PCR Multiplexing Based on a Single Fluorescent Channel Using Dynamic Melting Curve Analysis

Result code in IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216305%3A26620%2F20%3APU137798" target="_blank" >RIV/00216305:26620/20:PU137798 - isvavai.cz</a>

Result on the web

<a href="https://pubs.acs.org/doi/pdf/10.1021/acsomega.0c04766" target="_blank" >https://pubs.acs.org/doi/pdf/10.1021/acsomega.0c04766</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1021/acsomega.0c04766" target="_blank" >10.1021/acsomega.0c04766</a>

Result language

angličtina

Original language name

PCR Multiplexing Based on a Single Fluorescent Channel Using Dynamic Melting Curve Analysis

Original language description

Since its invention in 1986, the polymerase chain reaction (PCR), has become a well-established method for the detection and amplification of deoxyribonucleic acid (DNA) with a specific sequence. Incorporating fluorescent probes, known as TaqMan probes, or DNA intercalating dyes, such as SYBR Green, into the PCR mixture allows real-time monitoring of the reaction progress and extraction of quantitative information. Previously reported real-time PCR product detection using intercalating dyes required melting curve analysis (MCA) to be performed following thermal cycling. Here, we propose a technique to perform dynamic MCA during each thermal cycle, based on a continuous fluorescence monitoring method, providing qualitative and quantitative sample information. We applied the proposed method in multiplexing detection of hepatitis B virus DNA and complementary DNA of human immunodeficiency virus as well as glyceraldehyde 3-phosphate dehydrogenase in different concentration ratios. We extracted the DNA melting curve and its derivative from each PCR cycle during the transition from the elongation to the denaturation temperature with a set heating rate of 0.8 K· s−1and then used the data to construct individual PCR amplification curves for each gene to determine the initial concentration of DNA in the sample. Our proposed method allows researchers to look inside the PCR in each thermal cycle, determining the PCR product specificity in real time instead of waiting until the end of the PCR. Additionally, the slow transition rate from elongation to denaturation provides a dynamic multiplexing assay, allowing the detection of at least three genes in real time.

Czech name

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Czech description

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Type

J_imp - Article in a specialist periodical, which is included in the Web of Science database

CEP classification

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OECD FORD branch

10406 - Analytical chemistry

Project

Result was created during the realization of more than one project. More information in the Projects tab.

Continuities

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Publication year

2020

Confidentiality

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

Name of the periodical

ACS OMEGA

ISSN

2470-1343

e-ISSN

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Volume of the periodical

5

Issue of the periodical within the volume

46

Country of publishing house

US - UNITED STATES

Number of pages

7

Pages from-to

30267-30273

UT code for WoS article

000595527600064

EID of the result in the Scopus database

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