A primer set, kit, detection method and application for detecting a novel duck reovirus M3 gene

Abstract

The application discloses a primer group, a kit, a detection method and application for detecting a novel duck reovirus M3 gene, and relates to the technical field of animal epidemic disease molecular detection. The primer group comprises an upstream primer NDRV-M3-6-F, the nucleotide sequence of which is shown as SEQ ID NO:1; and a downstream primer NDRV-M3-6-R, the nucleotide sequence of which is shown as SEQ ID NO:2, and the primer group can be used for amplifying a fragment of a conservative region of a mu NS protein coding gene in the NDRV M3 gene. Sequence alignment results show that the upstream primer and the downstream primer have good conservation in the NDRV in a primer binding region, and there is a difference in the corresponding region of the MDRV. The primer group and the kit provided by the application can be used for detecting NDRV nucleic acid in an ex vivo sample and related epidemiological monitoring, and provide a new technical selection for NDRV nucleic acid detection.

Description

Technical Field

[0001] This invention relates to the field of molecular detection technology for animal diseases, specifically to a primer set, reagent kit, detection method, and application for detecting the novel duck reovirus M3 gene. Background Technology

[0002] Novel duck reovirus (NDRV) is an important viral pathogen affecting waterfowl such as ducks and geese. Infected animals can exhibit liver and spleen damage, hemorrhage and necrosis, and growth retardation, causing economic losses to the waterfowl farming industry. Therefore, establishing rapid and stable nucleic acid detection tools is of great significance for pathogen monitoring and epidemiological investigation.

[0003] Real-time quantitative PCR (RT-PCR) offers advantages such as rapid detection, high sensitivity, and ease of quantitative analysis, making it suitable for RNA virus nucleic acid detection. The performance of the detection system is closely related to the selection of target genes and primer design. If the primer binding region mutates in circulating strains, it may affect amplification efficiency or even lead to false negatives. Therefore, it is necessary to develop new nucleic acid detection primer combinations and kits targeting relatively conserved gene regions in NDRV. Among existing publicly available technologies, Shandong Provincial Standard DB37 / T 3821-2019 discloses an NDRV fluorescent PCR detection system based on the σC target, which can be used for NDRV nucleic acid detection. However, the actual detection performance of different target regions and primer combinations in samples from different sources may vary.

[0004] Therefore, developing novel primer combinations and detection kits targeting other conserved regions of NDRV can provide new technological options for NDRV nucleic acid detection. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a primer set, reagent kit, detection method, and application for detecting the novel duck reovirus M3 gene, thereby enabling the detection of NDRV nucleic acid in in vitro samples.

[0006] Therefore, in a first aspect, the present invention provides a primer set for detecting a novel duck reovirus, comprising: The upstream primer NDRV-M3-6-F has the nucleotide sequence shown in SEQ ID NO:1; The downstream primer NDRV-M3-6-R has the nucleotide sequence shown in SEQ ID NO:2.

[0007] Furthermore, the primer set was used to amplify the conserved region of the μNS protein encoded by the novel duck reovirus M3 gene.

[0008] A second aspect of the present invention provides a kit for detecting a novel duck reovirus, comprising the aforementioned primer set.

[0009] Furthermore, the kit also includes reverse transcription reagent, real-time quantitative PCR reaction solution containing SYBR Green I fluorescent dye, positive control, negative control, and instructions for use.

[0010] Further, the positive control is the recombinant plasmid pMD19-T-μNS; the recombinant plasmid pMD19-T-μNS contains a conserved region fragment of the novel duck reovirus M3 gene encoding the μNS protein, and the nucleotide sequence of the conserved region fragment is shown in SEQ ID NO:3.

