Triple RT-PCR primer set and kit for identification and detection of MDRV, GRV and NDRV

Abstract

The present invention relates to a triple RT-PCR primer set and a kit for identifying and detecting Muscovy duck reovirus, goose reovirus and novel duck reovirus, and the sequence of the primer set is as shown in SEQ ID NO.1-6. In the triple PCR reaction of the present invention, the addition ratio of the three groups of primers is 0.55:0.3:0.4. The present invention establishes a triple RT-PCR detection method for three waterfowl reoviruses. The optimization of primer design and reaction conditions is the key technology for the successful establishment of multiplex PCR. The main problem of multiplex PCR is the uneven amplification efficiency. The present invention narrows the gap in fragment size in primer selection. In the primer screening link, multiple pairs of primers are designed for amplifying MDRV, GRV and NDRV. Finally, three pairs of primers with similar amplified fragment sizes are screened out, thereby effectively reducing the impact of uneven amplification efficiency. The electrophoresis results show that the target bands are accurate, clear and have strong discrimination.

Description

Technical Field

[0001] The present invention belongs to the technical field of virus detection, and in particular relates to a triple RT-PCR primer set, a kit and a detection method for identifying and detecting Muscovy duck reovirus, goose reovirus and novel duck reovirus. Background Art

[0002] my country is a country that raises ducks and geese, and has a long history of waterfowl farming. In recent years, with the update of technology and equipment, as well as the expansion of scale and market of waterfowl farming in my country, waterfowl reovirus (WRV) has become a hazard that cannot be ignored in restricting the sustainable development of waterfowl farming. The diseases caused by it have posed a serious threat to the waterfowl farming industry. Waterfowl reovirus can be divided into three categories according to the host and genotype: Muscovy duck reovirus (MDRV), novel duck reovirus (NDRV) and goose reovirus (GRV).

[0003] Muscovy duck reovirus disease caused by Muscovy duck reovirus was first reported in South Africa in 1950. Since the 1990s, this disease has also appeared in Muscovy duck flocks in my country. The disease mainly infects young Muscovy ducks. After infection, the main clinical lesions are soft feet and diarrhea. The main lesions are a large number of gray-white needle-like necrotic spots on the surface and cut surface of the liver and spleen. Therefore, it is commonly known as Muscovy duck "liver white spot disease" or "flower liver disease". It has a high morbidity and mortality rate in Muscovy duck farming. The disease-resistant ducks become dead ducks. It has become one of the important diseases affecting the Muscovy duck farming industry.

[0004] Goose reovirus mainly attacks goslings under 3 weeks of age. The main symptoms of sick geese are listlessness, soft feet, and white loose feces. The incidence and mortality rates are closely related to age. The younger the age, the more serious the disease. Typical lesions in autopsy are a large number of white or yellow-white needle-like necrotic spots in the liver and spleen. The clinical symptoms are very similar to those of Muscovy duck reovirus disease, which seriously endangers the healthy development of my country's goose farming industry.

[0005] The new duck reovirus disease, commonly known as duck hemorrhagic necrotizing hepatitis, is a new infectious disease that has emerged in my country since 2005 and endangers the duck farming industry. Clinically, the disease is characterized by irregular necrosis and mixed hemorrhage of the liver, myocardial hemorrhage, enlarged plaque necrosis of the spleen, and hemorrhage in the kidney and bursa of Fabricius. It can occur in various breeds of ducks (such as Muscovy ducks, semi-Muscovy ducks, Mallards, Peking ducks, etc.); the age of onset is about 3 to 25 days, among which 5 to 10 days are the most common, the course of the disease is 5 to 7 days, the incidence rate is 5% to 20%, and the mortality rate is 2% to 15%. The younger the age or the time of concurrent infection, the higher the incidence rate and mortality rate, causing certain economic losses to the duck farming industry. It is different from Muscovy duck reovirus disease in serology, genotype, antigenicity, pathogenicity, etc.

