Nucleic Acids Detection for Mycobacterium tuberculosis Based on Gold Nanoparticles Counting and Rolling-Circle...

Tuberculosis (TB) is a common infectious disease caused by Mycobacterium tuberculosis, which usually disturbs the lungs, and remains the second leading cause of death from an infectious disease worldwide after the human immunodeficiency virus. Herein, we constructed a simple and sensitive method for Mycobacterium tuberculosis-specific DNA detection with the dark-field microscopic imaging of gold nanoparticles (AuNPs) counting strategy and rolling-circle amplification (RCA). Taking advantage of RCA amplification, one target molecule produced hundreds of general oligonucleotides, which could form the sandwich structure with capture-strand-modified magnetic beads and AuNPs. After magnetic separation, AuNPs were released and detected by dark-field imaging; about 10 fM Mycobacterium tuberculosis-specific DNA target can still be differentiated from the blank. No significant change of the absorbance signals was observed when the target DNA to genomic DNA ratio (in mass) was from 1:0 to 1:10⁶. The spike recovery results in genomic DNA from human and Klebsiella pneumoniae suggested that the proposed method has the feasibility for application with biological samples. This proposed method is performed on an entry-level dark-field microscope setup with only a 6 μL detection volume, which creates a new, simple, sensitive, and valuable tool for pathogen detection.