[Studies on detection methods of two DNA probes in Mycobacterium tuberculosis].
Two different biotinylated DNA probes which are highly specific to M. tuberculosis(MT) were made and studied. One probe is a 20 bp oligonucleotide labeled with biotin at 5' end, the other is a long DNA probe produced by PCR amplification procedure allowed for the incorporation of biotin labeled UTP. The two probes were hybridized with MT genome DNA and a 317 bp PCR product amplified from IS6110 sequence of MT, and then detected by alkaline phosphatase conjugates through colorimetric reaction. The detection sensitivity and specificity of the two probes were comparatively studied. The hybridization condition including concentration of probe, temperature of hybridization and washing filter thereafter were also investigated preliminary. The detection limit of the oligonucleotide probe and the 188 bp PCR probe were 100 ng and 6 ng of DNA respectively in detection of M. T genome, and 400 pg and 50 pg of DNA respectively in detection of PCR products of MT. The two probes can be only hybridized to MT and BCG, but not with other 24 mycobacterium or non-mycobacterium tested. The optimal hybridization temperature and washing filter temperature of oligonucleotide were 42 degrees C and 60 degrees C respectively; and that of 188 bp probe, 68 degrees C and 60 degrees C-68 degrees C. Generally the specificity of two probes were all high, but the sensitivity of 188 bp DNA probe was 7-16 times that of the oligonucleotide probe. The higher sensitivity, lower hybridization background and faster revelation of the 188 bp DNA probe made it a better choice in detection of MT.
Yang
H
H
Beijing Hospital National Centre for Clinical Laboratories, Beijing 100730.
Yang
S
S
Zhuang
Y
Y
Li
G
G
Li
B
B
D001710:Biotin; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013696:Temperature
D015342:DNA Probes; D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D001710:Biotin