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 nic translating the plasmid using biotinylated nucleotides. !he presence of specific hybrids can then be detected immunologically using anti"biotin antibodies follo#ed by signal amplification #ith an enzyme immunoassay. $n situ hybridization techniques have been developed to detect the site and temporal onset of % RNA synthesis in cell cultures using a biotinylated cDNA of the % RNA as a probe. % RNA #as detected in the perinuclear region of &'( )* cells by + hours post"infection, by )+ hours hybrids #ere found throughout the cytoplasm. A similar probe has been used to detect LaCrosse virus RNA in pools of infected mosquitoes. RNA #as e-tracted from mosquitoes. blotted onto nitrocellulose. and hybridized #ith the probe. /sing this technique. *00 ng of RNA #as readily detected. &iotinylated cDNA probes have also been prepared for dengue virus. 1ne probe #as capable of detecting picogram levels of dengue virus RNA. %ome signal #as detected in control cells. !o investigate this phenomenon. the three dengue specific probes #ere labeled #ith 2)P. !he use of isotopic probes revealed unequivocally that all probes contained dengue sequences.

Nucleic acid hybridization techniques offer a new approach to answer old, intractable questions in microbial ecology as well as new questions. These include characterization of the predominant, yet unculturable populations in nature, the role of the environment in gene expression, and the extent of gene exchange among communities in nature. The essence of this methodology is the denaturing and annealing of complementary strands of nucleic acid molecules. The specificity of this hybridization reaction can be controlled such that only identical, or nearly identical, sequences in a complex mixture of nucleic acids extracted from a population or community can anneal. Labeled DNA or RNA sequences (probes) are introduced into hybridization

reactions to identify and quantitate a particular organism containing the complementary target sequence. Methods for the recovery and purification of DNA from soils and sediments are given, as well as important considerations for the selection of probe and target sequences, and for the methods of detection and quantitation. Some advantages of this methodology include the abilities to: (1) detect populations without prior culturing of organisms, (2) detect specific organisms without the need for selectable markers, (3) detect multiple populations in the same analysis, and (4) detect genetic rearrangements or gene transfer in natural communities.