[0006] Polymerase chain reaction (PCR) is a technique for rapid in vitro DNA amplification. It is simple and convenient to operate, and reliable results can be obtained in 3-5 hours. These advantages have made PCR technology the most commonly used technology for pathogen detection. This is also true in the field of waterfowl reovirus diagnosis. For example, Lin Fengqiang et al. established a RT-PCR detection method for Muscovy duck reovirus in 2005 (Lin Fengqiang, Hu Qilin, Ouyang Suidong et al. Isolation and RT-PCR identification of Muscovy duck reovirus [J]. Advances in Veterinary Medicine, 2005(11):68-69.), and Wang Shao et al. established a RT-PCR detection method for a new type of duck reovirus in 2011 (Wang Shao, Chen Shaoying, Chen Shilong et al. Establishment and application of a new type of duck reovirus RT-PCR method [J]. Journal of Agricultural Biotechnology, 2011, 19(02):388-392.).

[0007] Multiplex PCR (Multiplex polymerase chain reaction, MPCR), also known as composite PCR, is different from single PCR. It can detect multiple different pathogens in a single sample at the same time. It has the advantages of high sensitivity, high efficiency, strong specificity, and high economic benefits. During the test, multiplex PCR will also greatly reduce the probability of false positives due to sample, reagent or primer contamination, so as to facilitate, quickly and accurately carry out relevant epidemiological monitoring.

[0008] Multiplex PCR has significant technical advantages, but there are also many technical problems to be overcome when establishing multiplex PCR. It not only needs to consider the specificity of the primers and the corresponding viruses to be detected, but also the mutual interference between the primers, the inability of the primers to bind to each other, and the size of the amplified products should not be too close when the annealing temperature and extension time are close. In addition, since MDRV, GRV and NDR are all reoviruses that harm waterfowl, the nucleotide sequence homology between them is relatively high, especially the nucleotide homology between MDRV and goose-derived Muscovy duck reovirus is more than 90%. Although single RT-PCR detection methods for MDRV, GRV and NDRV and double RT-PCR detection methods for MDRV and NDRV have been reported, it is very difficult to establish a detection method that can simultaneously differentiate and diagnose these three waterfowl reoviruses MDRV, GRV and NDRV, and the triple detection method has not been reported so far. Therefore, the establishment of a triple RT-PCR detection method for MDRV, GRV and NDRV can provide technical support for pathogen monitoring and disease prevention and control of the three waterfowl reoviruses in waterfowl farming, which has good innovation and clinical application value. Summary of the invention

[0009] The purpose of the present invention is to provide a triple RT-PCR primer set and a kit for detecting Muscovy duck reovirus, goose reovirus and novel duck reovirus, and at the same time, to establish a triple RT-PCR detection method for detecting and identifying Muscovy duck reovirus, goose reovirus and novel duck reovirus using the triple RT-PCR primer set to solve the above-mentioned existing technical problems. The triple RT-PCR detection method established by the present invention can perform rapid and accurate differential diagnosis of pathogens, and can provide an effective detection means for epidemiological investigation and scientific and precise prevention and control of MDRV, GRV and NDRV, three virus infections, and provide help for subsequent research on these three waterfowl reoviruses.

[0010] The purpose of the present invention is achieved through the following technical solutions:

[0011] A triple RT-PCR primer set for detecting Muscovy duck reovirus, goose reovirus and novel duck reovirus, the primer set comprising:

[0012] Primer pair sequence for amplifying MDRV virus:

[0013] MDRV-F: 5'-TTACCCTTCCCATCCGACCTC-3';

[0014] MDRV-R: 5'-GTTCATAGCGAAGCCCTGCT-3';

[0015] Primer pair sequence for amplifying GRV virus:

[0016] GRV-F: 5'-TGAAGTCCGACAACCCTACC-3';

[0017] GRV-R: 5'-CGTCATTGTCCACGGATCCA-3';

[0018] Primer pair sequence for amplifying NDRV virus:

[0019] NDRV-F: 5'-ATGTCGAGGACTGTTGGATG-3';

[0020] NDRV-R: 5'-CAGCACATGTAAAGCACGAC-3'.

[0021] A detection kit for detecting Muscovy duck reovirus, goose reovirus and novel duck reovirus comprises the primer set.

