Development of an EvaGreen based real-time RT-PCR assay for rapid detection, quantitation and diagnosis of goose calicivirus

Journal Pre-proof Development of an EvaGreen based real-time RT-PCR assay for rapid detection, quantitation and diagnosis of goose calicivirus Su Lin, Shizhong Zhang, Shao Wang, Kaichun Xie, Dandan Jiang, Shifeng Xiao, Xiuqin Chen, Shaoying Chen PII:

S0890-8508(19)30327-5

DOI:

https://doi.org/10.1016/j.mcp.2019.101489

Reference:

YMCPR 101489

To appear in:

Molecular and Cellular Probes

Received Date: 10 September 2019 Revised Date:

1 November 2019

Accepted Date: 16 November 2019

Please cite this article as: Lin S, Zhang S, Wang S, Xie K, Jiang D, Xiao S, Chen X, Chen S, Development of an EvaGreen based real-time RT-PCR assay for rapid detection, quantitation and diagnosis of goose calicivirus, Molecular and Cellular Probes (2019), doi: https://doi.org/10.1016/ j.mcp.2019.101489. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

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Development of an EvaGreen based real-time RT-PCR assay for rapid detection,

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quantitation and diagnosis of goose calicivirus

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Su Lin a,b†, Shizhong Zhang a†, Shao Wang a,b†*, Kaichun Xie c , Dandan Jiang a ,

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Shifeng Xiao a,b, Xiuqin Chen a,b, Shaoying Chen a,b*

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a

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Agriculture Sciences, Fuzhou, 350003, China.

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b

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China

Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of

Fujian Animal Diseases Control Technology Development Center, Fuzhou, 350013,

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c

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*Corresponding author, Institute of Animal Husbandry and Veterinary Medicine,

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Fujian Academy of Agricultural Sciences, Fuzhou, China.

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E-mail address: [email protected], [email protected]

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†

Animal Veterinary and Aquatic product Bureau, Nanping, 353000, China

These authors contributed equally to this work.

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Su Lin: [email protected]

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Shizhong Zhang: [email protected]

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Shao Wang: [email protected]

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Kaichun Xie: [email protected]

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Dandan Jiang: [email protected]

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Shifeng Xiao: [email protected]

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Xiuqin Chen: [email protected]

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Shaoying Chen: [email protected]

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Abstract: An unclassified calicivirus (CV) detected in geese was recently reported and proposed

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as a new member of the family Caliciviridae. There is limited information about the

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epidemiology, etiology and detection method of goose-origin CV (GCV) to date. In

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this study, an EvaGreen based fluorescence quantitative real-time RT-PCR assay was

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developed and optimized for the detection of GCVs. The assay sensitively detected

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GCV RNA template with a good linear standard curve We also demonstrated the

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specificity and reproducibility of the detection method for GCVs. Thus, the method

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developed in this study will benefit the investigation of possible sporadic outbreaks of

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CV infections in geese, as well as epidemiological and etiological studies of GCVs.

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Keywords: Goose calicivirus; RNA virus; EvaGreen real-time RT-PCR; viral

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detection

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Main text:

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Caliciviruses (CVs) are small, nonenveloped viruses belonging to the family

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Caliciviridae, which is currently classified into eleven genera: Lagovirus, Nebovirus,

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Norovirus, Sapovirus,Vesivirus, Bavovirus, Nacovirus, Recovirus, Salovirus, Valovirus

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and Minovirusand (Desselberger, 2019). The viruses are known to infect a wide

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variety of hosts, including human, fish, swine, feline and bird (Cubitt et al., 1985;

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Prasadet al., 1999; Chenet al., 2006; L' Hommeet al., 2009; Day et al., 2010; Wolf et

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al., 2011; Wolf et al., 2012; Mikalsen et al., 2014; Alkan et al., 2015; Mor et al., 2017).

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CVs possess a single-stranded, positive sense, RNA genome that consists of either

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two or three major open reading frames (ORFs) (Lee et al., 2017). In the case of

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goose caliciviruses (GCVs), two ORFs are described wherein ORF1 codes the

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non-structural (NS) proteins and the major structural capsid protein VP1, while ORF2

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codes the minor structural capsid protein VP2 (Wang et al., 2017).

