Isothermal amplification and rapid detection of Klebsiella pneumoniae based on the multiple cross displacement amplification (MCDA) and gold nanoparticle lateral flow biosensor (LFB)

Klebsiella pneumoniae ( K . pneumoniae ) is a frequent pathogen causing nosocomial infections and outbreaks. We developed a multiple cross displacement amplification (MCDA) assay for the detection of K . pneumoniae , which can get the positive results within 40 minutes’ isothermal amplification. Gold-nanoparticle lateral flow biosensor (LFB) and colorimetric indicators were used for the rapid readouts of MCDA amplification. The detection limit of this assay was 100 fg per reaction at 65°C, which was confirmed to be the optimal amplification temperature according to the real time turbidimeters. For specificity, all of the 30 clinical-source K . pneumoniae strains were positive for the MCDA, and all of the non- K . pneumoniae strains belonging to 31 different species were negative for this MCDA assay. To evaluate the practical applicability of this method, we assessed its detection limit for K . pneumoniae strains in sputum samples (24 CFU per reaction), and DNA templates of 100 sputum samples further underwent the MCDA-LFB tests. All of the sputum samples being positive for K . pneumoniae (30/100) with the culture method were successfully identified with the MCDA assay, the detection power of which was higher than that of polymerase chain reaction (PCR) (25/100). Thus, the MCDA test for K . pneumoniae combined with the gold nanoparticle LFB as the results readout scheme, are simple, specific, and sensitive methods for the rapid diagnosis of K . pneumoniae in clinical samples.