Establishment of a Rapid Detection Method for Highly Virulent Klebsiella Pneumoniae Based on Multienzyme Isothermal Rapid Amplification Technology.

Abstract

This study aimed to establish a method for the rapid detection of highly virulent Klebsiella pneumoniae (hvKP) by using multienzyme isothermal rapid amplification (MIRA) technology. The laboratory can quickly, accurately, and conveniently diagnose highly virulent Klebsiella pneumoniae infection. For this study, 7 laboratory standard strains and 184 clinical isolates (including 70 strains of Klebsiella pneumoniae) were collected and screened for highly virulent Klebsiella pneumoniae based on its colony morphology, wire drawing test, and next-generation sequencing (NGS) results. Based on the nucleic acid sequence of the peg344 gene of highly virulent Klebsiella pneumoniae on GenBank (no. AP006726.1), specific conserved regions were selected to design MIRA and real-time fluorescence quantitative PCR (qPCR) specific primers and probes. The MIRA and qPCR methods were used to detect the tested strain, and the specificity, sensitivity, and clinical performance of the MIRA method for detecting hvKP were evaluated. In total, 21 cases of hvKP were screened from clinical isolates. The MIRA detection method utilizes specific primers and probes to transmit significant fluorescence signals at 39°C, and the detection process takes 30 minutes. The specificity test results showed that only hvKP had a specific amplification curve, while the rest of non-highly virulent Klebsiella pneumoniae (non-hvKP) had no specific amplification curve. The sensitivity test results showed that the sensitivity of MIRA for detecting hvKP is 7 × 102 CFU/mL, which is consistent with the sensitivity of the real-time fluorescence qPCR method. A simultaneous detection of 184 clinical isolates was accomplished by using MIRA and qPCR methods. Twenty-one strains of hvKP have specific amplification curves, while the remaining 163 strains of non-hvKP have no specific amplification curves. The accuracy of both methods for detecting hvKP is 100%. The established multienzyme isothermal rapid amplification (MIRA) has the following characteristics: a short detection time, high sensitivity, and a strong specificity, and it can be used as a powerful tool for an early diagnosis and epidemiological monitoring of hvKp.