[Rapid differential diagnosis of Orthopoxviruses and Herpesviruses based upon multiplex real-time PCR].
OBJECTIVE
Variola virus, belonging to Orthopoxviridae family, is one of the most dangerous human pathogens that could be used as biological weapon. We have developed a new rapid assay, based upon Real-time PCR and melting temperatures analysis of amplicons, for the contemporary detection of Orthopoxvirus, VZV and HSV1-2, that are the most important infectious agents to be considered for differential diagnosis.


METHODS
The target for detection of orthopoxvirus DNA has been a region of the crmB gene which is common to Variola virus and to other old world orthopoxviruses pathogenic for humans. The targets for VZV and HSV1-2 have been ORF 29 and DNA polymerase, respectively. Suitability of the amplified fragments to RFLP or sequencing analysis, to recognize the involved viral species, has been also tested.


RESULTS
The selected primers have showed high sensitivity, specificity and compatibility with common amplification conditions. A mean melting temperature difference of 8.7 degree C was observed between the amplicons from the two virus types. Further identification of individual pathogens was made using RFLP analysis.


CONCLUSIONS
The PCR-based protocol set up in this study for presumptive differential diagnosis of variola and herpesviral infections is rapid and specific and it can be used also to detect other orthopoxviral infections, like monkeypox.
Sias
Catia
C
Laboratorio di Virologia, Istituto Nazionale per le Malattie Infettive L. Spallanzani, Roma, Italy.
Carletti
Fabrizio
F
Capobianchi
Maria R
MR
Travaglini
Damiano
D
Chiappini
Roberta
R
Horejsh
Douglas
D
Di Caro
Antonino
A
D023482:Bioterrorism; D003199:Computer Systems; D017931:DNA Primers; D004279:DNA, Viral / Q000097:blood*; D003937:Diagnosis, Differential; D006564:Herpesviridae / Q000235:genetics / Q000302:isolation & purification*; D006566:Herpesviridae Infections / Q000175:diagnosis* / Q000821:virology; D006801:Humans; D045908:Mpox, Monkeypox / Q000175:diagnosis / Q000821:virology; D008993:Monkeypox virus / Q000235:genetics / Q000302:isolation & purification; D009691:Nucleic Acid Denaturation; D018155:Orthopoxvirus / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D011213:Poxviridae Infections / Q000175:diagnosis* / Q000821:virology; D012680:Sensitivity and Specificity; D012899:Smallpox / Q000175:diagnosis / Q000821:virology; D013045:Species Specificity; D013997:Time Factors; D012901:Variola virus / Q000235:genetics / Q000302:isolation & purification; D014766:Viremia / Q000175:diagnosis* / Q000821:virology
D017931:DNA Primers; D004279:DNA, Viral