[0022] Novozymes III 1st Strand cDNA Synthesis Kit (+gDNAwiper) reverse transcription kit, refer to the instructions of the reverse transcription kit to reverse transcribe the viral RNA into cDNA samples.

[0023] The PCR reaction system of the detection kit is:

[0024] The following components are contained in the 20 μL total system: 10 μL of Dream Taq Green PCR Master Mix (2×), 0.55 μL of 10 μmol / L MDRV-F and MDRV-R mixed primers, 0.3 μL of 10 μmol / L GRV-F and GRV-R mixed primers, 0.4 μL of 10 μmol / L NDRV-F and NDRV-R mixed primers, 3 μL of pMD18-MDRV, pMD18-GRV and pMD18-NDRV plasmid mixed template (in the specific detection operation, the pMD18-MDRV, pMD18-GRV and pMD18-NDRV plasmid mixed template can be replaced by the nucleic acid template to be detected, i.e., 3 μL of the nucleic acid template to be detected), and double distilled water is added to make up to 20 μL.

[0025] The detection kit includes, in addition to the above-mentioned PCR reaction system, a positive control; the positive control is prepared by mixing equal amounts of positive plasmids pMD18-MDRV, pMD18-GRV and pMD18-NDRV containing MDRV, GRV and NDRV genome fragments respectively.

[0026] The optimized PCR reaction process of the detection kit is: 95°C pre-denaturation for 3 minutes, 95°C denaturation for 30 seconds, 57.7°C annealing for 30 seconds, 72°C extension for 1 minute, a total of 39 cycles, and 72°C extension for 5 minutes.

[0027] The detection kit can simultaneously identify and detect Muscovy duck reovirus, goose reovirus and novel duck reovirus, and the determination method is: testing the electrophoresis diagram of the amplified product, and judging whether the sample to be tested is a single infection or mixed infection of Muscovy duck reovirus, goose reovirus and novel duck reovirus by observing whether the electrophoresis diagram of the amplified product contains expected bands corresponding to the corresponding viruses; wherein, the length of the amplified product fragment corresponding to the MDRV virus is 708bp, the length of the amplified product fragment corresponding to the GRV virus is 324bp, and the length of the amplified product fragment corresponding to the NDRV virus is 198bp.

[0028] The present invention also provides the use of the triple RT-PCR primer set in preparing a detection reagent or a detection kit for identifying and detecting Muscovy duck reovirus, goose reovirus and novel duck reovirus.

[0029] The present invention also provides a triple RT-PCR detection method for identifying and detecting Muscovy duck reovirus, goose reovirus and novel duck reovirus using the triple RT-PCR primer set. The general process of the method is: the sample to be tested is reverse transcribed into a cDNA sample by referring to the instructions of the reverse transcription kit, and the cDNA sample after reverse transcription is used as a template to perform a triple RT-PCR reaction using the primer set; then, the amplified product is detected by agarose gel electrophoresis; wherein the addition amount of each primer pair sequence in the primer set is: primer pair sequence for amplifying MDRV virus: primer pair sequence for amplifying GRV virus: primer pair sequence for amplifying NDRV virus = 0.55:0.3:0.4.

[0030] The triple RT-PCR detection method for identifying and detecting Muscovy duck reovirus, goose reovirus and novel duck reovirus specifically comprises the following steps:

[0031] 1) Search for the conserved sequence of Muscovy duck reovirus MDRV, goose reovirus GRV and novel duck reovirus NDRV:

[0032] 2) According to the sequence information of the σNS gene of MDRV virus, the λAp gene of GRV virus and the σNS gene of NDRV virus published on GenBank, upstream and downstream primer pairs, i.e., the triple RT-PCR primer set, were designed respectively. The triple RT-PCR primer set includes:

[0033] Primer pair sequence for amplifying MDRV virus:

[0034] MDRV-F: 5'-TTACCCTTCCCATCCGACCTC-3'

[0035] MDRV-R: 5'-GTTCATAGCGAAGCCCTGCT-3';

[0036] Primer pair sequence for amplifying GRV virus:

[0037] GRV-F: 5'-TGAAGTCCGACAACCCTACC-3';

[0038] GRV-R: 5'-CGTCATTGTCCACGGATCCA-3';

[0039] Primer pair sequence for amplifying CRV virus:

[0040] NDRV-F: 5'-ATGTCGAGGACTGTTGGATG-3';

[0041] NDRV-R: 5'-CAGCACATGTAAAGCACGAC-3'.

