{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T01:08:53Z","timestamp":1790212133608,"version":"4.1.0"},"reference-count":42,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2023,2,9]],"date-time":"2023-02-09T00:00:00Z","timestamp":1675900800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["81802741"],"award-info":[{"award-number":["81802741"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:sec><jats:title>Introduction<\/jats:title><jats:p><jats:italic>Klebsiella pneumonia<\/jats:italic> (<jats:italic>K. pneumonia<\/jats:italic>) is a Gram-negative bacterium that opportunistically causes nosocomial infections in the lung, bloodstream, and urinary tract. Extended-spectrum \u03b2-Lactamases (ESBLs)-expressed <jats:italic>K. pneumonia<\/jats:italic> strains are widely reported to cause antibiotic resistance and therapy failure. Therefore, early identification of K. pneumonia, especially ESBL-positive strains, is essential in preventing severe infections. However, clinical detection of <jats:italic>K. pneumonia<\/jats:italic> requires a time-consuming process in agar disk diffusion. Nucleic acid detection, like qPCR, is precise but requires expensive equipment. Recent research reveals that collateral cleavage activity of CRISPR-LbCas12a has been applied in nucleic acid detection, and the unique testing model can accommodate various testing models.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>This study established a system that combined PCR with CRISPR-LbCas12a targeting the <jats:italic>K. pneumoniae<\/jats:italic> system. Additionally, this study summarized the antibiotic-resistant information of the past five years\u2019 <jats:italic>K. pneumoniae<\/jats:italic> clinic cases in Luohu Hospital and found that the ESBL-positive strains were growing. This study then designs a crRNA that targets <jats:italic>SHV<\/jats:italic> to detect ESBL-resistant <jats:italic>K. pneumoniae<\/jats:italic>. This work is to detect <jats:italic>K. pneumoniae<\/jats:italic> and ESBL-positive strains\u2019 nucleic acid using CRISPR-Cas12 technology. We compared PCR-LbCas12 workflow with PCR and qPCR techniques.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results and Discussion<\/jats:title><jats:p>This system showed excellent detection specificity and sensitivity in both bench work and clinical samples. Due to its advantages, its application can meet different detection requirements in health centers where qPCR is not accessible. The antibiotic-resistant information is valuable for further research.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fmicb.2023.1128261","type":"journal-article","created":{"date-parts":[[2023,2,9]],"date-time":"2023-02-09T11:37:45Z","timestamp":1675942665000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":10,"title":["Detection of Klebsiella pneumonia DNA and ESBL positive strains by PCR-based CRISPR-LbCas12a system"],"prefix":"10.3389","volume":"14","author":[{"given":"Shang","family":"Wang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shan","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ying","family":"Tang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guoyu","family":"Peng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tongyu","family":"Hao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xincheng","family":"Wu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiehong","family":"Wei","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinying","family":"Qiu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dewang","family":"Zhou","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shimao","family":"Zhu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuqing","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Song","family":"Wu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2023,2,9]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"198129","DOI":"10.1016\/j.virusres.2020.198129","article-title":"iSCAN: an RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2","volume":"288","author":"Ali","year":"2020","journal-title":"Virus Res."},{"key":"ref2","doi-asserted-by":"publisher","first-page":"548656","DOI":"10.1155\/2014\/548656","article-title":"Study of a natural mutant SHV-type beta-lactamase, SHV-104, from Klebsiella pneumoniae","volume":"2014","author":"Ben Achour","year":"2014","journal-title":"Int. J. Microbiol."