Development of a Magnetic Electrochemical Bar Code Array for Point Mutation Detection in the H5N1 Neuraminidase Gene

Ludmila Krejcova,Pavel Kopel,Libuse Trnkova,David Hynek,Rene Kizek,Miguel Rodrigo,Jaromir Hubalek,Vojtech Adam,Petr Babula

doi:10.3390/v5071719

Abstract

Since its first official detection in the Guangdong province of China in 1996, the highly pathogenic avian influenza virus of H5N1 subtype (HPAI H5N1) has reportedly been the cause of outbreaks in birds in more than 60 countries, 24 of which were European. The main issue is still to develop effective antiviral drugs. In this case, single point mutation in the neuraminidase gene, which causes resistance to antiviral drug and is, therefore, subjected to many studies including ours, was observed. In this study, we developed magnetic electrochemical bar code array for detection of single point mutations (mismatches in up to four nucleotides) in H5N1 neuraminidase gene. Paramagnetic particles Dynabeads® with covalently bound oligo (dT)25 were used as a tool for isolation of complementary H5N1 chains (H5N1 Zhejin, China and Aichi). For detection of H5N1 chains, oligonucleotide chains of lengths of 12 (+5 adenine) or 28 (+5 adenine) bp labeled with quantum dots (CdS, ZnS and/or PbS) were used. Individual probes hybridized to target molecules specifically with efficiency higher than 60%. The obtained signals identified mutations present in the sequence. Suggested experimental procedure allows obtaining further information from the redox signals of nucleic acids. Moreover, the used biosensor exhibits sequence specificity and low limits of detection of subnanogram quantities of target nucleic acids.

Highlights

Pathogenic avian influenza A (HPAI), subtype H5N1, represents a threat to the human population [1]
Mutations of the influenza virus in combination with the global increasing of the use of anti-influenza specific drugs allows for the selection of antiviral drug-resistant viruses
New assays are important for the surveillance of Highly pathogenic avian influenza A (HPAI) H5N1 and for decisions to use specific antiviral drugs in the treatment of human patients suspected or suffering from

Summary

Introduction

Pathogenic avian influenza A (HPAI), subtype H5N1, represents a threat to the human population [1]. HPAI viruses have caused millions of deaths in domestic poultry, and hundreds of deaths in humans [4]. Circulating of influenza viruses in wild animals poses the risk to human health [5,6]. Humans can be infected by animal subtypes, such as avian influenza virus H5N1 and H9N2 and swine influenza virus H1N1 and H3N2 [7,8,9]. H5N1 occurs in two distinct pathotypes in bird population, seasonal low pathogenic avian influenza (LPAI), and highly pathogenic avian influenza (HPAI) [11,12,13,14]. LPAI may become HPAI to poultry through mutations after introduction from wild birds to poultry, but only two subtypes (H5 and H7) can become HPAI [15,16,17]

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Results

Conclusion