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, p...

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Autores principales:	Monika Beinhauerova, Martina Beinhauerova, Sarah McCallum, Eric Sellal, Matteo Ricchi, Rory O’Brien, Beatrice Blanchard, Iva Slana, Vladimir Babak, Petr Kralik
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/98ef774986d04d299a97d5b9f83aa464
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spelling	oai:doaj.org-article:98ef774986d04d299a97d5b9f83aa4642021-12-02T15:02:57ZDevelopment of a reference standard for the detection and quantification of Mycobacterium avium subsp. paratuberculosis by quantitative PCR10.1038/s41598-021-90789-02045-2322https://doaj.org/article/98ef774986d04d299a97d5b9f83aa4642021-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-90789-0https://doaj.org/toc/2045-2322Abstract 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.Monika BeinhauerovaMartina BeinhauerovaSarah McCallumEric SellalMatteo RicchiRory O’BrienBeatrice BlanchardIva SlanaVladimir BabakPetr KralikNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Monika Beinhauerova Martina Beinhauerova Sarah McCallum Eric Sellal Matteo Ricchi Rory O’Brien Beatrice Blanchard Iva Slana Vladimir Babak Petr Kralik Development of a reference standard for the detection and quantification of Mycobacterium avium subsp. paratuberculosis by quantitative PCR
description	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.
format	article
author	Monika Beinhauerova Martina Beinhauerova Sarah McCallum Eric Sellal Matteo Ricchi Rory O’Brien Beatrice Blanchard Iva Slana Vladimir Babak Petr Kralik
author_facet	Monika Beinhauerova Martina Beinhauerova Sarah McCallum Eric Sellal Matteo Ricchi Rory O’Brien Beatrice Blanchard Iva Slana Vladimir Babak Petr Kralik
author_sort	Monika Beinhauerova
title	Development of a reference standard for the detection and quantification of Mycobacterium avium subsp. paratuberculosis by quantitative PCR
title_short	Development of a reference standard for the detection and quantification of Mycobacterium avium subsp. paratuberculosis by quantitative PCR
title_full	Development of a reference standard for the detection and quantification of Mycobacterium avium subsp. paratuberculosis by quantitative PCR
title_fullStr	Development of a reference standard for the detection and quantification of Mycobacterium avium subsp. paratuberculosis by quantitative PCR
title_full_unstemmed	Development of a reference standard for the detection and quantification of Mycobacterium avium subsp. paratuberculosis by quantitative PCR
title_sort	development of a reference standard for the detection and quantification of mycobacterium avium subsp. paratuberculosis by quantitative pcr
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/98ef774986d04d299a97d5b9f83aa464
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Development of a reference standard for the detection and quantification of Mycobacterium avium subsp. paratuberculosis by quantitative PCR

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