Identification of Mycobacterium tuberculosis by Dot Blot Hybridization Using Digoxigenin-Iabeled Probe

Abstract

Mycobacterium tuberculosis H37Rv ATCC 27294, restricted with EcoRI, was used to construct a genomic library in a bacteriophage expression vector, Lambda 9111. One 1.5-kb DNA fragment was selected by picoBlue immunoscreening kit on the basis of the ability to encode M. bovis-specific protein. Recombinant pBluescript with the 1.5-kb DNA was constructed into EcoRI site. Denatured DNA solution was blotted with BIO-DOT microfiltration apparatus(BIO-RAD) onto nylon membrane. The 1.5-kb probe, labeled with digoxigenin, reacted strongly with M. tuberculosis H37Rv, M. bovis, and 5 clinical isolates of M. tuberculosis. No hybridizations were observed with M. tuberculosis H37Ra, M. scrofulaceum, M. intracellulare, S. au reu s, S. epidennidis, S. marcescens, K. pneumoniae, E. coli, E. cloacae, P. vulgaris, and P. aeruginosa. When purified DNA from M. tuberculosis H37Rv was spotted onto nylon membrane, the 1.5-kb probe was able to detect 100 ng of DNA, which corresponds to 2 X 107 mycobacteria. This dot blot hybridization method using digoxigenin-Iabeled 1.5-kb probe may be applicable for identifying M. tuberculosis in hospital laboratories.