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

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.

Nucleic acid sensing by regenerable surface-associated isothermal rolling circle amplification.

A novel method for regenerating biosensors has been developed in which the highly specific detection of nucleic acid sequences is carried out using molecular padlock probe (MPP) technology and surface-associated rolling circle amplification (RCA). This technique has a low occurrence of false positive results when compared to polymerase chain reaction, and is an isothermal reaction, which is advantageous in systems requiring low power consumption such as remote field sensing applications. Gold-sputtered 96-well polystyrene microplates and a fluorescent label were used to explore the detection limits of the surface-associated RCA technique, specificity for different MPP, conditions for regeneration of the biomolecular sensing surface, and reproducibility of measurements on regenerated surfaces.