Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/7/2006
Publication Date: 10/9/2006
Citation: Callison, S.A., Riblet, S.M., Sun, S., Guillermo, Z., Williams, S., Resurreccion, R., Spackman, E., Garcia, M. 2006. Development and validation of a real-time Taqman(r) PCR assay for the detection and quantitation of infectious laryngotracheitis virus in poultry. Journal of Virological Methods. 139(1):31-38. Interpretive Summary: Infectious laryngotracheitis virus (ILTV) is an important disease agent in chickens. ILTV causes severe respiratory disease which causes financial loss to the US chicken industry. Rapid detection is critical to controlling the disease, however virus isolation is expensive and time consuming. This manuscript reports the development of a rapid, sensitive and highly specific diagnostic test for ILTV. This new test, real-time RT-PCR, was compared with virus isolation for ILTV detection in infected chickens and was shown to be reliable for detecting the virus in clinical samples.
Technical Abstract: In this study, we report the development and validation of a real-time PCR assay using a Taqman(R) labeled probe (ILTV assay) for the detection and quantification of infectious larygotracheitis virus (ILTV). The ILTV assay was highly specific with a detection limit of 25 viral template copies/amplification reaction and a quantification limit of 100 viral template copies/amplification reaction. From experimentally infected birds, the ILTV assay, virus isolation (VI), direct fluorescent antibody assay, and histopathology evaluated in this study performed well at early stages of infection. However, at late stages of infection, VI was not possible, and detection of viral DNA by the ILTV assay was more sensitive than detection of viral antigens by direct fluorescent antibody test. Furthermore, the ILTV assay was capable of quantifying viral template copies from tracheal tissues. During early acute stages of the infection, an average of 6.67 log10 viral template copies/amplification reaction were detected, while at late stages of infection an average of 2.86 to 3.27 log10 viral template copies/amplification reaction were detected. From natural outbreaks of ILT, a total of 246 tracheal swab samples were collected and tested by VI and the ILTV assay. Overall, the ILTV assay and VI agreed in 37% of the samples tested and the ILTV assay detected approximately 3.7 times more positives than VI. The ILTV assay was used to assess the viral load necessary to successfully isolate ILTV from 98 tracheal swabs collected from two flocks experiencing natural outbreaks of ILT. The ILTV assay results showed that a minimum of 5 log10 viral template copies/amplification reaction was needed from a tracheal swab sample for it to render a VI positive result. In conclusion, the ILTV assay was highly specific, sensitive, reproducible, and capable of reliably quantifying viral nucleic acid directly from clinical samples.