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.

Department of Chemistry and Biochemistry Faculty of Agronomy Mendel University in Brno Zemedelska 1 Brno CZ 61300 Czech Republic

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Shoham D. Influenza type A virus: An outstandingly protean pathogen and a potent modular weapon. Crit. Rev. Microbiol. 2013;39:123–138. doi: 10.3109/1040841X.2012.692355. PubMed DOI

Plourde J.R., Pyles J.A., Layton R.C., Vaughan S.E., Tipper J.L., Harrod K.S. Neurovirulence of H5N1 infection in ferrets is mediated by multifocal replication in distinct permissive neuronal cell regions. PLoS One. 2012;7:1–11. PubMed PMC

Gilbert M., Jambal L., Karesh W.B., Fine A., Shiilegdamba E., Dulam P., Sodnomdarjaa R., Ganzorig K., Batchuluun D., Tseveenmyadag N., et al. Highly pathogenic avian influenza virus among wild birds in mongolia. PLoS One. 2012;7:1–9. PubMed PMC

Wei K.F., Chen Y.F., Chen J., Wu L.J., Xie D.X. Evolution and adaptation of hemagglutinin gene of human H5N1 influenza virus. Virus Genes. 2012;44:450–458. PubMed

Abdelwhab E.M., Hafez H.M. Insight into alternative approaches for control of avian influenza in poultry, with emphasis on highly pathogenic H5N1. Viruses. 2012;4:3179–3208. PubMed PMC

Zhang J.F. Advances and future challenges in recombinant adenoviral vectored H5N1 influenza vaccines. Viruses. 2012;4:2711–2735. doi: 10.3390/v4112711. PubMed DOI PMC

Weinheimer V.K., Becher A., Tonnies M., Holland G., Knepper J., Bauer T.T., Schneider P., Neudecker J., Ruckert J.C., Szymanski K., et al. Influenza A viruses target type II pneumocytes in the human lung. J. Infect. Dis. 2012;206:1685–1694. doi: 10.1093/infdis/jis455. PubMed DOI PMC

Chen F., Yan Z.Q., Liu J., Ji J., Chang S., Liu D., Qin J.P., Ma J.Y., Bi Y.Z., Xie Q.M. Phylogenetic analysis of hemagglutinin genes of 40 H9N2 subtype avian influenza viruses isolated from poultry in China from 2010 to 2011. Virus Genes. 2012;45:69–75. doi: 10.1007/s11262-012-0742-9. PubMed DOI

Zhao J.Q., Wang X., Ragupathy V., Zhang P.H., Tang W., Ye Z.P., Eichelberger M., Hewlett I. Rapid detection and differentiation of swine-origin influenza A virus (H1N1/2009) from other seasonal influenza A viruses. Viruses. 2012;4:3012–3019. doi: 10.3390/v4113012. PubMed DOI PMC

Alberts B. INTRODUCTION H5N1. Science. 2012;336:1521–1521. doi: 10.1126/science.336.6088.1521. PubMed DOI

Takekawa J.Y., Prosser D.J., Newman S.H., Bin Muzaffar S., Hill N.J., Yan B.P., Xiao X.M., Lei F.M., Li T.X., Schwarzbach S.E., et al. Victims and vectors: Highly pathogenic avian influenza H5N1 and the ecology of wild birds. Avian Biol. Res. 2010;3:51–73. doi: 10.3184/175815510X12737339356701. DOI

Leung Y.H.C., Cheung P., Zhang L.J., Wu Y.O., Chow K.C., Ho C.K., Chow C.K., Ng C.F., Li B., Tsang C.L., et al. Influenza viruses in wild birds in Hong Kong, 2003–2010. Influenza Other Respir. Viruses. 2011;5:77–78.