[0011] Furthermore, the real-time quantitative PCR reaction system includes: 2×SYBR qPCR Mix 9μL~11μL, upstream primer NDRV-M3-6-F at a concentration of 9μmol / L~11μmol / L 0.36μL~0.44μL, downstream primer NDRV-M3-6-R at a concentration of 9μmol / L~11μmol / L 0.36μL~0.44μL, cDNA template obtained by reverse transcription of total RNA from the sample to be tested or recombinant plasmid pMD19-T-μNS template 0.8μL~1.2μL, ddH2O to make up to 18μL~22μL; The final concentration of the upstream primer NDRV-M3-6-F is 0.18 μmol / L to 0.22 μmol / L, and the final concentration of the downstream primer NDRV-M3-6-R is 0.18 μmol / L to 0.22 μmol / L.

[0012] Furthermore, The real-time quantitative PCR amplification program includes: Pre-denaturation at 94℃~96℃ for 28s~32s, one cycle; Denaturation at 94℃~96℃ for 8s~12s, annealing / extending at 55℃~57℃ for 28~32s, 40 cycles; The real-time quantitative PCR melting procedure includes: Hold at 94℃~96℃ for 14s~16s, then at 59℃~61℃ for 58s~62s, then raise to 94℃~96℃ and hold for 14s~16s, completing one cycle.

[0013] Furthermore, when using the recombinant plasmid pMD19-T-μNS as a template, the kit at 1.0 × 10⁻⁶... 1 ~1.0×10 7 It exhibits good linearity within the detection concentration range of copies / μL, with a minimum detectable concentration of 1.0 × 10⁻⁶. 1 copies / μL.

[0014] A third aspect of the present invention provides a novel real-time quantitative RT-PCR detection method for duck reovirus that is not intended for disease diagnosis or treatment, using the aforementioned kit, comprising: Total RNA was extracted from the sample to be tested and cDNA was obtained by reverse transcription. The cDNA was used as a template for real-time quantitative PCR amplification using SYBR Green I dye. The presence of a novel duck reovirus in the sample is determined based on the amplification and melting curves.

[0015] A fourth aspect of the invention provides the use of the primer set or the kit in the preparation of a detection reagent for in vitro detection of novel duck reovirus nucleic acid.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects: The primer set provided by this invention for detecting the novel duck reovirus M3 gene includes an upstream primer NDRV-M3-6-F, the nucleotide sequence of which is shown in SEQ ID NO:1; and a downstream primer NDRV-M3-6-R, the nucleotide sequence of which is shown in SEQ ID NO:2. These primers can be used to amplify the conserved region encoding the μNS protein in the NDRV M3 gene. Sequence alignment results show that the binding regions of both the upstream and downstream primers are well conserved in NDRV, but differ from the corresponding regions in Muscovy duck reovirus (MDRV). The primer set and kit provided by this invention can be used for the detection of NDRV nucleic acid in in vitro samples and related epidemiological monitoring, providing a new technical option for NDRV nucleic acid detection. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in the embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0018] Figure 1 This is an image showing the alignment results of the NDRV M3 target region primers in the corresponding sequences of NDRV and MDRV provided in this embodiment of the invention. Figure 1 In the middle, A represents the result of primer alignment in the MDRV strain. Figure 1 B represents the comparison result of primers in NDRV strains; Figure 2 This is a diagram showing the construction and sequencing identification results of the recombinant plasmid standard pMD19-T-μNS provided in this embodiment of the invention. Figure 2In the image, A represents the PCR amplification result of the M3 target fragment, M stands for DNA Marker, 1-4 represent the amplification products using NDRV-SD-1 cDNA as a template, and 5 represents the negative control. Figure 2 B represents the sequencing alignment result of the pMD19-T-μNS insert; Figure 3 The melting curve, amplification curve, and standard curve results provided in this embodiment of the invention are as follows: Figure 3 In the diagram, A represents the melting curve. Figure 3 B in the figure represents the amplification curve. Figure 3 In the middle, C represents the standard curve; Figure 4 This image shows the specific amplification results of the M3 target detection system provided in this embodiment of the invention for NDRV positive standards, nucleic acids of other avian disease-related pathogens, and negative materials. In this image, 1 represents the NDRV positive standard; 2-16 represent, in order, avian influenza virus positive nucleic acid, avian leukosis virus positive nucleic acid, Marek's disease virus positive nucleic acid, goose plague virus positive nucleic acid, duck plague bacillus positive nucleic acid, duck plague virus positive nucleic acid, negative chicken embryo allantoic fluid, negative chicken embryo tissue homogenate, negative goose embryo allantoic fluid, negative goose embryo tissue homogenate, negative duck embryo allantoic fluid, negative duck embryo tissue homogenate, negative CEF cells, avian reovirus S1133 strain nucleic acid, and Muscovy duck reovirus nucleic acid; NC represents the ddH2O negative control. Detailed Implementation

