Development and Application of Nucleic Acid Hybridization Techniques to Arbovirus Surveillance and Diagnosis

摘要

Nucleic acid hybridization techniques have been developed for detection of LaCrosse and dengue virus RNA in cells and in mosquitoes. RNA transcript or DNA probes of the respective viruses were prepared and labeled with either biotin or a radioisotope. Optimal conditions for cell fixation, permeabilization, retention of analyte, hybridization, and detection of hybrids for in situ hybridization were determined for detection of LaCrosse virus analyte in cells. LaCrosse RNA was first detected 4 hours post infection of cells, and as little as 700 copies of analyte could be readily detected. Both type-specific (a cDNA of the M RNA segment of a bunyavirus) and group-specific (a cDNA of the S RNA) have been developed. Radiolabeled probes have been emphasized for dengue virus detection. Studies demonstrated inefficient binding of RNA analyte to the substrate. Novel strategies to overcome this problem, UV crosslinking of analyte to substrate and sandwich hybridization, have been developed.