A TaqMan-based real-time PCR assay for specific detection of novel duck reovirus in China

Background In China, Newly emerging duck reovirus (NDRV) variants have been causing major disease problems in cherry valley ducks. NDRV has the potential to cause high morbidity and 5–50% mortality rates. Severe hemorrhagic-necrosis in the liver and spleen were commonly seen in NDRV affected ducks. The availability of upgraded methods for rapid diagnosis of newly emerging DRV variants is crucial for successful DRV infection control and prevention. Results In this study, we present a TaqMan-based real-time PCR assay (RT-qPCR) for the detection of NDRV infection. Using the conserved regions within the NDRV genome, we designed the specific primers and probe. The lower limit of detection for NDRV infection was 10 copies/μL (Ct values: 38.3) after the optimization of the RT-qPCR conditions. By cross-checking with other duck viral pathogens, no cross-reactivity was observed confirming the assay was highly specific for the detection of NDRV. Reproducibility of the RT-qPCR was confirmed by intra- and inter-assay variability was less than 2.91%(Intra-assay variability of Ct values: 0.07–1.48%; Interassay variability of Ct values: 0.49–2.91%). This RT-qPCR and conventional PCR (cPCR) detected one hundred and twenty samples of NDRV infection from different regions. The result shows that the positive rates were 94.17 and 84.17% respectively. The detection rate of RT-qPCR rapid detection assay was 10% higher than that of the cPCR method. Conclusion This research developed a highly sensitive, specific, reproducible and versatile of RT-qPCR for quantitatively detecting NDRV. It can be used to study the pathogenesis and epidemiology investigation of NDRV.

In recent years, a new duck reovirus disease was detected in China. The disease could affect different breeds of ducks and goslings. The main characteristic of the disease is hemorrhagic-necrosis in the liver and spleen [10,12,13,15,16]. The novel duck reovirus is distinct from previous MDRV isolates [9]. Thus, to distinguish it from the "classical" MDRV, the reovirus has been categorized as "novel" duck reovirus (NDRV) [15,17]. Recently, Related research found that a new variant of a duck orthoreovirus that is significantly different from any previously reported waterfowl-derived othoreovirus, causing duck spleen necrosis [18]. The complete sequences of the 10 genome segments of NDRV have been completely determined [11]. NDRV S1 segment is similar to avian reovirus (ARV), but it is distinct from classical MDRV. NDRV S1 contains three sequential overlapping ORFs, encoding p10, p18, and σC, but in MDRV p10 and σC proteins are encoded by the S4 segment and p18 is not present [10][11][12][13]16].
Rapid detection methods are the key for successful NDRV infection control. For many years now, quantitative real time PCR has been a standard diagnostic method, due to its rapid nature, sensitivity, reproducibility, and the reduced risk of false positives from the mispriming of the amplification primers. For viral epidemiological surveillance and pathogenesis studies, this method had been widely used [19][20][21][22]. Currently there are not any reports on a TaqMan-based realtime PCR assay for the specific detection of the novel reovirus infection. Thus, it is critical to develop the TaqMan-based real-time PCR assay of detecting NDRV infection.
In this study, we isolated novel reovirus distinct from previous duck reoviruses identified in China and developed a TaqMan probe-based RT-qPCR method which was developed for precise detection of NDRV infection based on specific primers and probe. The specific primers and probe were designed by targeting the conserved region of the NDRV S2 gene after bioinformatics analysis. The TaqMan-based real-time PCR assay was utilized extensively for virus pathogenesis studies and epidemiological investigations of NDRV.

The selection and design of primers and probe
The probe and primers used in the study were designed based on the S2 gene of NDRV (Table 1). The primers can amplify fragments of 85 bp in length. In Fig. 1, the S2 genome segment alignment of different avian orthoreoviruses were compared by mVISTA method and ClustalW method. The results showed that the primers and probes failed to align with sequences of other poultry reoviruses. Also, primers and probe were verified by the Basic Local Alignment Search Tool (BLAST, https://blast.ncbi.nlm.nih.gov/Blast.cgi) for specificity analysis [18].

