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

作者：McCarthy Erik L; Egeler Teressa J; Bickerstaff Lee E; da Cunha Mauricio Pereira; Millard Paul J^*

来源：Analytical and Bioanalytical Chemistry, 2006, 386(7-8): 1975-1984.

DOI：10.1007/s00216-006-0872-9

• bioanalytical methods
• biosensors
• fluorescence/luminescence
• virus
• molecular padlock

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.

• 出版日期2006-12