Rapid detection of Orthopoxvirus by semi-nested PCR directly from clinical specimens: a useful alternative for routine laboratories.
Orthopoxvirus (OPV) has been associated with worldwide exanthematic outbreaks, which have resulted in serious economic losses as well as impact on public health. Although the current classical and molecular methods are useful for the diagnosis of OPV, they are largely inaccessible to unsophisticated clinical laboratories. The major reason for the inaccessibility is that they require both virus isolation and DNA manipulation. In this report, a rapid, sensitive and low-cost semi-nested PCR method is described for the detection of OPV DNA directly from clinical specimens. A set of primers was designed to amplify the conserved OPV vgf gene. The most useful thermal and chemical conditions were selected and minimum non-inhibitory dilutions were determined. More than 100 Brazilian Vaccinia virus (VACV) field clinical specimens were tested using this semi-nested PCR in order to confirm its applicability. Cowpox virus was also detected by PCR from the ear scabs of scarified Balb/c mice. In addition, the method was highly sensitive for the detection of VACV DNA in murine blood and excreta, which are among the suggested reservoirs of OPV. Together, these data suggest that semi-nested PCR can be used for initial screening for OPV and as a routine diagnostic laboratory method.
Abrahão
Jônatas Santos
JS
Laboratório de Vírus, Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
Drumond
Betânia Paiva
BP
Trindade
Giliane de Souza
Gde S
da Silva-Fernandes
André Tavares
AT
Ferreira
Jaqueline Maria Siqueira
JM
Alves
Pedro Augusto
PA
Campos
Rafael Kroon
RK
Siqueira
Larissa
L
Bonjardim
Cláudio Antônio
CA
Ferreira
Paulo César Peregrino
PC
Kroon
Erna Geessien
EG
D000818:Animals; D001938:Brazil; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000821:virology; D019411:Clinical Laboratory Techniques / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004195:Disease Models, Animal; D005814:Genes, Viral; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D018155:Orthopoxvirus / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011213:Poxviridae Infections / Q000175:diagnosis* / Q000662:veterinary* / Q000821:virology; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004279:DNA, Viral