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

Result code in 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>

Result on the web

<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>

Result language

angličtina

Original language name

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

Original language description

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.

Czech name

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Czech description

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Type

J_x - Unclassified - Peer-reviewed scientific article (Jimp, Jsc and Jost)

CEP classification

EE - Microbiology, virology

OECD FORD branch

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Project

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Continuities

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Publication year

2016

Confidentiality

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

Name of the periodical

PLoS One

ISSN

1932-6203

e-ISSN

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Volume of the periodical

11

Issue of the periodical within the volume

3

Country of publishing house

US - UNITED STATES

Number of pages

20

Pages from-to

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UT code for WoS article

000373121800009

EID of the result in the Scopus database

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