Biosensor-Based Multiple Cross Displacement Amplification for the Rapid Detection of Mycobacterium leprae.

Abstract

Leprosy is an ancient disease caused by Mycobacterium leprae (ML) that remains a public health problem in poverty-stricken areas worldwide. Although many ML detection techniques have been used, a rapid and sensitive tool is essential for the early detection and treatment of leprosy. Herein, we developed a rapid ML detection technique by combining multiple cross displacement amplification (MCDA) with a nanoparticle-based lateral flow biosensor (LFB), termed ML-MCDA-LFB. MCDA induced a rapid isothermal reaction using specific primers targeting the RLEP gene, and the LFB enabled instant visual amplicon detection. The pure genomic DNA of ML and nucleic acids from various pathogens were employed to evaluate and optimize the ML-MCDA-LFB assay. The optimal conditions for ML-MCDA-LFB were 68 °C and 35 min, respectively. The limit of detection for pure ML genomic DNA was 150 fg per vessel, and the specificity of detection was 100% for the experimental strains. Additionally, the entire detection process could be performed within 40 min, including the isothermal amplification (35 min) and result confirmation (1-2 min). Hence, the ML-MCDA-LFB assay was shown to be a rapid, sensitive, and visual method for detecting ML and could be used as a potential tool for early clinical diagnosis and field screening of leprosy.