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"a025247","DOI":"10.1101\/cshperspect.a025247","article-title":"Beta-lactams and beta-lactamase inhibitors: an overview","volume":"6","author":"Bush","year":"2016","journal-title":"Cold Spring Harb. Perspect. Med."},{"key":"ref4","doi-asserted-by":"publisher","first-page":"743","DOI":"10.1093\/cid\/ciq238","article-title":"Foodborne nosocomial outbreak of SHV1 and CTX-M-15-producing Klebsiella pneumoniae: epidemiology and control","volume":"52","author":"Calbo","year":"2011","journal-title":"Clin. Infect. Dis."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"e02461-20","DOI":"10.1128\/AAC.02461-20","article-title":"Molecular characterization of baseline Enterobacterales and Pseudomonas aeruginosa isolates from a phase 3 nosocomial pneumonia (ASPECT-NP) clinical trial","volume":"65","author":"Castanheira","year":"2021","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref6","doi-asserted-by":"publisher","first-page":"4770","DOI":"10.1128\/AAC.00540-16","article-title":"Changes in the frequencies of beta-lactamase genes among Enterobacteriaceae isolates in U.S. hospitals, 2012 to 2014: activity of Ceftazidime-Avibactam tested against beta-lactamase-producing isolates","volume":"60","author":"Castanheira","year":"2016","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref7","doi-asserted-by":"publisher","first-page":"436","DOI":"10.1126\/science.aar6245","article-title":"CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity","volume":"360","author":"Chen","year":"2018","journal-title":"Science"},{"key":"ref8","doi-asserted-by":"publisher","first-page":"112642","DOI":"10.1016\/j.bios.2020.112642","article-title":"Contamination-free visual detection of SARS-CoV-2 with CRISPR\/Cas12a: a promising method in the point-of-care detection","volume":"169","author":"Chen","year":"2020","journal-title":"Biosens. Bioelectron."},{"key":"ref9","doi-asserted-by":"publisher","first-page":"2000045","DOI":"10.2807\/1560-7917.ES.2020.25.3.2000045","article-title":"Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR","volume":"25","author":"Corman","year":"2020","journal-title":"Euro Surveill."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"e0027621","DOI":"10.1128\/jcm.00276-21","article-title":"Update from the European Committee on antimicrobial susceptibility testing (EUCAST)","volume":"60","author":"Giske","year":"2022","journal-title":"J. Clin. Microbiol."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"439","DOI":"10.1126\/science.aaq0179","article-title":"Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6","volume":"360","author":"Gootenberg","year":"2018","journal-title":"Science"},{"key":"ref12","doi-asserted-by":"publisher","first-page":"1092","DOI":"10.1111\/jam.13706","article-title":"Comparison of false-negative rates and limits of detection following macrofoam-swab sampling of bacillus anthracis surrogates via rapid viability PCR and plate culture","volume":"124","author":"Hutchison","year":"2018","journal-title":"J. Appl. Microbiol."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"1204","DOI":"10.1089\/mdr.2018.0423","article-title":"Antibiotic resistance and clinical presentation of health care-associated Hypervirulent Klebsiella pneumoniae infection in Korea","volume":"25","author":"Hyun","year":"2019","journal-title":"Microb. Drug Resist."},{"key":"ref14","doi-asserted-by":"publisher","first-page":"83","DOI":"10.1186\/1471-2180-6-83","article-title":"Evaluation of BacLite rapid MRSA, a rapid culture based screening test for the detection of ciprofloxacin and methicillin resistant S. aureus (MRSA) from screening swabs","volume":"6","author":"Johnson","year":"2006","journal-title":"BMC Microbiol."},{"key":"ref15","doi-asserted-by":"publisher","first-page":"103721","DOI":"10.1016\/j.jevs.2021.103721","article-title":"Development and application of a multiplex real-time polymerase chain reaction assay for the simultaneous detection of bacterial Aetiologic agents associated with equine venereal diseases","volume":"105","author":"Kim","year":"2021","journal-title":"J. Equine. Vet. Sci."