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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RNA sequences derived from infectious hematopoeitic necrosis virus (IHNV) could be detected using a combination of surface-associated molecular padlock DNA probes (MPP) and rolling circle amplification (RCA) in microcapillary tubes. DNA oligonucleotides with base sequences identical to RNA obtained from IHNV were recognized by MPP. Circularized MPP were then captured on the inner surface of glass microcapillary tubes by immobilized DNA oligonucleotide primers. Extension of the immobilized primers by isothermal RCA gave rise to DNA concatamers, which were in turn bound by the fluorescent reporter SYBR Green II nucleic acid stain, and measured by microfluorimetry. Surface-associated molecular padlock technology, combined with isothermal RCA, exhibited high selectivity and sensitivity without thermal cycling. This technology is applicable to direct RNA and DNA detection, permitting detection of a variety of viral or bacterial pathogens.