Dual-probe real-time PCR assay for detection of variola or other orthopoxviruses with dried reagents.
A real-time, multiplexed polymerase chain reaction (PCR) assay based on dried PCR reagents was developed. Only variola virus could be specifically detected by a FAM (6-carboxyfluorescein)-labeled probe while camelpox, cowpox, monkeypox and vaccinia viruses could be detected by a TET (6-carboxytetramethylrhodamine)-labeled probe in a single PCR reaction. Approximately 25 copies of cloned variola virus DNA and 50 copies of genomic orthopoxviruses DNA could be detected with high reproducibility. The assay exhibited a dynamic range of seven orders of magnitude with a correlation coefficient value greater than 0.97. The sensitivity and specificity of the assay, as determined from 100 samples that contained nucleic acids from a multitude of bacterial and viral species were 96% and 98%, respectively. The limit of detection, sensitivity and specificity of the assay were comparable to standard real-time PCR assays with wet reagents. Employing a multiplexed format in this assay allows simultaneous discrimination of the variola virus from other closely related orthopoxviruses. Furthermore, the implementation of dried reagents in real-time PCR assays is an important step towards simplifying such assays and allowing their use in areas where cold storage is not easily accessible.
Aitichou
Mohamed
M
Virology Division, United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Frederick, Frederick, MD 21702-5011, USA. mohamed.aitichou@amedd.army.mil
Saleh
Sharron
S
Kyusung
Park
P
Huggins
John
J
O'Guinn
Monica
M
Jahrling
Peter
P
Ibrahim
Sofi
S
D015342:DNA Probes; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005612:Freeze Drying; D006801:Humans; D007202:Indicators and Reagents; D018155:Orthopoxvirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012235:Rhodamines; D012680:Sensitivity and Specificity; D012901:Variola virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*
C106141:6-carboxytetramethylrhodamine; D015342:DNA Probes; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D012235:Rhodamines; C024098:6-carboxyfluorescein