Development of a peptide-mediated capture PCR for detection of Mycobacterium avium subsp. paratuberculosis in milk

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 ISMav2-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.

FIG. 1.

Plate binding assay using M. paratuberculosis (M. ptb) and M. avium as solid-phase antigen and serial twofold dilutions of alkaline phosphatase-labeled phage fMP3 and control phages (initial phage library). The bars are the arithmetic means of the normalized optical densities at 405 nm (OD₄₀₅) obtained in three independent experiments, each performed in duplicate with the lines indicating the standard deviation.

FIG. 2.

Binding of phage fMP3 to M. paratuberculosis. Phage fMP3 (a and b) and the original phage library as control phages (c and d) were labeled with FITC and incubated with M. paratuberculosis. Specific binding of phage fMP3 to M. paratuberculosis was shown by comparing fluorescence (a and c) and light microscopy (b and d).

FIG. 3.

Capture of M. paratuberculosis using peptide-coated paramagnetic beads. Acridine orange stain of paramagnetic beads alone (a and b) and M. paratuberculosis incubated with coated paramagnetic beads carrying peptide aMP3 (c and d) or peptide aMPr (negative control; e and f). Specific binding of peptide aMP3 to M. paratuberculosis was shown by comparing fluorescence microscopy (a, c, and e) and light microscopy (b, d, and e).

FIG. 4.

Peptide-mediated capture PCR of M. paratuberculosis from milk spiked with M. paratuberculosis using ISMav2-derived primers. Blocks 1 to 6 correspond to milk samples containing 10⁵ to 10⁰ ml of M. paratuberculosis⁻¹. Lanes a to c contain the products of three parallel PCRs of the respective sample; lanes d contain the internal positive control (sample spiked with M. paratuberculosis DNA), and − indicates the position of the negative control (coated beads and milk only). The arrow to the left indicates the expected position of the PCR product of 318 bp in length.

FIG. 5.

Peptide-mediated capture PCR of M. paratuberculosis from bulk milk of ELISA-positive herds using ISMav2-derived primers. Blocks 1 to 7 correspond to milk samples from seven different ELISA-positive herds. Lanes a contain the products of the PCRs of the respective sample; lanes b contain the internal positive control (sample spiked with M. paratuberculosis DNA), and − indicates the position of the negative control (coated beads and milk only). The arrow to the left indicates the expected position of the PCR product of 318 bp in length.