Yaling Cao , Yuan Tian , Jing Huang , Ling Xu , Zihao Fan , Zhenzhen Pan , Sisi Chen , Yao Gao , Linlin Wei , Sujun Zheng , Xiangying Zhang , Yanhua Yu , Feng Ren
{"title":"CRISPR/ cas13辅助耐碳青霉烯肺炎克雷伯菌检测","authors":"Yaling Cao , Yuan Tian , Jing Huang , Ling Xu , Zihao Fan , Zhenzhen Pan , Sisi Chen , Yao Gao , Linlin Wei , Sujun Zheng , Xiangying Zhang , Yanhua Yu , Feng Ren","doi":"10.1016/j.jmii.2023.10.010","DOIUrl":null,"url":null,"abstract":"<div><h3>Background/Purpose</h3><p>Carbapenem-resistant <em>Klebsiella pneumoniae</em> (CRKP) is capable of causing serious community and hospital-acquired infections. However, currently, the identification of CRKP is complex and inefficient. Hence, this study aimed to develop methods for the early and effective identification of CRKP to allow reasonable antimicrobial therapy in a timely manner.</p></div><div><h3>Methods</h3><p><em>K. pneumoniae</em> (KP)-, <em>K. pneumoniae</em> carbapenemase (KPC)- and New Delhi metallo-β-lactamase (NDM)- specific CRISPR RNAs (crRNAs), polymerase chain reaction (PCR) primers and recombinase-aided amplification (RAA) primers were designed and screened in conserved sequence regions. We established fluorescence and lateral flow strip assays based on CRISPR/Cas13a combined with PCR and RAA, respectively, to assist in the detection of CRKP. Sixty-one clinical strains (including 51 CRKP strains and 10 carbapenem-sensitive strains) were collected for clinical validation.</p></div><div><h3>Results</h3><p>Using the PCR-CRISPR assay, the limit of detection (LOD) for KP and the blaKPC and blaNDM genes reached 1 copy/μL with the fluorescence signal readout. Using the RAA-CRISPR assay, the LOD could reach 10<sup>1</sup> copies/μL with both the fluorescence signal readout and the lateral flow strip readout. Additionally, the positivity rates of CRKP-positive samples detected by the PCR/RAA-CRISPR fluorescence and RAA-CRISPR lateral flow strip methods was 92.16% (47/51). The sensitivity and specificity reached 100% for KP and blaKPC and blaNDM gene detection. For detection in a simulated environmental sample, 1 CFU/cm<sup>2</sup> KP could be detected.</p></div><div><h3>Conclusion</h3><p>We established PCR/RAA-CRISPR assays for the detection of blaKPC and blaNDM carbapenemase genes, as well as KP, to facilitate the detection of CRKP.</p></div>","PeriodicalId":56117,"journal":{"name":"Journal of Microbiology Immunology and Infection","volume":"57 1","pages":"Pages 118-127"},"PeriodicalIF":4.5000,"publicationDate":"2024-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1684118223002001/pdfft?md5=fd891db6fca99d27517bdb4a7d05e2e9&pid=1-s2.0-S1684118223002001-main.pdf","citationCount":"0","resultStr":"{\"title\":\"CRISPR/Cas13-assisted carbapenem-resistant Klebsiella pneumoniae detection\",\"authors\":\"Yaling Cao , Yuan Tian , Jing Huang , Ling Xu , Zihao Fan , Zhenzhen Pan , Sisi Chen , Yao Gao , Linlin Wei , Sujun Zheng , Xiangying Zhang , Yanhua Yu , Feng Ren\",\"doi\":\"10.1016/j.jmii.2023.10.010\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><h3>Background/Purpose</h3><p>Carbapenem-resistant <em>Klebsiella pneumoniae</em> (CRKP) is capable of causing serious community and hospital-acquired infections. However, currently, the identification of CRKP is complex and inefficient. Hence, this study aimed to develop methods for the early and effective identification of CRKP to allow reasonable antimicrobial therapy in a timely manner.</p></div><div><h3>Methods</h3><p><em>K. pneumoniae</em> (KP)-, <em>K. pneumoniae</em> carbapenemase (KPC)- and New Delhi metallo-β-lactamase (NDM)- specific CRISPR RNAs (crRNAs), polymerase chain reaction (PCR) primers and recombinase-aided amplification (RAA) primers were designed and screened in conserved sequence regions. We established fluorescence and lateral flow strip assays based on CRISPR/Cas13a combined with PCR and RAA, respectively, to assist in the detection of CRKP. Sixty-one clinical strains (including 51 CRKP strains and 10 carbapenem-sensitive strains) were collected for clinical validation.