The application of polymerase chain reaction (PCR) and DNA probe on the detection of tuberculosis from fromalin-fixed tissue of zoo animal

• Main Authors: Chia-Hao Lee, 李家豪
• Other Authors: Chian-Ren Jeng;Victor Fei Pang
• Format: Others
• Language: zh-TW
• Published: 1996
• Online Access: http://ndltd.ncl.edu.tw/handle/47115140050632144207

碩士 === 國立臺灣大學 === 獸醫學系 === 84 === Mycobacterial infection is an important problem of zoonosis. O- pportunistic infections with atypical mycobacteria in the course of AIDS gained importance in the past decade. There have been c- ontinuous occurrences of tuberculosis in bovine and wild animals. We describe here a assay based on PCR amplification with 2 gro- ups of primers followed by Southern blot hybridization with AP- labelled DNA probe to detect and to group mycobacterial infection in formalin-fixed and tissues of zoo animal. A 383 bp fragment e- ncoding for the 65 kD Ag of M. tuberculosis, M. bovis, M. avium, M. paratuberculosis and M. fortuitum was dectected by PCR with 1- st group of primers. The specificity of PCR products was confirm- ed by Southern blot using a region-specific DNA probe. Using the 2nd group of primers, we achieved specific amplification of a 419 bp fragment of M. tuberculosis and M. bovis. The product was also confirmed by Southern blot with DNA probe. Serial dilution studi- es showed that our PCR method could detect DNA amplified from 1 pg DNA and the nucleic acid probe we used could detect DNA ampli- fied from 100 fg DNA. Fifty-three formalin-fixed tissue blocks of suspecious mycobac- terial infection cases were collected from Taipei City Zoo. The ratio of positive reaction for acid-fast staining was 60.4％. Th- e positive ratio of PCR-TB and Probe-TB were 69.8％ and 77.4％； the positive ratios of PCR-MT and Probe-MT were 60.4％ and Within the 32 cases showing positive result, there were 23 and 25 cases are belonged to the typical mycobacterial infection detect- ed by PCR and confirmed by Southern blot with AP- labelled nucleic acid probe. The result indicated that the methods of PCR and Sou- thern blot hybridization described above should be a important di agnostic method in detecting and grouping mycobacterial infection in formalin-fixed tissues.