The study aimed at direct detection of Mycobacterium avium subsp. Paratuberculosis (MAP) in milk
by evaluating a multiplex real-time PCR assay targeting IS900 and ISMAV2 sequences including
the amplification of PUC19-plasmid as internal control. The sensitivity of the assays was evaluated
by testing MAP isolates in broad linear range of DNA (50 ng – 5 fg/µl). For the validation of the
specificity, 6 MAP isolates and 22 isolates of genus Mycobacteriaceae were tested. Results revealed
that reproducible detection limit for real-time PCR targeting IS900 and ISMAV2 was 5 fg/µl and 50
fg/µl respectively. By targeting ISMAV2 sequence, 100% specificity was detected. However, a cross
reaction with 5 ng/µl of genome of 3 M. avian subspecies avium strains was detected by targeting
IS900 and negative in lower genome quantity (5pg/µl). To maximize the assay’s detection sensitivity,
an efficient strategy for MAP-DNA extraction from spiked milk was assessed. Targeting of IS900
was sensitive and targeting ISMAV2 was very specific. Therefore, a multiplex real-time PCR assay
targeting IS900 and ISMAV2 in combination with two commercial DNA extraction kits could be
an ideal sensitive and specific protocol for routine large-scale analysis of milk samples and other
clinical specimens from man and animals.