Peptide aMptD-mediated capture PCR for detection of Mycobacterium avium subsp. paratuberculosis in bulk milk samples

Abstract

A peptide-mediated capture PCR for the detection of Mycobacterium avium subsp. paratuberculosis in bulk milk samples was developed and characterized. Capture of the organism was performed using peptide aMptD, which had been shown to bind to the M. avium subsp. paratuberculosis MptD protein (J. Stratmann, B. Strommenger, R. Goethe, K. Dohmann, G. F. Gerlach, K. Stevenson, L. L. Li, Q. Zhang, V. Kapur, and T. J. Bull, Infect. Immun. 72:1265-1274, 2004). Consistent expression of the MptD receptor protein and binding of the aMptD ligand were demonstrated by capturing different Mycobacterium avium subsp. paratuberculosis type I and type II strains and subsequent PCR analysis using ISMav2-based primers. The analytical sensitivity of the method was determined to be 5 x 10(2) CFU ml(-1) for artificially contaminated milk. The specificity of aMptD binding was confirmed by culture and competitive capture assays, showing selective enrichment of M. avium subsp. paratuberculosis (at a concentration of 5 x 10(2) CFU ml(-1)) from samples containing 100- and 1,000-fold excesses of other mycobacterial species, including M. avium subsp. avium and M. avium subsp. hominissuis. The aMptD-mediated capture of M. avium subsp. paratuberculosis using paramagnetic beads, followed by culture, demonstrated the ability of this approach to capture viable target cells present in artificially contaminated milk. Surface plasmon resonance experiments revealed that the aMptD peptide is a high-affinity ligand with a calculated association rate constant of 9.28 x 10(3) and an association constant of 1.33 x 10(9). The potential use of the method on untreated raw milk in the field was investigated by testing 423 bulk milk samples obtained from different dairy farms in Germany, 23 of which tested positive. Taken together, the results imply that the peptide-mediated capture PCR might present a suitable test for paratuberculosis screening of dairy herds, as it has an analytical sensitivity sufficient for detection of M. avium subsp. paratuberculosis in bulk milk samples under field conditions, relies on a defined and validated ligand-receptor interaction, and is adaptable to routine diagnostic laboratory automation.

FIG. 1.

Restriction enzyme analysis and peptide aMptD-mediated capture PCR with different M. avium subsp. paratuberculosis strains. (A) Restriction enzyme digestion of the PCR-amplified mptD gene. Lanes 1, undigested PCR product; lanes 2, digest with AvaI; lanes 3, digest with BsrBI; lanes 4, digest with HinfI; lanes 5, digest with NarI; lanes 6, digest with NgoMIV; lanes 7, digest with MseI; lanes M, 100-bp DNA marker. The arrows to the right indicate the expected position of the undigested PCR product (in base pairs). (B) Peptide aMptD-mediated capture PCR with different M. avium subsp. paratuberculosis strains from artificially contaminated milk, using ISMav2-derived primers in spiked milk. Lane 1, negative control; lane 2, positive control; lane M, 100-bp DNA marker. Mycobacteria were spiked at different concentrations (a, 5 × 10³ ml⁻¹; b, 5 × 10² ml⁻¹). The arrow to the right indicates the expected position of the PCR product (in base pairs).

FIG. 2.

Competitive capture PCR amplification from artificially contaminated milk. Milk was artificially contaminated with a mixture of M. avium subsp. avium and M. avium subsp. paratuberculosis (A) or M. avium subsp. hominissuis and M. avium subsp. paratuberculosis (B). Lanes 1, negative control; lanes 2, M. avium subsp. paratuberculosis DNA; lanes 2, M. avium subsp. avium (A) or M. avium subsp. hominissuis DNA (B); lanes 4 to 8, capture from milk spiked with 10⁵ to 10¹ bacteria ml⁻¹ of M. avium subsp. avium (A) or M. avium subsp. hominissuis (B), with each sample containing 10³ bacteria ml⁻¹ of M. avium subsp. paratuberculosis. The arrows to the right indicate the expected positions of the PCR products obtained with ISMav2-derived primers (top), IS901-derived primers (bottom in panel A), or IS1245-derived primers (bottom in panel B).

FIG. 3.

Restriction enzyme analysis of PCR-amplified 16S RNA genes of different mycobacterial strains and competitive capture PCR amplification from milk artificially contaminated with a mixture of M. avium subsp. paratuberculosis and seven different mycobacterial species. (A) Restriction enzyme digestion of the PCR-amplified 16S RNA genes of the mycobacterial species indicated at the top. Lanes 1, undigested PCR product; lanes 2, digest with AvaI; lanes 3, digest with NciI; lanes 4, digest with Tsp45I; lane M, 100-bp DNA marker. The arrow on the right indicates the expected position of the PCR product (in base pairs). (B) Competitive capture PCR amplification from milk artificially contaminated with a mixture of M. avium subsp. paratuberculosis and seven different mycobacterial species, followed by restriction enzyme digestion. Lanes 1, undigested PCR product; lanes 2, digest with AvaI; lanes 3, digest with NciI; lanes 4, digest with Tsp45I. The number of bacteria ml⁻¹ (10⁴ or 10⁵) of each mycobacterial species in spiked milk is indicated, with each sample containing 10² bacteria ml⁻¹ of M. avium subsp. paratuberculosis. Lane M, 100-bp DNA marker. The arrow to the right indicates the expected position of the PCR product (in base pairs).

FIG. 4.

Plotted sensograms obtained using the BIAcore system. Peptides aMptD and aMpR were injected at increasing concentrations (3.125 μg ml⁻¹, 6.25 μg ml⁻¹, 12.5 μg ml⁻¹, and 25 μg ml⁻¹) over immobilized membranes of M. avium subsp. paratuberculosis and M. avium subsp. avium.

FIG. 5.

Peptide aMptD-mediated capture PCR. (A) Peptide-mediated capture PCR with artificially contaminated milk, using ISMav2-derived primers. Lane 1, negative control; lane 2, positive amplification control with M. avium subsp. paratuberculosis DNA as the template. The number (10⁵ to 10⁰) of M. avium subsp. paratuberculosis bacteria ml⁻¹ is indicated. The arrow on the right indicates the expected position of the PCR product (in base pairs). (B) Peptide-mediated capture PCR amplification from farm-derived bulk milk samples, using ISMav2-specific primers and incorporated IAC. Lanes 1 to 13, peptide-mediated capture PCR amplification from farm-derived bulk milk samples; lane 14, positive control (milk spiked with 10² bacteria of M. avium subsp. paratuberculosis ml⁻¹); lane 15, negative control. The arrows on the right indicate the expected positions of the PCR products (in base pairs); the arrows on the bottom indicate the positions of positive bulk milk samples.