Evaluation of PCR pncA-restriction fragment length polymorphism and PCR amplification of genomic regions of difference for the identification of M. bovis strains in lymph nodes cultures

Abstract

Background: A rapid accurate identification of Mycobacterium bovis is essential for surveillance purposes. Objectives: A PCR pncA-Restriction Fragment Length Polymorphism (RFLP) and a multiplex PCR based on the detection of 3 regions of difference (RD-PCR): RD9, RD4 and RD1 were evaluated for the identification of M. bovis in lymph nodes cultures, in Tunisia, during 2013-2015. Methods: Eighty-two M. tuberculosis complex strains were identified using the biochemical tests, GenoType MTBC assay, PCR pncA-RFLP and RD-PCR. Results: The PCR pncA-RFLP showed that 54 M. bovis strains, identified by GenoType MTBC, had a mutation at position 169 of pncA gene. Twenty-eight strains did not show any mutation at this position 27 M. tuberculosis isolates and one M. caprae. The PCR pncA-RFLP had a sensitivity of 100.0% (95%CI: 93.3 -100.0) and a specificity of 100.0% (95%CI: 87.9- 100.0) for identifying M. bovis. The RD-PCR showed that all M. bovis strains had the RD9 and RD4 deleted but presented RD1. RD-PCR also presented high sensitivity and specificity in detecting M. bovis strains (100.0%). Conclusions: PCR pncA-RFLP and RD-PCR represent very accurate and rapid tools to identify M. bovis. They can be easily implemented in each laboratory due to their low cost and easy use. Keywords: GenoType MTBC; lymph nodes; Mycobacterium bovis; PCR pncA-RFLP; RD-PCR.