Two Target Genes Based Multiple Cross Displacement Amplification Combined with Lateral Flow Biosensor for the Detection of Mycobacrterium Tuberculosis Complex

Abstract

Background: Mycobacterium tuberculosis complex (MTBC) causes tuberculosis (TB), which is a global public health problem that seriously endangers public health. Hence, development of a new and rapid method to detect MTBC is of great significance for prevention and treatment of TB. Results: In this study, a multiple cross displacement amplification combined with nanoparticle-based lateral flow biosensor (MCDA-LFB) was developed to simultaneously detect two target genes (IS6110 and mpb64) of MTBC. One suit of specific multiple cross displacement amplification (MCDA) primers, which was designed for IS6110 and mpb64 gene, respectively, was validated through using the genomic DNA extracted from reference strain H37Rv. The MCDA products were analyzed using real-time turbidity curve, colorimetric indicator (Malachite Green, MG) and LFB. The conditions of amplification temperature and time were optimized and the established MCDA-LFB method was applied to detect the sputum specimens and MTBC strains from clinical samples. The results show that two sets of MCDA primers for the IS6110 and mpb64 genes have detected MTBC validly. The MCDA reaction conditions were optimized at 67 °C for 35 min. The limit of detection of MCDA assay based on IS6110 and mpb64 genes was 100 fg of genomic DNA template per reaction. The specificity of MCDA-LFB detection was 100%, and no cross-reactions for non-MTBC strains detection. The positive rate of MCDA-LFB for the detection of MTBC strains was equal to that of semi-nested automatic real-time PCR (Xpert MTB/RIF), and had a higher positive rate than acid-fast staining (AFS) when used for the detection of sputum samples. The whole procedure of MCDA-LFB, including genomic template preparation, MCDA reaction and products analysis, was completed with 70 min.Conclusion: The simplicity, rapidity, sensitivity and reliability of the MCDA-LFB based on IS6110 and mpb64 gene of MTBC developed in this study make it potentially significant for the prevention and treatment of TB.

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

“…16 Considering its sensitivity and visualization capability, a nanoparticle-based LFB was employed to detect the amplicons produced by MCDA, which could be observed directly on the TLs of the LFB strips. 17,20,22 To develop this ML-MCDA-LFB technique, we first designed three sets of ML-MCDA primers for the RLEP gene and examined their amplification efficiency by real-time turbidimetry. The ML-MCDA primers with the highest efficiencies, RLEP-C1* and RLEP-D1*, were labeled with 6-FAM and biotin, respectively (Table 1).…”

“…16 Nanoparticle-based LFBs are designed to capture target nucleic acids using capture agents such as antifluorescein isothiocyanate (FITC) antibodies (Ab), antidigoxigenin (Dig) Ab, and biotin−bovine serum albumin (BSA). 22 LFB devices provide a convenient, visual, and sensitive method for verifying MCDA amplicons labeled using FITC/carboxyfluorescein (FAM). 17,22 Herein, we report an MCDA method combined with an LFB (MCDA-LFB) for rapidly detecting M. leprae.…”

“…22 LFB devices provide a convenient, visual, and sensitive method for verifying MCDA amplicons labeled using FITC/carboxyfluorescein (FAM). 17,22 Herein, we report an MCDA method combined with an LFB (MCDA-LFB) for rapidly detecting M. leprae. In this system, the MCDA reaction tube amplified the target M. leprae repeated element (RLEP) gene.…”