</p></div><div><h3>Results</h3><p>Using the PCR-CRISPR assay, the limit of detection (LOD) for KP and the blaKPC and blaNDM genes reached 1 copy/μL with the fluorescence signal readout. Using the RAA-CRISPR assay, the LOD could reach 10<sup>1</sup> copies/μL with both the fluorescence signal readout and the lateral flow strip readout. Additionally, the positivity rates of CRKP-positive samples detected by the PCR/RAA-CRISPR fluorescence and RAA-CRISPR lateral flow strip methods was 92.16% (47/51). The sensitivity and specificity reached 100% for KP and blaKPC and blaNDM gene detection. For detection in a simulated environmental sample, 1 CFU/cm<sup>2</sup> KP could be detected.</p></div><div><h3>Conclusion</h3><p>We established PCR/RAA-CRISPR assays for the detection of blaKPC and blaNDM carbapenemase genes, as well as KP, to facilitate the detection of CRKP.</p></div>\",\"PeriodicalId\":56117,\"journal\":{\"name\":\"Journal of Microbiology Immunology and Infection\",\"volume\":\"57 1\",\"pages\":\"Pages 118-127\"},\"PeriodicalIF\":4.5000,\"publicationDate\":\"2024-02-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.sciencedirect.com/science/article/pii/S1684118223002001/pdfft?md5=fd891db6fca99d27517bdb4a7d05e2e9&pid=1-s2.0-S1684118223002001-main.pdf\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of Microbiology Immunology and Infection\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S1684118223002001\",\"RegionNum\":2,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"IMMUNOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Microbiology Immunology and Infection","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1684118223002001","RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"IMMUNOLOGY","Score":null,"Total":0}
Carbapenem-resistant Klebsiella pneumoniae (CRKP) is capable of causing serious community and hospital-acquired infections. However, currently, the identification of CRKP is complex and inefficient. Hence, this study aimed to develop methods for the early and effective identification of CRKP to allow reasonable antimicrobial therapy in a timely manner.
Methods
K. pneumoniae (KP)-, K. pneumoniae carbapenemase (KPC)- and New Delhi metallo-β-lactamase (NDM)- specific CRISPR RNAs (crRNAs), polymerase chain reaction (PCR) primers and recombinase-aided amplification (RAA) primers were designed and screened in conserved sequence regions. We established fluorescence and lateral flow strip assays based on CRISPR/Cas13a combined with PCR and RAA, respectively, to assist in the detection of CRKP. Sixty-one clinical strains (including 51 CRKP strains and 10 carbapenem-sensitive strains) were collected for clinical validation.
Results
Using the PCR-CRISPR assay, the limit of detection (LOD) for KP and the blaKPC and blaNDM genes reached 1 copy/μL with the fluorescence signal readout. Using the RAA-CRISPR assay, the LOD could reach 101 copies/μL with both the fluorescence signal readout and the lateral flow strip readout. Additionally, the positivity rates of CRKP-positive samples detected by the PCR/RAA-CRISPR fluorescence and RAA-CRISPR lateral flow strip methods was 92.16% (47/51). The sensitivity and specificity reached 100% for KP and blaKPC and blaNDM gene detection. For detection in a simulated environmental sample, 1 CFU/cm2 KP could be detected.
Conclusion
We established PCR/RAA-CRISPR assays for the detection of blaKPC and blaNDM carbapenemase genes, as well as KP, to facilitate the detection of CRKP.
期刊介绍：
Journal of Microbiology Immunology and Infection is an open access journal, committed to disseminating information on the latest trends and advances in microbiology, immunology, infectious diseases and parasitology. Article types considered include perspectives, review articles, original articles, brief reports and correspondence.
With the aim of promoting effective and accurate scientific information, an expert panel of referees constitutes the backbone of the peer-review process in evaluating the quality and content of manuscripts submitted for publication.
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