Ethidium monoazide does not inhibit RT-PCR amplification of nonviable avian influenza RNA.
A critical obstacle to using PCR to quantify viral titers is the distinguishment of viable and nonviable genomic material. Pretreatments of ethidium monoazide (EMA) have been effective in preventing PCR amplification of DNA from nonviable bacteria. To test whether an EMA pretreatment could be used with RT-PCR to quantify viable RNA virions, avian influenza virus (AIV) survival was measured in water through 28d using cell culture titration and real-time RT-PCR with or without EMA pretreatment. Cell culture titration yielded significantly lower titers than both RT-PCR procedures, and there was no significant difference between RT-PCR results with or without EMA. Ineffective binding of EMA to AIV RNA may have allowed nonviable AIV RNA to amplify. Furthermore, since AIV inactivation may take place by means other than membrane disruption, any pretreatment distinguishing viable and nonviable AIV virions by membrane integrity may not be practical.
Graiver
David A
DA
Department of Civil Engineering, University of Nebraska-Lincoln, 203B Peter Kiewit Institute, Omaha, NE 68182-0178, United States.
Saunders
Samuel E
SE
Topliff
Christina L
CL
Kelling
Clayton L
CL
Bartelt-Hunt
Shannon L
SL
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D001386:Azides / Q000494:pharmacology*; D001717:Birds; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D009980:Influenza A virus / Q000235:genetics / Q000502:physiology*; D005585:Influenza in Birds / Q000821:virology; D050296:Microbial Viability / Q000187:drug effects*; D012367:RNA, Viral / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014773:Virology / Q000379:methods
D000998:Antiviral Agents; D001386:Azides; D004791:Enzyme Inhibitors; D012367:RNA, Viral; C014535:8-azidoethidium