Abstract
Mycobacterium kansasii is an opportunistic pathogen that causes both intrapulmonary and extrapulmonary infections. The symptoms of the pulmonary diseases caused by M. kansasii closely resemble Mycobacterium tuberculosis. Rapid and accurate differentiation of M. kansasii from M. tuberculosis, as well as other mycobacteria, is crucial for developing effective therapeutics and disease treatment. In this study, we combined loop-mediated isothermal amplification (LAMP) with lateral flow biosensors (LFB) to detect M. kansasii, by targeting the species-specific sequence of rpoB, a gene which encodes the β subunit of bacterial RNA polymerase. The assay was validated to ensure that it was highly selective by testing M. kansasii, M. tuberculosis, other species of respiratory bacteria, and other nontuberculous mycobacteria. The detection limit of the assay was 1 fg/μL of DNA and 50 CFU of bacilli in sputum. The M. kansasii-LAMP-LFB assay is a fast, cheap, and accurate method for detecting M. kansasii by constant temperature amplification and simple interpretation.
Highlights
Nontuberculous mycobacteria (NTM) comprise most mycobacteria except the Mycobacterium tuberculosis complex (MTBC) and Mycobacterium leprae
Once the biosensor had absorbed the entire quantity of running buffer (~ 2 min), the subsequent detection of the loop-mediated isothermal amplification (LAMP) products was computed in the form of red lines on the nitrocellulose filter membrane (NC membrane) of the lateral flow biosensor (LFB)
The artificial sputum used in this study was obtained from the National Tuberculosis Reference Laboratory of China (NTRL)
Summary
Nontuberculous mycobacteria (NTM) comprise most mycobacteria except the Mycobacterium tuberculosis complex (MTBC) and Mycobacterium leprae. Mycobacterium kansasii, an opportunistic pathogen, is a slow-growing NTM. As one of the most important pathogenic nontuberculous mycobacteria, the clinical symptoms of M. kansasii infection are similar to those seen in patients with tuberculosis and other nontuberculous mycobacterial infections, but the treatment is different. The traditional method of identifying M. kansasii is through biochemical identification after culturing, which is time intensive and yields in accurate results. Chromatography, including gas chromatography, high-pressure liquid chromatography, and time-of-flight mass spectrometry that have been used to detect M. kansasii, require expensive equipment and complex operation (George et al 2018; Lau et al 2015; Olivier and Loots 2012). The molecular methods, such as GeneChip, reverse hybridization, and sequencing technology that have been used to identify M. kansasii, are still relatively expensive because of the costly instruments required, time
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