Capua I., Alexander D.J. Ecology, epidemiology and human health implications of avian influenza viruses: Why do we need to share genetic data? Zoonoses Public Health. 2008;55:2–15. doi: 10.1111/j.1863-2378.2007.01081.x. PubMed DOI

Neumann G., Chen H., Gao G.F., Shu Y.L., Kawaoka Y. H5N1 influenza viruses: Outbreaks and biological properties. Cell Res. 2010;20:51–61. doi: 10.1038/cr.2009.124. PubMed DOI PMC

Bragstad K., Jorgensen P.H., Handberg K., Hammer A.S., Kabell S., Fomsgaard A. First introduction of highly pathogenic H5NI avian influenza A viruses in wild and domestic birds in Denmark, Northern Europe. Virol. J. 2007;4:1–10. doi: 10.1186/1743-422X-4-1. PubMed DOI PMC

Rebel J.M.J., Peeters B., Fijten H., Post J., Cornelissen J., Vervelde L. Highly pathogenic or low pathogenic avian influenza virus subtype H7N1 infection in chicken lungs: Small differences in general acute responses. Vet. Res. 2011;42:1–11. PubMed PMC

Comin A., Klinkenberg D., Marangon S., Toffan A., Stegeman A. Transmission dynamics of low pathogenicity avian influenza infections in Turkey flocks. PLoS One. 2011;6:1–9. PubMed PMC

Kabir S.M.L. Avian flu (H5N1): Threat of “global pandemic” is growing and it’s impact on the developing countries’ economy. Afr. J. Microbiol. Res. 2010;4:1192–1194.

Yamada S., Suzuki Y., Suzuki T., Le M.Q., Nidom C.A., Sakai-Tagawa Y., Muramoto Y., Ito M., Kiso M., Horimoto T., et al. Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses to human-type receptors. Nature. 2006;444:378–382. doi: 10.1038/nature05264. PubMed DOI

Fauci A.S., Collins F.S. Benefits and risks of influenza research: lessons learned. Science. 2012;336:1522–1523. doi: 10.1126/science.1224305. PubMed DOI PMC

Nguyen T., Rivailler P., Davis C.T., Hoa D.T., Balish A., Dang N.H., Jones J., Vui D.T., Simpson N., Huong N.T., et al. Evolution of highly pathogenic avian influenza (H5N1) virus populations in Vietnam between 2007 and 2010. Virology. 2012;432:405–416. doi: 10.1016/j.virol.2012.06.021. PubMed DOI

Tang D.J., Lam Y.M., Siu Y.L., Lam C.H., Chu S.L., Peiris J.S.M., Buchy P., Nal B., Bruzzone R. A single residue substitution in the receptor-binding domain of H5N1 hemagglutinin is critical for packaging into pseudotyped lentiviral particles. PLoS One. 2012;7:1–12. PubMed PMC

Park A.W., Glass K. Dynamic patterns of avian and human influenza in east and southeast Asia. Lancet Infect. Dis. 2007;7:543–548. doi: 10.1016/S1473-3099(07)70186-X. PubMed DOI

Rao S.S., Styles D., Kong W., Andrews C., Gorres J.P., Nabel G.J. A gene-based avian influenza vaccine in poultry. Poult. Sci. 2009;88:860–866. doi: 10.3382/ps.2008-00360. PubMed DOI PMC

Webster R.G., Govorkova E.A. Focus on research: H5N1 influenza–Continuing evolution and spread. N. Engl. J. Med. 2006;355:2174–2177. doi: 10.1056/NEJMp068205. PubMed DOI

Peiris J.S.M., de Jong M.D., Guan Y. Avian influenza virus (H5N1): A threat to human health. Clin. Microbiol. Rev. 2007;20:243–267. doi: 10.1128/CMR.00037-06. PubMed DOI PMC

Uyeki T.M., Bresee J.S. Detecting human-to-human transmission of avian influenza a (H5N1) Emerg. Infect. Dis. 2007;13:1969–1970. doi: 10.3201/eid1312.071153. PubMed DOI PMC

Hayden F.G., de Jong M.D. Emerging influenza antiviral resistance threats. J. Infect. Dis. 2011;203:6–10. doi: 10.1093/infdis/jiq012. PubMed DOI PMC

Cai Z.P., Ducatez M.F., Yang J.L., Zhang T., Long L.P., Boon A.C., Webby R.J., Wan X.F. Identifying antigenicity-associated sites in highly pathogenic H5N1 influenza virus hemagglutinin by using sparse learning. J. Mol. Biol. 2012;422:145–155. doi: 10.1016/j.jmb.2012.05.011. PubMed DOI PMC

