Identification of a double-stranded RNA virus by using polymerase chain reaction and magnetic separation of the synthesized DNA segments

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Abstract
A double-nested polymerase chain reaction assay (PCR), followed by magnetic separation of the PCR-synthesized DNa segments, was developed to detect a double-stranded RNA virus, infectious pancreatic necrosis virus from salmonid fish. Viral RNa was extracted from cell cultures and used for cDNA synthesis. The cDNA produced was used as a template in a double PCR. The sensitivity of the double PCR was approximately 0.8 pg of template double-stranded RNa. The DNA segment produced from the first PCR was also used as a template in a second PCR with a set of two 5''-labeled primers, one with biotin and the other with 32P. The PCR segment that was then synthesized was separated from the solution by using streptavidin-coated, superparamagnetic beads. The levels of radioactivity measured in the magnetically separated fractions were significantly higher in the positive samples than they were in the negative samples.