Location: Endemic Poultry Viral Diseases ResearchTitle: Development of a non invasion real-time PCR assay for the quantitation of chicken parvovirus in fecal swabs
Submitted to: American Association of Avian Pathologists
Publication Type: Proceedings
Publication Acceptance Date: 11/20/2014
Publication Date: N/A
Technical Abstract: The present study describes the development of a real time Taqman polymerase chain reaction (PCR) assay using a fluorescent labeled probe for the detection and quantitation of chicken parvovirus (ChPV) in feces. The primers and probes were designed based on the nucleotide sequence of the non structural (NS) gene of ChPV. A standard curve, plotted using 10-fold serial dilutions of a plasmid containing a synthetic NS amplicon, was found to be linear over eight 10-fold serial dilution range. Relative quantification of viral genome copies could be assessed based on the endogenous control alpha2-collagen gene of chicken (Gallus gallus domesticus) in a duplex reaction. The assay was highly sensitive with a quantitation limit of 50 viral genome copies per amplification reaction. In experimentally infected chickens, averages of 2.5 X 106 parvovirus genomes/swab were detected at 14 days post infection (dpi). At an early stage of infection (7 dpi) an average of 9.17 x 104 parvovirus genomes/swab were detected, however 5.9% of the samples determined negative at the early sampling time, originated for birds testing positive at the 14 dpi sampling time. Ninety-four percent of experimentally infected chickens tested positive at the later time point. In conclusion, the real time ChPV duplex assays, with alpha2-collagen detection as a DNA isolation control, was highly sensitive, reproducible, and capable of reliably quantifying virus nucleic acid directed from fecal samples.