Visual and Rapid Detection of Klebsiella pneumoniae by Magnetic Immunocapture-Loop-Mediated Isothermal Amplification Assay

Abstract

Background: Klebsiella pneumoniae is an important human pathogen that causes severe diseases including urinary tract infection, pneumonia, and bacteremia. However, a rapid and sensitive detection method remains to be developed. Objectives: This study aimed to develop a rapid, real-time, and visual detection method for K. pneumoniae. This is a primary screening method to improve the diagnosis of K. pneumoniae infection and save much precious time for clinical practice. Methods: Klebsiella pneumoniae was used as an antigen to produce a monoclonal antibodies (mAb). The mAb 1E6 recognizing outer membrane protein C on the surface of K. pneumoniae was screened by an indirect enzyme-linked immunosorbent assay (ELISA), Western Blot, and mass spectrometry assay, and then conjugated with the protein A/G coated magnetic beads to generate the immune magnetic beads (IMBs). Thereafter, the IMBs were used to capture K. pneumoniae, and the complex (beads-mAb-K. pneumoniae) was used for loop-mediated isothermal amplification (LAMP) assay. Results: We developed a rapid, real-time, and visual detection method employing an immunocapture loop-mediated isothermal amplification (IC-LAMP). The sensitivity of the IC-LAMP was 4 CFU mL-1. The process of K. pneumoniae detection lasted~60 minutes and had no cross-reaction to other microbial strains. Besides, the accuracy of IC-LAMP was further verified by examining 39 clinical isolates. Conclusions: Without the need for enrichment of bacteria and extraction of its genome, the IC-LAMP developed here could be used as a primary screening method supplementary to traditional detection methods to improve the diagnosis of K. pneumoniae infection for clinical practice.

Instrument-Free and Visual Detection of Salmonella Based on Magnetic Nanoparticles and an Antibody Probe Immunosensor

“…Thus, an instrument-free, rapid, and visual method remains to be developed. At present, many novelty biosensors are used for rapid and point-of-free detection of pathogenic microorganisms, including immunogold nanoparticles (IGNs) [16], lateral-flow strip immunoassays (LFSA) [17,18,19], and immunocapture loop-mediated isothermal amplification assay (IC-LAMP) [20,21]. However, both IGNs and LFSA are susceptible to either high pH or low pH and high salt ions, and have been associated with false positive results.…”