Development of an F57 sequence-based real-time PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in milk

Abstract

A light cycler-based real-time PCR (LC-PCR) assay that amplifies the F57 sequence of Mycobacterium avium subsp. paratuberculosis was developed. This assay also includes an internal amplification control template to monitor the amplification conditions in each reaction. The targeted F57 sequence element is unique for M.avium subsp. paratuberculosis and is not known to exist in any other bacterial species. The assay specificity was demonstrated by evaluation of 10 known M. avium subsp. paratuberculosis isolates and 33 other bacterial strains. The LC-PCR assay has a broad linear range (2 x 10(1) to 2 x10(6) copies) for quantitative estimation of the number of M. avium subsp. paratuberculosis F57 target copies in positive samples. To maximize the assay's detection sensitivity, an efficient strategy for isolation of M. avium subsp. paratuberculosis DNA from spiked milk samples was also developed. The integrated procedure combining optimal M. avium subsp. paratuberculosis DNA isolation and real-time PCR detection had a reproducible detection limit of about 10 M. avium subsp. paratuberculosis cells per ml when a starting sample volume of 10 ml of M. avium subsp. paratuberculosis-spiked milk was analyzed. The entire process can be completed within a single working day and is suitable for routine monitoring of milk samples for M. avium subsp. paratuberculosis contamination. The applicability of this protocol for naturally contaminated milk was also demonstrated using milk samples from symptomatic M. avium subsp. paratuberculosis-infected cows, as well as pooled samples from a dairy herd with a confirmed history of paratuberculosis.

FIG. 1.

Dynamics of F57 sequence and IC template coamplification under optimized LC-PCR assay conditions: graphic plot from the light cycler. The data show the results for coamplification of the F57 sequence standard dilutions and a constant amount of the IC template (2,000 copies) in the LC-PCR assay. The large graph shows M. avium subsp. paratuberculosis F57 amplification as monitored in the LC-Red 705 channel, and the inset shows IC template amplification as monitored in the LC-Red 640 channel. The inset shows that the amount of IC template used is large enough to effectively monitor amplification in the presence of small amounts of the M. avium subsp. paratuberculosis F57 target (0 to 2×10³ copies). No IC amplification was detected in the presence of large numbers of M. avium subsp. paratuberculosis F57 molecules (2 × 10⁴ to 2 × 10⁶ copies) due to competition. MAP, M. avium subsp. paratuberculosis.

FIG. 2.

Evaluation of the specificity of the M. avium subsp. paratuberculosis LC-PCR assay. Genomic DNA templates (1 ng) isolated from M. avium subsp. paratuberculosis ATCC 6783 and M. avium ATCC 19421 were amplified, and the amplicons were detected as outlined in Materials and Methods. The large graph shows F57 sequence amplification monitored by LC-Red 705 detection, and the inset shows IC amplification monitored by LC-Red 640 detection. In each reaction, the IC control template was included, and its amplification was confirmed (inset). MAP, M. avium subsp. paratuberculosis.

FIG. 3.

Influence of nonspecific DNA background on amplification of the F57 sequence by the LC-PCR assay. Tenfold serial dilutions of F57 sequence standard dilutions (2 × 10² to 2 × 10⁶) and the IC template (2,000 copies) were amplified alone (large graph) or after they were spiked into reaction mixtures containing 200 ng of nonspecific DNA (inset). The inset shows the influence of a DNA background mixture consisting of DNA templates from the following non-M. avium subsp. paratuberculosis mycobacteria: M. avium, M. bovis, M. intracellulare, M. phlei, M. scrofulaceum, M. terrae, and Mycobacterium strain 2333. MAP, M. avium subsp. paratuberculosis.

FIG. 4.

Quantitative estimation of M. avium subsp. paratuberculosis target copy numbers in defined amounts of purified M. avium subsp. paratuberculosis genomic DNA using the LC-PCR assay. F57 sequence-based quantification standards (2 × 10¹ to 2 × 10⁶ copies) and quasi-unknown samples A and B containing 15 pg and 1.5 pg of M. avium subsp. paratuberculosis DNA, respectively, were amplified. Based on theoretical calculations, samples A and B contained about 3,000 and 300 M. avium subsp. paratuberculosis genome copies, respectively. The target copy numbers in these samples were estimated from the standard curve generated from amplification of the F57 standards. The results obtained from these quantitative assays are summarized in Table 4. MAP, M. avium subsp. paratuberculosis.