Rapid and Visual Detection of the Emerging Novel Duck Reovirus by Using a Specic and Sensitive Reverse Transcription Recombinase Polymerase Amplication Method

spleen necrosis disease (DSND) caused by Novel Duck Reovirus (NDRV), is an emerging infectious disease that causes severely threaten to duck industry. Currently, the for is it to and accurate molecular diagnosis techniques of viral pathogens Recombinase polymerase Amplication (RPA) is a new of rapid and cost-effective been reported for ducklings [14, 15]. But RT-LAMP method requires high temperature (60–65 ℃ ), six primers and more dicult downstream analysis, such as cloning and direct sequencing, limitations that are overcome by the RT-RPA procedure. In our study, we rst developed a new method for rapid detection of NDRV based on an isothermal gene amplication, with high sensitivity and specicity. primer optimization carry in a for 30 using Basic Kit Then the different reaction temperatures (33 ℃ to 43 ℃ ), different reaction times (5 min to 40 min) and different primer concentrations (0.32 µM to 0.48 µM) were evaluated respectively. For RT-RPA amplication, 2.0 µl of the standard plasmid DNA was mix with 2.4 µl of each NDRV-RPA primer (10 µM), 29.5 µl of rehydration buffer, 11.2 µl of ddH 2 O and 2.5 µl of magnesium acetate (280 mM). RPA amplicons were puried using a Universal DNA Purication Kit (TIANGEN, Beijing, China) and aliquots (5 µl) of the puried RPA products analyzed by 2% agarose gel electrophoresis or added SYBR Green I to RT-RPA amplication products for visual detection. PCR; ELISA:enzyme-linked assay; RT-LAMP:reverse amplication; DHAV:Duck Hepatitis Virus; NDV:Newcastle Disease Virus; DTMUV:Duck Tanzuru virus; DPV:Duck parvovirus; IBDV:Infectious Bursal Disease Viruses; IBV:Infectious Bronchitis Virus; aMPV:Avian Metapneumovirus.


Background
Novel duck reovirus (NDRV), an avian Orthoreovirus, is an emerging important viral pathogen causing Duck Spleen Necrosis Disease (DSND) and infecting a variety of duck species recently [1][2][3][4]. The emerging NDRV can cause severe damage to the immune organs, and co-infected with Salmonella spp. can greatly enhance its pathogenesis and further raise the risk of missed and/or mistaken diagnoses [4]. Compared with all previously reported duck reovirus (DRV) infections, NDRV infection exhibits higher virulence and widely host species [5] and the viral S3 gene have been used to distinguish NDRV from the Muscovy duck reovirus (MDRV) or broiler/layer-origin reovirus (ARV) [6,7]. Even though vaccination against ARVs or MDRVs could reduce the risk of the reoviruses infections, an outbreak of the emerging, highly contagious, and fatal disease caused by NDRV still brings severely threaten to duck industry [1,8]. This especially has a profound effect on the food supply in China because about 4.43 billion meat-type ducks were produced in 2019, up an increase of 33.2% over 2018, and because ducks are one of the alternatives for the shorter supply of pork due to the outbreaks of African swine fever over the country [9]. So, it is pivotal to develop a simple, rapid and accurate on-site detection method to assist with the diagnosis and effective control of this disease for the duck industry.
Traditional NDRV diagnostic techniques, such as electron microscopy, virus isolation and immuno uorescence assay, could identify the pathogen in tissues and/or cell culture. Compared with traditional methods, reverse transcription polymerase chain reaction (RT-PCR) and quantitative PCR (qRT-PCR) techniques to detect NDRV with higher sensitivity and speci city [10][11][12]. RT-PCR and qRT-PCR for detecting NDRV in tissue samples requires expensive thermal cycler equipment and is time-consuming. In addition, the enzyme-linked immunosorbent assay (ELISA) have been reported to be effective in detecting NDRV [13], but it relies on high antibodies quality and speci city and easy to produce false positives. The reverse transcription loop-mediated isothermal ampli cation (RT-LAMP) was established to detect NDRV [14,15], which needs six primers, high temperature reaction (60-65 ℃) and long reaction time (50-65 min) makes it di cult as an on-site facilities rapid detection method [16].
Recombinase polymerase ampli cation (RPA) is an emerging next-generation molecular diagnostic technique, which considered as a simple, rapid and cost-effective isothermal ampli cation method [17]. RPA is a considerably simpler technique and does not require a thermal cycler and could be completed at a low temperature (37 ℃ to 42 ℃) in less than 20 min in time [18,19]. RPA has been widely applied in the clinical diagnosis of animal and plant viral including porcine circovirus-2 [20], porcine parvovirus [21], foot-and-mouth disease virus [22,23], avian in uenza [24], potato virus Y [25], maize chlorotic mottle virus [26] and apple stem grooving virus [27]. To our knowledge, the RT-RPA method for the rapid detection of NDRV in ducks has not yet been reported.
This study aims to establish and apply the RT-RPA as a reliable and alternative method for the rapid and speci c detection of NDRV. Initially, the RT-RPA primers were designed and the reaction conditions were optimized for the detection of NDRV. Secondary, the sensitivity and speci city of the RT-RPA were conducted with NDRV and other common waterfowl-origin viruses. Lastly, the performance of this method was conducted with eld and experimental challenged samples for the detection of NDRV. This method could provide a simple, rapid and reliable on-site detection method for NDRV in both eld and experimental sample tests.