[0042] 3) Perform triple RT-PCR reaction: using the sample to be tested as a template and the above three sets of sequences as primers, perform triple RT-PCR reaction, and detect the amplified products by agarose gel electrophoresis; in the reaction system, the addition amount of the three sets of primers conforms to the primer pair for amplifying MDRV virus: the primer pair for amplifying GRV virus: the primer pair for amplifying NDRV virus = 0.55:0.3:0.4.

[0043] Among them, after optimization, the PCR reaction process of step 3) is: 95℃ pre-denaturation for 3min, 95℃ denaturation for 30sec, 57.7℃ annealing for 30sec, 72℃ extension for 1min, a total of 39 cycles, and 72℃ extension for 5min;

[0044] The total volume of the PCR reaction system in step 3) is 20 μL, including: 10 μL of Dream Taq Green PCR MasterMix (2×), 0.55 μL of MDRV-F and MDRV-R mixed primers at a concentration of 10 μmol / L; 0.3 μL of GRV-F and GRV-R mixed primers at a concentration of 10 μmol / L; 0.4 μL of NDRV-F and NDRV-R mixed primers at a concentration of 10 μmol / L; 3 μL of pMD18-MDRV, pMD18-GRV and pMD18-NDRV plasmid mixed template (in the specific detection operation, pMD18-MDRV, pMD18-GRV and pMD18-NDRV plasmid mixed template can be replaced with the nucleic acid template to be detected, that is, 3 μL of the nucleic acid template to be detected), and double distilled water is added to make up to 20 μL.

[0045] Further, in step 3), the length of the amplified product fragment of the MDRV virus is 708bp, the length of the amplified product fragment of the GRV virus is 324bp, and the length of the amplified product fragment of the NDRV virus is 198bp. By observing whether the electrophoresis diagram of the amplified product contains the expected bands corresponding to the corresponding viruses, it is determined whether the sample to be tested is a single infection or mixed infection of Muscovy duck reovirus, goose reovirus and new duck reovirus.

[0046] In addition, the Gene Bank sequence number of the MDRV virus in step 2) is DQ066923.1;

[0047] The Gene Bank sequence number of the GRV virus in step 2) is MZ546418.1.

[0048] The Gene Bank sequence number of the NDRV virus in step 2) is OM930743.1.

[0049] Compared with the prior art, the advantages of the present invention are:

[0050] 1. The present invention establishes a triple RT-PCR detection method for three waterfowl reoviruses (MDRV, GRV and NDRV). The optimization of primer design and reaction conditions is the key technology for the successful establishment of multiplex PCR, and the main problem of multiplex PCR is the uneven amplification efficiency. The present invention tries to narrow the gap in fragment size in primer selection. In the primer screening link, multiple pairs of primers are designed for amplifying MDRV, GRV and NDRV, and finally three pairs of primers with similar amplified fragment sizes are screened out, thereby effectively reducing the impact of uneven amplification efficiency. The electrophoresis results show that the target bands are accurate, clear and have strong discrimination.

[0051] 2. The present invention further optimizes the reaction system by constructing positive standard plasmids of three pathogens and provides a suitable reaction system. This method can simultaneously detect three different pathogens in a single sample and has the advantages of high sensitivity, strong specificity, time saving and labor saving. In addition, triple PCR can also reduce the false positive rate caused by contamination during the test.

[0052] 3. In the epidemiological monitoring of waterfowl diseases, it was found that MDRV, GRV and NDRV were infected to varying degrees in waterfowl farms across my country, and waterfowl reovirus infection would damage the immune organs of poultry, thereby causing secondary mixed infection with other pathogens. Therefore, the triple RT-PCR system established in this study can be well applied to clinical testing and provide technical support for pathogen monitoring of the three viruses in waterfowl farming and disease prevention and control.