[0019] To better understand the above technical solutions, the technical solutions of the embodiments of this application will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the embodiments of this application and the specific features in the embodiments are detailed descriptions of the technical solutions of the embodiments of this application, rather than limitations on the technical solutions of this application. In the absence of conflict, the embodiments of this application and the technical features in the embodiments can be combined with each other.

[0020] A first aspect of this invention provides a primer set for detecting a novel duck reovirus, comprising: The upstream primer NDRV-M3-6-F has the nucleotide sequence shown in SEQ ID NO:1; The downstream primer NDRV-M3-6-R has the nucleotide sequence shown in SEQ ID NO:2.

[0021] In some embodiments, the primer set is used to amplify the conserved region of the novel duck reovirus M3 gene encoding the μNS protein, and the amplification product is 163 bp in length.

[0022] Specifically, the primer set targets the conserved region of the NDRV M3 gene. Sequence alignment results show that the binding regions of both upstream and downstream primers are well conserved in NDRV, but differ from the corresponding regions in MDRV.

[0023] Analysis was performed using recombinant plasmid standards, and the established amplification system was at 1.0 × 10⁻⁶. 1 ~1.0×10 7 It exhibits good linearity within the range of copies / μL, with a regression equation of y = -3.4791x + 36.184 and R0. 2 =0.9985; based on the slope, the amplification efficiency is approximately 93.8%.

[0024] When evaluated using recombinant plasmid standards, the lowest detectable concentration is 1.0 × 10⁻⁶. 1 copies / μL; at 1.0×10 3 ~1.0×10 7 Within the range of copies / μL, the coefficients of variation for both intra-batch and inter-batch repeatability tests were less than 1%.

[0025] Specific detection results showed that NDRV positive standards showed specific amplification signals, while avian influenza virus, avian leukosis virus, Marek's disease virus, goose plague virus, duck plague bacillus, duck plague virus, avian reovirus S1133 strain, Muscovy duck reovirus, as well as negative embryonic material and negative CEF cells did not show specific amplification signals.

[0026] In the comparative detection of NDRV-infected CEF cell samples, the M3 target detection system of this invention obtained positive amplification results in samples at an early time point after infection. In the detection of NDRV samples from three different sources, the M3 target detection system of this invention obtained positive amplification results in all samples. In particular, for the NDRV-YZ sample, for which the σC open control detection system did not obtain a detectable positive amplification result, the M3 target detection system of this invention still obtained a positive amplification result, indicating that it has good detection applicability in the tested samples.

[0027] A second aspect of this invention provides a kit for detecting a novel duck reovirus, comprising a primer set.

[0028] In some embodiments, the kit may also include reverse transcription reagent, real-time quantitative PCR reaction solution containing SYBR Green I fluorescent dye, positive control, negative control, and instructions for use.

[0029] In some embodiments, the positive control is the recombinant plasmid pMD19-T-μNS; the recombinant plasmid pMD19-T-μNS contains a conserved region fragment of the μNS protein encoded by the novel duck reovirus M3 gene, and the nucleotide sequence of the conserved region fragment is shown in SEQ ID NO:3.

