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中华实验和临床感染病杂志(电子版) ›› 2025, Vol. 19 ›› Issue (02) : 70 -76. doi: 10.3877/cma.j.issn.1674-1358.2025.02.002

论著

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
曹亚玲1, 房忠军2, 徐玲1, 姜彬彬3, 张向颖1, 黄晶4, 任锋1,()   
  1. 1. 100069 北京,首都医科大学附属北京佑安医院/北京肝病研究所
    2. 100069 北京,首都医科大学附属北京佑安医院检验科
    3. 102600 北京,北京市大兴区黄村医院检验科
    4. 100730 北京,中国医学科学院北京协和医院医院感染管理处
  • 收稿日期:2024-09-27 出版日期:2025-04-15
  • 通信作者: 任锋
  • 基金资助:
    国家自然科学基金(No. 82002243)北京自然科学基金和北京市教委联合资助重点项目(No.KZ202010025035)北京市自然科学基金-昌平创新联合基金(No. L234046)北京市医院管理中心“青苗”计划专项(No. QML20201702)北京市医管局“登峰”人才计划(No. DFL20221503)高层次公共卫生技术人才建设项目(No. 学科带头人-02-13)

Establishment and evaluation of a recombinase aided amplification-clustered regularly interspaced short palindromic repeats/associated protein system for detecting Klebsiella pneumoniae

Yaling Cao1, Zhongjun Fang2, Ling Xu1, Binbin Jiang3, Xiangying Zhang1, Jing Huang4, Feng Ren1,()   

  1. 1. Beijing Institute of Hepatology/Beijing Youan Hospital, Capital Medical University, Beijing 100069, China
    2. Department of Clinical Laboratory, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China
    3. Department of Clinical Laboratory, Beijing Daxing District Huangcun Hospital, Beijing 102600, China
    4. Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Department of Infection Control, Beijing 100730, China
  • Received:2024-09-27 Published:2025-04-15
  • Corresponding author: Feng Ren
引用本文:

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.

Yaling Cao, Zhongjun Fang, Ling Xu, Binbin Jiang, Xiangying Zhang, Jing Huang, Feng Ren. Establishment and evaluation of a recombinase aided amplification-clustered regularly interspaced short palindromic repeats/associated protein system for detecting Klebsiella pneumoniae[J/OL]. Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition), 2025, 19(02): 70-76.

目的

建立基于重组酶介导等温扩增技术(RAA)-规律间隔成簇短回文重复序列/相关蛋白(CRISPR-Cas13a)系统检测肺炎克雷伯菌(KP)的方法。

方法

设计筛选检测KP特异的CRISPR RNA(crRNA)和RAA引物对,建立基于RAA-CRISPR-Cas13a技术快速准确检测KP的方法。RAA-CRISPR系统和荧光定量PCR(RT-qPCR)方法比较使用合成的KP质粒。从铜绿假单胞菌、鲍曼不动杆菌、大肠埃希菌和肺炎链球菌临床菌株中提取基因组DNA,进行RAA-CRISPR检测,评估该方法的特异性。收集2023年4月至2024年3月中国医学科学院北京协和医院50份临床样本(包含30份细菌培养KP阳性样本和20份KP阴性样本),分别使用RAA-CRISPR和RT-qPCR方法进行KP检测,比较两种方法的灵敏度、阳性符合率和阴性符合率。结果数据应用方差分析和配对t检验方法进行分析。

结果

使用KP阳性质粒筛选出检测KP效果最佳的crRNA和RAA引物,建立了CRISPR-Cas13a检测KP的方法。CRISPR-Cas13a检测KP质粒的灵敏度可达1拷贝/μl,高于RT-qPCR(10拷贝/μl);特异性评价结果显示,未与非靶标菌株产生交叉反应,提示CRISPR-Cas13a系统特异性较好。样本检测以细菌培养为金标准,RAA-CRISPR和RT-qPCR两种方法的灵敏度分别为100%(30/30)和83.3%(25/30);两种检测方法的阳性符合率分别为100%(30/30)和83.3%(25/30),阴性符合率均为100%。

结论

本研究将RAA扩增技术与CRISPR-Cas13a技术相结合,建立了准确检测KP的方法,该方法可精准检测KP,有助于临床诊断KP感染以进行及时有效地治疗。

Objective

To establish a method for detection of Klebsiella pneumoniae (KP) based on recombinase aided amplification (RAA)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (CRISPR-Cas13a) system.