Phylogenetic analysis of σC genes
From GenBank (www.ncbi.nlm.nih.gov), thirteen ARVs, four MDRVs, and four DRVs strains were downloaded to compare the difference among SDHZYC and other DRV, ARV and MDRV strains. The SDHZYC (GenBank accession number MK789277) strain was a new clone from a field isolate. By constructing the phylogenetic tree of σC genes (Fig. 2) and homology analysis, we observed that the SDHZYC strains grouped with China with ARV and 51% with MDRV. These suggest that the genetic evolutionary relationship of the SDHZYC strain is more similar to China DRV strains, and NDRV is caused by a mutation in DRV [18].

Standard curve, sensitivity and repeatability
The results are showed in Fig. 3A, the triplicate standard curve plots indicate a linear correlation between the Log of the copy number and the CT. The standard curve was Y = − 3.3468X + 41.681, of which Y = threshold cycle and X = log sta. The linear correlation (R 2 ) of the standard curve was 0.9988. The concentration of plasmid was from 1.0 × 10 8 to 1.0 × 10 2 copies/μL. The range of DNA copy numbers of the standard curve was from 1.0 × 10 7.9 to 1.0 × 10 2.2 copies/μL. To evaluate the sensitivity of the RT-qPCR assay, the DNA standards plasmid was diluted from 1.0 × 10 9 copies/μL to 1.0 × 10 0 copies/μL. After confirmation, the lowest detection limit of the RT-qPCR was 1.0 × 10 1 copies/μL (Ct values: 38.3) (Fig. 3B1). By comparison, the lowest detection standard of conventional PCR only was 1.0 × 10 4 copies/μL (Fig. 3B2).
On 3 different days, 10-fold serial dilutions of standard NDRV plasmid DNA (concentration from 1.0 × 10 8 to 1.0 × 10 1 copies/μL) were used to test the intra-and inter-assay reproducibility. All samples were detected in triplicate [19]. In the detection of the intra-assay, the CVs ranged from 0.07 to 1.48%, and the result of the inter-assay CVs ranged from 0.49 to 2.91% (Table 2). It shows that the repeatability of RT-qPCR is high. Eight different avian viruses were used to test the specificity of RT-qPCR detection. After the detection, NDRV develops a strong response signal. But H9N2 AIV, DTMUV, GPV, N-GPV, DHAV-1, DHAV-3, DuCV, DPV, and Nuclease-free water were not amplified (Fig. 4). The results show that the PCR is specific for NDRV when tested against the listed pathogens.

Experimentally infected ducklings
The major pathological changes of the ducklings include enlarged liver hepatomegaly with bleeding and necrosis, brittle texture, red darken, splenomegaly, patchy hemorrhagic necrosis [23] (Fig. 5). Ducks in groups 2 did not show clinical signs. The most important purpose of using experimentally infected ducklings is to validate the RT-qPCR using clinical samples of RT-qPCR. Therefore, we collected thirtynine samples respectively from different affected organs at 24 h, 48 h and 72 h, including heart, liver, spleen, lung, kidney, pancreas, stomach, brain, intestinal, bursa, thymus, stool and serum. The result was showed in Table 3. After conventional RT-PCR assay and real-time PCR assay, thirty-nine samples of group 2 are negative. Compare to conventional RT-PCR assay, real-time PCR assay detected NDRV earlier, and more frequently. This means that real-time PCR assay had high sensitivity and is appropriate for the detection of NDRV.

Clinical sample detection
One hundred and twenty clinical spleen samples from the cherry valley ducklings with NDRV were identified by the TaqMan based real-time PCR and conventional PCR assays. Of these, 113 samples were RT-qPCR positive, and only 101 samples were cPCR positive. The results are presented in Table 4. Statistical analysis showed a difference between the two methods was significant (P < 0.05) in the detection of clinical samples. The positive rate of NDRV was 84.17% according to the detection of conventional PCR. However, the positive rate of NDRV was 94.17% through the detection of the RT-qPCR assay established in the study. In the same run described above, samples from non-inoculated SPF chicken embryo were tested negative by RT-qPCR and cPCR. The Copy number of clinical samples were shown in Table 5.

