cas12a快速检测新型冠状病毒方法的建立与评价[J]. 临床检验杂志, 2024, 42(4): 246-251.
19
LI D, HUANG X, RAO H, et al. Klebsiella pneumoniae bacteremia mortality: A systematic review and meta-analysis[J]. Frontiers in cellular and infection microbiology, 2023, 13: 1157010.
20
GIACOBBE D R, MARELLI C, CATTARDICO G, et al. Mortality in KPC-producing Klebsiella pneumoniae bloodstream infections: A changing landscape[J]. The journal of antimicrobial chemotherapy, 2023, 78(10): 2505-2514.
21
SUN X, ZOU X, ZHOU B, et al. Comparison of bloodstream and non-bloodstream infections caused by carbapenem-resistant Klebsiella pneumoniae in the intensive care unit: A 9-year respective study[J]. Frontiers in medicine, 2023, 10: 1230721.
22
PU D, ZHAO J, CHANG K, et al. "Superbugs" with hypervirulence and carbapenem resistance in Klebsiella pneumoniae: The rise of such emerging nosocomial pathogens in China[J]. Science bulletin, 2023, 68(21): 2658-2670.
23
YANG X, SUN Q, LI J, et al. Molecular epidemiology of carbapenem-resistant hypervirulent Klebsiella pneumoniae in China[J]. Emerging microbes & infections, 2022, 11(1): 841-849.
24
LIAO W, LIU Y, ZHANG W. Virulence evolution, molecular mechanisms of resistance and prevalence of ST11 carbapenem-resistant Klebsiella pneumoniae in China: A review over the last 10 years[J]. Journal of global antimicrobial resistance, 2020, 23: 174-180.
25
HU Y, LIU C, SHEN Z, et al. Prevalence, risk factors and molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae in patients from Zhejiang, China, 2008-2018[J]. Emerging microbes & infections, 2020, 9(1): 1771-1779.
26
POTRON A, FOURNIER D, EMERAUD C, et al. Evaluation of the immunochromatographic NG-Test Carba 5 for rapid identification of carbapenemase in nonfermenters[J]. Antimicrob agents chemother, 2019, 63(9): e00968-19.
27
HONG J S, KIM D, YOON E J, et al. Performance evaluation of the PANA RealTyper™ CRE Kit for detecting carbapenemase genes in Gram-negative bacilli[J]. Journal of global antimicrobial resistance, 2019, 18: 100-103.
28
HUANG Y, LI J, WANG Q, et al. Rapid detection of KPC-producing Klebsiella pneumoniae in China based on MALDI-TOF MS[J]. Journal of microbiological methods, 2022, 192: 106385.
29
曹亚玲, 田原, 范子豪, 等. 基于RAA-CRISPR-Cas13a检测KPC型碳青霉烯酶基因方法的建立及评价[J]. 中华检验医学杂志, 2024, 47(2): 159-164.
30
XU H, TANG H, LI R, et al. A new method based on LAMP-CRISPR-Cas12a-Lateral flow immunochromatographic strip for detection[J]. Infection and drug resistance, 2022, 15: 685-696.
[1] 曹亚玲, 房忠军, 徐玲, 姜彬彬, 张向颖, 黄晶, 任锋. 基于重组酶介导等温扩增技术-规律间隔成簇短回文重复序列/相关蛋白系统检测肺炎克雷伯菌方法的建立及评价[J/OL]. 中华实验和临床感染病杂志(电子版), 2025, 19(02): 70-76.
[2] 宋江勤, 向健, 杨昊, 周锦. 荧光定量PCR技术检测肺外结核患者样本临床应用[J/OL]. 中华实验和临床感染病杂志(电子版), 2018, 12(03): 251-255.
[3] 中国医师协会结直肠肿瘤专业委员会免疫学组. 结直肠癌免疫治疗专家共识(2025版)[J/OL]. 中华结直肠疾病电子杂志, 2025, 14(04): 289-298.
[4] 王雯邈, 董林, 李文斌, 邹霜梅, 吕宁. Lynch综合征相关结直肠癌的遗传基因及分子病理筛查策略[J/OL]. 中华结直肠疾病电子杂志, 2018, 07(02): 176-180.
[5] 李志强, 余娟平, 杨军, 李晓华. 基于分子检测技术的实验室质量改进与管理[J/OL]. 中华临床实验室管理电子杂志, 2018, 06(04): 240-244.
阅读次数
全文


摘要


AI


AI小编
你好!我是《中华医学电子期刊资源库》AI小编,有什么可以帮您的吗?