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Published on: June 28, 2013
Gene for A-type inclusion body protein is useful for a polymerase chain reaction assay to differentiate
H Meyer1, S L Ropp, J J Esposito
1Institute of Microbiology, Federal Armed Forces Medical Academy, Munich, Germany.
Abstract:
Orthopoxvirus species were identified and differentiated by polymerase chain reaction amplification of genome DNA using a single primer-pair based on sequences coding for the major protein component of the cowpox virus acidophilic-type inclusion body (ATI). DNA available for 6 of 8 Old World (cowpox, variola, monkeypox, camelpox, ectromelia and vaccinia viruses) and 3 New World (skunkpox, volepox, and raccoonpox) resulted in amplicons that ranged in size from 510 to 1673 base pairs depending on the species, except for raccoonpox virus DNA which did not amplify. XbaI digest gel electrophoresis profiles of the amplicons improved resolution of the differences.
Insights
This study developed a polymerase chain reaction (PCR) method to identify and differentiate Orthopoxvirus species using a single primer pair. The method successfully amplified DNA from several Old and New World orthopoxviruses, aiding in their distinction.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Orthopoxviruses are a genus of double-stranded DNA viruses known for causing various diseases in mammals.
- Accurate identification and differentiation of Orthopoxvirus species are crucial for disease control and public health surveillance.
- Existing methods for differentiating these viruses can be complex and time-consuming.
Purpose of the Study:
- To develop a rapid and reliable method for identifying and differentiating Orthopoxvirus species.
- To utilize polymerase chain reaction (PCR) amplification targeting specific viral gene sequences.
- To assess the efficacy of the developed PCR method across a range of Old and New World orthopoxviruses.
Main Methods:
- DNA extraction from various Orthopoxvirus species.
- Polymerase chain reaction (PCR) amplification using a single primer pair targeting sequences of the cowpox virus acidophilic-type inclusion body (ATI) major protein component.
- Analysis of PCR amplicon sizes (510–1673 base pairs) for differentiation.
- XbaI digest gel electrophoresis of amplicons to enhance resolution of differences.
Main Results:
- Successful amplification of genome DNA from six Old World (cowpox, variola, monkeypox, camelpox, ectromelia, vaccinia) and two New World (skunkpox, volepox) Orthopoxvirus species.
- Amplicons varied in size (510–1673 bp) depending on the species, allowing for differentiation.
- Raccoonpox virus DNA did not amplify under the tested conditions.
- XbaI digestion of amplicons improved the resolution of species-specific differences.
Conclusions:
- A single primer-pair PCR method is effective for identifying and differentiating multiple Orthopoxvirus species.
- The PCR assay provides a valuable tool for rapid viral identification in diagnostic and research settings.
- Further optimization may be needed for complete coverage of all relevant Orthopoxvirus species, such as raccoonpox virus.