},{"key":"ref16","doi-asserted-by":"publisher","first-page":"431","DOI":"10.1016\/j.ymeth.2021.04.007","article-title":"CRISPR-Cas systems for diagnosing infectious diseases","volume":"203","author":"Kostyusheva","year":"2021","journal-title":"Methods"},{"key":"ref17","doi-asserted-by":"publisher","first-page":"6","DOI":"10.1186\/1746-6148-5-6","article-title":"A comparative study of cultural methods for the detection of salmonella in feed and feed ingredients","volume":"5","author":"Koyuncu","year":"2009","journal-title":"BMC Vet. Res."},{"key":"ref18","doi-asserted-by":"publisher","first-page":"491","DOI":"10.1038\/s41422-018-0022-x","article-title":"CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA","volume":"28","author":"Li","year":"2018","journal-title":"Cell Res."},{"key":"ref19","doi-asserted-by":"publisher","first-page":"2228","DOI":"10.1021\/acssynbio.9b00209","article-title":"HOLMESv2: a CRISPR-Cas12b-assisted platform for nucleic acid detection and DNA methylation quantitation","volume":"8","author":"Li","year":"2019","journal-title":"ACS Synth. Biol."},{"key":"ref20","doi-asserted-by":"publisher","first-page":"219","DOI":"10.1093\/cid\/ciz174","article-title":"A one health study of the genetic relatedness of Klebsiella pneumoniae and their mobile elements in the east of England","volume":"70","author":"Ludden","year":"2020","journal-title":"Clin. Infect. Dis."},{"key":"ref21","doi-asserted-by":"publisher","first-page":"2920","DOI":"10.1021\/acssensors.1c00686","article-title":"CRISPR-Cas12a-powered dual-mode biosensor for ultrasensitive and cross-validating detection of pathogenic bacteria","volume":"6","author":"Ma","year":"2021","journal-title":"ACS Sens."},{"key":"ref22","doi-asserted-by":"publisher","first-page":"1198","DOI":"10.1056\/NEJMoa1306801","article-title":"Multistate point-prevalence survey of health care-associated infections","volume":"370","author":"Magill","year":"2014","journal-title":"N. Engl. J. Med."},{"key":"ref23","doi-asserted-by":"publisher","first-page":"1086","DOI":"10.3892\/etm.2021.10520","article-title":"Insights into multidrug-resistant K. pneumoniae urinary tract infections: from susceptibility to mortality","volume":"22","author":"Miftode","year":"2021","journal-title":"Exp. Ther. Med."},{"key":"ref24","doi-asserted-by":"publisher","first-page":"113012","DOI":"10.1016\/j.bios.2021.113012","article-title":"CRISPR-based detection of SARS-CoV-2: a review from sample to result","volume":"178","author":"Nouri","year":"2021","journal-title":"Biosens. Bioelectron."},{"key":"ref25","doi-asserted-by":"publisher","first-page":"26","DOI":"10.7326\/0003-4819-140-1-200401060-00008","article-title":"International prospective study of Klebsiella pneumoniae bacteremia: implications of extended-spectrum beta-lactamase production in nosocomial infections","volume":"140","author":"Paterson","year":"2004","journal-title":"Ann. Intern. Med."},{"key":"ref26","doi-asserted-by":"publisher","first-page":"542","DOI":"10.3390\/microorganisms8040542","article-title":"Isolation and characterization of a novel phage for controlling multidrug-resistant Klebsiella pneumoniae","volume":"8","author":"Peng","year":"2020","journal-title":"Microorganisms"},{"key":"ref27","doi-asserted-by":"publisher","first-page":"934","DOI":"10.3390\/jcm8070934","article-title":"Treatment options for Colistin resistant Klebsiella pneumoniae: present and future","volume":"8","author":"Petrosillo","year":"2019","journal-title":"J. Clin. Med."},{"key":"ref28","doi-asserted-by":"publisher","first-page":"589","DOI":"10.1128\/CMR.11.4.589","article-title":"Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors","volume":"11","author":"Podschun","year":"1998","journal-title":"Clin. Microbiol. Rev."},{"key":"ref29","doi-asserted-by":"publisher","first-page":"8509650","DOI":"10.1155\/2020\/8509650","article-title":"Antibacterial potential of Jatropha sp. latex against multidrug-resistant bacteria. Int","volume":"2020","author":"Prastiyanto","year":"2020","journal-title":"J. Microbiol."},{"key":"ref30","doi-asserted-by":"publisher","first-page":"29518","DOI":"10.1073\/pnas.2010254117","article-title":"Electric field-driven microfluidics for rapid CRISPR-based diagnostics and its application to detection of SARS-CoV-2","volume":"117","author":"Ramachandran","year":"2020","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"ref31","doi-asserted-by":"publisher","first-page":"983","DOI":"10.1099\/jmm.0.030577-0","article-title":"Role of SHV beta-lactamase variants in resistance of clinical Klebsiella pneumoniae strains to beta-lactams in an Algerian hospital","volume":"60","author":"Ramdani-Bouguessa","year":"2011","journal-title":"J. Med. Microbiol."