RT-RPA primers evaluation
Analysis of the RT-RPA primer sequences using BLASTn indicated 100% query coverage with that of NDRV strains. No matches were detected with other waterfowl-origin viruses and bacteria. Only two reactions with the standard plasmid DNA generated amplicons of the expected size, while no ampli cation was detected by RPA analysis in blank controls (Fig. 1.). So, the RPA3 primer-set could be utilized in the later assays.
Temperature range, incubation time and primer concentration of the RT-RPA Initially, we assessed a series of temperature (33-43 ℃) during incubation for 30 minutes. Ampli cation signals can be detected at 33, 35, 37, 39, 41 and 43 ℃, among which the ampli cation signal was in the highest at 37 ℃ ( Fig. 2A). Then the performance of RT-RPA assay was tested at 37 ℃ with the incubations for 5, 10, 20, 30 and 40 min, respectively. Ampli cation signals could be detected at 37 ℃ with the incubation for 5 min, and the signal intensity increased with time increasing (Fig. 2B). However, there was no signi cant difference in the products yielded among the 20-, 30-and 40-min reactions, so the optimal incubation time was considered to be 20 min in the following experiments. As shown in Fig. 2C, the most suitable primer concentrations for RT-RPA assays were performed with 0.48 µM each from here onwards.
Clear DNA band was visualized by using the puri ed RPA products Although DNA products directly from RPA could be visualized in agarose gels through electrophoresis, better results could be obtained by using the puri ed products before electrophoresis, due mainly to the presence of proteins in the RPA reaction mixtures. It has been reported that the presence of enzymes in the mixture can inhibit DNA migration in the gel [28]. Although better results could be achieved by usage of Universal DNA Puri cation Kit, it increased the cost of RPA-based assays. However, Jiao et al. reported that the phenol/chloroform/isoamyl alcohol-or heat-treated RPA DNA products showed stronger DNA bands in the gels when compared to those puri ed by using the Universal DNA Puri cation Kit [26].

Analytical speci city, sensitivity and repeatability
To test the speci city of the RT-RPA, the reactions were conducted with NDRV and 11 other waterfowl-origin viruses DNAs as the templates and ddH 2 O as the blank control. As clearly shown in Fig. 3A, only NDRV was used as template for the positive reaction, and no other virus were ampli ed. The RT-RPA assay showed high sensitivity, with the detection limit is 3.48 × 10 − 6 ng/µl, which had 10 × higher sensitivity than that found with the conventional RT-PCR (3.48 × 10 − 5 ng/µl) ( Fig. 3B and C). Three positive NDRV samples were randomly selected for repeatability test, indicating that the RT-RPA method was stable and repeatable ( Fig. 3D).