Methods

CrRNA and RAA primer pairs were designed and screened for the detection of KP gene, while a rapid and accurate method for the detection of KP gene was developed based on RAA-CRISPR-Cas13a technology. The RAA-CRISPR and real time fluorescence quantitative PCR (RT-qPCR) methods were compared by the synthesized KP plasmid. Genomic DNA was extracted from clinical strains of Pseudomonas aeruginosaAcinetobacter baumanniiEscherichia coli and Streptococcus pneumoniae for RAA-CRISPR detection to evaluate the specificity of this method.Total of 50 clinical samples (including 30 KP culture-positive samples and 20 KP-negative samples) collected from Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, from April 2023 to March 2024 were detected for KP through both RAA-CRISPR and RT-qPCR. The sensitivity, positive agreement rate and negative agreement rate of both methods were compared, respectively. Statistical analysis of data was performed by ANOVA and paired t-tests.

Results

The optimal crRNA and RAA primers for KP detection were selected by KP-positive plasmids, and CRISPR-Cas13a-based KP detection method was established successfully. The detection sensitivity of CRISPR-Cas13a for KP plasmids reached 1 copy/μl, higher than that of RT-qPCR (10 copies/μl). The specific evaluation results showed that there was no cross-reactivity with nontarget strains, indicating that CRISPR-Cas13a system was specific. Among the sample detection, the sensitivity of RAA-CRISPR and RT-qPCR were 100% (30/30) and 83.3% (25/30), respectively, using bacterial culture and mass spectrometry technology as the gold standard. The positive concordance rate of both detection were 100% (30/30) and 83.3% (25/30), and the negative concordance rate was 100%.

Conclusions

A method was established to accurately detect KP gene by RAA amplification technology and CRISPR-Cas13a system. This method can accurately detect KP gene, which can help diagnose KP infection for timely and effective treatment.

