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.

Adiagene Ploufragan France

Biobest Laboratories Milton Bridge UK

BioSellal Dardilly France

Department of Animal Origin Food and Gastronomic Sciences University of Veterinary and Pharmaceutical Sciences Brno Czech Republic

Department of Experimental Biology Faculty of Science Masaryk University Brno Czech Republic

Department of Microbiology and Antimicrobial Resistance Veterinary Research Institute Brno Czech Republic

Disease Research Ltd Mosgiel New Zealand

Istituto Zooprofilattico Sperimentale della Lombardia e dell´Emilia Romagna Piacenza Italy

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Stabel JR. Johne's disease: A hidden threat. J. Dairy Sci. 1998;81:283–288. doi: 10.3168/jds.S0022-0302(98)75577-8. PubMed DOI

Manning EJB, Collins MT. Mycobacterium avium subsp paratuberculosis: Pathogen, pathogenesis and diagnosis. Rev. Sci. Tech. 2001;20:133–150. doi: 10.20506/rst.20.1.1275. PubMed DOI

Whittington RJ, Sergeant ES. Progress towards understanding the spread, detection and control of Mycobacterium avium subsp paratuberculosis in animal populations. Aust. Vet. J. 2001;79:267–278. doi: 10.1111/j.1751-0813.2001.tb11980.x. PubMed DOI

Feller M, et al. Mycobacterium avium subspecies paratuberculosis and Crohn's disease: A systematic review and meta-analysis. Lancet Infect. Dis. 2007;7:607–613. doi: 10.1016/S1473-3099(07)70211-6. PubMed DOI

Ayele WY, Svastova P, Roubal P, Bartos M, Pavlik I. Mycobacterium avium subspecies paratuberculosis cultured from locally and commercially pasteurized cow's milk in the Czech Republic. Appl. Environ. Microbiol. 2005;71:1210–1214. doi: 10.1128/Aem.71.3.1210-1214.2005. PubMed DOI PMC

Ellingson JLE, et al. Detection of viable Mycobacterium avium subsp paratuberculosis in retail pasteurized whole milk by two culture methods and PCR. J. Food Prot. 2005;68:966–972. doi: 10.4315/0362-028X-68.5.966. PubMed DOI

Paolicchi F, Cirone K, Morsella C, Gioffre A. First isolation of Mycobacterium avium subsp paratuberculosis from commercial pasteurized milk in Argentina. Braz. J. Microbiol. 2012;43:1034–1037. doi: 10.1590/S1517-83822012000300028. PubMed DOI PMC

Timms VJ, Gehringer MM, Mitchell HM, Daskalopoulos G, Neilan BA. How accurately can we detect Mycobacterium avium subsp. paratuberculosis infection? J. Microbiol. Methods. 2011;85:1–8. doi: 10.1016/j.mimet.2011.01.026. PubMed DOI

Kawaji S, Taylor DL, Mori Y, Whittington RJ. Detection of Mycobacterium avium subsp paratuberculosis in ovine faeces by direct quantitative PCR has similar or greater sensitivity compared to radiometric culture. Vet. Microbiol. 2007;125:36–48. doi: 10.1016/j.vetmic.2007.05.002. PubMed DOI

Slana I, Kralik P, Kralova A, Pavlik I. On-farm spread of Mycobacterium avium subsp paratuberculosis in raw milk studied by IS900 and F57 competitive real time quantitative PCR and culture examination. Int. J. Food Microbiol. 2008;128:250–257. doi: 10.1016/j.ijfoodmicro.2008.08.013. PubMed DOI

Elguezabal N, et al. Estimation of Mycobacterium avium subsp paratuberculosis growth parameters: Strain characterization and comparison of methods. Appl. Environ. Microbiol. 2011;77:8615–8624. doi: 10.1128/AEM.05818-11. PubMed DOI PMC

Dhanasekaran S, Doherty TM, Kenneth J, Grp TTS. Comparison of different standards for real-time PCR-based absolute quantification. J. Immunol. Methods. 2010;354:34–39. doi: 10.1016/j.jim.2010.01.004. PubMed DOI

Chaitanya RK, Reddy YK, Raj GD, Thangavelu A. Quantification of Mycobacterium avium subsp. paratuberculosis from the tissues of challenged mice using SYBR Green real time PCR assay for the assessment of vaccine efficacy. Indian J. Anim. Res. 2019;53:944–948.

Song N, et al. Detection of circulating Mycobacterium tuberculosis-specific DNA by droplet digital PCR for vaccine evaluation in challenged monkeys and TB diagnosis. Emerg. Microbes Infect. 2018;7:78. PubMed PMC

Mitchell RM, Gollnick NS, Sreevatsan S, Russell DG, Schukken YH. Quantification of Mycobacterium avium subsp. paratuberculosis (MAP) survival in monocyte-derived macrophages. Vet. Immunol. Immunopathol. 2011;139:73–78. doi: 10.1016/j.vetimm.2010.08.003. PubMed DOI

Pavsic J, et al. Standardization of nucleic acid tests for clinical measurements of bacteria and viruses. J. Clin. Microbiol. 2015;53:2008–2014. doi: 10.1128/JCM.02136-14. PubMed DOI PMC

Whitlock, R. H. et al. "Cattle shedding MAP: a new paradigm, passive shedding or active shedding?". In Johne’s Disease Integrated Program 4th Annual Conference, Lansing, Michigan, 72–82 (2018).