[0053] 4. The present invention designs multiple pairs of primers for amplifying MDRV, GRV and NDRV, and finally screens out three pairs of primers with similar amplified fragment sizes, thereby effectively reducing the impact of uneven amplification efficiency.

[0054] 5. The present invention can be used to simultaneously detect three waterfowl reoviruses (MDRV, GRV and NDRV) in a single sample. The target band is accurate, clear and highly distinguishable. It has the advantages of high sensitivity, strong specificity, time saving and labor saving, and can also reduce the false positive rate caused by contamination.

[0055] 6. Using the present invention, when multiple diseases are mixed and infected at the same time, the multiplex PCR detection method makes the diagnosis of mixed infection convenient and feasible, and can also achieve rapid and accurate detection of pathogens with low virus content. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is the electrophoresis diagram of triple RT-PCR condition optimization when the annealing temperature is 57.7°C in the embodiment of the present invention, wherein M: DNA marker DL2000; 1: MDRV; 2: GRV; 3: NDRV; 4-11: 46.2°C, 48.6°C, 51°C, 53.5°C, 54.9°C, 57.7°C, 59.9°C, 61°C; 12: negative control.

[0057] Figure 2 This is a specific identification electrophoresis diagram in the embodiment of the present invention, wherein M: DNA marker DL2000; 1: MDRV, GRV, NDRV mixture; 2: MDRV; 3: GRV; 4: NDRV; 5: cGPV; 6: MDPV; 7: MDGPV; 8: SBDSV; 9: DAdV B2; 10: AIV H9 subtype; 11: DEV; 12: DTMUV; 13: negative control

[0058] Figure 3 This is the electrophoresis diagram of sensitivity identification in the embodiment of the present invention. M: DNA marker DL2000; 1-9: 3 positive plasmids from 10 9 ~10 1 The copy number after serial dilution, where

[0059] 1. The copy numbers of MDRV, GRV, and NDRV are: 6.72×10 9 copies·μL -1 , 6.68×10 9 copies·μL -1 , 6.11×10 9 copies·μL -1 ;

[0060] 2. The copy numbers of MDRV, GRV, and NDRV are: 6.72×10 8 copies·μL -1 , 6.68×10 8 copies·μL -1 , 6.11×10 8 copies·μL -1 ;

[0061] 3. The copy numbers of MDRV, GRV, and NDRV are: 6.72×10 7 copies·μL -1 ; 6.68×10 7 copies·μL -1 ; 6.11×10 7 copies·μL -1 ;

[0062] 4. The copy numbers of MDRV, GRV, and NDRV are: 6.72×10 6 copies·μL -1 ; 6.68×10 6 copies·μL -1 ; 6.11×10 6 copies·μL -1 ;

[0063] 5. The copy numbers of MDRV, GRV, and NDRV are: 6.72×10 5 copies·μL -1 ; 6.68×10 5 copies·μL -1; 6.11×10 5 copies·μL -1 ;

[0064] 6. The copy numbers of MDRV, GRV, and NDRV are: 6.72×10 4 copies·μL -1 ; 6.68×10 4 copies·μL -1 ; 6.11×10 4 copies·μL -1 ;

[0065] 7. The copy numbers of MDRV, GRV, and NDRV are: 6.72×10 3 copies·μL -1 ; 6.68×10 3 copies·μL -1 ; 6.11×10 3 copies·μL -1 ;

[0066] 8. The copy numbers of MDRV, GRV, and NDRV are: 6.72×10 2 copies·μL -1 ; 6.68×10 2 copies·μL -1 ; 6.11×10 2 copies·μL -1 ;

[0067] 9. The copy numbers of MDRV, GRV, and NDRV are: 6.72×10 1 copies·μL -1 ; 6.68×10 1 copies·μL -1 ; 6.11×10 1 copies·μL -1 ;

[0068] 10: Negative control. DETAILED DESCRIPTION

[0069] The present invention is described in detail below with reference to the accompanying drawings and embodiments:

