Publication detail

Single Fluorescence Channel-based Multiplex Detection of Avian Influenza Virus by Quantitative PCR with Intercalating Dye

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

7

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"
}