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The emergence of GCV in China in 2014 and 2017 caused concern among veterinary

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practitioners (Liao et al, 2014; Wang et al, 2017). To date, the pathogeny and

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epidemiology information of CVs in waterfowl is limited. Goose exposure to

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calicivirus can be confirmed by using the random polymerase chain reaction (PCR)

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and DNA-sequencing based assay. Although this method is reliable, it is also time

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consuming, cumbersome, and difficult when testing a large number of clinical

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samples. However, avian calicivirus, which seem to be prevalent in growth-stunted

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chickens and turkeys (Day et al, 2013), is a new problem threatening the waterfowl

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production industry. Therefore, a fast and sensitive diagnostic method to detect GCVs

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is thought to be great importance. Real-time reverse transcription-PCR (RT-PCR) is 3

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relatively rapid and precise for RNA identification. It has become a widely used

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method for gene detection and quantitation in RNA viruses like CVs (Oka et al., 2006;

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Park et al., 2009). The real-time fluorescent quantitative PCR can be carried out using

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either probes or DNA dyes. Among the latter, EvaGreen is newly developed and

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found to be the better alternative to the commonly used SYBR Green (Mao et al, 2007;

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Eischeid, 2011). The aim of this study was to develop a rapid, simple, specific and

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sensitive EvaGreen based real-time RT-PCR assay for the detection of GCVs.

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One GCV isolate, MA (GenBank accession number: MN068022), found in a sample

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of kindney tissue from a gosling with gout-malabsorption syndrome in the Fujian

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province of China, was selected for the development of the real-time RT-PCR and

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also used as the positive control in field sample tests in this experimental work. As

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negative controls, different waterfowl-origin virus strains, including goose parvovirus

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(GPV), Muscovy duck parvovirus (MDPV), Novel duck reovirus (NDRV), Muscovy

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duck reovirus (MDRV), duck hepatitis virus (DHV), duck plague virus (DPV), and

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duck tembusu virus (DTMUV), were included. Viral RNA extraction was carried out

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using TRIzol® (Invitrogen, Carlsbad, CA, USA) following the methods recommended

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by the manufacturer. The RNA concentration was measured on a Nanodrop 1000

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spectrophotometer (Technilab, USA) and genomic copies were deduced. Aliquots of

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the RNA template were stored at -80 °C before dilution until use. The GCV-specific

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primers were designed based on waterfowl-origin calicivirus non-structural

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polyprotein (NS) gene sequences from GenBank (GenBank accession numbers

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KJ473715, KY399947, MH453811, MK204416, MN068022, MN175552, and 4

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MN175556) using the Clustal W method of Lasergene 7.0 MegAlign program

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(DNASTAR Inc., Madison, WI, USA). The primer pairs were chosen according to the

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sequences of GCV isolate accession number MN068022. The F1 primer 5’-

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GAGCGCACGCCTTCTA-3’ (covering nucleotides 2051–2066), and R1 primer 5’-

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ACACCTGGGTTCTTCTTCAT-3’ (complementary to nucleotides 2116–2135), were

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used

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TCCCGCGTCATTTTGGCAACTACAAAC-3’ (covering nucleotides 1993–2019),

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and R2 primer 5’- AGGTGTGAACAGTCTGCCTTGA-3’ (complementary to

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nucleotides 2156–2177), were designed for the construction of the calibrator plasmid,

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as a template for in vitro RNA transcriptions. The F1/R1 and F2 /R2 primer sets were

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designed to amplify a PCR product of 85 bp and 185 bp, respectively. BLAST

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sequence analyses were also performed to confirm the specificity of the designed

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for

real-time

amplification

of

GCV.

The

F2

primer

5’-

primers. The presence of the GCV was demonstrated by amplification of a 185 nucleotide (nt)

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NS sequence using a RT-PCR assay with primers F2 and R2. The purified RT-PCR

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product of 185 bp was cloned into the vector pGEM-T vector (Promega, Madison, WI,

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USA) according to the instructions of the manufacturer. The resulting plasmid was

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used to transform competent Escherichia coli DH5α cells and purified using a

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QIAGEN plasmid purification kit (Qiagen, Germany) according to the manufacturer's

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instructions. The DNA sequence of the recombinant clone was verified by sequencing.

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The obtained positive recombinant plasmid with the target gene was designed as

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pGEM-NS185 and subsequently used as the standard plasmid DNA template for in

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vitro RNA transcripts, which was carried out with the Guide-itTM sgRNA In Vitro

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Transcription Kit (Takara, Dalian, China) following the manufacturer’s instructions. 5

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The real-time RT-PCR assays optimized for the detection of GCV were performed

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using a Mastercycler® ep realplex system (Eppendorf, Germany) and One-Step

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EvaGreen qRT-PCR Kit (ABM, Richmond, Canada). The one-step real time RT-PCR

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was performed in 20 µl reaction volume containing 10 µl of EvaGreen qPCR

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MasterMix, 0.4 µl of qRT-PCR Enzyme Mix (50×), 0.5 µl each of 10 µM F1 forward

