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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

Imen Bouzouita
Henda Draoui
Samia Mahdhi
Leila Essalah
Leila Slim Saidi


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.

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eISSN: 1729-0503
print ISSN: 1680-6905