Efficacy of infected tissue sample brief-culture on Lowenstein-Jensen media as pre-polymerase chain reaction (Pre-PCR) to diagnosis of Mycobacterium

Mycobacterium Tuberculosis (Mtb), the causative agent of Tuberculosis does not retain any bacteriological stain due to the high lipid content in its wall. Classic methods like acid-fast staining or Ziehl-Neelsen staining are used for the diagnosis. Nucleic acid amplification techniques such as PCR are very useful in the rapid diagnosis of infections by M. tuberculosis but the sensitivity of polymerase chain reaction PCR is considerably lower because of the presence of inhibitory substances in tissues. The aim of this study was to improve the sensitivity of PCR in inhibiting tissue samples. Thirty-six lymph nodes isolated from Inoculated guinea pigs with BCG vaccine. After drinding and suspending samples in sterile distilled water; they were cultured on Lowenstein-Jensen. DNA extraction had been done after 5 days by cetyltrimethylammonium bromide (CTAB) method. Finally, PCR had been done for each extracted DNA. Sensitivity of PCR before culture was 27.8% but 62.5% following brief-culture. The sensitivity elevated by short term culture on LJ and then PCR. It is due to diffusion of inhibitory substances into the substrate, as well as to increase the number of bacteria after the brief-culture.   Key words: Mycobacteria, tuberculosis, polymerase chain reaction (PCR), brief-culture.