In addition to classical clinical diagnostic methods, poxviruses can be diagnosed using various methods on the molecular level.

Real-time PCR (rtPCR)
At present, the most sensitive and rapid test assay for poxviruses is performed by detecting viral nucleic acid by using real-time PCR.

Genome sequencing
By DNA sequencing (Illumina, AmpliSeq), sequencing of the viral genome is possible, enabling an exact identification of the virus.

Electron microscopy (EM)
There is a close cooperation with the Consultant Laboratory for Diagnostic Electron Microscopy in Infectious Diseases headed by Dr. Michael Laue. EM diagnostics is a rapid method which is sufficiently sensitive when investigating clinical samples to differentiate parapoxviruses from other poxviruses. However, no typing is possible by EM.

Cell culture
In order to characterize orthopoxviruses biologically, it is possible to propagate them in cell culture or on the chorioallantois membrane (CPE, CAM). However, this is time consuming and therefore of little importance in primary diagnostics. It is possible to use cell culture as confirmation of virus propagation using specific antibodies and the immunofluorescence assay.

Serology
Antibodies against orthopoxviruses can be quantified by immunofluorescence assay, ELISA or neutralization test. Because of antigenic similarity, antibodies against different orthopoxviruses cannot be differentiated by standard laboratory methods. Furthermore, a conclusion on the infection status of the patient can only be made with time course evaluation or unequivocal IgM detection.

Table 1: Detection of human pathogenic poxviruses by rtPCR

Genus	Species differentiation
Orthopoxvirus (generic)	Variola virus
	Cowpox virus
	Mpox virus
Parapoxvirus (generic)	Orf virus
	Pseudo cowpox virus
	Bovine papular stomatitis virus
Mollusci­poxvirus	Molluscum contagiosum virus
Yatapoxvirus	Tanapoxvirus
	Yabapoxvirus