Extract from the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Updated: 23.07.2004
c) Nucleic acid recognition methodsreference 25
The PCR has been widely evaluated for the detection of M. tuberculosis complex in clinical samples (mainly sputum) in human patients and has recently been used for the diagnosis of tuberculosis in animals. A number of commercially available kits and various ‘in-house’ methods have been evaluated for the detection of the M. tuberculosis complex in fresh and fixed tissues. Various primers have been used, including primers that have amplified sequences from 16S–23S rRNA, the insertion sequences IS6110 and IS1081, and genes coding for M.-tuberculosis-complex-specific proteins, such as MPB70 and the 38 kDa antigen b. Amplification products have been analysed by hybridisation with probes or by gel electrophoresis. Commercial kits and the in-house methods, in fresh, frozen or boric acid-preserved tissues, have shown variable and less than satisfactory results in interlaboratory comparisons (25). False-positive and false-negative results, particularly in specimens containing low numbers of bacilli, have reduced the reliability of this test. Variability in results has been attributed to the low copy number of the target sequence per bacillus combined with a low number of bacilli. Variability has also been attributed to decontamination methods, DNA extraction procedures, techniques for the elimination of polymerase enzyme inhibitors, internal and external controls and procedures for the prevention of cross-contamination. Improvement in the reliability of PCR as a practical test for the detection of M. tuberculosis complex in fresh clinical specimens will require the development of standardised and robust procedures. PCR is not only used for direct detection in material, or strain characterisation or differentiation within the TB complex, but it is also widely used as a method of initial identification (selection of organism being done on the basis of colony morphology and AF staining). Commercial kits that are quite ‘robust’ are available, for example the Gen probe accuprobe. Although the number of species that can be identified by these kits is limited, their primers for the M. tuberculosis complex are widely used in both the human and veterinary field. Laboratories have also developed their own ‘in-house’ methods. Cross contamination is the greatest problem with this type of application and this is why proper controls have to be set up with each amplification. This type of application has been overlooked. Usually some biochemical tests are done to confirm the finding. However, PCR is now being used on a routine basis to detect the M. tuberculosis group and distinguish it from M. avium in formalin-fixed, paraffin-embedded tissues (23, 24). Optimal results are obtained when both PCR and isolation methods are used. ......
25. Noredhoek G.T., van Embden J.D.A. & Kolk A.H.J. (1996). Reliability of nucleic acid amplification for detection of Mycobacterium tuberculosis: an international collaborative quality control study among 30 laboratories. J. Clin. Microbiol., 34, 2522–2525.