图1 RAA引物筛选 注:在检测进行60 min时,RAA-2引物对的荧光值最高。每对引物进行3次重复,**P < 0.01、***P < 0.001,NC为阴性对照(以水为模版进行RAA-CRISPR系统检测)
图2 crRNA的筛选 注:在检测进行30 min时,crRNA1荧光值最高。每个crRNA检测进行3次重复,***P < 0.001,NC:阴性对照(以水为模板进行RAA-CRISPR检测)
表1 CRISPR-Cas13a系统和 RT-qPCR方法检测KP阳性质粒灵敏度(±s
图3 RAA-CRISPR系统检测KP的特异性 注:KP:肺炎克雷伯菌;PA:铜绿假单胞菌;AB:鲍曼不动杆菌;E. coli:大肠埃希菌;SP:肺炎链球菌;NC:阴性对照(以水为模板进行RAA-CRISPR检测)。***P < 0.001;ns:P >0.05,差异无统计学意义
表2 CRISPR-Cas13a系统和 RT-qPCR方法检测50份临床标本中KP
图4 RAA-CRISPR系统对临床样本中的KP检测 注:1~30为KP阳性样本(1~10为痰液样本,11~28为血液样本,29~30为脑脊液样本),31~50为KP阴性菌株
[1]
Liu C, Dong N, Huang X, et al. Emergence of the clinical rdar morphotype carbapenem-resistant and hypervirulent Klebsiella pneumoniae with enhanced adaption to hospital environment[J]. Sci Total Environ,2023,889:164302.
[2]
Yang X, Dong N, Chan EW, et al. Carbapenem resistance-encoding and virulence-encoding conjugative plasmids in Klebsiella pneumoniae[J]. Trends Microbiol,2021,29(1):65-83.
[3]
Li ZJ, Zhang HY, Ren LL, et al. Etiological and epidemiological features of acute respiratory infections in China[J]. Nat Commun,2021,12(1):5026.
[4]
Karampatakis T, Tsergouli K, Behzadi P. Carbapenem-resistant Klebsiella pneumoniae: virulence factors, molecular epidemiology and latest updates in treatment options[J]. Antibiotics (Basel),2023,12(2):234.
[5]
Pu D, Zhao J, Lu B, et al. Within-host resistance evolution of a fatal ST11 hypervirulent carbapenem-resistant Klebsiella pneumoniae[J].Int J Antimicrob Agents,2023,61(4):106747.
[6]
Mukherjee S, Mitra S, Dutta S, et al. Neonatal sepsis: The impact of carbapenem-resistant and hypervirulent Klebsiella pneumoniae[J].Front Med (Lausanne),2021,8:634349.
[7]
Yang X, Sun Q, Li J, et al. Molecular epidemiology of carbapenemresistant hypervirulent Klebsiella pneumoniae in China[J]. Emerg Microbes Infect,2022,11(1):841-849.
[8]
Jin X, Chen Q, Shen F, et al. Resistance evolution of hypervirulent carbapenem-resistant Klebsiella pneumoniae ST11 during treatment with tigecycline and polymyxin[J]. Emerg Microbes Infect,2021,10(1):1129-1136.
[9]
Gootenberg JS, Abudayyeh OO, Lee JW, et al. Nucleic acid detection with CRISPR-Cas13a/C2c2[J]. Science,2017,356(6336):438-442.
[10]
Kellner MJ, Koob JG, Gootenberg JS, et al. SHERLOCK: nucleic acid detection with CRISPR nucleases[J]. Nat Protoc,2019,14(10):2986-3012.
[11]
Kaminski MM, Abudayyeh OO, Gootenberg JS, et al. CRISPR-based diagnostics[J]. Nat Biomed Eng,2021,5(7):643-656.
[12]
Chen JS, Ma E, Harrington LB, et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity[J].Science,2018,360(6387):436-439.
[13]
Dong D, Liu W, Li H, et al. Survey and rapid detection of Klebsiella pneumoniae in clinical samples targeting the rcsA gene in Beijing,China[J]. Front Microbiol,2015,6:519.
[14]
Hartman LJ, Selby EB, Whitehouse CA, et al. Rapid real-time PCR assays for detection of Klebsiella pneumoniae with the rmpA or magA genes associated with the hypermucoviscosity phenotype: screening of nonhuman primates[J]. J Mol Diagn,2009,11(5):464-471.
[15]
Huang Y, Li J, Wang Q, et al. Rapid detection of KPC-producing Klebsiella pneumoniae in China based on MALDI-TOF MS[J]. J Microbiol Methods,2022,192:106385.
[16]
Wang J, Xia C, Wu Y, et al. Rapid detection of carbapenem-resistant Klebsiella pneumoniae using machine learning and MALDI-TOF MS platform[J]. Infect Drug Resist,2022,15:3703-3710.
[17]
Kurupati P, Chow C, Kumarasinghe G, et al. Rapid detection of Klebsiella pneumoniae from blood culture bottles by real-time PCR[J]. J Clin Microbiol,2004,42(3):1337-1340.
[18]
Feng J, Cui X, Du B, et al. Detection and quantification of Klebsiella pneumoniae in fecal samples using digital droplet PCR in comparison with real-time PCR[J]. Microbiol Spectr,2023,11(4):e0424922.
[19]
Qiu X, Liu X, Ma X, et al. One-pot isothermal LAMP-CRISPRbased assay for Klebsiella pneumoniae detection[J]. Microbiol Spectr,2022,10(4):e0154522.
[20]
Tominaga T. Rapid detection of Klebsiella pneumoniae Klebsiella oxytocaRaoultella ornithinolytica and other related bacteria in food by lateral-flow test strip immunoassays[J]. J Microbiol Methods,2018,147:43-49.
[21]
Eisenstein M. Seven technologies to watch in 2022[J].Nature,2022,601(7894):658-661.
[22]
Abudayyeh OO, Gootenberg JS, Konermann S, et al. C2c2 is a single component programmable RNA-guided RNA-targeting CRISPR effector[J]. Science,2016,353(6299):f5573.
[23]
Gootenberg JS, Abudayyeh OO, Kellner MJ, et al. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6[J]. Science,2018,360(6387):439-444.
[24]
王俊文, 田原, 范子豪, 等. 基于规律成簇的间隔短回文重复序列及其相关蛋白技术检测乙型肝炎病毒共价闭合环状DNA方法的建立[J/CD].中华实验和临床感染病杂志(电子版),2022,16(5):320-327.
[25]
Tan M, Liang L, Liao C, et al. A rapid and ultra-sensitive dual readout platform for Klebsiella pneumoniae detection based on RPA-CRISPR/Cas12a[J]. Front Cell Infect Microbiol,2024,14:1362513.
[26]
Liu Y, Xu H, Liu C, et al. CRISPR-Cas13a nanomachine based simple technology for avian influenza A (H7N9) virus on-site detection[J]. J Biomed Nanotechnol,2019,15(4):790-798.
[27]
Zou Y, Mason MG, Wang Y, et al. Nucleic acid purification from plants, animals, and microbes in under 30 seconds[J]. PLoS Biol,2017,15(11):e2003916.
[28]
Wang Y, Ke Y, Liu W, et al. A one-pot toolbox based on Cas12a/crRNA enables rapid foodborne pathogen detection at attomolar level[J]. ACS Sens,2020,5(5):1427-1435.
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