Detection of vaccinia virus, herpes simplex virus, varicella-zoster virus, and Bacillus anthracis DNA by LightCycler polymerase chain reaction after autoclaving: implications for biosafety of bioterrorism agents.
OBJECTIVE
To determine whether autoclaving suspensions of vaccinia virus, herpes simplex virus (HSV), varicella-zoster virus (VZV), and Bacillus anthracis inactivate infectivity of these agents but allow detection of target DNA by LightCycler polymerase chain reaction (PCR).


METHODS
Swabs were inserted into tubes containing serial 10-fold dilutions (10(-1) to 10(-5); 500 microL; 6 samples per dilution) of vaccinia virus, HSV, VZV, or a single suspension of 10(8) colony-forming units of B anthracis (2 samples). One half of the samples were autoclaved, and the remainder were not. An aliquot of each not autoclaved sample served as a control for infectivity.


RESULTS
Autoclaving swabs saturated with suspensions of vaccinia virus, HSV, or VZV eliminated the infectivity of these agents; however, DNA was detectable in most autoclaved samples in dilutions of 10(-1) to 10(-4) by LightCycler PCR. All not autoclaved specimens were detected by culture (infectivity) except for VZV and, in most dilutions of 10(-1) to 10(-3), by assay of target DNA by LightCycler PCR. Similarly positive results were obtained for PCR assessment of sporulated B anthracis.


CONCLUSIONS
Standard autoclaving procedures eliminated the infectivity of viruses (and B anthracis), but target DNA was often retained for detection by LightCycler PCR. Current recommendations indicate that the laboratory diagnosis of smallpox virus infection be performed only within Biosafety Level 4 facilities. We suggest that, in addition to the requirement for immediate coordination with public health officials, the federal government consider expanding the existing guidelines for processing these specimens to encourage immediate collection, autoclaving, and testing by LightCycler PCR to differentiate smallpox virus from other dermal pathogens such as HSV and VZV by specific qualified laboratories.
Espy
Mark J
MJ
Division of Clinical Microbiology, Mayo Clinic, Rochester, MN 55905, USA.
Uhl
James R
JR
Sloan
Lynne M
LM
Rosenblatt
Jon E
JE
Cockerill
Franklin R
FR
Smith
Thomas F
TF
D001408:Bacillus anthracis / Q000235:genetics / Q000302:isolation & purification*; D023482:Bioterrorism; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004279:DNA, Viral / Q000302:isolation & purification*; D017408:Guidelines as Topic; D014645:Herpesvirus 3, Human / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000295:instrumentation*; D018139:Simplexvirus / Q000235:genetics / Q000302:isolation & purification*; D013242:Sterilization; D014481:United States; D014616:Vaccinia virus / Q000235:genetics / Q000302:isolation & purification*
D004269:DNA, Bacterial; D004279:DNA, Viral