Development and Application of Nucleic Acid Hybridization Techniques to Arbovirus Surveillance and Diagnosis.
Abstract
Nucleic acid hybridization techniques have been developed to detect dengue and LaCrosse arbovirus RNA in cells and in cell and tissue suspensions. Probes are comprised of cDNAs of portions of the respective virus genomes cloned into plasmids. Nonisotopic probes are prepared by nick translating the plasmid using biotinylated nucleotides. The presence of specific hybrids can then be detected immunologically using anti-biotin antibodies followed by signal amplification with an enzyme immunoassay. In situ hybridization techniques have been developed to detect the site and temporal onset of S RNA synthesis in cell cultures using a biotinylated cDNA of the S RNA as a probe. S RNA was detected in the perinuclear region of BHK 21 cells by 4 hours post-infection; by 24 hours hybrids were found throughout the cytoplasm. A similar probe has been used to detect LaCrosse virus RNA in pools of infected mosquitoes. RNA was extracted from mosquitoes, blotted onto nitrocellulose, and hybridized with the probe. Using this technique, 100 ng of RNA was readily detected. Biotinylated cDNA probes have also been prepared for dengue virus. One probe was capable of detecting picogram levels of dengue virus RNA. Some signal was detected in control cells. To investigate this phenomenon, the three dengue specific probes were labeled with 32P. The use of isotopic probes revealed unequivocally that all probes contained dengue sequences.
Document Details
- Document Type
- Technical Report
- Publication Date
- Nov 04, 1986
- Accession Number
- ADA177420
Entities
People
- Barry J. Beaty
- Carol D. Blair
Organizations
- Colorado State University