Development of methods for detection and quantification of avian influenza and Newcastle disease viruses in compost by real-time reverse transcription polymerase chain reaction and virus isolation.
Composting has been used for disposal of poultry carcasses and manure following outbreaks caused by avian influenza virus (AIV) and Newcastle disease virus (NDV), but methods are needed to test for survival of the viruses in compost to ensure biosecurity. Methods developed in the present study include extracting viruses from compost and purifying viral RNA. The extracted viruses were detected by virus isolation using embryonated chicken eggs, and the purified RNA was detected by real-time reverse transcription PCR (RRT-PCR). The virus isolation and the RRT-PCR methods were evaluated with 3 compost preparations that were produced from chicken manure mixed with corn silage, wood shavings, or wheat straw. The detection limits of both methods were 1,700 and 1,000 embryo lethal doses of AIV and NDV per gram of compost, respectively. The copy number of viral RNA quantified by RRT-PCR was highly correlated with the amount of virus in compost. The results suggested that the RRT-PCR method may be used as an alternative to the virus isolation method for rapid detection and accurate quantification of AIV and NDV in compost.
Guan
J
J
Canadian Food Inspection Agency, 3851 Fallowfield Road, Nepean, Ontario, Canada, K2H 8P9. guanj@inspection.gc.ca
Chan
M
M
Ma
B
B
Grenier
C
C
Wilkie
D C
DC
Pasick
J
J
Brooks
B W
BW
Spencer
J L
JL
D000818:Animals; D002645:Chickens; D005243:Feces / Q000821:virology; D009980:Influenza A virus / Q000235:genetics / Q000302:isolation & purification*; D008372:Manure / Q000821:virology; D009522:Newcastle disease virus / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000737:chemistry / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012987:Soil / Q000032:analysis; D012988:Soil Microbiology; D013047:Specific Pathogen-Free Organisms
D008372:Manure; D012367:RNA, Viral; D012987:Soil