[0070] Viral RNA extraction kit FastPure Viral DNA / RNA Mini Kit, reverse transcription kit III 1st Strand cDNA Synthesis Kit (+gDNAwiper), DNA fragment gel recovery kit FastPure Gel DNA Extraction Mini Kit and RNase-free ddH2O were purchased from Novazonics; DreamTaq Green PCR Master Mix (2×) was purchased from Thermo Biotechnology; nucleic acid molecular weight standard Marker (DL-2000) and pMD18-T Vector Cloning Kit were purchased from TaKaRa; small amount of plasmid extraction kit was purchased from Axygen; agarose was purchased from BIOWEST; anhydrous ethanol was purchased from Xilong Scientific Co., Ltd.; LB liquid culture medium and LB solid culture medium containing ampicillin were prepared by the laboratory according to the conventional ratio in the field.

[0071] Example A triple RT-PCR detection method for Muscovy duck reovirus, goose reovirus and novel duck reovirus was established, comprising the following steps:

[0072] 1. Primer Design

[0073] According to the sequence information of the σNS gene of MDRV virus published on GenBank, Gene Bank sequence number DQ066923.1; the λAp gene of GRV virus, Gene BANK sequence number MZ546418.1 and the σNS gene of NDRV virus, Gene BANK sequence number OM930743.1, primers were designed and synthesized, respectively, see Table 1.

[0074] Table 1

[0075]

[0076] 2. Extract RNA and synthesize cDNA.

[0077] 2.1 Sample processing

[0078] Diseased specimens were collected from goose and duck farms in Fujian and Jiangsu. The collected organs were ground, diluted with Hank's solution, frozen and thawed three times, vortexed for 1 min, and heated at 3000 r / min. -l Centrifuge for 10 min, take 200 μL of supernatant and store it at -20°C for RNA extraction.

[0079] 2.2 RNA extraction

[0080] The total viral RNA in the above samples was extracted using the Novagen FastPure Viral DNA / RNA Mini Kit. The total viral RNA in the above samples was extracted according to the kit instructions. RNase-free EP tubes and pipette tips were used in the present embodiment. The specific operation was as follows:

[0081] 1) Take 200 μL of sample, add 500 μL of Buffer VL, vortex mix for 15-30 seconds, and centrifuge the mixture to the bottom of the tube;

[0082] 2) Transfer the mixture to FastPure RNA Columns, centrifuge at 12000 rpm (13400 × g) for 1 min, and discard the filtrate;

[0083] 3) Add 600 μL Buffer RW and centrifuge at 12000 rpm (13400 × g) for 30 seconds;

[0084] 4) Discard the filtrate and repeat step 3, centrifuging the empty column at 12000 rpm (13400 × g) for 2 min;

[0085] 5) Take 50 μL RNase-free ddH2O, place at room temperature for 1 min, centrifuge at 12000 rpm (13400 × g) for 2 min to elute RNA;

[0086] 2.3 Synthesis of cDNA

[0087] By Novozyme III 1st Strand cDNA Synthesis Kit (+gDNAwiper) Reverse Transcription Kit Instructions Reverse transcription into cDNA samples, the steps are as follows:

[0088] 1) RNA template denaturation: Prepare the following mixture in an RNase-free centrifuge tube: Take 8 μL of viral RNA, heat at 65°C for 5 min, quickly cool on ice, and let stand on ice for 2 min.

[0089] 2) Genomic DNA removal: Add 2 μL of 5x gDNAwiper Mix to the RNA solution in the previous step, mix gently by pipetting, and heat at 42°C for 2 min.

[0090] 3) Prepare the first-strand cDNA synthesis reaction solution: 10μL of the previous mixture; 2μL of 10×RT Mix; 2μL of HiScriptⅢEnzyme Mix; 1μL of Random hexamers; 5μL of RNase-free ddH2O, and mix them by gently pipetting. Perform the first-strand cDNA synthesis reaction under the following conditions: 25℃5min; 37℃45min; 85℃5sec. The obtained product is cDNA.

