This study describes a peptide-mediated capture PCR assay for the specific detection of M. paratuberculosis
in milk and is the first utilization of phage display technology for developing a diagnostic test. The procedure is simple and rapid and can be standardized and automated and is, therefore, suitable for routine application in diagnostic laboratories. Such a diagnostic test is urgently required to implement effective control measures, since the prevalence of M. paratuberculosis
in dairy cattle is increasing and since there are rising concerns over putative wildlife reservoirs (3
) and the zoonotic potential of the organism (17
). As voluntary eradication and certification programs instituted in several European countries (22
) and in many states within the United States have not improved the general situation (34
), mandatory control programs need to be devised (19
). For dairy cattle, the most economic and efficient control regime would involve regular monitoring of herd milk samples for the presence of M. paratuberculosis
Present techniques for the milk-based diagnosis of paratuberculosis mainly rely on the detection of antibodies in milk (21
). However, as a positive antibody response in a herd does not necessarily indicate shedding of the organism, mandatory control measures should be based on the direct detection of the organism. Recent studies have shown that this can be done effectively using immunomagnetic separation (IMS) and subsequent PCR analyses (14
). Since, however, the method was performed with a polyclonal antibody, it would be difficult to standardize and certify it for routine applications.
In order to solve the problem of standardization, we propose the use of high-affinity peptides as an alternative to antibodies in routine diagnostic microbiology. In this study we used a commercially available phage-peptide library to identify a peptide that would specifically bind M. paratuberculosis
. However it was necessary to introduce a final high-stringency wash with a chaotropic buffer during biopanning in order to identify specific phages with sufficient affinity to be used for mycobacterial capture in milk. This might have been due to the fact that milk contains a large variety of different proteins and peptides that likely coat the mycobacterial surface. This hypothesis is supported by the fact that IMS of M. paratuberculosis
in whole milk has been shown to require a longer time than in PBS (14
A special processing of milk samples is necessary to gain optimal results for PCR-based assays. Milk is a substrate that is known to be inhibitory to the PCR (5
); therefore, an elimination of milk constituents facilitates the prevention of false negative results. When the Gene Clean Kit is used, those constituents are mostly removed, and false-negative results are eliminated by incorporating an internal control as is recommended for diagnostic PCR (18
). The failure of one of three PCRs in spiked milk samples containing 105
bacteria per ml as well as the differences in strength of the PCR products are likely to be caused by residual inhibitory substances commonly observed in milk (23
). The failure of two of three PCRs in spiked milk containing 101
bacteria per ml are likely due to the lack of bacterial DNA in these vials.
The sensitivity that we obtained with a peptide-mediated capture PCR in artificially contaminated milk was comparable to the IMS results obtained with a polyclonal serum and subsequent culture of the organism (15
). Thus, for the IMS PCR using IS900
primers, a detection limit of 103
mycobacteria per 50 ml was estimated, and we could detect 10 mycobacteria per ml. We used primers directed against a different insertion element of M. paratuberculosis
, as unspecific reactions have been reported for IS900
). Furthermore, ISMav2
is present in only three copies on the genome, and therefore, positive results obtained with this element potentially allow a detection of the organism also when using single-copy genes.
Binding of a peptide or an antibody to cultured bacteria does not necessarily ensure that it will detect bacteria shed by the infected animal, as the synthesis of surface structures is dependent on the environment (12
). We showed that the peptide-mediated capture PCR also could be used successfully on milk samples of naturally infected animals as well as on bulk milk samples. Therefore, the peptide apparently binds to a structure expressed on the mycobacterial surface upon growth in vitro and in vivo. Due to the lack of toxic reagents, the preparation protocol is likely to facilitate not only PCR detection but also culturing of the affinity-purified pathogen.
In summary, we have, for the first time, applied phage display technology to microbial diagnostics, developing a peptide-mediated capture PCR for the detection of M. paratuberculosis
in milk. Due to the defined sequence of the synthetic peptide, this assay can be standardized completely, allowing certification for routine applications. In addition, all steps of the method can be automated such that it could be applied to high-throughput screening when combined with a molecular beacon approach as described recently (11