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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 detectingpicogram 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

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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 geneticrearrangements or gene transfer in natural communities.