[0030] In some embodiments, the real-time quantitative PCR reaction system includes: 2×SYBR qPCR Mix 9μL~11μL, upstream primer NDRV-M3-6-F (concentration 9μmol / L~11μmol / L) 0.36μL~0.44μL, downstream primer NDRV-M3-6-R (concentration 9μmol / L~11μmol / L) 0.36μL~0.44μL, cDNA template obtained by reverse transcription of total RNA from the sample to be tested or recombinant plasmid pMD19-T-μNS template 0.8μL~1.2μL, ddH2O to make up to 18μL~22μL; The final concentration of the upstream primer NDRV-M3-6-F was 0.18 μmol / L to 0.22 μmol / L, and the final concentration of the downstream primer NDRV-M3-6-R was 0.18 μmol / L to 0.22 μmol / L.

[0031] Preferably, the real-time quantitative PCR reaction system includes: 10 μL of 2×SYBR qPCR Mix, 0.4 μL of 10 μmol / L upstream primer NDRV-M3-6-F, 0.4 μL of 10 μmol / L downstream primer NDRV-M3-6-R, 1.0 μL of cDNA template obtained by reverse transcription of total RNA from the sample to be tested or recombinant plasmid pMD19-T-μNS template, and ddH2O to a final volume of 20 μL; The final concentration of the upstream primer NDRV-M3-6-F was 0.2 μmol / L, and the final concentration of the downstream primer NDRV-M3-6-R was 0.2 μmol / L.

[0032] In some embodiments, The real-time quantitative PCR amplification program includes: Pre-denaturation at 94℃~96℃ for 28s~32s, one cycle; Denaturation at 94℃~96℃ for 8s~12s, annealing / extending at 55℃~57℃ for 28~32s, 40 cycles; The real-time quantitative PCR melting curve program includes: Hold at 94℃~96℃ for 14s~16s, then at 59℃~61℃ for 58s~62s, then raise to 94℃~96℃ and hold for 14s~16s, completing one cycle.

[0033] Preferably, the real-time quantitative PCR amplification program includes: Pre-denaturation at 95℃ for 30 seconds, one cycle; 95℃ denaturation for 10s, 56℃ annealing / extending for 30s, 40 cycles; The real-time quantitative PCR melting procedure includes: Hold at 95℃ for 15 seconds, then at 60℃ for 60 seconds, then at 95℃ for 15 seconds, for one cycle.

[0034] In some embodiments, when the recombinant plasmid pMD19-T-μNS is used as a template, the kit is 1.0 × 10 1 ~1.0×10 7 It exhibits good linearity within the detection concentration range of copies / μL, with a minimum detectable concentration of 1.0 × 10⁻⁶. 1 copies / μL.

[0035] A third aspect of this invention provides a novel real-time quantitative RT-PCR detection method for duck reovirus that is not intended for disease diagnosis or treatment, using a kit for detection, comprising: Total RNA was extracted from the sample to be tested and cDNA was obtained by reverse transcription. Real-time quantitative PCR amplification using SYBR Green I dye as a template was performed using cDNA as a template. The presence of a novel duck reovirus in the sample is determined based on the amplification and melting curves.

[0036] A fourth aspect of the present invention provides the use of primer sets or kits in the preparation of detection reagents for in vitro detection of novel duck reovirus nucleic acid.

[0037] Example 1: M3 target selection and specific primer design Based on the alignment analysis of the NDRV M3 gene sequence, regions with good conservation were selected as amplification target areas. The M3 gene encodes the non-structural protein μNS. Real-time quantitative RT-PCR primer pairs NDRV-M3-6-F / NDRV-M3-6-R were designed around the conserved region, and their sequences are shown in Table 1.

[0038] Table 1 Primer pairs for real-time quantitative RT-PCR of the conserved region of the M3 gene.