Visualization of ampli cation products of RT-RPA reactions
Aliquots (5 µl) of the ampli cation products were analyzed respectively by 2% agarose gel electrophoresis and directly SYBR Green I staining. After the ampli cation reactions of RT-RPA, the tubes were spun brie y and then the SYBR Green I was added and mixed with the ampli ed product. At 37 ℃, 3 min incubation for RPA reaction containing positive templates turned green under the ultraviolet light, and the signal intensity increased with time increasing. And the negative sample retained its original orange color. Thus, our results indicate that the sensitivity of the SYBR Green I staining detection is the same as that of the agarose gel electrophoresis (Fig. 4.) but with much shorter time.
Performance of the RT-RPA assay on the eld and experimentally infected samples The performance of the NDRV RT-RPA for rapid detection was tested and validated with spleen samples collected from the clinical ducklings with suspected NDRV infection (n = 8) and the experimentally infected ducklings (n = 8) and the healthy uninfected control ducklings (n = 4). As shown in Fig. 5., the NDRV RT-RPA method can 100% accurately identify all the samples. The performance of RT-RPA was consistent with the conventional RT-PCR assays (16 positive and 4 negative cases), and the results were also identical to that of the qRT-PCR. In addition, the samples collected from the eld and experimentally infected ducklings were all positive while the samples from the healthy ducklings were all negative by NDRV RT-RPA assay.

Discussion
An emerging novel duck reovirus (NDRV) disease, called Duck Spleen Necrosis Disease, was recently found in China and the pathogen, distinct from the MDRV isolates previous, was identi ed to be a Orthoreovirus [1,3].
Importantly, Wang et al. recently report that the emerging NDRV XT18 has extensive tissue tropism and could cause severe damage to the immune organs [3,8]. Furthermore, the NDRV co-infection with other pathogens can result in serious duck diseases [4]. At present, with the frequent trade of agricultural products between countries or regions, the possibility of virus transmission is greatly increased. So, it is essential to develop molecular diagnosis techniques for the purpose of correct and rapid detection of viral pathogens in order to prevent further disease transmission or outbreaks.
Multiple diagnostic techniques, including RT-PCR, RT-LAMP and several serological assays, have been used to detect viruses in various samples [11,13,14]. Although test results obtained through RT-PCR are reliable, this technique is too costly and time-consuming for companies or persons who have limited laboratory equipment, including a thermal cycler, and can only conduct mostly eld surveys [26]. RPA is a rapid and simple isothermal gene ampli cation method and does not require thermal cycler and high temperatures that can overcome the shortcomings of conventional PCR-based methods. At 37 ℃, the reaction can be completed within 20 minutes with only a pair of primers and a simple device such as water bath and heating block. Rapid and speci c detection of NDRV using the RT-LAMP procedure has also been reported for ducklings [14,15]. But RT-LAMP method requires high temperature (60-65 ℃), six primers and more di cult downstream analysis, such as cloning and direct sequencing, limitations that are overcome by the RT-RPA procedure. In our study, we rst developed a new method for rapid detection of NDRV based on an isothermal gene ampli cation, with high sensitivity and speci city.
Currently, all kinds of modi ed RPA assay have been applied to the diseases diagnosis in the eld of human and veterinary medicine, as well as the detection of pathogenic bacteria in the food safety and agriculture respectively [29]. The RT-RPA assays has some advantages over the ELISA, conventional RT-PCR and RT-LAMP currently used for detection NDRV due to its short reaction time and only require a single constant temperature (33 to 43 ℃). The RT-RPA assays was enough to produce su cient amount of NDRV amplicon at 37 ℃ incubated for 3 min for rapid detection of the virus. On the contrary, conventional RT-PCR requires more than 95 min to complete before gel electrophoresis [11] and 60-65 min was required for RT-LAMP [14]; and two days were require for ELISA detection [13]. In addition, the high speci city and ampli cation of the RT-RPA method also allows for the easy and rapid visualization of the ampli ed products without the need for gel electrophoresis, thus making it a very simple and rapid diagnostic tool.
Compared with the conventional RT-PCR, RT-RPA assays has higher sensitivity and speci city in detecting NDRV. This method has high speci city and no cross-reactivity was detected with other waterfowl-origin viruses. Furthermore, the detection limit of RT-RPA was 3.48 × 10 − 6 ng/µl with the standard plasmid DNA, which is a 10-fold higher sensitivity rate than that of the conventional RT-PCR previously reported. RT-LAMP and RT-PCR are both reliable methods for detecting NDRV with high sensitivity and speci city. However, aerosol pollution and false positives are prone to occur in RT-LAMP reactions [30]. The RPA method developed in our study did not produce this pollution and/or false positives, possibly since its reagents used are provided in a lyophilized pellet [31]. Finally, we evaluated the newly established RT-RPA method in this study by using eld and experimental infected samples, and found that the test results were highly consistent those with the conventional RT-PCR and qRT-PCR assays.