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and R1 reverse primers, 1 µl RNA template and 7.6 µl of Nuclease-free water. The

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thermal cycle conditions included 15 min at 42 °C, 5 min at 95°C, followed by 40

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cycles of 95 °C for 10 s, 55 °C for 10 s and 72 °C for 10 s. After the RT-PCR cycles,

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a melting curve was generated (with the annealing temperatures 55°C to 95°C at a

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linear transition rate of 0.1°C/s, where the reaction was held at 16°C upon completion)

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to discriminate between the specific amplicons and the non-specific amplification

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products. The Tm value was defined as the peak of the curve. The specific single peak

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of melting curve corresponding to the GCV RNA template was detected at Tm =

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82.7±0.33°C.

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The real-time RT-PCR standard curve was generated under optimized conditions.

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The GCV RNA template was diluted in a 10-fold dilution series ranging from 3 × 105

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to 3 × 101 copies/µl and tested in triplicate within the real-time RT-PCR assay. As

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shown in Fig.1 A and B, the standard curve covered a linear range of five orders of

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magnitude, for which the slopes were -3.547 and R2 = 0.999. It was demonstrated that

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the newly established real-time RT-PCR assay detected the standard RNA to minimal

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3×101 copies from serial diluted samples and was feasible for the quantification of

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GCV in samples. To assess the reproducibility of this new assay, the intra-

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(within-run) and inter- (between runs) assay reproducibility were evaluated using

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10-fold serial dilutions of the GCV RNA template (3 × 105 to 3 × 103 copies/µl),

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tested in triplicate on three different days. The intra- and inter-assay coefficients of 6

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variation for cycle threshold values were in the range of 0.20-0.62% and 1.05-2.21%,

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respectively (Table 1), demonstrating the good reproducibility of the assay. To verify

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the specificity of the primers F1/R1 in the current one-step real-time RT-PCR,

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amplification of RNA and DNA extracted from seven other waterfowl-origin viruses

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(namely, GPV, MDPV, NDRV, MDRV, DHV, DPV, and DTMUV) were tested. In

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addition, the GCV isolate MA and ultrapure water were used as positive and negative

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controls, respectively. No positive results were obtained from the other seven viruses

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and negative control. As expected, only GCV was detected as a single melting peak,

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demonstrating the high specificity of the primers used (Fig. 2).

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In addition, the sensitivity and specificity of the EvaGreen based RT-PCR was

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compared with another conventional RT-PCR. Fresh tissues (such as liver, spleen,

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kidney, intestine, etc.) were collected from diseased goslings (n=76) in 2018 from

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different goose flocks in Fujian province. The geese were 5 to 12 days old and

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suffered mostly from diarrhea and/or severe depression (ruffled feathers and

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reluctance to move) or showed clinical signs of gouty arthritis and/or kidney disease.

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Total RNA was extracted from the GCV stock, liver, spleen, and kidney samples using

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RNAiso Plus reagents (Takara, Dalian, China) according to the manufacturer’s

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instructions. The conventional RT-PCR was performed in a single-step with the

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SuperScript III One-Step RT-PCR System with Platinum® Taq DNA Polymerase kit

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(Invitrogen, Carlsbad, USA). The One-step RT-PCR was conducted in a 50 µl reaction

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volume, containing 25 µl 2× Reaction Mix, 1µl (10 uM) each of the Primer (F2 and

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R2), 5 µl RNA, and 1 µl SuperScript III RT/Platinum Taq Mix and 17 µl diethyl

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pyrocarbonate-treated water. Amplification occurred in a 2720 thermal cycler 7

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(Applied Biosystems®, California, USA) starting with a reverse transcription step of

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45 °C for 30 min, then an initial denaturing step of 94 °C for 2 min, followed by 40

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cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at

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72 °C for 30 s. There was a single final extension step at 72 °C for 7 min. The results

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showed that the conventional one-step RT-PCR was highly specific in detecting the

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GCV RNA as indicated by the amplified product of 185-bp fragment but it was far

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less sensitive in the GCV RNA when compared to that of the one-step real time

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RT-PCR assay. The positive rate of GCV infection was 71.1% (54/76) by the

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conventional RT-PCR, and 100% (76/76) by the real-time RT-PCR assay established

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in the study. All samples positive for GCV in the conventional RT-PCR assay were as

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well as positive in the real-time RT-PCR assay; on the contrary, about one third of the

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samples that were negative for GCV in the conventional RT-PCR assay were detected

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positively in the real-time RT-PCR assay. For those tissue samples that were positive

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both in real-time RT-PCR and conventional RT-PCR assays, the copy number of

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GCV RNA template ranged from 103.71 to104.88 copies/g. The tissue samples that

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presented positive results exclusively in the real-time RT-PCR showed copy numbers

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ranging from 102.55 to 102.92 copies/g. The real-time RT-PCR result herein suggested

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that liver, spleen, kidney, and intestine, should be equally important to focus in the

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detection of GCV. However, GCV loads in different tissues still need further research.

