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

J 2021

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

BEINHAUEROVÁ, Monika; Martina BEINHAUEROVÁ; Sarah MCCALLUM; Eric SELLAL; Matteo RICCHI et al.

Základní údaje

Originální název

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

Autoři

BEINHAUEROVÁ, Monika; Martina BEINHAUEROVÁ; Sarah MCCALLUM; Eric SELLAL; Matteo RICCHI; Rory O’BRIEN; Beatrice BLANCHARD; Iva SLANA; Vladimir BABAK a Petr KRALIK

Vydání

Scientific Reports, Nature Research, 2021, 2045-2322

Další údaje

Jazyk

angličtina

Typ výsledku

Článek v odborném periodiku

Obor

10606 Microbiology

Stát vydavatele

Německo

Utajení

není předmětem státního či obchodního tajemství

Odkazy

URL

Impakt faktor

Impact factor: 4.997

Označené pro přenos do RIV

Ano

Kód RIV

RIV/00216224:14310/21:00121710

Organizační jednotka

Přírodovědecká fakulta

Klíčová slova anglicky

reference standard; Mycobacterium avium subsp. paratuberculosis; MAP; qPCR; detection; quantification; stability; comparability

Štítky

14314010, rivok

Příznaky

Mezinárodní význam, Recenzováno

Změněno: 16. 5. 2022 11:22, Mgr. Marie Novosadová Šípková, DiS.

Anotace

V originále

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.

Citovat

BEINHAUEROVÁ, Monika; Martina BEINHAUEROVÁ; Sarah MCCALLUM; Eric SELLAL; Matteo RICCHI; Rory O’BRIEN; Beatrice BLANCHARD; Iva SLANA; Vladimir BABAK a Petr KRALIK. Development of a reference standard for the detection and quantification of Mycobacterium avium subsp. paratuberculosis by quantitative PCR. Scientific Reports. Nature Research, 2021, roč. 11, č. 1, s. 1-12. ISSN 2045-2322. Dostupné z: https://doi.org/10.1038/s41598-021-90789-0.

@article{1773517,
   author = {Beinhauerová, Monika and Beinhauerová, Martina and McCallum, Sarah and Sellal, Eric and Ricchi, Matteo and O’Brien, Rory and Blanchard, Beatrice and Slana, Iva and Babak, Vladimir and Kralik, Petr},
   article_number = {1},
   doi = {https://doi.org/10.1038/s41598-021-90789-0},
   keywords = {reference standard; Mycobacterium avium subsp. paratuberculosis; MAP; qPCR; detection; quantification; stability; comparability},
   language = {eng},
   issn = {2045-2322},
   journal = {Scientific Reports},
   title = {Development of a reference standard for the detection and quantification of Mycobacterium avium subsp. paratuberculosis by quantitative PCR},
   url = {https://doi.org/10.1038/s41598-021-90789-0},
   volume = {11},
   year = {2021}
}

TY  - JOUR
ID  - 1773517
AU  - Beinhauerová, Monika - Beinhauerová, Martina - McCallum, Sarah - Sellal, Eric - Ricchi, Matteo - O’Brien, Rory - Blanchard, Beatrice - Slana, Iva - Babak, Vladimir - Kralik, Petr
PY  - 2021
TI  - Development of a reference standard for the detection and quantification of Mycobacterium avium subsp. paratuberculosis by quantitative PCR
JF  - Scientific Reports
VL  - 11
IS  - 1
SP  - 1-12
EP  - 1-12
PB  - Nature Research
SN  - 20452322
KW  - reference standard
KW  - Mycobacterium avium subsp. paratuberculosis
KW  - MAP
KW  - qPCR
KW  - detection
KW  - quantification
KW  - stability
KW  - comparability
UR  - https://doi.org/10.1038/s41598-021-90789-0
N2  - 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.
ER  -

BEINHAUEROVÁ, Monika; Martina BEINHAUEROVÁ; Sarah MCCALLUM; Eric SELLAL; Matteo RICCHI; Rory O’BRIEN; Beatrice BLANCHARD; Iva SLANA; Vladimir BABAK a Petr KRALIK. Development of a reference standard for the detection and quantification of Mycobacterium avium subsp. paratuberculosis by quantitative PCR. \textit{Scientific Reports}. Nature Research, 2021, roč.~11, č.~1, s.~1-12. ISSN~2045-2322. Dostupné z: https://doi.org/10.1038/s41598-021-90789-0.

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