Inactivation of orthopoxvirus for diagnostic PCR analysis

Abstract

Diagnoses of ongoing viral infections commonly rely on PCR methodology. Sample material that may contain hazardous virus should be efficiently inactivated in biological containment or bed-side before diagnostic PCR analysis. Surprisingly little documentation is available for inactivation of human viral pathogens by inactivation reagents that allow for subsequent PCR diagnostics. It is now shown that pathogenic DNA viruses (orthopoxvirus) are completely inactivated by a commercially available Roche MagNA Pure lysis/binding buffer as evaluated by subsequent cell culture. However, inactivation reagents are typically toxic and therefore problematic in cell culture. Using the relatively large orthopoxvirus, a method was developed in which virus is precipitated by high-speed centrifugation after inactivation but prior to application onto the target cells, thereby eliminating the cytotoxic effect of the lysis buffer. The results from quantitative PCR analysis indicate that the viral DNA from the completely inactivated virus particles, remain associated to macromolecules and aggregates. The use of inactivation buffers for bed-side inactivation of special patient samples taken for PCR diagnostics should be considered in cases where high containment would otherwise be required.