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

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Authors
Kim, Eui Chong
Issue Date
1992-09
Publisher
Seoul National University College of Medicine
Citation
Seoul J Med 1992;33(3):213-218
Keywords
Mycobacterium tuberculosisDot blot hybridizationDigoxigeninLambda gt 11Expression library
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.
ISSN
0582-6802
Language
English
URI
http://hdl.handle.net/10371/6239
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College of Medicine/School of Medicine (의과대학/대학원)Dept. of Medicine (의학과)The Seoul Journal of MedicineThe Seoul Journal of Medicine Vol. 33 No.3 (1992)
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