{"title":"基于 RPA-CRISPR/Cas12a 的快速、超灵敏肺炎克雷伯菌检测双读出平台","authors":"Meiying Tan, Lina Liang, Chuan Liao, Zihan Zhou, Shaoping Long, Xueli Yi, Chunfang Wang, Caiheng Wei, Jinyuan Cai, Xuebin Li, Guijiang Wei","doi":"10.3389/fcimb.2024.1362513","DOIUrl":null,"url":null,"abstract":"The bacterium <jats:italic>Klebsiella pneumoniae</jats:italic> (<jats:italic>Kp</jats:italic>) was the primary pathogen of hospital-acquired infection, but the current detection method could not rapidly and conveniently identify <jats:italic>Kp</jats:italic>. Recombinase polymerase amplification (RPA) was a fast and convenient isothermal amplification technology, and the clustered regularly interspaced short palindromic repeats (CRISPR) system could rapidly amplify the signal of RPA and improve its limit of detection (LOD). In this study, we designed three pairs of RPA primers for the rcsA gene of <jats:italic>Kp</jats:italic>, amplified the RPA signal through single-strand DNA reporter cleavage by CRISPR/Cas12a, and finally analyzed the cleavage signal using fluorescence detection (FD) and lateral flow test strips (LFTS). Our results indicated that the RPA-CRISPR/Cas12a platform could specifically identify <jats:italic>Kp</jats:italic> from eleven common clinical pathogens. The LOD of FD and LFTS were 1 fg/μL and 10 fg/μL, respectively. In clinical sample testing, the RPA-CRISPR/Cas12a platform was consistent with the culture method and qPCR method, and its sensitivity and specificity were 100% (16/16) and 100% (9/9), respectively. With the advantages of detection speed, simplicity, and accuracy, the RPA-CRISPR/Cas12a platform was expected to be a convenient tool for the early clinical detection of <jats:italic>Kp</jats:italic>.","PeriodicalId":12458,"journal":{"name":"Frontiers in Cellular and Infection Microbiology","volume":"16 1","pages":""},"PeriodicalIF":4.8000,"publicationDate":"2024-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"A rapid and ultra-sensitive dual readout platform for Klebsiella pneumoniae detection based on RPA-CRISPR/Cas12a\",\"authors\":\"Meiying Tan, Lina Liang, Chuan Liao, Zihan Zhou, Shaoping Long, Xueli Yi, Chunfang Wang, Caiheng Wei, Jinyuan Cai, Xuebin Li, Guijiang Wei\",\"doi\":\"10.3389/fcimb.2024.1362513\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"The bacterium <jats:italic>Klebsiella pneumoniae</jats:italic> (<jats:italic>Kp</jats:italic>) was the primary pathogen of hospital-acquired infection, but the current detection method could not rapidly and conveniently identify <jats:italic>Kp</jats:italic>. Recombinase polymerase amplification (RPA) was a fast and convenient isothermal amplification technology, and the clustered regularly interspaced short palindromic repeats (CRISPR) system could rapidly amplify the signal of RPA and improve its limit of detection (LOD). In this study, we designed three pairs of RPA primers for the rcsA gene of <jats:italic>Kp</jats:italic>, amplified the RPA signal through single-strand DNA reporter cleavage by CRISPR/Cas12a, and finally analyzed the cleavage signal using fluorescence detection (FD) and lateral flow test strips (LFTS). Our results indicated that the RPA-CRISPR/Cas12a platform could specifically identify <jats:italic>Kp</jats:italic> from eleven common clinical pathogens. The LOD of FD and LFTS were 1 fg/μL and 10 fg/μL, respectively. In clinical sample testing, the RPA-CRISPR/Cas12a platform was consistent with the culture method and qPCR method, and its sensitivity and specificity were 100% (16/16) and 100% (9/9), respectively. With the advantages of detection speed, simplicity, and accuracy, the RPA-CRISPR/Cas12a platform was expected to be a convenient tool for the early clinical detection of <jats:italic>Kp</jats:italic>.\",\"PeriodicalId\":12458,\"journal\":{\"name\":\"Frontiers in Cellular and Infection Microbiology\",\"volume\":\"16 1\",\"pages\":\"\"},\"PeriodicalIF\":4.8000,\"publicationDate\":\"2024-06-27\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Frontiers in Cellular and Infection Microbiology\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.3389/fcimb.2024.1362513\",\"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":"Frontiers in Cellular and Infection Microbiology","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.3389/fcimb.2024.1362513","RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"IMMUNOLOGY","Score":null,"Total":0}
A rapid and ultra-sensitive dual readout platform for Klebsiella pneumoniae detection based on RPA-CRISPR/Cas12a
The bacterium Klebsiella pneumoniae (Kp) was the primary pathogen of hospital-acquired infection, but the current detection method could not rapidly and conveniently identify Kp. Recombinase polymerase amplification (RPA) was a fast and convenient isothermal amplification technology, and the clustered regularly interspaced short palindromic repeats (CRISPR) system could rapidly amplify the signal of RPA and improve its limit of detection (LOD). In this study, we designed three pairs of RPA primers for the rcsA gene of Kp, amplified the RPA signal through single-strand DNA reporter cleavage by CRISPR/Cas12a, and finally analyzed the cleavage signal using fluorescence detection (FD) and lateral flow test strips (LFTS). Our results indicated that the RPA-CRISPR/Cas12a platform could specifically identify Kp from eleven common clinical pathogens. The LOD of FD and LFTS were 1 fg/μL and 10 fg/μL, respectively. In clinical sample testing, the RPA-CRISPR/Cas12a platform was consistent with the culture method and qPCR method, and its sensitivity and specificity were 100% (16/16) and 100% (9/9), respectively. With the advantages of detection speed, simplicity, and accuracy, the RPA-CRISPR/Cas12a platform was expected to be a convenient tool for the early clinical detection of Kp.
期刊介绍:
Frontiers in Cellular and Infection Microbiology is a leading specialty journal, publishing rigorously peer-reviewed research across all pathogenic microorganisms and their interaction with their hosts. Chief Editor Yousef Abu Kwaik, University of Louisville is supported by an outstanding Editorial Board of international experts. This multidisciplinary open-access journal is at the forefront of disseminating and communicating scientific knowledge and impactful discoveries to researchers, academics, clinicians and the public worldwide.
Frontiers in Cellular and Infection Microbiology includes research on bacteria, fungi, parasites, viruses, endosymbionts, prions and all microbial pathogens as well as the microbiota and its effect on health and disease in various hosts. The research approaches include molecular microbiology, cellular microbiology, gene regulation, proteomics, signal transduction, pathogenic evolution, genomics, structural biology, and virulence factors as well as model hosts. Areas of research to counteract infectious agents by the host include the host innate and adaptive immune responses as well as metabolic restrictions to various pathogenic microorganisms, vaccine design and development against various pathogenic microorganisms, and the mechanisms of antibiotic resistance and its countermeasures.