[0091] 3. Construction of virus-positive plasmid standards

[0092] Using the above cDNA as a template, PCR amplification was performed using the primers in Table 1, and the PCR amplification product was recovered. The recovered target fragment was cloned into the pMD-18-T vector and transformed into the DH5α competent cell. A single colony was picked for bacterial liquid PCR identification, and the suspected positive plasmid was sent to the company for sequencing. Sequence comparison was performed in the NCBI database, and the positive plasmid with correct sequencing was stored at -20°C for future use.

[0093] Among them, the PCR reaction system was 20 μL, Dream Taq Green PCR Master Mix (2×) 10 μL, upstream and downstream primers 0.5 μL each, 1 μL template, and ddH2O was used to make up to 20 μL. PCR reaction conditions: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 sec; annealing at 56°C for 30 sec; extension at 72°C for 1 min for a total of 39 cycles; complete extension at 72°C for 5 min;

[0094] The concentrations of the positive plasmids pMD18-MDRV, pMD18-GRV, and pMD18-NDRV containing MDRV, GRV, and NDRV genome fragments were detected by a micro-nucleic acid concentration analyzer, and the concentrations were 250 ng·μL -1 , 210.5ng·μL -1 , 202ng·μL -1 According to the formula: plasmid copy number (copies / μL) = C × 10 -9 ×6.02×10 23 / (M×660), where C is the plasmid concentration (ng / μL) and M is the number of bases in the plasmid. The calculated copy numbers of the plasmid standards are 6.72×10 10 copies·μL -1 , 6.68×10 10 copies·μL -1 With 6.11×10 10 copies·μL -1 . Dilute the three plasmid standard solutions 10-fold to 10 9 , 108 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 copies / μL -1 , and then mix them in equal amounts to obtain the positive control plasmid standard.

[0095] 4. Optimize PCR reaction system

[0096] The PCR reaction system was 20 μL, to which 10 μL of Dream Taq Green PCR Master Mix (2×) and 3 μL of pMD18-MDRV, pMD18-GRV and pMD18-NDRV positive mixed plasmid template were added. The starting concentration of the primers was 10 μmol / L, and the primer volume was adjusted between 0.5 μL-1.0 μL to determine the most suitable primer volume; based on the determination of the optimal primer amount, the annealing temperature of the reaction was further optimized, and the annealing temperature was optimized in the range of 46.2°C-61°C to determine the most suitable annealing temperature.

[0097] Annealing temperature is 57.7℃, the target band can be amplified, and the three target bands can be clearly distinguished, see Figure 1 .

[0098] By optimizing the primer concentration and annealing temperature, the final PCR reaction system was determined as follows: Dream TaqGreen PCR Master Mix (2×) 10 μL, MDRV-F / MDRV-R (10 μmol / L) mixed and added 0.55 μL, GRV-F / GRV-R (10 μmol / L) mixed and added 0.3 μL, NDRV-F / NDRV-R (10 μmol / L) mixed and added 0.4 μL, template 3 μL, add double distilled water to make up to 20 μL;

[0099] The optimal reaction conditions were: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 sec, annealing at 57.7°C for 30 sec, extension at 72°C for 1 min, a total of 39 cycles, and extension at 72°C for 5 min.

[0100] 5. Specificity test

[0101] According to step 2.2, viral DNA of goose plague virus (cGPV), Muscovy duck parvovirus (MDPV), Muscovy duck gosling plague virus (MDGPV), duck short-beak dwarf syndrome virus (SBDSV), duck plague virus (DEV), duck adenovirus B2 (DAdV B2) were extracted; according to step 2.3, viral nucleic acids of Muscovy duck reovirus (MDRV), goose reovirus (GRV), novel duck reovirus (NDRV), avian influenza virus H9 subtype (AIV H9), duck Tembusu virus (DTMUV) were extracted and purified according to Novazonic III 1st Strand cDNA Synthesis Kit (+gDNA wiper) Reverse Transcription Kit Instructions Reverse transcription into cDNA samples.

