Studies On Application Of Two DNA Probes In Detecting M.TB

To find out a more sensible, more special and more practical method to detect M.TB directly, the conditions of hybridization by nonisotope labeled nucleic probes were studied.We produced and labeled two M.TB probes: TB42 probe, an oligonucleotide probe labeled by biotin at 5' end; 188 bp PCR probe,biotin-labeled in several bases, which was amplified by PCR. They were detected by colorimetric or chemiluminescent method,through biotin-streptavidin system in dot blotting.We Studied dot-blotting conditions of those two probes,and their sensitivity and their specificity.The results showed: hybridization temperature 42℃ washing membrane temperature 60℃ ,and probe concentration 150ng/ml were the best hybridization condition of TB42 oligonucleotide probe;hybridization temmperature 68 °C ,washing membrane temperature 60℃,probe concentration 150ng/ml were the best condition for 188bp PCR probe.The sensitivity of TB42 probe and PCR probe to detect genome DNA, and 317bp DNA of M.TB were 110ng and7ng,200pg and 50pg respectively visualized by colorimetric system.The sensitivity of TB42 probe and 188bp probe to detect genome DNA and 317bp DNA visualized by chemiluminescence were 7ng and 0.88ng, 25pg and 25pg respectively.These results showed that chemiluminescent system were more sensible than colorimetric one.21 mycobacterium and 9 nonmycobacterium were hybridized with two probes respectively, only M.TB and BCG were positive, while other bacteria tested were negative. The Specificity of the two probes was high.To study the practicality of the two probes, we collected 110...