Development and application of a dual RT-qPCR assay for differentiating of goose astrovirus genotypes 1 and 2

Abstract

Goose astrovirus (GAstV) is an important pathogen causing joint and visceral gout in goslings. Its high incidence and mortality had caused enormous economic losses to the goose farming industry. The study aimed to develop a method for the simultaneous and differential detection of goose astrovirus genotype 1 (GAstV-1) and genotype 2 (GAstV-2). Specific primers and TaqMan probes were designed and synthesized based on the ORF1b gene. Through optimizing the concentrations of primers and probes, annealing temperature, and evaluating the specificity, sensitivity and repeatability, a dual real-time fluorescent RT-PCR (RT-qPCR) assay for GAstV-1 and GAstV-2 was developed. The results demonstrated that the specific amplification curves for GAstV-1 and GAstV-2 were produced in the assay, with no amplifications for other tested common avian pathogens. Utilizing the in vitro transcribed RNA as a template, the detection limits for both GAstV-1 and GAstV-2 were determined to be 1.0 × 10² copies/reaction. Standard curves were generated correlating the logarithmic concentration values with Ct values, with the amplification efficiencies and R² values of 96.06 % and 0.999 for GAstV-1, 90.39 % and 0.998 for GAstV-2, respectively. The coefficients of variation both within and between groups were below 3 %, indicating a good repeatability. The developed dual RT-qPCR assay was further evaluated on 104 liver samples from goslings exhibiting gout symptoms and those without gout symptoms. The GAstV-1 and GAstV-2 were detected in 19 (18.27 %, 19/104) and 69 (66.35 %, 69/104) samples, respectively, in which 16 samples (15.38 %, 16/104) carried both GAstV-1 and GAstV-2 nucleic acids. The developed dual RT-qPCR assay in this study performed well on the clinical samples, thereby providing a robust technical support for the epidemiological investigation of GAstV infection and the prevention and control of gosling gout.

Keywords: Differential detection; Dual real-time fluorescent RT-PCR; GAstV-1; GAstV-2; ORF1b gene.