Rapid Detection and Differentiation of Legionella pneumophila and Non-Legionella pneumophila Species by Using Recombinase Polymerase Amplification Combined With EuNPs-Based Lateral Flow Immunochromatography

Abstract

Legionella, a waterborne pathogen, is the main cause of Legionnaires’ disease. Therefore, timely and accurate detection and differentiation of Legionella pneumophila and non-Legionella pneumophila species is crucial. In this study, we develop an easy and rapid recombinase polymerase amplification assay combined with EuNPs-based lateral flow immunochromatography (EuNPs-LFIC-RPA) to specifically distinguish Legionella pneumophila and non-Legionella pneumophila. We designed primers based on the mip gene of Legionella pneumophila and the 5S rRNA gene of non-Legionella pneumophila. The recombinase polymerase amplification reaction could go to completion in 10 min at 37°C, and the amplification products could be detected within 5 min with EuNPs-LFIC strips. Using a florescent test strip reader, the quantitative results were achieved by reading the colored signal intensities on the strips. The sensitivity was 1.6 × 101 CFU/ml, and a linear standard linear curve plotted from the test strip reader had a correlation coefficient for the determination of Legionella pneumophila (R2 = 0.9516). Completed concordance for the presence or absence of Legionella pneumophila by EuNPs-LFIC-RPA and qPCR was 97.32% (κ = 0.79, 95% CI), according to an analysis of practical water samples (n = 112). In short, this work shows the feasibility of EuNPs-LFIC-RPA for efficient and rapid monitoring of Legionella pneumophila and non-Legionella pneumophila in water samples.

Advances in the application of molecular diagnostic techniques for the detection of infectious disease pathogens (Review)

“…The amplified products can often be monitored by turbidity, color change, lateral flow test strips and fluorescence curves, making it suitable for resource-poor areas and primary healthcare units. To date, loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA) and nucleic acid sequence-based amplification (NASBA) have been used for rapid field detection of infectious disease pathogens ( 55 ). However, the target product of the isothermal amplification reaction is short and susceptible to contamination by exogenous genetic material, which can result in false-positives.…”

Genomic characterization and assessment of pathogenic potential of Legionella spp. isolates from environmental monitoring

“…A major factor for limited availability of epidemiological data of non-pneumophila species is under-diagnosis due to mild symptoms or tests specifically targeting L. pneumophila such as frequently used urinary antigen test ( Chambers et al, 2021 ). For the above-mentioned reasons, it was noted that correct identification and typing of strains isolated from patients and the environment is crucial for understanding the pathogenic role of bacteria belonging to the Legionella genus ( Mazzotta et al, 2021 ; Du et al, 2022 ; Wroblewski et al, 2022 ). Whole-genome sequencing (WGS) analysis is a highly discriminatory method capable of assessing genetic relatedness among isolates and therefore constitutes an important tool for outbreak investigations as well as pathogen surveillance ( Quainoo et al, 2017 ; Xu et al, 2021 ).…”