Detection of viral sequences of low reiteration frequency by in situ hybridization.

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Abstract
The sensitivity of in situ hybridization was increased at least 10-fold by hybridizing in c[complementary]DNA excess, by increasing the diffusion of the cDNA through the cells, by hybridizing at optimum temperature and by stabilizing hybrids during autoradiography. Saturation of intracellular RNA with [3H]cDNA was achieved. The assay is quantitative. In situ hybridization was used to detect and quantitate visna virus RNA in infected [sheep choroid plexus] cells. By using [3H]cDNA with specific activity of 2 .times. 108 dpm/.mu.g and conditions that reduce background to negligible levels, 10-20 copies of viral RNA/cell can be detected and quantitated after 2 days of autoradiographic exposure.