Colorimetric detection of the tuberculosis complex using cycling probe technology and hybridization in microplates

Cycling probe technology (CPT) is a simple signal amplification method for the detection of specific target DNA sequences. CPT uses a chimeric DNA-RNA -DNA probe that is cut by RNase H when bound to its complementary target se quence. In this study, a hybridization assay was developed to detect biotin ylated CPT products that result from the amplification of a Mycobacterium t uberculosis complex sequence. The chimeric probe was specifically designed to avoid the formation of secondary structures. The chosen capture probe wa s perfectly complementary to and was the same size as OL2, one of the two C PT products. The assay was based on the observation that a long sequence, s uch as the initial probe, was destabilized when bound to a small capture pr obe as a result of steric hindrance. The capture probe preferentially bound OL2 rather than the long initial probe. We added a prehybridization step w ith a helper DNA to enhance this discrimination between the two sequences. Colorimetric detection was performed using a peroxidase-streptavidin conjug ate. After optimization, the non-isotopic hybridization assay allowed the d etection of around 10 amol of target DNA. Besides being faster and easier t o perform, this detection method was compared to electrophoresis separation and gave similar results.