Conclusions
To our knowledge, this is the rst report of the newly RT-RPA method for use-friendly and reliable detection and diagnosis of NDRV infection. It is believed that the RT-RPA assay, as a next-generation gene ampli cation technology, is an ideal for rapid and e cient detection of NDRV especially suitable for laboratories or eld Co., Ltd., China). Another 12 samples collected from the NDRV experimentally infected ducklings (n = 8) and non-infected ducklings (n = 4) were also used in the study (the local commercial duck hatchery). All the ducks used in the study were euthanized using the carbon dioxide method. The extraction of total RNA/DNA as noted in our previous description [4].

Primer's design and evaluation
The primers were designed according to the manufacturer's protocol suggested in the TwistAmp™ Basic Kit. Based on the alignment of 6 different NDRV virus sequences representing the known NDRV strains, a total of 3 primer pairs were designed to target the S3 gene. In addition, the primers NDRV-S3F/R were used to amplify the S3 gene of NDRV to construct the plasmid as the standard positive DNA sample. The speci city of the primer was assessed using the Primer-Blast program with the NCBI. The RPA primers and PCR primers were synthesized by BGI Technology Co., Ltd., Beijing, China. The sequences of all primers used in this study are shown in Table 1.

Recombinant plasmid construction
A recombinant plasmid used in the study was constructed by using the S3 gene of a Guangxi NDRV reference strain DRV/GX-Y7/2018/China (GenBank ID: MN747004-MN747013). The target gene was ampli ed, puri ed and cloned as the descriptions by our group have described previously [4]. Plasmid DNA was puri ed using the Endo-free Plasmid Midi Kit (Cwbio, Beijing, China) and quanti ed by BioDrop spectrophotometry (BioDrop, Cambridge, England). The positive clone was veri ed by sequencing (BGI, Guangzhou, China) and the sequence obtained was analyzed using the BLAST nucleotide option in the GenBank database. and qRT-PCR according to the developed detection methods, respectively [11,12]. Three repeated tests were carried out to evaluate the reliability of the newly established RT-RPA method.

Testing of the eld and experimental challenged samples
To determine the practical application of RT-RPA on-site eld diagnostics, a total of 8 natural eld samples suspected having NDRV infection were collected from Cherry Valley ducklings in Guangxi, China. Also, another 12 samples collected respectively from NDRV experimentally infected 8 ducklings and non-infected 4 ducklings were used. For the sake of comparison, the same samples were also subjected to NDRV detection by conventional RT-PCR and qRT-PCR assays respectively, as noted in previously described [11,12].

Availability of data and materials
The datasets during in our study are available from the corresponding author upon request.

Competing interests
All the authors in this study declare that there is no potential competing interest.

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