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7th Pastor & Wife Appreciation Celebration Group

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Martin Novikov
Martin Novikov

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The use of targeted regions of MAP such as the previously mentioned multi-copy IS900 is commonly used in molecular MAP diagnostics, detecting MAP in culture, faeces, tissue, milk, milk powder and cheese. This is followed by f57, a single copy gene [63]. qPCR approaches have also been employed that target IS1311, mbtA gene, IS_MAP04 and IS_MAP02 [59, 97]. In a recent study carried out by Butot and colleagues, it was found that IS900 targets were preferred in terms of sensitivity and presented with the lowest levels of variability between laboratories, for the detection of MAP in milk in comparison to f57 and phage based techniques. A number of commonly used primers can be found in Table 1. They state this is a consequence of the use of standardised reagents using commercial kits that are available for DNA extraction and qPCR assays. Digital PCR (dPCR), also known as third generation PCR, is said to be more precise and predict absolute numbers of microbes shed in faeces and is also resistant to inhibitors. Furthermore, a calibration curve is not necessary to provide a copy number, an issue that is often faced with qPCR. However, similar hurdles may be encountered during optimisation including choice of master mix, template properties and positioning of primers using the BioMark dPCR system. Nonetheless, dPCR may be an alternative to qPCR in the validation of tests and biomarkers. Such investigations have been carried out by Devonshire et al. (2015) with regards to M. tuberculosis [102]. More recent advances in rapid disease identification using PCR-like, lab-on-a-chip, portable devices are paving the way for cheaper and faster disease diagnosis. TriSilix, a disposable, silicon-based integrated Point-of-Need transductor, has been tested to quantitatively detect MAP using IS900 primers, with the limit of detection equivalent to a single bacterium [103]. In that instance, DNA was extracted from pure cultures of MAP K-10 reference strain but further tests are needed with more complex matrices such as milk and faeces that contain inhibitors that may hamper results.




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