Kralik P, Pribylova-Dziedzinska R, Kralova A, Kovarcik K, Slana I. Evidence of passive faecal shedding of Mycobacterium avium subsp. paratuberculosis in a Limousin cattle herd. Vet. J. 2014;201:91–94. doi: 10.1016/j.tvjl.2014.02.011. PubMed DOI

Kralik P, Beran V, Pavlik I. Enumeration of Mycobacterium avium subsp. paratuberculosis by quantitative real-time PCR, culture on solid media and optical densitometry. BMC Res. Notes. 2012;5:114. doi: 10.1186/1756-0500-5-114. PubMed DOI PMC

Butot S, et al. Estimation of performance characteristics of analytical methods for Mycobacterium avium subsp. paratuberculosis detection in dairy products. Front. Microbiol. 2019;10:509. doi: 10.3389/fmicb.2019.00509. PubMed DOI PMC

Ravva SV, Stanker LH. Real-time quantitative PCR detection of Mycobacterium avium subsp. paratuberculosis and differentiation from other mycobacteria using SYBR Green and TaqMan assays. J. Microbiol. Methods. 2005;63:305–317. doi: 10.1016/j.mimet.2005.04.004. PubMed DOI

Stanley EC, et al. Development of a new, combined rapid method using phage and PCR for detection and identification of viable Mycobacterium paratuberculosis bacteria within 48 hours. Appl. Environ. Microbiol. 2007;73:1851–1857. doi: 10.1128/AEM.01722-06. PubMed DOI PMC

Madej RM, et al. International standards and reference materials for quantitative molecular infectious disease testing. J. Mol. Diagn. 2010;12:133–143. doi: 10.2353/jmoldx.2010.090067. PubMed DOI PMC

Holmes H, Davis G, Heath A, Hewlett I, Lelie N. An international collaborative study to establish the 1st international standard for HIV-1 RNA for use in nucleic acid-based techniques. J. Virol. Methods. 2001;92:141–150. doi: 10.1016/S0166-0934(00)00283-4. PubMed DOI

Saldanha J, et al. An international collaborative study to establish a World Health Organization international standard for hepatitis B virus DNA nucleic acid amplification techniques. Vox Sang. 2001;80:63–71. doi: 10.1046/j.1423-0410.2001.00003.x. PubMed DOI

Saldanha J, Lelie N, Yu MW, Heath A, Grp BCS. Establishment of the first World Health Organization international standard for human parvovirus B19 DNA nucleic acid amplification techniques. Vox Sang. 2002;82:24–31. doi: 10.1046/j.1423-0410.2002.00132.x. PubMed DOI

Saldanha J, et al. World Health Organization collaborative study to establish a replacement WHO international standard for hepatitis C virus RNA nucleic acid amplification technology assays. Vox Sang. 2005;88:202–204. doi: 10.1111/j.1423-0410.2005.00606.x. PubMed DOI

Saldanha J, et al. A World Health Organization international standard for hepatitis A virus RNA nucleic acid amplification technology assays. Vox Sang. 2005;89:52–58. doi: 10.1111/j.1423-0410.2005.00633.x. PubMed DOI

Sweeney RW, et al. Tissue predilection sites and effect of dose on Mycobacterium avium subs. paratuberculosis organism recovery in a short-term bovine experimental oral infection model. Res. Vet. Sci. 2006;80:253–259. doi: 10.1016/j.rvsc.2005.07.007. PubMed DOI

World Health Organization. Recommendations for the preparation, characterization and establishment of international and other biological reference standards. WHO Technical Report Services No. 932 (2006). https://www.who.int/bloodproducts/publications/TRS932Annex2_Inter_biolefstandardsrev2004.pdf?ua=1 Accessed 18 Dec 2020.

Huggett JF, et al. The digital MIQE guidelines: Minimum information for publication of quantitative digital PCR experiments. Clin. Chem. 2013;59:892–902. doi: 10.1373/clinchem.2013.206375. PubMed DOI

Kuypers J, Jerome KR. Applications of digital PCR for clinical microbiology. J. Clin. Microbiol. 2017;55:1621–1628. doi: 10.1128/JCM.00211-17. PubMed DOI PMC

Beinhauerova M, Babak V, Bertasi B, Boniotti MB, Kralik P. Utilization of digital PCR in quantity verification of plasmid standards used in quantitative PCR. Front. Mol. Biosci. 2020;7:155. doi: 10.3389/fmolb.2020.00155. PubMed DOI PMC

Taylor SC, Laperriere G, Germain H. Droplet digital PCR versus qPCR for gene expression analysis with low abundant targets: From variable nonsense to publication quality data. Sci. Rep. 2017;7:2409. doi: 10.1038/s41598-017-02217-x. PubMed DOI PMC

Foddai A, Strain S, Whitlock RH, Elliott CT, Grant IR. Application of a peptide-mediated magnetic separation-phage assay for detection of viable Mycobacterium avium subsp paratuberculosis to bovine bulk tank milk and feces samples. J. Clin. Microbiol. 2011;49:2017–2019. doi: 10.1128/JCM.00429-11. PubMed DOI PMC

Saldanha J, Lelie N, Heath A, Grp WCS. Establishment of the first international standard for nucleic acid amplification technology (NAT) assays for HCV RNA. Vox Sang. 1999;76:149–158. doi: 10.1046/j.1423-0410.1999.7630149.x. PubMed DOI

Preiksaitis JK, et al. Interlaboratory comparison of Epstein-Barr virus viral load assays. Am. J. Transplant. 2009;9:269–279. doi: 10.1111/j.1600-6143.2008.02514.x. PubMed DOI