[0039] Sequence alignment results showed that the binding sites of both upstream and downstream primers were relatively conserved in NDRV. Figure 1 (B); In the corresponding MDRV sequence, there are significant differences in the downstream primer binding sites ( Figure 1(A). Therefore, the primer pair described is suitable for the detection of NDRV target nucleic acids, but not for use as universal detection primers for MDRV.

[0040] Example 2 Construction and Identification of Recombinant Plasmid Standards Total RNA from NDRV was extracted using AG RNAex Pro RNA extraction reagent, and the obtained RNA was reverse transcribed into cDNA. Using the cDNA as a template, the M3 gene fragment containing the detection target region was amplified using NDRV-NS-F / NDRV-NS-R primers. The primer sequences are shown in Table 2. The PCR product was recovered, purified, and ligated into the pMD19-T vector to construct the recombinant plasmid pMD19-T-μNS(…). Figure 2 The PCR-positive recombinant plasmid was sent to Genewiz Biotechnology Co., Ltd. for sequencing verification. The M3 insert confirmed by sequencing was 237 bp in length, and its nucleotide sequence is shown in SEQ ID NO:3.

[0041] Table 2 Primer pair sequences for amplification of the standard plasmid M3 insert fragment

[0042] The concentration of recombinant plasmids confirmed by sequencing was determined using a NanoDrop ND-2000C micro spectrophotometer, and the copy number of standard plasmids was calculated using the following formula: Copy number (copies / μL) = [plasmid concentration (ng / μL) × 6.02 × 10] 23 ] / [660×total plasmid length (bp)×10 9 The total length of the plasmid is the sum of the length of the pMD19-T vector sequence and the length of the M3 insert fragment.

[0043] The obtained recombinant plasmid standards were used for subsequent standard curve establishment, amplification sensitivity and repeatability evaluation.

[0044] Example 3: Two-step dye real-time fluorescence quantitative RT-PCR detection system When the sample to be tested was RNA, reverse transcription was performed using the Novizan HiScript III RT SuperMix for qPCR (+gDNA wiper) kit. First, genomic DNA removal was performed: template RNA (1 pg~1 μg total RNA), 4 μL of 4×gDNA wiper Mix, and RNase-free ddH2O were added to an RNase-free centrifuge tube to a final volume of 16 μL. After gentle mixing, the mixture was incubated at 42℃ for 2 min. Then, 4 μL of 5×HiScript III qRTSuperMix was added to the above reaction solution to a total volume of 20 μL. After gentle mixing, reverse transcription was performed at 37℃ for 15 min and 85℃ for 5 s. The resulting cDNA was used directly as a template for SYBR Green I dye-based real-time quantitative PCR amplification. The real-time quantitative PCR reaction system is shown in Table 3, and the amplification and melting curve programs are shown in Table 4.

[0045] Table 3 Real-time quantitative PCR reaction system

[0046] Table 4 Real-time quantitative PCR amplification and melting procedures

[0047] The criteria for determining the results are as follows: when the sample shows a typical S-shaped amplification curve and the melting curve shows a single specific melting peak consistent with the positive control, it is determined to be positive for NDRV nucleic acid; when there is no typical amplification curve or no specific melting peak consistent with the positive control, it is determined to be negative.

[0048] Example 4: Optimization of primer concentration and annealing temperature Recombinant plasmid standards were used as templates, and the final primer concentration and annealing temperature were combined for screening. Results showed that under the conditions of a final primer concentration of 0.2 μmol / L and an annealing temperature of 56℃, the average Ct value of the positive template was 23.38 ± 0.15, no amplification signal was detected in the negative control, and the melting curve showed a single specific peak. Figure 3 (A). Therefore, 56℃ and 0.2μmol / L were determined to be the preferred amplification conditions for this detection system.

