Real-time PCR to identify variola virus or other human pathogenic orthopox viruses.
BACKGROUND
Variola virus (family Poxviridae, genus Orthopoxvirus) and the closely related cowpox, vaccinia, and monkeypox viruses can infect humans. Efforts are mounting to replenish the smallpox vaccine stocks, optimize diagnostic methods for poxviruses, and develop new antivirals against smallpox, because it is feared that variola virus might be used as a weapon of bioterrorism.


METHODS
We developed an assay for the detection of variola virus DNA. The assay is based on TaqMan chemistry targeting the 14-kD protein gene. For the 1st stage of the assay we used genus consensus primers and a mixture of 2 probes (14-kD POX and 14-kD VAR) spanning the 14-kD protein-encoding gene for detection of all human pathogenic orthopoxviruses. We then tested positive samples with the specific orthopoxvirus-specific probe 14-kD POX to identify monkeypox, cowpox, and vaccinia viruses and with the 14-kD VAR probe to identify variola viruses. The assay was established on 4 different PCR cycler platforms. It was assessed in a study with 85 different orthopoxvirus species and strains that included variola, camelpox, cowpox, monkeypox, and vaccinia viruses at concentrations ranging from 100 ng/L to 1 microg/L.


RESULTS
The assay detected as little as 0.05 fg of DNA, corresponding to 25 copies of DNA, and enabled differentiation of variola virus from the other orthopoxviruses.


CONCLUSIONS
This real-time PCR assay provides a rapid method for the early detection and differentiation of smallpox and other human pathogenic orthopoxvirus infections.
Scaramozzino
Natale
N
Laboratoire de Virologie, Centre de Recherches du Service de Santé des Armées (CRSSA) Emile Pardé, Grenoble, France.
Ferrier-Rembert
Audrey
A
Favier
Anne-Laure
AL
Rothlisberger
Corinne
C
Richard
Stéphane
S
Crance
Jean-Marc
JM
Meyer
Hermann
H
Garin
Daniel
D
D001483:Base Sequence; D004279:DNA, Viral / Q000032:analysis*; D016679:Genome, Viral; D006801:Humans; D018155:Orthopoxvirus / Q000145:classification* / Q000235:genetics; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D012901:Variola virus / Q000145:classification / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics*; D014773:Virology / Q000379:methods
D004279:DNA, Viral; D014764:Viral Proteins