Development of an isothermal recombinase‐aided amplification assay for the rapid and visualized detection of Klebsiella pneumoniae

Abstract

BACKGROUND: Klebsiella pneumoniae is a zoonotic opportunistic pathogen, leading to severe infections in dairy cows and humans. Efficient, on-site and accurate detection of K. pneumoniae is necessary to reduce the harm of cow mastitis and human infections. The objective of this study was to establish a recombinase-aided amplification (RAA) method combined with lateral flow dipstick (LFD) for rapid detection of K. pneumoniae. RESULTS:The primer concentration, incubation temperature and incubation time of the RAA reaction were optimized. When the primer concentration was 100 nmol L -1 , the strongest band could be obtained by incubation at 37 °C for 20 min. The RAA-LFD method had high specificity to K. pneumoniae and showed no cross-reaction with other pathogens. In addition, the detection limit of RAA-LFD for K. pneumoniae was 20 fg genomic DNA and 2.5 × 10 2 CFU mL −1 of bacteria in pure culture, which is 100 times higher than that of polymerase chain reaction (PCR) detection. Moreover, the RAA-LFD method can detect K. pneumoniae at initial concentrations as low as 2.5 CFU per 25 mL in artificially spiked milk samples after at least incubation for 6 h. Importantly, RAA-LFD had a high agreement with a test accuracy of 96.9%, compared with the biochemical identification method. Also, the detection accuracy of RAA-LFD was higher than that of the PCR assay (95.3%). CONCLUSIONS:The results demonstrated that the RAA-LFD assay is an accurate, sensitive, simple and point-of-use detection method for K. pneumoniae, which could be used as a potential application in the research laboratory and for disease diagnosis.

Rapid on-site nucleic acid testing: On-chip sample preparation, amplification, and detection, and their integration into all-in-one systems

“… Xia et al (2014) were able to amplify DNA to a detectable range within 10 min; in an efficiency comparison with the qPCR method, the same amplification result was achieved by qPCR after 134 min. Another method, known as “recombinase-assisted amplification” (RAA), makes use of a similar underlying principle to RPA; the main difference lies in the source of enzymes ( Hou et al, 2022 ). In this review, we also include studies using RAA under the category of RPA.…”

Research progress on detection techniques for point-of-care testing of foodborne pathogens

“…RAA is a technique for nucleic acid amplification using recombinant enzyme, single-chain binding protein, and DNA polymerase under isothermal conditions (optimal temperature 37°C) ( Feng et al, 2022a ; Hou et al, 2022 ; Zhang et al, 2022 ). The established RAA method can effectively shorten the detection time and does not require temperature change during nucleic acid amplification ( Aman et al, 2020 ; Teklemariam et al, 2020 ; Li et al, 2021a ).…”

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 ].…”