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AHRBERG, C. MANZ, A. NEUŽIL, P.
Original Title
Single Fluorescence Channel-based Multiplex Detection of Avian Influenza Virus by Quantitative PCR with Intercalating Dye
Type
journal article in Web of Science
Language
English
Original Abstract
Since its invention in 1985 the polymerase chain reaction (PCR) has become a well-established method for amplification and detection of segments of double-stranded DNA. Incorporation of fluorogenic probe or DNA intercalating dyes (such as SYBR Green) into the PCR mixture allowed real-time reaction monitoring and extraction of quantitative information (qPCR). Probes with different excitation spectra enable multiplex qPCR of several DNA segments using multi-channel optical detection systems. Here we show multiplex qPCR using an economical EvaGreen-based system with single optical channel detection. Previously reported non quantitative multiplex realtime PCR techniques based on intercalating dyes were conducted once the PCR is completed by performing melting curve analysis (MCA). The technique presented in this paper is both qualitative and quantitative as it provides information about the presence of multiple DNA strands as well as the number of starting copies in the tested sample. Besides important internal control, multiplex qPCR also allows detecting concentrations of more than one DNA strand within the same sample. Detection of the avian influenza virus H7N9 by PCR is a well established method. Multiplex qPCR greatly enhances its specificity as it is capable of distinguishing both haemagglutinin (HA) and neuraminidase (NA) genes as well as their ratio.
Keywords
multiplexing, dynamic melting curve analysis, PCR
Authors
AHRBERG, C.; MANZ, A.; NEUŽIL, P.
RIV year
2015
Released
19. 6. 2015
Publisher
Nature Publishing Group
ISBN
2045-2322
Periodical
Scientific Reports
Year of study
5
Number
11479
State
United Kingdom of Great Britain and Northern Ireland
Pages from
1
Pages to
7
Pages count
URL
http://www.nature.com/articles/srep11479
Full text in the Digital Library
http://hdl.handle.net/11012/61763
BibTex
@article{BUT115139, author="Christian D. {Ahrberg} and Andreas {Manz} and Pavel {Neužil}", title="Single Fluorescence Channel-based Multiplex Detection of Avian Influenza Virus by Quantitative PCR with Intercalating Dye", journal="Scientific Reports", year="2015", volume="5", number="11479", pages="1--7", doi="10.1038/srep11479", issn="2045-2322", url="http://www.nature.com/articles/srep11479" }