Development of a duplex TaqMan real-time RT-PCR assay for simultaneous detection of goose astrovirus genotypes 1 and 2

Zewen Yi; Rui Ding; Ruibing Cao; Weidong Sun; Mingzhi Sun; Yongyi Dong; Buriro Rehana; Endong Bao; Yingjun Lv

doi:10.1016/j.jviromet.2022.114542

Development of a duplex TaqMan real-time RT-PCR assay for simultaneous detection of goose astrovirus genotypes 1 and 2

J Virol Methods. 2022 Aug:306:114542. doi: 10.1016/j.jviromet.2022.114542. Epub 2022 May 13.

Authors

Zewen Yi¹, Rui Ding¹, Ruibing Cao¹, Weidong Sun¹, Mingzhi Sun², Yongyi Dong³, Buriro Rehana⁴, Endong Bao¹, Yingjun Lv⁵

Affiliations

¹ MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Weigang 1, Nanjing 210095, China.
² College of Life Sciences, Nanjing Agricultural University, Weigang 1, Nanjing 210095, China.
³ Animal Disease Control Center, Caochangmen street 124, Nanjing 210036, China.
⁴ Department of Veterinary Pharmacology, Sindh Agriculture University, Tando Jam, 70060 Hyderabad, Pakistan.
⁵ MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Weigang 1, Nanjing 210095, China. Electronic address: lyj@njau.edu.cn.

PMID: 35577125
DOI: 10.1016/j.jviromet.2022.114542

Abstract

Goose astrovirus (GAstV) is a novel pathogen that was discovered in 2018. It has two genotypes, GAstV-1 and GAstV-2, and both can cause visceral gout of goslings and result in significant economic losses. The present work aimed to develop a duplex TaqMan real-time quantitative reverse transcription PCR (RT-qPCR) assay to distinguish the two genotypes. MegAlign software was used to design two pairs of primers and a pair of matched probes based on the open reading frame 2 (ORF2) sequence with the greatest difference between GAstV-1 and GAstV-2, and primer and probe concentrations and annealing temperatures were optimised. Fluorescence signals were obtained for GAstV-1 and GAstV-2 in the FAM and VIC channels, respectively, but no fluorescent signal was observed for other pathogens. The detection limit for GAstV-1 and GAstV-2 was 33.3 and 33.7 DNA copies/μL, respectively. Intra- and inter-assay variability tests revealed excellent reproducibility. Furthermore, the assay detected GAstV-1 and GAstV-2 in allantoic fluids (100% positive) spiked with viruses, and 70 clinical gout gosling samples were examined, of which 11.4% were positive for GAstV-1, 74.3% were positive for GAstV-2%, and 5.7% were positive for mixed infection. In summary, the developed duplex RT-qPCR assay has high specificity, sensitivity, and reproducibility, and can be used in the clinic for detection of GAstV-1 and GAstV-2.

Keywords: Duplex TaqMan real-time RT-PCR; GAstV-1; GAstV-2; Goose astrovirus.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Astroviridae Infections* / diagnosis
Astroviridae Infections* / veterinary
Avastrovirus* / genetics
Geese
Genotype
Gout*
Real-Time Polymerase Chain Reaction
Reproducibility of Results
Reverse Transcriptase Polymerase Chain Reaction
Sensitivity and Specificity