The Development and Application of Quantitative Reverse Transcriptase Polymerase Chain Reaction for the Detection of Avian Reoviruses
Type of DegreeDissertation
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We developed a TaqMan probe based real-time RT-PCR for qualitative and quantitative detection of avian reoviruses (ARVs). The primer-probe set was designed from the conserved region of ARV S4 genome segment. ARV strains S1133, 2408, CO8, 1733, JR1, ss412, and two vaccine strains (ChickVac™ and V.A. Vac®) were tested. Specificity tests indicated that the real-time RT-PCR had high specificity in the detection of 8 ARVs with no cross-reaction with other avian viruses. TaqMan real-time PCR successfully detected CO8 and ss412, which belonged in different serological subgroups compared with the other 6 strains. Full -length ARV S4 gene was cloned and in vitro transcription performed to produce a pure ARV S4 RNA standard, which was used for sensitivity tests. Compared to traditional RT-PCR, real-time RT-PCR was about 150 times more sensitive. The detection limit of the real-time RT-PCR was approximately 25 ARV genome copies, which was equivalent to 5 copies/µl. Statistical analyses indicated excellent reproducibility. Correlations between ARV genome copies and virus titer (EID50 and TCID50) were determined using 7-day chicken embryos and chicken embryo kidney cell culture. Results indicated that for ARV strain 2408, 1 EID50 was equivalent to 3.9 ± 0.8 ARV genome copies, and 1 TCID50 was equivalent to 2.9 ±0.3 ARV genome copies. The TaqMan probe-based real-time RT-PCR developed was used to monitor ARV shedding in feces of commercial and specific-pathogen-free chickens infected with ARV 2408. ARV was detected in cloacal swabs at 1 day post-inoculation and throughout the tested period. Commercial chickens, which had high maternal ARV antibody titer, showed minimal clinical signs and low ARV excretion in the feces, whereas the SPF chickens had 30% mortality, more severe clinical signs, and higher virus shedding. A SYBR-Green I based real-time PCR was developed to differentiate closely related ARV strains. Three primers sets were designed to amplify three partially overlapping regions, which covered a majority of mutation sites. Subsequently, melting curve analyses were performed to determine the unique melting peak temperature (Tm) of each region. Results indicated that each ARV strain had a specific profile of Tm combination within the three regions. Strains CO8 and ss412 demonstrated more variations in Tm profiles than other tested strains, indicating that they may belong to different subgroups.