Prototype of the reference standard for the quantification of Mycobacterium avium subsp. paratuberculosis by quantitative PCR in faeces

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00027162%3A_____%2F18%3AN0000076" target="_blank" >RIV/00027162:_____/18:N0000076 - isvavai.cz</a>

Výsledek na webu

<a href="http://www.paratuberculosis.net/proceedings/proc14.pdf" target="_blank" >http://www.paratuberculosis.net/proceedings/proc14.pdf</a>

DOI - Digital Object Identifier

—

Jazyk výsledku

angličtina

Název v původním jazyce

Prototype of the reference standard for the quantification of Mycobacterium avium subsp. paratuberculosis by quantitative PCR in faeces

Popis výsledku v původním jazyce

14th International Colloquium on Paratuberculosis, 4.-8.6.2018, Mexiko. Quantitative PCR (qPCR) is nowadays a frequently employed method for the direct detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP). However, the quantity of MAP assessed by qPCR is dependent on the type of the qPCR quantification standard and the way how DNA quantity of the standard is determined. The most common way is the absorbance determination, which can, however, differ among different types of DNA standards (plasmid, isolated DNA). This leads to the situation that quantities of MAP determined by the different qPCR assays in different laboratories are not comparable with each other. The objective of this study was to prepare the prototype of the reference standard for the quantification of MAP in faeces by qPCR. This standard would make possible to compare the results between individual laboratories across the world. Supernatant of the 40% faceal suspension was artificially contaminated with MAP reference strain, mixed with the matrix and lyophilized. Different types of matrices were tested. Repeatability and stability of the MAP reference standard in time were tested by qPCR. This first generation of the MAP reference standard was prepared to assess the suitability of selected procedure for the generation of the worldwide qPCR standard. Our laboratory is ready to serve as the supplier of the MAP reference standard for diagnostic laboratories. This effort should bring the unification of the DNA quantification standards (independently on the DNA isolation and qPCR) in qPCR detection and quantification of MAP. And should lead to the situation that it will be possible to directly compare quantitative data from different qPCR assays and different laboratories.

Název v anglickém jazyce

Prototype of the reference standard for the quantification of Mycobacterium avium subsp. paratuberculosis by quantitative PCR in faeces

Popis výsledku anglicky

14th International Colloquium on Paratuberculosis, 4.-8.6.2018, Mexiko. Quantitative PCR (qPCR) is nowadays a frequently employed method for the direct detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP). However, the quantity of MAP assessed by qPCR is dependent on the type of the qPCR quantification standard and the way how DNA quantity of the standard is determined. The most common way is the absorbance determination, which can, however, differ among different types of DNA standards (plasmid, isolated DNA). This leads to the situation that quantities of MAP determined by the different qPCR assays in different laboratories are not comparable with each other. The objective of this study was to prepare the prototype of the reference standard for the quantification of MAP in faeces by qPCR. This standard would make possible to compare the results between individual laboratories across the world. Supernatant of the 40% faceal suspension was artificially contaminated with MAP reference strain, mixed with the matrix and lyophilized. Different types of matrices were tested. Repeatability and stability of the MAP reference standard in time were tested by qPCR. This first generation of the MAP reference standard was prepared to assess the suitability of selected procedure for the generation of the worldwide qPCR standard. Our laboratory is ready to serve as the supplier of the MAP reference standard for diagnostic laboratories. This effort should bring the unification of the DNA quantification standards (independently on the DNA isolation and qPCR) in qPCR detection and quantification of MAP. And should lead to the situation that it will be possible to directly compare quantitative data from different qPCR assays and different laboratories.

Druh

O - Ostatní výsledky

CEP obor

—

OECD FORD obor

10606 - Microbiology

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

2018

Kód důvěrnosti údajů

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