Direct detection of Mycobacterium avium subsp. paratuberculosis in bovine milk by multiplex real-time PCR
2013
Selim, A. (Benha University, Toukh (Egypt). Faculty of Veterinary Medicine) | El-Haig, M. (Suez Canal University, Ismailia (Egypt). Faculty of Veterinary Medicine, Department of Internal Medicine and Infectious Diseases) | Galila, E. S. (Benha University, Toukh (Egypt). Faculty of Veterinary Medicine)
This study aimed to 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 Mycobacteriacea 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.
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