Rapid Detection of Klebsiella pneumoniae in Infants With Severe Infection Disease by Instant Nanofluorescent Microspheres-Immunochromatography Combined With Recombinase Polymerase Isothermal Amplification Technology.

BACKGROUND

Klebsiella pneumoniae (KP) is an important and virulent pathogen causing acute respiratory infections in infants. Currently, traditional methods for detecting Klebsiella pneumoniae in babies are labour-intensive and time-consuming, and their accuracy and sensitivity require improvement. Recombinant enzyme polymerase amplification technology (RPA) has the advantages of good specificity, high sensitivity, and isothermal amplification. It can be combined with fluorescence immunochromatography test strip (FICTS) based on nanofluorescent microspheres (nFM) to achieve rapid, low-cost, high sensitivity, and specific detection of Klebsiella pneumoniae in infants.

METHODS

mdh was selected as a genus-specific gene from Klebsiella pneumoniae, and recombinant polymerase isothermal amplification-nanofluorescence microsphere immunochromatography (RPA-FICTS) was established by using standard strains. The sensitivity and specificity of this detection technology can be improved by the magnetic bead purification step of RPA nucleic acid amplification products. Subsequently, rapid quantitative detection of K. pneumoniae in infant samples was performed using fluorescent immunochromatographic test strips. A total of 19 clinical samples of Klebsiella pneumoniae (KP) and 7 clinical samples of non-Klebsiella pneumoniae (nKP) were detected by the new detection technology platform established in this paper, and the specificity and sensitivity of RPA-FICTS were verified by agarose gel electrophoresis.

RESULTS

Under the optimal detection conditions, the maximum detection limits of RPA-GE (agarose gel electrophoresis) and RPA-FICTS are 9.03 × 10-3 and 9.03 × 10-8 ng/μL. RPA-FICTS can quickly detect Klebsiella pneumoniae and specifically distinguish between Klebsiella pneumoniae and non-Klebsiella pneumoniae. Compared with traditional detection methods, this method has a shorter detection time (55 min), strong specificity, high sensitivity, and simple operation, providing a new method for rapid detection of human Klebsiella pneumoniae.