Development of a reference standard for the detection and quantification of Mycobacterium avium subsp. paratuberculosis by quantitative PCR

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.

The Development of 3D Bovine Intestinal Organoid Derived Models to Investigate Mycobacterium Avium ssp Paratuberculosis Pathogenesis

“…There are several advantages and disadvantages of the different methods of MAP quantification used by different groups around the world. The gold standard is CFU analysis, but this has limitations due to the length of incubation time to obtain colonies, dormant non-replicative MAP that do not form colonies ( 38 ), and bacterial culture can lead to an underestimation of the number of viable bacteria due to the propensity of MAP to form aggregates ( 39 ) While methods based on confocal microscopy ( 21 ) or qPCR of genome copy number ( 40 ) may not differentiate between live and dead bacteria, these may be more appropriate depending on the research question being asked.…”

Isothermal inactivation of Mycobacterium avium subsp. paratuberculosis in curd simulating the stretching phase in pasta-filata cheese process

“…Briefly, decimal dilutions of the suspensions were streaked on Herrold’s egg yolk medium supplemented with Mycobactin (2 mg/l), Nalidixic acid, Vancomycin and Fungizone (HEY ANV) and analysed by f57-qPCR with the genomic equivalent method. The standard for the latter method was obtained from DNA isolated from a lyophilised MAP isolate in the framework of determining some reference material for DNA from MAP ( Beinhauerova et al, 2021 ).…”

Alternatives to antibiotics in veterinary medicine: considerations for the management of Johne's disease

“…Feces-based PCR tests are rapid and comparably sensitive to fecal culture, and the MAP-specific genetic elements, such as the insertion element IS 900 (gold standard), f57, and open reading frame MAP0865, provide prime targets for analysis (Semret et al ., 2006; Imirzalioglu et al ., 2011; Cunha et al ., 2020; Ramovic et al ., 2020; Elsohaby et al ., 2021). Quantitative real-time PCR (qPCR) methods for IS 900 detection have improved MAP sensitivity compared to conventional PCR (Sonawane and Tripathi, 2013; Albuquerque et al ., 2017; Beinhauerova et al ., 2021) and Acharya et al . (2017) have provided solutions to PCR inhibition that improved qPCR sensitivity to 80% compared to fecal culture.…”

“…qPCR has also been shown to be highly effective at detecting infective MAP in environmental samples (Albuquerque et al ., 2017; Ramovic et al ., 2020). However, fecal-qPCR tests can be liable to return false positives due to passive MAP shedding, which can lead to premature culling (Forde et al ., 2015; Corbett et al ., 2019; Whittington et al ., 2019; Beinhauerova et al ., 2021).…”