MeltMan: optimization, evaluation, and universal application of a qPCR system integrating the TaqMan qPCR and melting analysis into a single assay

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F75010330%3A_____%2F16%3A00011368" target="_blank" >RIV/75010330:_____/16:00011368 - isvavai.cz</a>

Výsledek na webu

<a href="http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0151204" target="_blank" >http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0151204</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1371/journal.pone.0151204" target="_blank" >10.1371/journal.pone.0151204</a>

Jazyk výsledku

angličtina

Název v původním jazyce

MeltMan: optimization, evaluation, and universal application of a qPCR system integrating the TaqMan qPCR and melting analysis into a single assay

Popis výsledku v původním jazyce

In the present work, we optimised and evaluated a qPCR system integrating 6-FAM (6-carboxyfluorescein)-labelled TaqMan probes and melting analysis using the SYTO 82 (S82) DNA binding dye in a single reaction. We investigated the influence of the S82 on various TaqMan and melting analysis parameters and defined its optimal concentration. In the next step, the method was evaluated in 36 different TaqMan assays with a total of 729 paired reactions using various DNA and RNA templates, including field specimens. In addition, the melting profiles of interest were correlated with the electrophoretic patterns. We proved that the S82 is fully compatible with the FAM-TaqMan system. Further, the advantages of this approach in routine diagnostic TaqMan qPCR were illustrated with practical examples. These included solving problems with flat or other atypical amplification curves or even false negativity as a result of probe binding failure. Our data clearly show that the integration of the TaqMan qPCR and melting analysis into a single assay provides an additional control option as well as the opportunity to perform more complex analyses, get more data from the reactions, and obtain analysis results with higher confidence.

Název v anglickém jazyce

MeltMan: optimization, evaluation, and universal application of a qPCR system integrating the TaqMan qPCR and melting analysis into a single assay

Popis výsledku anglicky

In the present work, we optimised and evaluated a qPCR system integrating 6-FAM (6-carboxyfluorescein)-labelled TaqMan probes and melting analysis using the SYTO 82 (S82) DNA binding dye in a single reaction. We investigated the influence of the S82 on various TaqMan and melting analysis parameters and defined its optimal concentration. In the next step, the method was evaluated in 36 different TaqMan assays with a total of 729 paired reactions using various DNA and RNA templates, including field specimens. In addition, the melting profiles of interest were correlated with the electrophoretic patterns. We proved that the S82 is fully compatible with the FAM-TaqMan system. Further, the advantages of this approach in routine diagnostic TaqMan qPCR were illustrated with practical examples. These included solving problems with flat or other atypical amplification curves or even false negativity as a result of probe binding failure. Our data clearly show that the integration of the TaqMan qPCR and melting analysis into a single assay provides an additional control option as well as the opportunity to perform more complex analyses, get more data from the reactions, and obtain analysis results with higher confidence.

Druh

J_x - Nezařazeno - Článek v odborném periodiku (Jimp, Jsc a Jost)

CEP obor

EE - Mikrobiologie, virologie

OECD FORD obor

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Projekt

—

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2016

Kód důvěrnosti údajů

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

Název periodika

PLoS One

ISSN

1932-6203

e-ISSN

—

Svazek periodika

11

Číslo periodika v rámci svazku

3

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

20

Strana od-do

—

Kód UT WoS článku

000373121800009

EID výsledku v databázi Scopus

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