Virus strains
The novel goose astrovirus genotypes 2 (GoAstV-2) was isolated from goose kidneys with gout [40]. GoAstV-2, Goose astrovirus genotypes 1 (GoAstV-1), goose parvovirus (GPV), Duck tembusu virus (DTMUV), and duck reovirus (DREOV) were preserved in our laboratory and stored at − 80°C before use. The nucleic acids of Newcastle disease virus (NDV), avian influenza virus (AIV), and fowl adenovirus (FAdV) were obtained from Guangdong Laboratory Animals Monitoring Institute.
Design Of Real-time RT-RPA Probes And Primers
The ORF2 complete gene sequences of GoAstV-2 were downloaded from Genbank and identified using the MegAlign software. According to the design manual from TwistDx Co. Ltd., two exo probes were designed and carefully screened based on the conserved region of the ORF2 sequence. The TwistAmpexo probe length was 46–52 nucleotides. A pair of T residues in close proximity to one another (with only 1–5 intervening nucleotides) were found in a probe sequence, and the relevant T residues were replaced by dT-fluorophore residues and dT-quencher residues. One base was replaced by the THF residue, and the 3’-end was blocked with a C3-spacer [41, 42]. Three pairs of primers were designed to match probe 1 and two pairs of primers were suitable for probe 2. The sequences of all primers and probes are listed in Table 3 and were synthesized by Sangon Biotech (Shanghai, China) Co. Ltd.
Table 3
The sequences of primers and probes used in this study
Method
|
Name
|
Sequence (5′–3′)
|
Amplicon size (bp)
|
Source
|
Real-time
RT-RPA
|
GoAstV-F1
|
TAATGACAAAATGACTACCACAATAACACT
|
161
|
this study
|
GoAstV-R1
|
TATATTGTGAAGCCCTTATACTTAGAGGAG
|
GoAstV-F2
|
CGTTAATGACAAAATGACTACCACAATAAC
|
165
|
GoAstV-R2
|
GTTATATTGTGAAGCCCTTATACTTAGAGG
|
GoAstV-F3
|
AAAGAAGTGAAAGGTTTGAAGAAAAGAGTA
|
241
|
GoAstV-R3
|
CAAGGCGGATATGCAGCTTTCTGATCCTCC
|
GoAstV-probe1
|
CAACAGACACACTCGACCGGAAGCATAAA[FAM-dt] [THF]C[BHQ1-dT] TCACAAATCCACTC (C3-spacer)
|
|
GoAstV-F4
|
AGGTCAAGATACAATGCAAATATAACCTTC
|
126
|
GoAstV-R4
|
AAAAATAACTCCTGTTTGTGAAAGTGTCAT
|
GoAstV-F5
|
GAATACCACAGGCAGACTCCAGGTCAAGAT
|
141
|
GoAstV-R5
|
TAACTCCTGTTTGTGAAAGTGTCATAACAG
|
GoAstV-probe2
|
ATGTTGGCTATCGTGGAAGGACTTCAACA[FAM-dt] [THF]A[BHQ1-dT] TTACACTTGGGACA (C3-spacer)
|
|
RT-PCR
|
GoAstV-F
|
CAGTATCTGGCATCGCCTCAT
|
406
|
GoAstV-R
|
CCTGGGAACAGAACCTGAACT
|
RT-qPCR
|
qF
|
GGCCAATATTCAACAACA
|
172
|
Chunhe Wan et al., 2019
|
qR
|
CCTTCCTTATTGACACAAG
|
qP
|
TGTGTAATGTCTGGCTCACCCA
|
Construction Of The RNA Standard
The partial sequences (406 bp) of the nucleocapsid gene were amplified by the primers GoAstV-F and GoAstV-R using a one-step RT-PCR kit (Takara, Dalian, China). The amplification products were purified by a gel extraction kit (Omega, USA) and inserted into the pGEM-T vector (Promega, USA). The positive recombinant plasmid pGEM-T-cap was sequenced by Sangon Biotech (Shanghai, China) and linearized by SalI (Takara, Dalian, China). The purified linear DNA was in vitro transcribed to RNA using a T7 RiboMAX™ Express RNAi System (Promega, USA) following the manufacturer’s manual. The double-stranded RNA was purified using a Qiagen Viral RNA mini kit (Qiagen, Germany). The RNA standard concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher, USA), and the copy number was calculated using a previously described formula [21].
Goastv-2 Real-time RT-RPA Assay
The real-time RT-RPA reaction was performed in a 50 µL volume with a fluorescent RT-RPA kit (Zongce, Hangzhou, China). The reaction tube contained lyophilized enzyme pellets comprising 25 µL of rehydration buffer (A buffer), 2 µL of each primer (10 µmol/L), 0.6 µL of the RT-RPA probe (10 µmol/L), 2.5 µL of magnesium acetate (B buffer), 2 µL of the RNA/DNA template, and 15.9 µL of RNase-free H2O and incubated at 39°C for 20 min using a Deaou-308C instrument (DEAOU Biotechnology, Guangzhou, China). The fluorescence signal was collected and the time of the amplification curve was calculated based on the threshold. If the amplification curve was generated within 20 min, the sample was set as positive; otherwise, the sample was negative.
Specificity Analysis Of The Real-time RT-RPA Assay
The nucleic acid of GoAstV-2 and other avian viruses, including GoAstV-1, DTMUV, DREOV, GPV, AIV, NDV, and FAdV, and distilled water were tested to evaluate the specificity of the GoAstV-2 RT-RPA assay using the above mentioned procedure. GoAstV-2 and distilled water were used as positive and negative controls, respectively.
Sensitivity And Repeatability Analysis Of The Real-time RT-RPA Assay
To analyze the limit of detection of the GoAstV-2 RT-RPA assay, the in vitro transcribed RNA standard was serially diluted ten-fold in EASY Dilution solution ranging from 105 copies/µL to 100 copies/µL. RNase-free H2O, instead of the RNA standard, was tested as a negative control. All reactions were performed in triplicate.
To validate the repeatability of the method, six 10-fold gradient dilutions (105, 104, 103, 102, 101, and 100 copies/µL) of RNA standard were carried out five times using the developed RT-RPA assay under optimized reaction conditions, and the coefficient of variation (CV) values of the intra- and inter-assay were calculated.
Clinical Sample Analysis Using The RT-RPA And Real-time RT-PCR Assays
A total of 270 goose clinical samples, including 60 tissue samples artificially infected with GoAstV-2, 120 clinical tissue samples (kidney, spleen, and liver), and 90 cloacal swabs were collected from geese farms in Jiangxi Province, China. The tissue samples were homogenized with phosphate-buffered saline (PBS; pH 7.2) at a ratio of tissue:PBS of 1:10 and centrifuged at 4°C at 5000 g for 15 min. The supernatants were collected and stored at − 80°C. The cloacal swabs were placed in a 2 mL tube with 1 mL of PBS and mixed. All the samples were treated with lysis buffer at a ratio of 1:1 and centrifuged, and then were used as templates in the real-time RT-RPA assay. To further validate the results of the assay detection, all the samples were also tested by real-time RT-PCR as previously described [17].Total RNA and DNA were also extracted from the supernatants of the tissue homogenates and the cloacal swabs using a TIANamp Virus Genomic DNA/RNA kit (Tiangen Biotech, Beijing, China) in accordance with the manufacturer’s protocol and stored at − 80°C until used for RT-qPCR.