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Direct detection of Mycobacterium avium subsp. Paratuberculosis in bovine milk by multiplex Real-time PCR

Abdelfatah Selim
Faculty of Veterinary Medicine, Benha University, P.O. Box 13736, Toukh, Egypt
Abstract :
The study aimed at direct detection of
Mycobacterium avium
) 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
isolates in broad linear range of DNA (50 ng – 5 fg/μl). For the validation of the
specificity, 6
isolates and 22 isolates of genus
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
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
-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.
Keywords :
nternal control / milk / multiplex real-time PCR / paratuberculosis

Date Deposited : 02 Apr 2015 10:23

Last Modified : 02 Apr 2015 10:23

Official URL:

Volume 31, Number 4, - 2013

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