Zhao G., Zhong L., Lu X.L., Hu J., Gu X.B., Kai Y., Song Q.Q., Sun Q., Liu J.B., Peng D.X., et al. Characterisation of a highly pathogenic H5N1 clade 2.3.2 influenza virus isolated from swans in Shanghai, China. Virus Genes. 2012;44:55–62. doi: 10.1007/s11262-011-0667-8. PubMed DOI

Nidom C.A., Yamada S., Nidom R.V., Rahmawati K., Alamudi M.Y., Kholik, Indrasari S., Hayati R.S., Horimoto K.I., Kawaoka Y. Genetic characterization of H5N1 influenza viruses isolated from chickens in Indonesia in 2010. Virus Genes. 2012;44:459–465. doi: 10.1007/s11262-012-0722-0. PubMed DOI PMC

Huang K., Zhu H.V., Fan X.H., Wang J., Cheung C.L., Duan L., Hong W.S., Liu Y.M., Li L.F., Smith D.K., et al. Establishment and lineage replacement of H6 influenza viruses in domestic ducks in Southern China. J. Virol. 2012;86:6075–6083. doi: 10.1128/JVI.06389-11. PubMed DOI PMC

Miyoshi-Akiyama T., Akasaka Y., Oogane T., Kondo Y., Matsushita T., Funatogawa K., Kirikae T. Development and evaluation of a line probe assay for rapid typing of influenza viruses and detection of the H274Y mutation. J. Virol. Methods. 2012;185:276–280. doi: 10.1016/j.jviromet.2012.06.018. PubMed DOI

Redlberger-Fritz M., Aberle S.W., Strassl R., Popow-Kraupp T. Rapid identification of neuraminidase inhibitor resistance mutations in seasonal influenza virus A(H1N1), A(H1N1)2009, and A(H3N2) subtypes by melting point analysis. Eur. J. Clin. Microbiol. Infect. Dis. 2012;31:1593–1601. doi: 10.1007/s10096-011-1482-9. PubMed DOI

Bao J.R., Huard T.K., Piscitelli A.E., Tummala P.R., Aleemi V.E., Coon S.L., Master R.N., Lewinski M.A., Clark R.B. Reverse-transcription polymerase chain reaction/pyrosequencing to characterize neuraminidase H275 residue of influenza A 2009 H1N1 virus for rapid and specific detection of the viral oseltamivir resistance marker in a clinical laboratory. Diagn. Microbiol. Infect. Dis. 2011;71:396–402. doi: 10.1016/j.diagmicrobio.2011.09.003. PubMed DOI PMC

Deng Y.M., Caldwell N., Hurt A., Shaw T., Kelso A., Chidlow G., Williams S., Smith D., Barr I. A comparison of pyrosequencing and neuraminidase inhibition assays for the detection of oseltamivir-resistant pandemic influenza A(H1N1) 2009 viruses. Antivir. Res. 2011;90:87–91. doi: 10.1016/j.antiviral.2011.02.014. PubMed DOI

Leang S.K., Deng Y.M., Shaw R., Caldwell N., Iannello P., Komadina N., Buchy P., Chittaganpitch M., Dwyer D.E., Fagan P., et al. Influenza antiviral resistance in the Asia-Pacific region during 2011. Antivir. Res. 2013;97:206–210. doi: 10.1016/j.antiviral.2012.12.016. PubMed DOI

Chairat K., Tarning J., White N.J., Lindegardh N. Pharmacokinetic properties of anti-influenza neuraminidase inhibitors. J. Clin. Pharmacol. 2013;53:119–139. PubMed

Li H., Shih W.Y., Shih W.H. Synthesis and characterization of aqueous carboxyl-capped CdS quantum dots for bioapplications. Ind. Eng. Chem. Res. 2007;46:2013–2019.

Hennequin B., Turyanska L., Ben T., Beltran A.M., Molina S.I., Li M., Mann S., Patane A., Thomas N.R. Aqueous near-infrared fluorescent composites based on apoferritin-encapsulated PbS quantum dots. Adv. Mater. 2008;20:3592–3596. doi: 10.1002/adma.200800530. DOI

Genbank. [(accessed on 1 July 2013)]. Available online: http://www.ncbi.nlm.nih.gov/genbank/

Krejcova L., Huska D., Hynek D., Kopel P., Adam V., Hubalek J., Trnkova L., Kizek R. Using of paramagnetic microparticles and quantum dots for isolation and electrochemical detection of influenza viruses’ specific nucleic acids. Int. J. Electrochem. Sci. . 2013 in press.

Krejcova L., Hynek D., Kopel P., Adam V., Hubalek J., Trnkova L., Kizek R. Paramagnetic particles isolation of influenza oligonucleotide labelled with CdS QDs. Chromatographia. 2013;76:355–362. doi: 10.1007/s10337-012-2327-0. DOI

Huska D., Adam V., Babula P., Trnkova L., Hubalek J., Zehnalek J., Havel L., Kizek R. Microfluidic robotic device coupled with electrochemical sensor field for handling of paramagnetic micro-particles as a tool for determination of plant mRNA. Microchim. Acta. 2011;173:189–197. doi: 10.1007/s00604-011-0545-z. DOI

Huska D., Hubalek J., Adam V., Vajtr D., Horna A., Trnkova L., Havel L., Kizek R. Automated nucleic acids isolation using paramagnetic microparticles coupled with electrochemical detection. Talanta. 2009;79:402–411. PubMed

Krejcova L., Dospivova D., Ryvolova M., Kopel P., Hynek D., Krizkova S., Hubalek J., Adam V., Kizek R. Paramagnetic particles coupled with an automated flow injection analysis as a tool for influenza viral protein detection. Electrophoresis. 2012;33:3195–3204. PubMed

Zitka O., Krizkova S., Krejcova L., Hynek D., Gumulec J., Masarik M., Sochor J., Adam V., Hubalek J., Trnkova L., et al. Microfluidic tool based on the antibody-modified paramagnetic particles for detection of 8-hydroxy-2'-deoxyguanosine in urine of prostate cancer patients. Electrophoresis. 2011;32:3207–3220. doi: 10.1002/elps.201100430. PubMed DOI

Prasek J., Huska D., Jasek O., Zajickova L., Trnkova L., Adam V., Kizek R., Hubalek J. Carbon composite micro- and nano-tubes based electrodes for detection of nucleic acids. Nanoscale Res. Lett. 2011;6:1–5. PubMed PMC

Huska D., Zitka O., Krystofova O., Adam V., Babula P., Zehnalek J., Bartusek K., Beklova M., Havel L., Kizek R. Effects of cadmium(II) ions on early somatic embryos of Norway spruce studied by using electrochemical techniques and nuclear magnetic resonance. Int. J. Electrochem. Sci. 2010;5:1535–1549.

Chomoucka J., Drbohlavova J., Masarik M., Ryvolova M., Huska D., Prasek J., Horna A., Trnkova L., Provaznik I., Adam V., et al. Nanotechnologies for society. New designs and applications of nanosensors and nanobiosensors in medicine and environmental analysis. Int. J. Nanotechnol. 2012;9:746–783.

Berton M., Turelli P., Trono D., Stein C.A., Allemann E., Gurny R. Inhibition of HIV-1 in cell culture by oligonucleotide-loaded nanoparticles. Pharm. Res. 2001;18:1096–1101. PubMed

Schneider T., Becker A., Ringe K., Reinhold A., Firsching R., Sabel B.A. Brain tumor therapy by combined vaccination and antisense oligonucleotide delivery with nanoparticles. J. Neuroimmunol. 2008;195:21–27. PubMed

Cai H., Zhu N.N., Jiang Y., He P.G., Fang Y.Z. Cu@Au alloy nanoparticle as oligonucleotides labels for electrochemical stripping detection of DNA hybridization. Biosens. Bioelectron. 2003;18:1311–1319. PubMed

Sun W., Zhong J.H., Qin P., Jiao K. Electrochemical biosensor for the detection of cauliflower mosaic virus 35 S gene sequences using lead sulfide nanoparticles as oligonucleotide labels. Anal. Biochem. 2008;377:115–119. PubMed

Roh C., Lee H.Y., Kim S.E., Jo S.K. A highly sensitive and selective viral protein detection method based on RNA oligonucleotide nanoparticle. Int. J. Nanomed. 2010;5:323–329. PubMed PMC

Bandyopadhyay A., Chatterjee S., Sarkar K. Rapid isolation of genomic DNA from E. coli XL1 Blue strain approaching bare magnetic nanoparticles. Curr. Sci. 2011;101:210–214.

Trachtova S., Kaman O., Spanova A., Veverka P., Pollert E., Rittich B. Silica-coated La0.75Sr0.25MnO3 nanoparticles for magnetically driven DNA isolation. J. Sep. Sci. 2011;34:3077–3082. doi: 10.1002/jssc.201100442. PubMed DOI

Zhang T., Zhao P.S., Zhang W., Liang M., Gao Y.W., Yang S.T., Wang T.C., Qin C., Wang C.Y., Xia X.Z. Antisense oligonucleotide inhibits avian influenza virus H5N1 replication by single chain antibody delivery system. Vaccine. 2011;29:1558–1564. PubMed

Malecka K., Grabowska I., Radecki J., Stachyra A., Gora-Sochacka A., Sirko A., Radecka H. Voltammetric detection of a specific DNA sequence of avian influenza virus H5N1 using HS-ssDNA probe deposited onto gold electrode. Electroanalysis. 2012;24:439–446.

Ganbold E.O., Kang T., Lee K., Lee S.Y., Joo S.W. Aggregation effects of gold nanoparticles for single-base mismatch detection in influenza A (H1N1) DNA sequences using fluorescence and Raman measurements. Colloid Surf. B-Biointerfaces. 2012;93:148–153. PubMed

Liu X.G., Cheng Z.Q., Fan H., Ai S.Y., Han R.X. Electrochemical detection of avian influenza virus H5N1 gene sequence using a DNA aptamer immobilized onto a hybrid nanomaterial-modified electrode. Electrochim. Acta. 2011;56:6266–6270. doi: 10.1016/j.electacta.2011.05.055. DOI

Lai W.A., Lin C.H., Yang Y.S., Lu M.S.C. Ultrasensitive and label-free detection of pathogenic avian influenza DNA by using CMOS impedimetric sensors. Biosens. Bioelectron. 2012;35:456–460. PubMed

Tian J.P., Zhao H.M., Liu M., Chen Y.Q., Quan X. Detection of influenza A virus based on fluorescence resonance energy transfer from quantum dots to carbon nanotubes. Anal. Chim. Acta. 2012;723:83–87. PubMed

Chung D.J., Kim K.C., Choi S.H. Electrochemical DNA biosensor based on avidin-biotin conjugation for influenza virus (type A) detection. Appl. Surf. Sci. 2011;257:9390–9396. doi: 10.1016/j.apsusc.2011.06.015. DOI

Fan H., Ju P., Ai S.Y. Controllable synthesis of CdSe nanostructures with tunable morphology and their application in DNA biosensor of Avian Influenza Virus. Sens. Actuator B-Chem. 2010;149:98–104.

Adam V., Huska D., Hubalek J., Kizek R. Easy to use and rapid isolation and detection of a viral nucleic acid by using paramagnetic microparticles and carbon nanotubes-based screen-printed electrodes. Microfluid. Nanofluid. 2010;8:329–339. doi: 10.1007/s10404-009-0464-z. DOI

Chen X.J., Xie H., Seow Z.Y., Gao Z.Q. An ultrasensitive DNA biosensor based on enzyme-catalyzed deposition of cupric hexacyanoferrate nanoparticles. Biosens. Bioelectron. 2010;25:1420–1426. PubMed

Lim S.H., Buchy P., Mardy S., Kang M.S., Yu A.D.C. Specific nucleic acid detection using photophysical properties of quantum dot probes. Anal. Chem. 2010;82:886–891. PubMed

Tam P.D., Hieu V.N., Chien N.D., Le A.T., Tuan M.A. DNA sensor development based on multi-wall carbon nanotubes for label-free influenza virus (type A) detection. J. Immunol. Methods. 2009;350:118–124. doi: 10.1016/j.jim.2009.08.002. PubMed DOI

Kim S.A., Kim S.J., Lee S.H., Park T.H., Byun K.M., Kim S.G., Shuler M.L. Detection of avian influenza-DNA hybridization using wavelength-scanning surface plasmon resonance biosensor. J. Opt. Soc. Korea. 2009;13:392–397.