A Recombinase Aided Amplification Assay for Rapid Detection of the Klebsiella pneumoniae Carbapenemase Gene and Its Characteristics in Klebsiella pneumoniae

Abstract

Klebsiella pneumoniae carbapenemase genes (blaKPC) play an important role in carbapenem-resistant Enterobacteriaceae in China. A rapid detection method for blaKPC genes and investigations into the molecular characteristics of blaKPC positive Klebsiella pneumoniae were necessary. In this study, an easy and rapid recombinase aided amplification assay (RAA) for blaKPC was established. This protocol could be completed at 39°C in 15–20 min. The sensitivity of this assay was determined as 48 copies per reaction, and the specificity was 100%. The blaKPC RAA method could be used for clinical diagnosis and epidemiological investigation. Among 801 fecal samples from inpatients, 34 blaKPC positive isolates were identified from each sample, of which 23 isolates were K. pneumoniae. ST11 with blaKPC-2 was the most prevalent type. All these strains were multidrug resistant and carried various virulence genes. Fecal carriage of blaKPC positive carbapenem-resistant K.pneumoniae poses significant challenges for public health control.

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 ].…”

Rapid detection of Burkholderia cepacia complex carrying the 16S rRNA gene in clinical specimens by recombinase-aided amplification

“…Recombinase-aided amplification (RAA) is a rapid, specific, sensitive and reliable isothermal gene amplification technology ( Wang et al., 2021 ). At present, RAA has been widely used in the detection of various pathogens such as SARS-CoV-2, adenovirus, hepatitis B virus, Escherichia coli , Klebsiella pneumoniae , Mycoplasma pneumoniae , Salmonella and Vibrio parahaemolyticus ( Zhang et al., 2017 ; Shen et al., 2019 ; Wang et al., 2019 ; Xue et al., 2020 ; Mu et al., 2021 ; Zhang et al., 2021 ; Zheng et al., 2021 ; Feng et al., 2022 ). RAA achieves DNA amplification by employing recombinase UvsX, DNA polymerase and single-stranded DNA binding protein at 35°C–42°C, replacing the traditional thermal cycling process ( Fan et al., 2020 ).…”

Rapid Detection of Multi-Resistance Strains Carrying mcr-1 Gene Using Recombinase-Aided Amplification Directly on Clinical Samples

“…RAA assay is a highly efficient method for the rapid detection of specific target genes. Based on isothermal amplification technology, the RAA assay can be completed within 15–30 min at 39°C and has been widely used in clinical applications, such as for identifying New Delhi Metallo-β-Lactamase Gene ( Feng et al, 2021 ), bla KPC ( Zhang et al, 2021 ) and other applications ( Fan et al, 2019 ; Qi et al, 2019 ; Shen et al, 2019 ; Xue et al, 2020a , b ). Here, an RAA assay was developed to detect the mcr -1 gene in clinical samples, which was proven to have high specificity and sensitivity.…”