[0049] Example 5: Establishment of Standard Curve and Evaluation of Amplification Sensitivity The recombinant plasmid standard was serially diluted 10-fold, and a 1.0 × 10⁻⁶ sample was selected. 1 ~1.0×10 7Using copies / μL as template, three replicates were set up for each dilution, with ddH2O as a negative control, and real-time quantitative PCR amplification was performed. A standard curve was plotted with the logarithm of the standard copy number on the x-axis and the Ct value on the y-axis.

[0050] The results are as follows Figure 3 China B and Figure 3 As shown in Figure C, the standard curve linear regression equation is y = -3.4791x + 36.184, and the coefficient of determination R0 is... 2 =0.9985; based on the slope of the standard curve, the amplification efficiency is approximately 93.8%. This indicates that the system exhibits good linear amplification within the detection range.

[0051] Table 5. Evaluation of amplification sensitivity of plasmid standards at different concentrations

[0052] As shown in Table 5, the lowest detectable concentration in the above plasmid standard detection experiments was 1.0 × 10⁻⁶. 1 The result represents the analytical sensitivity of the amplification system of this invention to plasmid standards; the detection limits for intact RNA extraction and reverse transcription should be further validated using RNA standards or quantified viral RNA samples.

[0053] Example 6 Repeatability Evaluation In intra-batch repeatability tests, 1.0 × 10⁻⁶ was selected. 3 ~1.0×10 7 Plasmid standards of copies / μL were prepared, and each concentration was amplified in triplicate. In batch-to-batch repeatability tests, the above concentrations were amplified independently three times at 3-day intervals, with three replicates for each concentration.

[0054] Table 6. Repeatability evaluation of plasmid standards at different concentrations

[0055] The results are shown in Table 6. At 1.0 × 10 3 ~1.0×10 7 Within the range of copies / μL, the coefficients of variation for both intra-batch and inter-batch repeatability tests were less than 1%, indicating that the amplification system has good repeatability for plasmid standards.

[0056] Example 7 Specificity Validation Scheme The specificity was validated using the optimized quantitative real-time PCR method, with a concentration of 1.0 × 10⁻⁶. 3A pMD19-T-μNS recombinant plasmid standard of copies / μL was used as a positive control, and ddH2O was used as a negative control. The templates to be tested included positive nucleic acids of avian influenza virus, avian leukosis virus, Marek's disease virus, goose plague virus, duck plague bacillus, duck plague virus, avian reovirus S1133 strain, Muscovy duck reovirus, as well as negative chicken embryo allantoic fluid, negative chicken embryo tissue homogenate, negative goose embryo allantoic fluid, negative goose embryo tissue homogenate, negative duck embryo allantoic fluid, negative duck embryo tissue homogenate, and negative CEF cell samples.

[0057] Test results as follows Figure 4 As shown, the NDRV positive standard exhibited a specific amplification curve; however, no specific amplification signals were observed for avian influenza virus, avian leukosis virus, Marek's disease virus, goose plague virus, duck plague bacillus, duck plague virus, avian reovirus S1133 strain, Muscovy duck reovirus, as well as negative chicken embryos, negative goose embryos, negative duck embryos, negative CEF cells, and ddH2O. This indicates that the M3 target detection system established in this invention has good specificity.

[0058] Example 8: Comparison of detection performance in early time point samples of NDRV-infected CEF cells To evaluate the detection performance of the M3 target detection system of this invention in early NDRV-infected cell samples, the σC target fluorescent PCR system disclosed in Shandong Provincial Standard DB37 / T 3821—2019 was used as a control system.

[0059] 8.1 σC Public Control Detection System The σC open control system uses upstream primer P3 and downstream primer P4, the sequences of which are shown in Table 7. The reaction system is shown in Table 8. The amplification reaction program is as follows: pre-denaturation at 95℃ for 45s; followed by 95℃ for 10s, 56℃ for 10s, and 72℃ for 15s, for a total of 40 cycles. Fluorescence signals are collected during the 72℃ extension phase of each cycle.

[0060] Table 7. Primer pair sequences for the σC open control system in real-time quantitative PCR.

