AUTHOR=Wang Shang , Wang Shan , Tang Ying , Peng Guoyu , Hao Tongyu , Wu Xincheng , Wei Jiehong , Qiu Xinying , Zhou Dewang , Zhu Shimao , Li Yuqing , Wu Song 

TITLE=Detection of Klebsiella pneumonia DNA and ESBL positive strains by PCR-based CRISPR-LbCas12a system

JOURNAL=Frontiers in Microbiology

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2023.1128261

DOI=10.3389/fmicb.2023.1128261

ISSN=1664-302X

ABSTRACT=<sec><title>Introduction</title><p><italic>Klebsiella pneumonia</italic> (<italic>K. pneumonia</italic>) is a Gram-negative bacterium that opportunistically causes nosocomial infections in the lung, bloodstream, and urinary tract. Extended-spectrum β-Lactamases (ESBLs)-expressed <italic>K. pneumonia</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 <italic>K. pneumonia</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.</p></sec><sec><title>Methods</title><p>This study established a system that combined PCR with CRISPR-LbCas12a targeting the <italic>K. pneumoniae</italic> system. Additionally, this study summarized the antibiotic-resistant information of the past five years’ <italic>K. pneumoniae</italic> clinic cases in Luohu Hospital and found that the ESBL-positive strains were growing. This study then designs a crRNA that targets <italic>SHV</italic> to detect ESBL-resistant <italic>K. pneumoniae</italic>. This work is to detect <italic>K. pneumoniae</italic> and ESBL-positive strains’ nucleic acid using CRISPR-Cas12 technology. We compared PCR-LbCas12 workflow with PCR and qPCR techniques.</p></sec><sec><title>Results and Discussion</title><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.</p></sec>