Development and validation of a real-time Taqman PCR assay for the detection and quantitation of infectious laryngotracheitis virus in poultry.
In this study, the development and validation of a real-time (ReTi) PCR assay is described using a Taqman labeled probe for the detection and quantitation of infectious larygotracheitis virus (ILTV) in chickens. The ReTi ILTV assay was highly specific with a quantitation limit of 100 viral template copies per amplification reaction. In experimentally infected, birds during early acute stages of infection, an average of 6.67 log(10) viral template copies/amplification reaction were detected, while at chronic late stages of infection an average of 2.86-3.27 log(10) viral template copies/amplification reaction were detected. A total of 246 tracheal swab samples collected from natural outbreaks of the disease were tested by virus isolation and the ReTi ILTV assay. Both assays agreed in 37% of the samples tested and the ReTi ILTV assay detected approximately 3.7 times more positives samples than virus isolation. A minimum of 5 log(10) viral template copies/amplification reaction were required from a tracheal swab to render a virus isolation positive result. In conclusion, the ReTi ILTV assay was highly specific, sensitive, reproducible, and capable of reliably quantifying viral nucleic acid directly from clinical samples.
Callison
S A
SA
Department of Population Health, Poultry Diagnostic and Research Center, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602, USA.
Riblet
S M
SM
Oldoni
I
I
Sun
S
S
Zavala
G
G
Williams
S
S
Resurreccion
R S
RS
Spackman
E
E
García
M
M
D000818:Animals; D001483:Base Sequence; D001352:Herpesvirus 1, Gallid / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011200:Poultry / Q000821:virology*; D012680:Sensitivity and Specificity