[0061] Table 8. σC Public Control System Real-Time Quantitative PCR Reaction System

[0062] 8.2 NDRV infection of CEF cells and sample collection at time points Chicken embryo fibroblasts (CEF) were used as the infected cell model. NDRV-SD-1 was seeded into 12-well plates containing a confluent monolayer of CEF cells at an MOI of 0.01. A mock uninfected control group was also set up. The mock group was operated on separate culture plates to reduce the risk of cross-contamination.

[0063] After virus inoculation, the culture plate was placed in a 37°C, 5% CO2 incubation environment for 60 min for adsorption. During adsorption, the culture plate was gently shaken once every 15 min to ensure that the virus solution evenly covered the cell surface.

[0064] After adsorption is complete, the virus solution in each well is gently aspirated. Then, pre-cooled PBS buffer is added to each well, and the cells are gently washed twice. After each wash, the PBS is slowly aspirated to reduce the impact of unadsorbed or loosely bound free viruses on the cell surface on the test results.

[0065] Immediately after rinsing, cell samples from the 0h group were collected. Specifically, 500 μL of pre-chilled PBS buffer was added to each well, and cells were gently scraped using a sterile cell scraper. The cell suspension was transferred to a sterile centrifuge tube and stored at -80°C. The 0h samples represent the baseline level of cell-associated viral nucleic acids after virus adsorption and rinsing.

[0066] Except for the 0h group, all other groups were incubated with fresh maintenance medium after rinsing and cultured at 37℃ and 5% CO2. Cell samples were collected at 20 min, 40 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, and 36 h. At the corresponding time points, the maintenance medium was discarded, the cells were gently rinsed once with PBS, and then 500 μL of pre-cooled PBS buffer was added to each well. The cell suspension was collected using a sterile cell scraper and immediately stored at -80℃.

[0067] After all time points of sample collection were completed, total RNA was extracted and reverse transcribed using the HiScript III RT SuperMix for qPCR (+gDNA wiper) kit. The resulting cDNA was used as amplification template for the M3 target detection system and the σC open control detection system of this invention, respectively.

[0068] 8.3 Test Results The test results of the samples at each time point are shown in Table 9.

[0069] Table 9. Detection results of samples at various time points between the M3 target detection system and the publicly available σC control system.

[0070] The results showed that, under the NDRV-infected CEF cell samples and detection conditions, the M3 target detection system of this invention obtained identifiable positive amplification results in samples from 0h to 2h time points; the σC published control detection system obtained positive amplification results in samples from 0h time points, but did not obtain identifiable positive amplification results in samples from 20min to 2h time points; from 4h onwards, both detection systems could detect NDRV nucleic acid signals.

[0071] The above results indicate that the M3 target detection system of the present invention can be used for nucleic acid detection in NDRV infection-related CEF cell samples, and shows good detection applicability in the early post-infection time point samples detected in this embodiment.

[0072] This embodiment compares the actual detection performance of the M3 target detection system of this invention with the publicly available σC control detection system in the same batch of infected cell samples. Since the primers, reaction systems, and amplification procedures used in the two detection systems are different, and this embodiment did not perform absolute quantitative analysis of the viral nucleic acid content in the samples at each time point, the results are not intended to demonstrate that the detection system of this invention has a definite fold increase in analytical sensitivity compared to the publicly available σC control system.

[0073] Example 9: Detection performance of NDRV samples from different sources The M3 target detection system and σC control detection system of this invention were used to detect NDRV samples from different sources. The existing detection results are shown in Table 10.

[0074] Table 10. Detection results of the M3 target detection system and the publicly available σC control system for NDRV samples from different sources.

[0075] Since this embodiment did not perform sequencing analysis on the σC target region of the NDRV-YZ sample, the results are only used to illustrate the actual detection performance of the M3 target detection system of the present invention and the publicly disclosed σC control detection system in the tested samples, and do not involve the determination of the reason why the publicly disclosed σC control detection system was not detected in the NDRV-YZ sample.

