Alkaline hydrolysis to remove potentially infectious viral RNA contaminants from DNA.
BACKGROUND
Diagnostics and research of high-consequence animal disease agents is often limited to laboratories with a high level of biosecurity that restrict the transport of biological material. Often, sharing of DNA with external partners is needed to support diagnostics, forensics, or research. Even in the absence of virus, RNA from positive-sense single stranded RNA (+ssRNA) viruses that may contaminate otherwise purified DNA preparations continues to pose a threat due to its potential to be infectious via direct translation to yield viral proteins. While the risk of animal infection or accidental reconstitution and release of a virus from RNA is very low, the high impact of an animal disease event associated with the accidental release of some + ssRNA viruses, such as classical swine fever or foot-and-mouth disease viruses, necessitates the precaution of having procedures to ensure the complete inactivation of viruses and + ssRNA viral genomes. RNA and DNA are differentially susceptible to enzymatic degradations; however, such procedures are susceptible to unintended DNA damage and/or failure due to enzyme or cofactor instabilities. Therefore, we describe the development and verification of a robust and simple chemical and physical method to selectively degrade RNA from purified DNA preparations. The procedure employs incubation of DNA in 0.25 N sodium hydroxide at 65 °C for 1 h followed by neutralization and boiling for 10 min to hydrolyze contaminating RNA and inactivate animal disease viruses from DNA preparations. Additional critical quality control elements include use of a synthetic control RNA (SCR) and an SCR-specific real-time RT-PCR to track effectiveness of the procedure in a parallel treated control sample, and a pH check of reagents to ensure proper neutralization of alkaline conditions.


RESULTS
The new procedure reduced intact RNA beyond the limit of detection by realtime RT-PCR and inactivated viruses by in vitro culture infectivity assays.


CONCLUSIONS
Treated DNA, while denatured, remains suitable for most common molecular biology procedures including PCR, transformation of E. coli, and molecular sequencing. The procedure ensures not only the inactivation of a variety of viruses but also the degradation through hydrolysis of potentially contaminating infectious + ssRNA viral genomes.
Foreign Animal Disease Diagnostic Laboratory, NVSL, STAS, VS, APHIS, USDA, Plum Island Animal Disease Center, PO Box 848, Greenport, NY, 11944-0848, USA.
Karissa A
KA
Lemire
Foreign Animal Disease Diagnostic Laboratory, NVSL, STAS, VS, APHIS, USDA, Plum Island Animal Disease Center, PO Box 848, Greenport, NY, 11944-0848, USA.
Yelitza Y
YY
Rodriguez
Foreign Animal Disease Diagnostic Laboratory, NVSL, STAS, VS, APHIS, USDA, Plum Island Animal Disease Center, PO Box 848, Greenport, NY, 11944-0848, USA. michael.t.mcintosh@aphis.usda.gov.
Michael T
MT
McIntosh
D000818:Animals; D004247:DNA / Q000302:isolation & purification*; D004202:Disinfectants / Q000494:pharmacology*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D012367:RNA, Viral / Q000187:drug effects*; D012972:Sodium Hydroxide / Q000494:pharmacology*; D013997:Time Factors; D038301:Virus Inactivation
D004202:Disinfectants; D012367:RNA, Viral; D012972:Sodium Hydroxide; D004247:DNA