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The partial NS gene sequences of GCV were identified by sequencing of RT-PCR

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amplified products. The detected GCV contigs displayed similarity to calicivirus

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isolates calicivirus goose/N/China/2012 (56.5–56.8% nucleotide identity), calicivirus 8

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Dabbling duck/MW20/Australia/2013 (63.1–63.8% nucleotide identity), calicivirus

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goose/H146/China/2014 (88.1-88.2% nucleotide identity) , calicivirus Mallard

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duck/B6/Canada/2015 (57.1-57.3% nucleotide identity), calicivirus American black

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duck/B76/Canada/2015 (56.1-56.2% nucleotide identity) , and calicivirus Pink-eared

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duck/MW23/Australia/2017 (55.6-55.7% nucleotide identity). This means that

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waterfowl-origin caliciviruses are highly diverse genetically, and more than one

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calicivirus genus may exist in the same host species (Wang et al, 2017; de et al,

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2019).

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To the best of our knowledge, there are no reports available for the real-time

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RT-PCR assay for the detection of GCV RNA. In this study, an EvaGreen-based

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real-time RT-PCR is developed, allowing the detection and quantitation of GCVs.

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The diagnostic tool showed a good specificity and a higher sensitivity than

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conventional RT-PCR. Moreover, the sensitivity of this assay established was able to

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test as few as 3×101 copies from GCV RNA standard samples. Reproducibility of the

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developed method was also validated by the intra- and inter-assay. In conclusion, this

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study reports the development of a rapid, sensitive, specific, and reproducible

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EvaGreen-based real-time RT-PCR assay for detection, diagnosis and quantitation of

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GCVs. The assay may be useful in the investigation of sporadic cases and unexpected

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outbreaks of GCV infections, as well as in epidemiological surveys and etiological

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studies.

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Funding

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This work was supported by the Fujian Public Welfare Project (2019R1026-3).

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Conflict of interest 9

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All authors declare that they have no conflict of interests with any organization.

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Reference

207

[1] Alkan F. Karayel, İ. Catella, C. Bodnar, L. Lanave, G. Bányai, K. Di Martino B.,

208

Decaro N. Buonavoglia, C. Martella V., Identification of a bovine enteric calicivirus,

209

Kırklareli virus, distantly related to neboviruses, in calves with enteritis in Turkey, J.

210

Clin. Microbiol. 53 (2015) 3614-3617.

211

[2] Chen R., Neill J.D., Estes M.K., Prasad B.V., X-ray structure of a native

212

calicivirus: structural insights into antigenic diversity and host specificity, Proc. Natl.

213

Acad. Sci. USA 103 (2006) 8048-8053.

214

[3] Cubitt W.D., Barrett A.D., Propagation and preliminary characterization of a

215

chicken candidate calicivirus, J Gen Virol. 66 (1985) 1431-1438.

216

[4] Day J.M., Ballard L.L., Duke M.V., Schefflerm B.E., Zsak L., Metagenomic

217

analysis of the turkey gut RNA virus community, Virology Journal 7 (2010) 313.

218

[5] Day J.M., Zsak L., Recent progress in the characterization of avian enteric viruses,

219

Avian Dis. 57 (2013) 573-580.

220

[6] de Souza W.M., Fumagalli M.J., de Araujo J., Ometto T., Modha S., Thomazelli

221

L.M., Durigon E.L., Murcia P.R., Figueiredo LTM., Discovery of novel astrovirus and

222

calicivirus identified in ruddy turnstones in Brazil, Sci Rep. 9 (2019) 5556.

223

[7] Desselberger U., Caliciviridae Other Than Noroviruses, Viruses. 11 (2019) E286.

224

[8] Eischeid A.C., SYTO dyes and EvaGreen outperform SYBR Green in real-time

225

PCR, BMC Res Notes 4 (2011) 263-267.

226

[9] L' Homme, Y. Sansregret, R. Plante-Fortier, E. Lamontagne, A.M. Ouardani, M.

10

227

Lacroix G., Simard C., Genomic characterization of swine caliciviruses representing a

228

new genus of Caliciviridae, Virus Genes 39 (2009) 66-75.

229

[10] Lee J.H., Chung M.S., Kim K.H., Structure and Function of Caliciviral RNA

230

Polymerases,Viruses. 9 (2017) E329.