[0076] It will be readily understood by those skilled in the art that the above-described advantageous methods can be freely combined and superimposed without conflict. The above are merely preferred embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application. The above are merely preferred embodiments of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of this application, and these improvements and modifications should also be considered within the protection scope of this application.

Claims

1. A primer set for detecting a novel duck reovirus, characterized in that, include: The upstream primer NDRV-M3-6-F has the nucleotide sequence shown in SEQ ID NO:1; The downstream primer NDRV-M3-6-R has the nucleotide sequence shown in SEQ ID NO:

2.

2. The primer set according to claim 1, characterized in that, The primer set was used to amplify the conserved region of the μNS protein encoded by the novel duck reovirus M3 gene.

3. A reagent kit for detecting a novel duck reovirus, characterized in that, Includes the primer set as described in claim 1 or 2.

4. The reagent kit according to claim 3, characterized in that, The kit also includes reverse transcription reagent, real-time quantitative PCR reaction solution containing SYBR Green I fluorescent dye, positive control, negative control, and instructions for use.

5. The reagent kit according to claim 4, characterized in that, The positive control is the recombinant plasmid pMD19-T-μNS; the recombinant plasmid pMD19-T-μNS contains a conserved region fragment of the μNS protein encoded by the novel duck reovirus M3 gene, and the nucleotide sequence of the conserved region fragment is shown in SEQ ID NO:

3.

6. The reagent kit according to claim 4, characterized in that, The real-time quantitative PCR reaction system includes: 2×SYBR qPCR Mix 9μL~11μL, upstream primer NDRV-M3-6-F at a concentration of 9μmol / L~11μmol / L 0.36μL~0.44μL, downstream primer NDRV-M3-6-R at a concentration of 9μmol / L~11μmol / L 0.36μL~0.44μL, cDNA template obtained by reverse transcription of total RNA from the sample to be tested or recombinant plasmid pMD19-T-μNS template 0.8μL~1.2μL, ddH2O to make up to 18μL~22μL; The final concentration of the upstream primer NDRV-M3-6-F is 0.18 μmol / L to 0.22 μmol / L, and the final concentration of the downstream primer NDRV-M3-6-R is 0.18 μmol / L to 0.22 μmol / L.

7. The reagent kit according to claim 4, characterized in that, The real-time quantitative PCR amplification program includes: Pre-denaturation at 94℃~96℃ for 28s~32s, one cycle; Denaturation at 94℃~96℃ for 8s~12s, annealing / extending at 55℃~57℃ for 28~32s, 40 cycles; The real-time quantitative PCR melting procedure includes: Hold at 94℃~96℃ for 14s~16s, then at 59℃~61℃ for 58s~62s, then raise to 94℃~96℃ and hold for 14s~16s, completing one cycle.

8. The reagent kit according to any one of claims 3 to 7, characterized in that, When using the recombinant plasmid pMD19-T-μNS as a template, the kit at 1.0 × 10⁻⁶... 1 ~1.0×10 7 It exhibits good linearity within the detection concentration range of copies / μL, with a minimum detectable concentration of 1.0 × 10⁻⁶. 1 copies / μL.

9. A novel real-time quantitative RT-PCR detection method for duck reovirus, not intended for disease diagnosis or treatment, characterized in that... The detection is performed using the kit according to any one of claims 3 to 8, comprising: Total RNA was extracted from the sample to be tested and cDNA was obtained by reverse transcription. The cDNA was used as a template for real-time quantitative PCR amplification using SYBR Green I dye. The presence of a novel duck reovirus in the sample is determined based on the amplification and melting curves.

10. The use of the primer set according to claim 1 or 2 or the kit according to any one of claims 3 to 8 in the preparation of a detection reagent for in vitro detection of novel duck reovirus nucleic acid.

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