},{"key":"ref32","doi-asserted-by":"publisher","DOI":"10.3390\/diagnostics11091646","article-title":"RT-LAMP CRISPR-Cas12\/13-based SARS-CoV-2 detection methods","volume":"11","author":"Selvam","year":"2021","journal-title":"Diagnostics"},{"key":"ref33","doi-asserted-by":"publisher","first-page":"32","DOI":"10.1186\/s12941-021-00437-8","article-title":"Molecular characteristics of antibiotic-resistant Escherichia coli and Klebsiella pneumoniae strains isolated from hospitalized patients in Tehran","volume":"20","author":"Sharahi","year":"2021","journal-title":"Iran. Ann. Clin. Microbiol. Antimicrob."},{"key":"ref34","doi-asserted-by":"publisher","first-page":"2512","DOI":"10.1093\/jac\/dkx189","article-title":"Clinical sensitivity and specificity of the check-points check-direct ESBL screen for BD MAX, a real-time PCR for direct ESBL detection from rectal swabs","volume":"72","author":"Souverein","year":"2017","journal-title":"J. Antimicrob. Chemother."},{"key":"ref35","doi-asserted-by":"publisher","first-page":"19","DOI":"10.1007\/5584_2017_38","article-title":"Mortality due to nosocomial infection with Klebsiella pneumoniae ESBL<sup\/>","volume":"1022","author":"Starzyk-Luszcz","year":"2017","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref36","doi-asserted-by":"publisher","first-page":"226","DOI":"10.1111\/vox.12410","article-title":"Investigation of bacterial inactivation in apheresis platelets with 24 or 30 hours between inoculation and inactivation","volume":"111","author":"Wagner","year":"2016","journal-title":"Vox Sang."},{"key":"ref37","doi-asserted-by":"publisher","first-page":"113663","DOI":"10.1016\/j.bios.2021.113663","article-title":"A finger-driven disposable micro-platform based on isothermal amplification for the application of multiplexed and point-of-care diagnosis of tuberculosis","volume":"195","author":"Wang","year":"2022","journal-title":"Biosens. Bioelectron."},{"key":"ref38","doi-asserted-by":"publisher","first-page":"88","DOI":"10.1126\/science.aav7271","article-title":"Functionally diverse type V CRISPR-Cas systems","volume":"363","author":"Yan","year":"2019","journal-title":"Science"},{"key":"ref39","doi-asserted-by":"publisher","first-page":"801292","DOI":"10.3389\/fmicb.2021.801292","article-title":"High prevalence of antimicrobial resistance and Integron gene cassettes in multi-drug-resistant Klebsiella pneumoniae isolates from captive Giant pandas (Ailuropoda melanoleuca)","volume":"12","author":"Yan","year":"2021","journal-title":"Front. Microbiol."},{"key":"ref40","doi-asserted-by":"publisher","first-page":"2657","DOI":"10.2147\/IDR.S314490","article-title":"Molecular mechanism of Polymyxin resistance in multidrug-resistant Klebsiella pneumoniae and Escherichia coli isolates from Henan Province, China: a multicenter study","volume":"14","author":"Yan","year":"2021","journal-title":"Infect. Drug Resist."},{"key":"ref41","doi-asserted-by":"publisher","first-page":"1033","DOI":"10.1111\/tbed.13787","article-title":"Isolation and characterization of multidrug-resistant Klebsiella pneumoniae from raw cow milk in Jiangsu and Shandong provinces, China","volume":"68","author":"Yang","year":"2021","journal-title":"Transbound. Emerg. Dis."},{"key":"ref42","doi-asserted-by":"crossref","first-page":"937","DOI":"10.3390\/ijerph18030937","article-title":"Characterization of extended-Spectrum Beta-lactamase-producing Escherichia coli isolates from Jurong Lake, Singapore with whole-genome-sequencing","volume":"18","author":"Zhong","year":"2021","journal-title":"Int. J. Environ. Res. Public Health"}],"container-title":["Frontiers in Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2023.1128261\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,2,9]],"date-time":"2023-02-09T11:37:50Z","timestamp":1675942670000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2023.1128261\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,2,9]]},"references-count":42,"alternative-id":["10.3389\/fmicb.2023.1128261"],"URL":"https:\/\/doi.org\/10.3389\/fmicb.2023.1128261","relation":{},"ISSN":["1664-302X"],"issn-type":[{"value":"1664-302X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,2,9]]},"article-number":"1128261"}}