Rapid, specific, and sensitive detection of the ureR_1 gene in Klebsiella pneumoniae by loop-mediated isothermal amplification method

Abstract

Klebsiella pneumoniae is one of the main pathogenic bacteria that causes nosocomial infections, such as pneumonia, urinary tract infection, and sepsis. Therefore, the rapid and accurate detection of K. pneumoniae is important for the timely treatment of infectious patients. This study aimed to establish a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of K. pneumoniae -specific gene ureR_1 (Gene ID: 11847803). The ureR_1 gene was obtained through local and online BLAST, and the specific primers were designed for its detection. Positive reactions were observed on all 140 K. pneumoniae clinical isolates while all the 82 non- K. pneumoniae clinical isolates were negative. Plasmids with the specific gene and the mouse blood with K. pneumoniae were used for sensitivity analysis. The detection limit of the LAMP was 1 bacterium/reaction. The results showed that the LAMP targeted to ureR_1 is a fast, specific, sensitive, inexpensive, and suitable method for the detection of K. pneumoniae .

Development of Loop-Mediated Isothermal Amplification Rapid Diagnostic Assays for the Detection of Klebsiella pneumoniae and Carbapenemase Genes in Clinical Samples

“…Furthermore, the assays described here demonstrated a limit of detection 100 times lower than the routine PCR method used in Ruijin Hospital, and LAMP results were obtained in a much shorter time frame: 1 h from sample processing to results versus 2 h 30 min for PCR detection and 24–48 h for culture-based methods. Several LAMP assays have been developed for the detection of K. pneumoniae ( Kang et al, 2012 ; Dong et al, 2015 ; Barnes et al, 2018 ; Li et al, 2019 ; Tian et al, 2019 ; Vergara et al, 2020 ) using different target genes, including KPN_04473 fimD ( Kang et al, 2012 ; Barnes et al, 2018 ; Vergara et al, 2020 ), ure1 ( Li et al, 2019 ), celB ( Tian et al, 2019 ), and rscA ( Dong et al, 2015 ). These different target genes were chosen following bibliographic reviews or sequences alignment and online BLAST searches.…”

“…LAMP is a single-step nucleic acid amplification technique that is rapid, sensitive, and cost-effective. This technique can amplify a few copies of DNA into billions of copies within 30 min ( Notomi et al, 2000 ; Diribe et al, 2014 ) and has been used to detect a wide range of viral, bacterial, and parasitic pathogens ( Wong Y.-P. et al, 2018 ; Li et al, 2019 ; Tian et al, 2019 ; Vergara et al, 2020 ). LAMP is less sensitive to PCR inhibitors in poorly or nonprocessed samples, such as blood, sputum, and urine, making it suitable for use as a point-of-care diagnostic ( Notomi et al, 2000 ; Nagamine et al, 2002 ; Kaneko et al, 2005 ; Parida et al, 2008 ; Francois et al, 2011 ; Dhama et al, 2014 ; Gadkar et al, 2018 ; Moehling et al, 2021 ).…”