Rapid Detection of Klebsiella pneumoniae Carrying Virulence Gene rmpA2 by Recombinase Polymerase Amplification Combined With Lateral Flow Strips

Abstract

Highly virulent Klebsiella pneumoniae often causes invasive infections with high morbidity and mortality rates, posing an immense clinical challenge. Rapid and accurate detection of pathogenic bacteria is of great significance for treatment and preventive control. Conventional detection by polymerase chain reaction (PCR) is limited by a dependence on laboratory equipment and professional staff. Recombinase polymerase amplification (RPA) combined with a lateral flow strip (LFS) can rapidly amplify and visualize target genes in a short period of time. The aim of this study was to develop an RPA-LFS technique for detection of the K. pneumoniae virulence gene rmpA2. Primers were designed against conserved sequences specific to the virulence gene, and primer probe design was optimized by introducing base substitution to obtain a specific and sensitive primer-probe combination for clinical detection. We tested 65 actual samples collected from clinics to evaluate the performance of the newly established RPA-LFS system in comparison with conventional PCR methods and qPCR methods. The RPA-LFS assay was performed at for 25 min a constant temperature of 37°C, and results could be observed without instrumentation. The system could specifically identify highly virulent K. pneumoniae carrying the virulence gene rmpA2 with a minimum detection limit of 10−1 ng/μL and 10 copies/μL. For the 65 clinical samples tested, The RPA-LFS assay results were in complete agreement with the qPCR results and PCR results. The RPA-LFS assay provides a rapid, accurate, and simple method for identification of highly virulent K. pneumoniae carrying rmpA2.

Meat Authenticity Made Easy: DNA Extraction-Free Rapid Onsite Detection of Duck and Pork Ingredients in Beef and Lamb Using Dual-Recombinase-Aided Amplification and Multiplex Lateral Flow Strips

“…However, modifying forward and reverse primers can lead to false-positive LFS results, often requiring over a 1000-fold dilution for accuracy . Many RAA-LFS assays include CRISPR-Cas steps to counter high-concentration primer interference. , Commercial RAA nfo kits with modified probes reduce false positives but may still vary with different modifications …”

“…33 Many RAA-LFS assays include CRISPR-Cas steps to counter high-concentration primer interference. 34,35 Commercial RAA nfo kits with modified probes reduce false positives but may still vary with different modifications. 36 Additionally, the LFS used here does not need extra buffers.…”

Unique combination of Recombinase Polymerase Amplification with label-free citrate-stabilised silver nanoparticle assay offers a highly sensitive, fast, accurate and visual molecular detection ofKlebsiella pneumoniae

“…In comparison, RPA provides further advantage of rapid (15-30 min) isothermal amplification at lower temperatures (∼37-42°C) permitting quicker detection 23 . RPA when combined with Lateral Flow Strip (LFS) system is capable of detecting as low as 100-1000 bacteria, highlighting scope for improving the sensitivity of detection post RPA-based DNA amplification 18, 19 . A recent study highlights utility of tailor-made plasmonic aptamer-gold nanoparticle (AuNP) assay for detection of Klebsiella without the need of amplification, however, the sensitivity of this methods is ∼3400 bacteria 24 .…”

A Multiplex Recombinase‐Aided qPCR Assay for Highly Sensitive and Rapid Detection of khe, blaKPC‐2, and blaNDM‐1 Genes in Klebsiella pneumoniae

“…Molecular diagnostic methods such as qPCR are used to detect K. pneumoniae [ 11 ], but these methods are not sensitive enough and take a long time to detect. The development of fast isothermal amplification assay, such as recombinase polymerase amplification (RPA) [ 12 ], recombinase‐aided amplification (RAA) [ 13 , 14 , 15 ], and loop‐mediated isothermal amplification (LAMP) [ 1 , 16 ], have been increasingly applied for detecting a wide range of pathogens but still difficult to detect the targets in a multiplex and high‐throughput format [ 17 ].…”