Choice of Target Genes for PCR
The gene target in any pathogen PCR detection assay needs to be specific for that pathogen. The discovery of IS900, a multicopy insertion sequence (17 copies/MAP genome), has provided a sensitive and specific target for molecular
Table 19.2.
Genes from Mycobacterium avium subsp. paratuberculosis (MAP) used as target genes for PCR-based diagnostic tests for paratuberculosis, with examples of published assays for each gene (adapted from Bolske and Herthnek, 2012).| Gene Copy no.a | Selected references regarding validation and use in PCR assays |
| IS900 17 | Kawaji et al. (2007); Donaghy et al. (2011); Schwalm et al. (2018); Arsenault et al. (2019) |
| ISMap02 6 ISMav2 3 | Stabel and Bannantine (2005); Irenge et al. (2009) Strommenger et al. (2001); Sting et al. (2014) |
| f57 1 | Ricchi et al. (2014); Schonenbrucher et al. (2008) |
| hspX 1 MAP2765c 1 | Ellingson et al. (1998); Alinovi et al. (2009) Imirzalioglu et al. (2011) |
| MAP0865 1 | Imirzalioglu et al. (2011) |
’Number of copies in the MAP genome.
detection of MAP DNA (Green et al., 1989; Collins et al., 1993) and continues to be used in many PCR assays. The advantage of using a multicopy gene is it provides higher analytical sensitivity of the PCR assay as compared with targeting a single-copy gene. However, a number of sequences with similarity to the IS 900 sequence have been identified, following nonspecific amplification using primers thought to be specific for the MAP IS900; Strain 2333 in Sweden was the most similar, having 94% sequence homology to MAP IS900 (Cousins et al., 1999; Englund et al., 2002; Taddei et al., 2008). These bacterial species are not commonly identified in the field and are usually phenotypically distinct in terms of growth in culture (e.g. faster growth on media without mycobactin), however it is advisable that the analytical specificity of IS900 PCR assays be confirmed (Kawaji et al., 2007).
Various other MAP-specific genes have been identified and used as targets in PCR-based diagnostic assays (Table 19.2); these may allow for increased specificity of the PCR assays, however, there are fewer copy numbers compared with IS900 within the MAP genome (Bannantine et al., 2002).
19.4