RESUMO
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.
Assuntos
Sistemas CRISPR-Cas , Klebsiella pneumoniae , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico , Sensibilidade e Especificidade , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Humanos , Infecções por Klebsiella/diagnóstico , Infecções por Klebsiella/microbiologia , Recombinases/metabolismo , Recombinases/genética , Técnicas de Diagnóstico Molecular/métodos , Proteínas de Bactérias/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Proteínas Associadas a CRISPR/genética , DNA Bacteriano/genética , EndodesoxirribonucleasesRESUMO
Introduction: Salmonella was one of the most common bacteria that caused foodborne illness, with S. typhimurium (Salmonella typhimurium) and S. enteritidis (Salmonella enteritidis) infections accounting for more than 75% of human salmonella infections. Methods: In this study, we developed a method of dual recombinase polymerase amplification (RPA) combined with a lateral flow dipstick for the rapid detection of S. typhimurium and S. enteritidis in clinical specimens (stool). Results: The entire reaction process, including amplification and result reading, could be completed within 65 min. The detection limits of S. typhimurium and S. enteritidis in pure culture samples were 5.23 × 101 CFU/mL and 3.59 × 101 CFU/mL, respectively. The detection limits of S. typhimurium and S. enteritidis in artificially contaminated samples were 8.30 × 101 CFU/mL and 2.70 × 102 CFU/mL, respectively. In addition, the method had no cross-reaction with other pathogenic microorganisms. The results in clinical samples were fully consistent with those obtained using Bacterial Analysis Manual, with sensitivity and specificity were 100% (8/8) and 100% (17/17) for S. typhimurium and 100% (4/4) and 100% (21/21) for S. enteritidis, respectively. Discussion: The detection limits of S. typhimurium and S. enteritidis in artificially contaminated samples were higher than those in pure culture samples, which might be attributed to the inherent complex composition of artificially contaminated samples. In addition, the detection limits of S. typhimurium and S. enteritidis in the same sample were also different, which might be attributed to different amplification efficiency of two target genes in the same reaction system. Conclusion: This assay had potential application outdoors, as it could be performed within 1 h at 38°C without a complex instrument, and the results could be observed with the naked eye. In conclusion, the dual RPA-LFD assay established in this study had practical significance for the rapid detection of S. typhimurium and S. enteritidis in the future.