Tuberculosis detection via rolling circle amplification.

Abstract

Hybridization-based assays for DNA detection often use single-stranded DNA (ssDNA) probes to capture ssDNA targets in solution. Unfortunately, these assays are often not able to detect double-stranded DNA (dsDNA). Here, we achieve highly sensitive dsDNA target detection by including short oligonucleotide sequences during denaturing and cooling. After performing an isothermal nucleic acid amplification technique (Rolling Circle Amplification, RCA), these captured dsDNA targets are labeled, allowing single amplified molecules to be imaged and counted. This detection method was first applied to the detection of PCR-generated (polymerase chain reaction) dsDNA targets, yielding a limit of detection of 4.25 fM. As an application of the developed assay, the detection of extracted Mycobacterium tuberculosis (M. tb.) genomic DNA was attempted. A M. tb.-specific target was detected with high specificity compared to similar bacteria, and a detection limit of 10 000 colony forming units (cfu) ml-1 was achieved, close to the sensitivity required for clinical diagnosis.