Creation and stability testing of the reference standard for the detection and quantification of Mycobacterium avium subsp. paratuberculosis in faeces by qPCR

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00027162%3A_____%2F19%3AN0000259" target="_blank" >RIV/00027162:_____/19:N0000259 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00027162:_____/19:N0000324

Výsledek na webu

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DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

Creation and stability testing of the reference standard for the detection and quantification of Mycobacterium avium subsp. paratuberculosis in faeces by qPCR

Popis výsledku v původním jazyce

XXVIII. Tomasek days of young microbiologists, 6th and 7th June 2019 at Brno – lecture. Quantitative PCR (qPCR) is nowadays one of the most widely used direct methods for the detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP). However, the amount of MAP determined by qPCR depends on the type of quantification standard used (plasmid, isolated DNA), which in practice can lead to a situation where it is not possible to compare the resulting data of different qPCR tests in different laboratories. The creation of the reference standard should bring the unification of quantification standards in MAP detection and allow comparison of quantitative data between different laboratories. The aim of this study was the preparation of reference standard prototype for quantification of MAP in faeces by qPCR method, to find a suitable storage matrix and test stability and reproducibility over time. With regard to stability, storage and possible shipment, a lyophilized suspension of faeces artificially contaminated with a reference MAP strain was chosen as the most suitable form of standard. In total, five types of lyophilization matrices were used and their effects on MAP stability were monitored at two-weekly intervals. DNA from the lyophilized standard was isolated using a standardized protocol, and MAP quantification was performed using qPCR. The most suitable lyophilization matrix was selected and is used in further testing to monitor the effect of different storage conditions on standard stability.

Název v anglickém jazyce

Creation and stability testing of the reference standard for the detection and quantification of Mycobacterium avium subsp. paratuberculosis in faeces by qPCR

Popis výsledku anglicky

XXVIII. Tomasek days of young microbiologists, 6th and 7th June 2019 at Brno – lecture. Quantitative PCR (qPCR) is nowadays one of the most widely used direct methods for the detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP). However, the amount of MAP determined by qPCR depends on the type of quantification standard used (plasmid, isolated DNA), which in practice can lead to a situation where it is not possible to compare the resulting data of different qPCR tests in different laboratories. The creation of the reference standard should bring the unification of quantification standards in MAP detection and allow comparison of quantitative data between different laboratories. The aim of this study was the preparation of reference standard prototype for quantification of MAP in faeces by qPCR method, to find a suitable storage matrix and test stability and reproducibility over time. With regard to stability, storage and possible shipment, a lyophilized suspension of faeces artificially contaminated with a reference MAP strain was chosen as the most suitable form of standard. In total, five types of lyophilization matrices were used and their effects on MAP stability were monitored at two-weekly intervals. DNA from the lyophilized standard was isolated using a standardized protocol, and MAP quantification was performed using qPCR. The most suitable lyophilization matrix was selected and is used in further testing to monitor the effect of different storage conditions on standard stability.

Druh

O - Ostatní výsledky

CEP obor

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OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

<a href="/cs/project/VI20152020044" target="_blank" >VI20152020044: Multiplexní xMAP technologie pro komplexní detekci patogenních agens významných z pohledu zajištění ochrany zdraví lidí a zvířat</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)<br>I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2019

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů