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Detection and identification of IHN and ISA viruses by isothermal DNA amplification in microcapillary tubes

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Abstract

Unique base sequences derived from RNA of both infectious hematopoietic necrosis virus (IHNV) and infectious salmon anemia virus (ISAV) were detected and identified using a combination of surface-associated molecular padlock DNA probes (MPPs) and rolling circle amplification (RCA) in microcapillary tubes. DNA oligonucleotides with base sequences identical to RNA obtained from IHNV or ISAV were recognized by MPPs. Circularized MPPs were then captured on the inner surfaces of glass microcapillary tubes by immobilized DNA oligonucleotide primers. Extension of the immobilized primers by isothermal RCA produced DNA concatamers, which were labeled with fluorescent SYBR Green II nucleic acid stain, and measured by microfluorimetry. Molecular padlock probes, combined with this method of surface-associated isothermal RCA, exhibited high selectivity without the need for thermal cycling. This method is applicable to the design of low-power field sensors capable of multiplex detection of viral, bacterial, and protozoan pathogens within localized regions of microcapillary tubes.

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Acknowledgements

We gratefully acknowledge Drs. Carl Tripp and Ben McCool for their help with procedures for amine derivatization of glass microcapillaries, and Dr. Carol Kim for helpful discussion and reading of the manuscript. This work was supported in part by Maine Sea Grant NA16RG1034 and National Science Foundation grants ECS-0329913 and EEC-0452021.

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Authors and Affiliations

  1. Department of Chemical and Biological Engineering and the Laboratory for Surface Science and Technology (LASST), University of Maine, 245 ESRB-Barrows, Orono, ME, 04469, USA

    Erik L. McCarthy, Teressa J. Egeler, Lee E. Bickerstaff & Paul J. Millard

  2. Department of Electrical and Computer Engineering and the Laboratory for Surface Science and Technology (LASST), University of Maine, Orono, ME, 04469, USA

    Mauricio Pereira da Cunha

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  1. Erik L. McCarthy
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  2. Teressa J. Egeler
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  3. Lee E. Bickerstaff
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  4. Mauricio Pereira da Cunha
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  5. Paul J. Millard
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Correspondence to Paul J. Millard.

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McCarthy, E.L., Egeler, T.J., Bickerstaff, L.E. et al. Detection and identification of IHN and ISA viruses by isothermal DNA amplification in microcapillary tubes. Anal Bioanal Chem 386, 1975–1984 (2006). https://doi.org/10.1007/s00216-006-0872-9

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  • DOI: https://doi.org/10.1007/s00216-006-0872-9

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