Abstract

The lower sensitivity and specificity, moreover the time required for reporting the results of the traditional methods make them of low value for diagnosis of Mycobacterium bovis.Therefore, the purpose of the prospective study was targeted to amplify a 560 bp fragment of the 16S rRNA which is conserved in all Mycobacterium species and 270 bp region nested within the first 16S rRNA and conserved only in Mycobacterium tuberculosiscomplex from the extracted DNA of 520 milk samples collected from tuberculin positive and negative animals as well as market milk samples during the period of 2008 to 2010. Amplification of 560 bp fragment was observed with the extracted DNA of 33 (6.35%) out of 520 examined milk samples, out of them 23 (4.42%) milk samples were positive by Ziehl-Neelsen stain. Amplification of 270 bp fragment was observed with 22 (4.23%) milk samples, while the other 11 samples were negative. Amplification of 500 bp fragment which is specific for M. bovis revealed positive results with 16 (3.06%) milk samples only. Polymerase chain reaction (PCR) could detect all samples yielding M. bovis with bacteriological examination in addition to, 8 milk samples with negative bacteriological examination. The results revealed that nested PCR could be used for rapid and specific detection of M. tuberculosis complex in dairy product after extraction of DNA by Trizole reagent, which seems to have the highest ability for purification of high molecular weight DNA from clinical samples.

 Key words: Mycobacterium bovis, nested polymerase chain reaction, milk samples,Mycobacterium tuberculosis complex.