Assessment of mycobacterial infection and multiplication in macrophages by polymerase chain reaction

The amplification of mycobacterium-specific DNA sequences from samples obtained from infected patients by the polymerase chain reaction (PCR) has been useful in the clinical diagnosis of mycobacterial diseases. Using 20 bp oligonucleotide primers that recognize a 123 bp repeated sequence present in M. bovis and M. tuberculosis DNA, we describe in detail the conditions of the PCR reaction that allow an assessment of the mycobacterial content of infected macrophages. The results of the highly reproducible, time-efficient PCR technique show good correlation with the widely used colony forming unit (CFU) and [3H]uracil incorporation methods for the detection of Mycobacterium. Our method allows an assessment of the level of M. bovis BCG infection from a variety of sources, including peritoneal macrophages and macrophage lines, within a few hours, making it the assay of choice for rapid determination of the level of mycobacterial growth in infected cells, in experimental models of mycobacterial infection.