231

[11] Liao Q., Wang X., Wang D., Zhang D., Complete genome sequence of a novel

232

calicivirus from a goose, Arch Virol. 159 (2014) 2529-2531.

233

[12] Mao F., Leung W.Y., Xin X., Characterization of EvaGreen and the implication

234

of its physicochemical properties for qPCR applications, BMC Biotechnol. 7 (2007)

235

76-91.

236

[13] Mikalsen A.B., Nilsen P., Frøystad-Saugen M., Lindmo K., Eliassen T.M., Rode

237

M., Evensen O., Characterization of a novel calicivirus causing systemic infection in

238

atlantic salmon (Salmo salar L.): proposal for a new genus of Caliciviridae, PLoS

239

One. 9 (2014) e107132.

240

[14] Mor S.K., Phelps N.B.D., Ng T.F.F., Subramaniam K., Primus A., Armien A.G.,

241

McCann R., Puzach C., Waltzek T.B., Goyal S.M., Genomic characterization of a

242

novel calicivirus, FHMCV-2012, from baitfish in the USA, Arch. Virol. 162 (2017)

243

3619-3627.

244

[15] Oka T., Katayama K., Hansman G.S., Kageyama T., Ogawa S., Wu F.T., White

245

P.A.,

246

transcription-polymerase chain reaction, J. Med. Virol. 78 (2006) 1347-1353.

247

[16] Park S.I., Park D.H., Saif L.J., Jeong Y.J., Shin D.J., Chun Y.H., Park S.J., Kim

248

H.J., Hosmillo M., Kwon H.J., Kang M.I., Cho K.O., Development of SYBR Green

Takeda

N.,

Detection

of

human

11

sapovirus

by

real-time

reverse

249

real-time RT-PCR for rapid detection, quantitation and diagnosis of unclassified

250

bovine enteric calicivirus, J Virol Methods. 159 (2009) 64-68.

251

[17] Prasad B.V., Hardy M.E., Dokland T., Bella J., Rossmann M.G., Estes M.K.,

252

X-ray crystallographic structure of the Norwalk virus capsid, Science 286 (1999)

253

287-290.

254

[18] Wang F., Wang M., Dong Y., Zhang B., Zhang, D., Genetic characterization of a

255

novel calicivirus from a goose, Arch Virol. 162 (2017) 2115-2118.

256

[19] Wolf S., Reetz J., Hoffmann K., Gründel A., Schwarz B.A., Hänel I., Otto P.H.,

257

Discovery and genetic characterization of novel caliciviruses in German and Dutch

258

poultry, Arch Virol. 157 (2012) 1499-1507.

259

[20] Wolf S., Reetz J., Otto P., Genetic characterization of a novel calicivirus from

260

chicken, Arch. Virol. 156 (2011) 1143-1150.

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Table 1 Variance analysis of Ct values for real-time RT-PCR assay Intra-assay variability Concentration of

cycle threshold

RNA templates

(Ct)

(copies/µl)

coefficient of variation

cycle threshold (Ct)

coefficient of variation

Mean

SD

(%)

Mean

SD

(%)

1×105

21.26

0.12

0.55

20.89

0.46

2.21

1×104

24.88

0.05

0.20

25.17

0.45

1.81

1×103

28.45

0.18

0.62

28.59

0.30

1.05

263 264

Inter-assay variability

Figure legends 12

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FIG. 1. Detection of 10-fold serial dilutions (3 × 105 to 3 × 101 copies/µl) of the

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template GCV RNA using the EvaGreen based real-time RT-PCR. A: GCV real-time

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RT-PCR amplification curve. B: Standard curve where each dot represents the cycle

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threshold value for the template GCV RNA at a given copy number. The x-axis

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indicates the copy number, ranging from 3 × 105 to 3 × 101 copies/µl, used in the

270

experiments. The y-axis represents the corresponding cycle threshold (Ct) values.

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Each data point was amplified in triplicate of each dilution, and the equation below

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the graph is the standard formulate for regression analysis to calculate viral copy

273

number.

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FIG. 2. Specificity of the EvaGreen based real-time RT-PCR in different viruses.

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GCV isolate MA and DNA or RNA from seven other waterfowl-origin viruses and

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RNA-free water were subjected to GCV-specific real time RT-PCR.

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FIG. 1.

279

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280 281 282 283 284

FIG. 2.

285 286 287

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Highlights: 1. An EvaGreen based real-time RT-PCR assay was developed for detection and quantitation of newly emerging goose caliciviruses 2. The assay provides quantitative data that may be used to inform epidemiological and etiological studies