Many phenotypic and genotypic methods involving virological, immunological, and molecular approaches have been used to identify Orthopoxvirus. The molecular approaches, including DNA sequencing, polymerase chain reaction (PCR), restriction fragment-length polymorphism (RFLP), real-time PCR, and microarrays, are more sensitive and specific than the virological and immunological approaches. Of these approaches, sequencing provides the highest level of specificity for species or strain identification but current sequencing techniques are not yet practical as rapid diagnostic tools in most laboratories. Restriction fragment-length polymorphism analysis1-8-1 and microarray genotyping also provide high levels of specificity, and when combined with PCR, these approaches can offer high levels of sensitivity as well. Real-time PCR methods provide exquisite levels of sensitivity and specificity.[10,11] The basic concept behind real-time PCR is the measurement, by fluorescence detection, of the amount of nucleic acids produced during every cycle of the PCR. Several detection chemistries, e.g., intercalating dyes (SYBR Green), hydrolysis probes [5' nuclease or Taqman, minor groove-binding proteins (MGB)], hybridization probes [fluorescence resonance energy transfer (FRET)], and molecular beacons, are used. Several instruments for real-time PCR, e.g., the ABI 7700, Smart Cycler, LightCycler, RAPID, Opticon, MX 4000, Rotor-Gene, and others, are commercially available. When combined with portable analytical platforms such as the Smart Cycler or LightCycler, realtime PCR systems can be readily deployed to field sites for rapid testing.
In the following section, we describe an approach for genome typing of orthopox viruses by using long polymerase chain reaction (LPCR) amplification, followed by RFLP analysis.
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