Development of a Peptide-Mediated Capture PCR for Detection of Mycobacterium avium subsp. paratuberculosis in Milk

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Abstract
Based on phage display technology, a peptide-mediated magnetic separation technique was developed to facilitate selective isolation of Mycobacterium avium subsp. paratuberculosis ( M. paratuberculosis ) from bulk milk of naturally infected dairy herds. Nine recombinant bacteriophages binding to M. paratuberculosis were isolated from a commercial phage-peptide library encoding random 12-mer peptides. Nucleotide sequencing revealed the deduced sequence of the binding peptides. One peptide with the sequence NYVIHDVPRHPA, designated aMP3, was chemically synthesized with an amino-terminal biotin residue attached via an amino-hexacarbonic acid spacer molecule. Paramagnetic beads coated with the phage or with peptide aMP3 enabled the capture of M. paratuberculosis from milk. Combining this peptide-mediated magnetic separation with an IS Mav2 -based PCR allowed the detection of M. paratuberculosis in artificially spiked milk down to a concentration of 10 1 ml −1 . Experiments using milk from naturally infected cows and bulk milk samples from infected herds demonstrated that the peptide-mediated capture PCR is sufficiently sensitive to detect single strong shedders in pooled milk samples. The method, for the first time, applies phage display technology to microbial diagnostics and has potential value as a completely standardizable tool for the routine M. paratuberculosis screening of bulk milk samples at acceptable costs.

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