DETECTION OF MYCOBACTERIUM-BOVIS IN TISSUES BY POLYMERASE CHAIN-REACTION

A polymerase chain reaction (PCR) test was developed to detect Mycobac terium bovis in tissues. The test was based on amplification of a 248 bp segment of the insertion sequence, IS1081, present in six copies in strains of M. bovis and other members of the tuberculosis complex. Th e procedure involved digestion with proteinase K, lysis with sodium do decyl sulphate, and extraction with hexadecyl tetramethyl ammonium bro mide and phenol:chloroform:iso-amyl alcohol. When agarose gel electrop horesis was used for detection, the method was able to detect 1 fg of pure DNA, or 0.2 genome equivalents. It could also detect as few as 10 organisms from pure cultures and between 200-500 organisms from tissu es spiked with cultured organisms. Detection by hybridization was only marginally more sensitive. The method was tested on 110 selected tiss ues recovered post mortem from a variety of animals. Fifty three of 58 samples diagnosed as M. bovis culture positive, including all samples containing microscopically visible acid-fast bacilli, were positive o n duplicate testing by PCR. Five of 52 culture negative samples were a lso positive by PCR including three which contained large numbers of a cid-fast organisms. Ten of the culture negative samples came from anim als in a herd known to be free of bovine tuberculosis and all these we re negative by PCR.