Two target genes based multiple cross displacement amplification combined with a lateral flow biosensor for the detection of Mycobacterium tuberculosis complex

Abstract

Background: Tuberculosis (TB) is a serious chronic infectious disease caused by Mycobacterium tuberculosis complex (MTBC). Hence, the development of a novel, simple, rapid and sensitive method to detect MTBC is of great significance for the prevention and treatment of TB. Results: In this study, multiple cross displacement amplification (MCDA) combined with a nanoparticle-based lateral flow biosensor (LFB) was developed to simultaneously detect two target genes (IS6110 and mpb64) of MTBC (MCDA-LFB). One suite of specific MCDA primers designed for the IS6110 and mpb64 genes was validated using genomic DNA extracted from the reference strain H37Rv. The MCDA amplicons were analyzed using a real-time turbidimeter, colorimetric indicator (malachite green, MG) and LFBs. The optimal amplification temperature and time were confirmed, and the MCDA-LFB method established in the current report was evaluated by detecting various pathogens (i.e., reference strains, isolates and clinical sputum samples). The results showed that the two sets of MCDA primers targeting the IS6110 and mpb64 genes could effectively detect MTBC strains. The optimal reaction conditions for the MCDA assay were determined to be 67 °C for 35 min. The MCDA assay limit of detection (LoD) was 100 fg per reaction for pure genomic DNA. The specificity of the MCDA-LFB assay was 100%, and there were no cross-reactions for non-MTBC strains. For sputum samples and MTBC strain detection, the positive rate of MCDA-LFB for the detection of MTBC strains was consistent with seminested automatic real-time PCR (Xpert MTB/RIF) and higher than acid-fast staining (AFS) and culture assays when used for sputum samples. The MCDA-LFB assay was a rapid tool, and the whole procedure for MCDA-LFB, including DNA template preparation, MCDA reaction and amplification product analysis, was completed within 70 min. Conclusion: The MCDA-LFB assay targeting the IS6110 and mpb64 genes is a simple, rapid, sensitive and reliable detection method, and it has potential significance for the prevention and treatment of TB.