Abstract:
Objective A universal one-step RT-PCR detection method on goose astrovirus (GAstV) was developed.
Methods Primers were designed based on the conserved region of the RNA polymerase gene (RbRp) of GAstV and reaction conditions optimized for the methodology development. Specificity, sensitivity, and reproducibility of the assay were scrutinized with a verification test conducted on clinical specimens.
Results On assay specificity, the amplified target fragments of GAstV-1 and GAstV-2 were determined to be 870 bp and 873 bp, respectively, while the results were negative on other common goose viruses. For the sensitivity of detection, the minimum detectable limits were 8.3×103 copies/μL and 8.9×103 copies·μL−1, respectively, on GAstV-1 and GAstV-2. On 3 different templates tested at 3 different times, consistent and reproducible results were obtained. On 72 clinical diseased tissue specimens, the new assay rendered a positive rate of 84.72% on GAstV, which was consistent with what delivered by dual RT-PCR tests. A sequence analysis on 4 amplification products showed GAstV-1 and GAstV-2 to locate in two completely different branches of the evolutionary tree.
Conclusion The newly established simple and rapid universal one-step RT-PCR method was highly specific, sensitive, accurate, and repeatable in detecting GAstV. It was considered appropriate for disease diagnosis, customs quarantine, and molecular epidemiological studies related to the virus.
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