Abstract
Quantitative PCR (qPCR) has become a frequently employed direct method for the detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP). The quantity of MAP determined by qPCR, however, may be affected by the type of qPCR quantification standard used (PCR product, plasmid, genomic DNA) and the way in which standard DNA quantity is determined (absorbance, fluorescence). In practice, this can be reflected in the inability to properly compare quantitative data from the same qPCR assays in different laboratories. Thus, the aim of this study was to prepare a prototype of an international MAP reference standard, which could be used to calibrate routinely used qPCR quantification standards in various laboratories to promote clinical data comparability. Considering stability, storage and shipment issues, a lyophilised fecal suspension artificially contaminated with a MAP reference strain was chosen as the most suitable form of the standard. The effect of five types of lyophilisation matrices on standard stability was monitored on 2-weeks interval basis for 4 months by F57 qPCR. The lyophilisation matrix with 10% skimmed milk provided the best recovery and stability in time and was thus selected for subsequent comparative testing of the standard involving six diagnostic and research laboratories, where DNA isolation and qPCR assay procedures were performed with the parallel use of the identical supplied genomic DNA solution. Furthermore, the effect of storage conditions on the standard stability was tested for at least 6 months. The storage at room temperature in the dark and under light, at + 4 °C, − 20 °C and − 80 °C showed no significant changes in the stability, and also no substantial changes in MAP viability were found using phage amplification assay. The prepared MAP quantification standard provided homogeneous and reproducible results demonstrating its suitability for utilisation as an international reference qPCR standard.
Highlights
Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of a chronic and infectious disease called paratuberculosis or Johne’s d isease[1]
The quantitative polymerase chain reaction (qPCR) detection and quantification are most commonly based on amplification of MAP specific targets IS900 and F579–11
The IS900 sequence is present in 14–20 copies in the MAP genome and is a preferable target for the sensitive detection of MAP in comparison with the single copy element F57, which in turn is preferred for MAP quantification[10]
Summary
Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of a chronic and infectious disease called paratuberculosis or Johne’s d isease[1]. The determination of MAP quantity by qPCR, in faeces, is essential for the correct diagnosis and classification of the infection status of animals The reason for such precision is the phenomenon of “passive shedding” described initially by culture in heavily infected cattle herds in the U SA17. This concept is based on findings that a high percentage of MAP shedders in infected herds shed low numbers of MAP in their faeces and after the removal of heavy shedders from the herd the overall percentage of MAP shedding animals decreases. This threshold cannot be applied automatically; other factors like the size of the herd, prevalence of paratuberculosis or housing conditions must be considered
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