For many years the dairy industry has been faced with a significant problem regarding M. avium
. This organism causes chronic disease in cattle, is difficult to grow, has been shown to survive pasteurization when it is present at high concentrations (8
), and has been implicated as a potential factor in the development of Crohn's disease. Consequently, the demand for rapid and sensitive milk screening assays should be a priority in the dairy farming and milk-producing industries. Ideally, any analytical method should be able to establish presence or absence, microbial load, and viability. Unfortunately, within the shelf life of liquid milk these factors are difficult to establish for M. avium
due to its slow growth. In order to address the quantitative difficulties, it was our intention to take technology previously developed in our laboratory (25
) and design a real-time PCR assay for M. avium
following extraction from milk which would be rapid, sensitive, and ultimately quantitative. Recently, two such assays have been reported for M. avium
from bovine fecal samples, which illustrated the potential and application of this technology for slow-growing pathogens (7
In this study, the target chosen was a region of the IS900
transposon as this region is the most widely used target for M. avium
from milk (5
) and also multiple copies (14 to 18 copies) are present in each cell, thereby increasing the sensitivity of the assay. We decided to use fluorescent probes in place of SYBR Green due to the specificity of the probes and the greater sensitivity afforded by them. After the various PCR parameters were optimized, the assay proved to be sensitive, quantitative, and reproducible, providing a suitable alternative to previously developed methods. As expected, the real-time PCR worked exceptionally well with purified template (Fig. ) (sensitivity, five copies, which is equivalent to less than one organism), was reproducible (Fig. ), and was completed in less than 30 min, which is significantly faster than conventional PCR assays. Furthermore, the analysis of each sample was carried out in real time and in closed capillaries, offering the advantages of convenience and assurance of sample integrity. However, despite the apparent benefits of this procedure with purified DNA, the diagnostic applications are limited in the absence of a robust and effective procedure for extraction of M. avium
DNA from milk.
To this end, several extraction strategies were evaluated, and despite significant variation in yield, a strong PCR signal was observed for most methods with a moderate inoculum of M. avium
CFU/ml) (Fig. ). This allowed us to refine the strategy and improve the sensitivity even further. Surprisingly, pretreatment of the milk with enzymes, solvents, and clarifying agents reduced the efficiency, and simple lysis solutions proved to be the most effective treatment. In particular, the use of GITC in the lysis mixture (adopted from the method of Odumeru et al. [24
]) and inclusion of nucleic acid spin columns were extremely useful in purifying the DNA.
Therefore, the combination of centrifugation, harsh lysis, physical grinding, boiling, nucleic acid purification, and real-time PCR pushed the detection limit to 40 CFU/ml of milk, which is comparable to or better than the detection limits in previously reported studies (5
). Furthermore, results could be obtained in less than 3 h, which is significantly faster than results previously obtained in studies with milk. The most notable advantage of this strategy over previously reported methods is the ability to quantitate the initial titer of M. avium
in milk by using predetermined standards. Quantitation of DNA from microbes by using real-time PCR has been reported previously (13
) and is based on determining the crossing point for each sample (the cycle number at which the fluorescence is notably increased above a baseline level). This crossing point or threshold is unique for a particular concentration of DNA and can be used to construct a standard curve that is used to determine the corresponding concentrations of unknown samples. By using software available with the Lightcycler system, the crossing points of standards were plotted and used to accurately measure the amounts of DNA targets (Fig. ) or the numbers of CFU per milliliter (Table ). In both cases, quantitation was linear over a broad range and the method could be used for heavily or mildly contaminated products.
Prior to this study it was difficult to accurately determine the microbial load of M. avium
in milk as conventional PCR is not quantitative and decontamination procedures necessary to eliminate other organisms during culturing are lethal for a variable proportion of the M. avium
population. Determining the presence and number of M. avium
organisms in a milk sample provides a useful means for identifying contaminated product. The presence of M. avium
in milk samples as determined by PCR has been reported by many workers, including workers in the United Kingdom (21
), the United States (26
), Canada (8
), and Switzerland (5
), providing evidence that M. avium
DNA or whole cells are entering the human food chain. Using the optimized assay described above should facilitate rapid, sensitive, and quantitative screening for M. avium
in milk, as shown in Table . Although this assay was evaluated solely with spiked milk, the initial experimental results indicate that our extraction and detection methodology is equally effective in detecting M. avium
in naturally contaminated milk (data not shown). Further laboratory work and surveillance are being performed in order to prove this observation.
In summary, we developed and optimized a real-time PCR assay for M. avium subsp. paratuberculosis with fluorescence resonance energy transfer probes which, when combined with a strategic extraction method, generate qualitative or quantitative data for the presence of M. avium subsp. paratuberculosis in milk in less than 3 h. Implementation of this assay could lead to early identification of contaminated product and allow control measures to be put in place to protect the public from unnecessary exposure to M. avium subsp. paratuberculosis until further unambiguous evidence is made available regarding the dangers associated with this organism for humans.