Detection of Mycobacterium tuberculosis on tissue section slides by in-situ polymerase chain reaction and fluorescent in-situ hybridization

• Main Authors: Ming-jih Tsai, 蔡明志
• Other Authors: Chan-ping Lee
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/20751125799765949866

碩士 === 慈濟大學 === 醫學研究所 === 89 === Diagnosis of tuberculosis on formalin-fixed paraffin-embedded (FFPE) archival tissues represents a major challenge in molecular diagnosis. The sensitivity of the conventional histochemical demonstration of acid-fast bacteria can be as low as 20%-30%, this method neither provides us the way to distinguish Mycobacterium tuberculosis (MTB) from Mycobacterium bovis (M. bovis) nor from other mycobacterial strains. To overcome these problems, here we study the in-situ polymerase chain reaction (IS-PCR) technology for detection of mycobacteria in FFPE tissue section on slide. We first study tube PCR conditions including the characterization of specific target genes, and IS-PCR conditions using bacterial smears (E. coli and MTB) fixed on superfrost plus slides. Then we use two model systems, the elastin gene in sheep lung tissue and the ?-globin gene in human intestine tissue, to determine the various parameters important for IS-PCR. The parameters include the time of proteinase K treatment, the optimized conditions for IS-PCR, the selection of thermal cycler, the way to seal the slide, how to prevent the evaporation of reagents and loss of localization and PCR products, how to use the biotin-dUTP tag, how to select the color-formation substrate (Fast-Red rather than NBT/BCIP), and in conjunction with a proper counter-stain for the signal detection. Taken together, this technique allows us to detect E. coli and M. tuberculosis on smeared slides and M. tuberculosis complex on 1 intestine, 2 lung and 1 lymph node FFPE tissues from patients with TB infections. In addition, we also studied to improve signal sensitivity using fluorescent in-situ hybridization. We used the oligo probe and amplicon probe to detect specific target genes on bacterial smear and FFPE tissue sections. We showed that the amplicon probe gives stronger signal than the oligo probe.