Discussion
In China, NDRV has emerged in recent years and is a current common genotype [13,16]. Recently, a group of newly emerging DRVs [24][25][26] was confirmed and characterized in Cherry Valley duck in China [18]. The NDRV from the mainly infected ducklings caused Fig. 2 Phylogenetic relationship between SDHZYC(•) and other avian reovirus strains based on the σC gene in the phylogenetic tree. The tree was constructed using the neighbour-joining algorithm of MEGA5.0, and 1000 bootstrap replicates were performed to assign confidences to the groupings. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Note: The SDHZYC strain was marked with filled circles hemorrhage and necrosis in the liver. There are several notable different properties between classical MDRV and NDRV, including different antigenicity by cross-neutralization tests, host species differences, pathogenic properties, protein profiles [27][28][29], electropherotypes, and genomic coding assignments [10-13, 16, 30]. As fatal pathogenic viruses that can kill ducklings within 72 h, NDRV had caused huge economic losses for the duck industry over the past several decades [31]. Therefore, an easy rapid highly sensitive and specific method for NDRV detection is crucially required to develop [23].
In this study, we designed the probe and primers used in the study based on the S2 gene of NDRV. By using the mVISTA online program, we found that primers (NDRV-F and NDRV-R) and probe (NDRV-P) distinguished duck reovirus from other reoviruses. Then, a TaqMan-based real-time PCR for detecting NDRV infection was established. Verified by a series of experiments, the RT-qPCR has high sensitivity, specificity, and reproducibility. The sensitivity of the RT-qPCR was evaluated using ten-fold diluted DNA standard plasmid, and the lowest amount of detection for NDRV was found was 1.0 × 10 1 copies/μL (Ct values: 38.3). It's thousands of times higher than conventional PCR (1.0 × 10 4 copies/ μL). In subsequent experiments, the RT-qPCR showed high analytical specificity because other duck-derived pathogens were not detected, including Avian influenza virus (H9N2 AIV), Duck Tembusu virus (DTMUV), Goose parvovirus (GPV), Novel goose parvovirus (N-GPV), Duck hepatitis virus type 1 and 3 (DHAV-1 and DHAV-3), Duck circovirus (DuCV), Duck Plague Virus (DPV). The RT-qPCR assay was also found to be highly reproducible. The variability of intra-assay and interassay were ≤ 1.48 and 2.91%, respectively.
The performance of the RT-qPCR assay used as a diagnostic tool to rapidly detect the NDRV is confirmed by the tested results using one hundred and twenty clinical specimens from suspected cases of infected ducks from different regions of China. These clinical samples, spleen samples, were obtained from different duck farms and laboratory diagnostic cases. Comparative analysis of the conventional PCR and RT-qPCR assay using clinical samples showed significant differences. The positive rate

Conclusions
The RT-qPCR could be used as a reliable tool for the rapid detection of NDRV clinical samples, thereby facilitating epidemiological investigations of animals infected with NDRV.

Virus isolation
In this study, we isolated the reovirus from Cherry Valley duck [18] in Shandong province, China. The DRV field strain were isolated from spleen tissues of sick bird which showed symptoms of NDRV infections. The necrotic spleen tissue was extracted from sick birds, homogenized in phosphate-buffered saline (PBS, pH 7.2), freeze-thawed three times, and centrifuged at 8000×g for 15 min [18,32].  [32].

Experimental infection of ducklings
Eighteen 1-day-old cherry valley ducklings have divided ducklings into 2 groups (9 ducklings in each group) randomly. To study the NDRV infection, group 1 was intramuscularly injected with 0.2 mL (10 6.367 ELD 50 ) of the NDRV cell fluid. As the control group, group 2 was treated with sterile DMEM (500 mL, Catalog 01-172-1ACS; BI, Shanghai, China) in the same way. All ducklings were purchased from the commercial hatchery of Yike Company Limited in Xintai County. All experiments with ducks were fed and managed at Shandong Agricultural University according to the established humane procedures and biosecurity guidelines. Water and food were fed ad libitum and were provided living conditions of 40-60% relative humidity and a 12/12 h light/ dark cycle every day. All ducklings were observed and euthanatized using intravenous pentobarbital sodium (New Asia Pharmaceutical, Hainan, China) for 72 h post-infection (hpi) [23].

