Establishment of a detection method for Klebsiella pneumoniae based on ERA-CRISPR/Cas12a

OBJECTIVE To establish a nucleic acid detection method for Klebsiella pneumoniae (KP) based on enzymatic recombinant isothermal amplification - clustered regularly interspaced short palindromic repeats (ERA-CRISPR/Cas12a) protein. METHODS Three CRISPR RNAs (crRNAs) were designed for KP (fimI) gene. The plasmids containing the KP gene were used as templates for ERA amplification, and the best crRNA was selected by CRISPR/Cas12a fluorescence reaction. Three pairs of ERA primers were designed and 9 primer combinations were constructed. One set of primers was selected as the best one for ERA-CRISPR/Cas12a reaction. A complete detection system was established by combining the easy-readout and sensitive enhanced (ERASE) nucleic acid test strip technology. Tenfold gradient dilution of fimI gene plasmid was used as a template to detect the sensitivity of the method. At the same time, the specificity of the method in detection of KP and other four types of common gram-negative bacilli was evaluated. The nucleic acid test was performed for 34 clinical isolates, and the result was compared with the result of quantitative real-time PCR (qPCR). RESULTS The ERA-CRISPR/Cas12a detection system was successfully established. The result of sensitivity experiment showed that the minimum detection limit of detection (LOD) of the plasmid template was 1 copy/μl, with the ERASE dipstick 10 copies/μl, both were superior to qPCR (100 copies/μl). The result of the specificity test showed that only KP was tested positive. All of the KP strains were detected from the 34 clinical isolates, which was consistent with the results of qPCR. CONCLUSIONS A simple, economical, highly sensitive and specific nucleic acid detection method is successfully established for KP. The method is suitable for nucleic acid detection in primary health institutions in economically underdeveloped areas.