Nucleic Acidbased methods

The detection of virus nucleic acid has revolutionized diagnostics. The use of molecular techniques has identified viruses that cannot infect cells in culture or inoculated animals. Most specimens such as blood, tissues, urine, stool, CSF and respiratory secretions can be treated and the nucleic acid extracted, inactivating the virus and reducing concern about transport or contact in the laboratory Detection of virus in the blood is diagnostic of an active infection, and the sensitivity of PCR has enhanced our capability to detect very low levels of virus in persistent infections; the universal genetic code allows investigators to detect virus sequence from host insects, animals, bacteria, and fungi where serological reagents may not be available. The specificity of PCR is dependent on the selection of the primer sets. Primers are designed to anneal to conserved regions in a virus genome. Genes that encode proteins essential for the virus life cycle such as polymerases, helicases, and integrases as well as RNA elements in untranslated regions are maintained even in viruses with high mutation rates. There are good PCR amplification targets. To identify a new virus, universal primer sets are designed based on alignments of virus family sequences and selected to minimize base pair mismatches. Primers containing degenerate bases are used to widen the net and find a genetically distant virus. For DNA viruses, PCR can be applied directly. RNA viruses require an initial reverse transcription RT-PCR reaction before amplification. Complex variations on the basic technique of PCR including utilization of multiplex primers, TaqMan probes, and molecular beacons can enhance the ability to identify viral sequences.

PCR products are either sequenced directly or cloned into intermediate vectors for sequencing. Multiple PCR isolates are sequenced in order to verify the authentic virus sequence, as mutations arise during PCR amplification. Sequencing virus directly from clinical samples without amplification in cell culture minimizes cell culture adaptation artifacts. The unknown virus sequences are entered into sequence databases to search for any similarity with known virus genomes. The database should include sequences from clinical isolates, field samples from the natural hosts, and nonpathogenic viruses to assist identification of a novel emerging virus. As with any assay, molecular diagnosis of virus nucleic acid is confirmed with other diagnostic methods such as serology and virus isolation.

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