Sequence analysis
According to reports [31], the sigma C is the major antigenic determinant of avian reovirus. Structural protein Sigma C was the main protein of avian  reovirus. It is in the shell of the virus, carried the surface antigen of virus type-specific neutralization reaction. It is related to the adsorption, proliferation, and syncytial formation of the virus. Therefore, it is of great significance to analyze the genetic evolution of Sigma C protein. The S1 segment encoding sigma C gene of NDRV was amplified by primers σC fulllength (Forward primer) and σC full-length (Reverse primer) ( Table 1). Amplified PCR products were separated on a 1% agarose gel and then purified using the Agarose Gel DNA Purification Kit (200 preps, Catalog D2500-02; OMEGA, Georgia, USA). The PCR products were cloned into the pMD18-T vector (20 preps, Catalog 6011; Takara, Beijing, China) and transformed the positive recombinant plasmid into DH5α competent cells (10 × 100 μL, Catalog BC102-01; Biomed, Beijing, China). Then, the samples were sent to the Beijing Genomics Institute to be sequenced. The sequencing sequences were assembled into a complete 966 bp sequence using the SeqMan program of the DNAstar software package (version 7.1) (DNAstar, Madison, WI, USA). Afterward, the sequence was aligned with other reovirus sequences using the MegAlign program of the DNA star package [34].
Utilizing the neighbor-joining method, a phylogenetic tree was constructed with MEGA 6.0 and performed 1000 bootstrap replicates. In Table 6, all the reference avian reovirus isolates were listed.

RT-qPCR assay for NDRV
Based on the obtained fluorescence and lowest threshold cycle (Ct), the concentrations of the primers, probe, and templates were optimized [35]. The optimized RT-qPCR of NDRV was reacted in a 20 μL system (One Step PrimeScript™ RT-PCR Kit; Takara, Beijing, China Standard plasmid preparation, construct standard curves and sensitivity The forward primer (RT-qPCR-F) and reverse primer (RT-qPCR-R) were used to amplify the partial S2 gene (85 bp) of NDRV. The PCR products were separated by electrophoresis on 1.0% agarose gel. The PCR product was cloned into pMD18-T (the plasmid vector) and then was verified by sequencing [36]. The plasmid of pMD18-NDRV was serially diluted from 1.0 × 10 10 copies/μL to 1.0 × 10 1 copies/μL by 10 × Tris-EDTA Buffer (pH 7.4), and stored at − 20°C. The 10 × Tris-EDTA Buffer (pH 7.4) is prepared by Tris-EDTA Buffer 10 × Powder, pH 7.4 (10 pouches, Catalog T9111; Takara, Beijing, China) dissolved in water. Then, it was used to construct the standard curve [19] and confirm the detection limit of RT-qPCR.

Conventional PCR for NDRV
Meanwhile, conventional PCR (cPCR) was conducted [37] under the same circumstances. The primers used for the cPCR were showed in Table 1. The reaction conditions include 95°C for 5 min, 35 cycles at 95°C for 30 s, 55°C for 30 s and 72°C for 30 s; and 72°C for 5 min at last. The sensitivity of the cPCR was confirmed by agarose gel electrophoresis. Nucleasefree water was used as the negative control in RT-qPCR determination. And all reactions were repeated three times.

Repeatability analysis of the RT-qPCR assay
To evaluate the coefficient of variation (CV) of the RT-qPCR, the 10-fold dilutions of pMD18-S2 (concentration of 1.0 × 10 8 to 1.0 × 10 1 copies/μL) were tested. In checking to see the intra-batch repeatability, triplicates of each dilution were detected, and according to the formula of the geometric mean Cq values / standard deviation calculated the coefficients of variation (CV). The coefficient of variation for inter-assay repeatability shows the differences among the measures at different times [33]. Three repeats were performed for each of the inter and intra assay analysis.

Clinical samples detection
During 2017-2018, we collected 120 samples of spleen from suspected cases of infected ducks. According to the survey, these samples were collected from different regions of Weifang (Shandong), Linyi (Shandong), Dangshan (Anhui), and Xuzhou (Jiangsu). One hundred and twenty clinical spleen samples were detected with the TaqMan based real-time PCR and conventional PCR assays (Primers and probe were presented in Table 1). The tissue samples from SPF duck embryo were used as controls.

Statistical analysis
Statistically significant differences in mean detection rates were determined by one-way ANOVA assessment using GraphPad Prism version 6 (GraphPad Software Inc., San Diego, Calif.) when different types of samples were tested. At *P < 0.05, the difference was considered significant.