[0102] The PCR reaction conditions optimized in step 4 were used: 95°C pre-denaturation for 3 min, 95°C denaturation for 30 sec, 57.7°C annealing for 30 sec, 72°C extension for 1 min, 39 cycles in total, and 72°C extension for 5 min to detect pMD18-MDRV, pMD18-GRV, pMD18-NDRV positive plasmid templates and cGPV, MDPV, MDGPV, SBDSV, DEV, DAdV B2 viral DNA templates and AIV H9, DTMUV cDNA templates. The specificity of the established triple RT-PCR system was tested, and the results are shown in Figure 2 .

[0103] Depend on Figure 2 It can be seen that the expected bands can be obtained by detecting the positive mixed plasmids of pMD18-MDRV, pMD18-GRV and pMD18-NDRV; while no bands appeared in the viral DNA detection of cGPV, MDPV, MDGPV, SBDSV, DEV, and DAdV B2, and no bands appeared in the cDNA template detection of AIVH9 and DTMUV, indicating that this method has good specificity.

[0104] 6. Sensitivity test

[0105] The positive control plasmid standard solutions pMD18-MDRV, pMD18-GRV and pMD18-NDRV diluted and mixed in equal amounts in step 3 were added to the PCR system in step 4, and amplified using the optimal primer volume and the most suitable reaction temperature to test the sensitivity of the PCR system. The results are shown in Figure 3 .

[0106] from Figure 3 As can be seen in the figure, using the triple RT-PCR amplification system, the pMD18-MDRV, pMD18-GRV, and pMD18-NDRV positive plasmid templates were diluted to 6.72×103 copies·μL -1 , 6.68×10 3 copies·μL -1 With 6.11×10 3 copies·μL -1 After the amplification, the corresponding target band can still be amplified, indicating good sensitivity.

[0107] The above-mentioned embodiments are intended to illustrate that the present invention can be implemented or used by professionals in the field. Modifications to the above-mentioned embodiments will be obvious to professionals in the field. Therefore, the present invention includes but is not limited to the above-mentioned embodiments. Any method, process, or product that complies with the claims or description of the specification and complies with the principles and novelty and creative features disclosed herein falls within the scope of protection of the present invention.

Claims

1. A triple RT-PCR primer set for detecting Muscovy duck reovirus, goose reovirus and novel duck reovirus, characterized in that: The primer set comprises: Primer pair sequence for amplifying MDRV virus: MDRV-F: 5'-TTACCCTTCCCATCCGACCTC-3'; MDRV-R: 5'-GTTCATAGCGAAGCCCTGCT-3'; Primer pair sequence for amplifying GRV virus: GRV-F: 5'-TGAAGTCCGACAACCCTACC-3'; GRV-R: 5'-CGTCATTGTCCACGGATCCA-3'; Primer pair sequence for amplifying NDRV virus: NDRV-F: 5'-ATGTCGAGGACTGTTGGATG-3'; NDRV-R: 5'-CAGCACATGTAAAGCACGAC-3'.

2. A detection kit for detecting Muscovy duck reovirus, goose reovirus and novel duck reovirus, characterized in that: It comprises the primer set according to claim 1.

3. The detection kit according to claim 2, characterized in that: It also includes a positive control; the positive control is prepared by mixing equal amounts of positive plasmids pMD18-MDRV, pMD18-GRV and pMD18-NDRV containing MDRV, GRV and NDRV genome fragments respectively.

4. The detection kit according to claim 3, characterized in that: The PCR reaction system is: The total system of 20 μL contains the following ingredients: 2×Dream Taq Green PCR Master Mix 10μL, MDRV-F and MDRV-R mixed primers at a concentration of 10μmol / L 0.55μL, GRV-F and GRV-R mixed primers at a concentration of 10μmol / L 0.3μL, NDRV-F and NDRV-R mixed primers at a concentration of 10μmol / L 0.4μL, pMD18-MDRV, pMD18-GRV and pMD18-NDRV plasmid mixed template 3μL, add double distilled water to make up to 20μL.

5. The detection kit according to claim 2, characterized in that: The PCR reaction process was as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 57.7°C for 30 s, extension at 72°C for 1 min, a total of 39 cycles, and extension at 72°C for 5 min.

6. A triple RT-PCR detection method for identifying and detecting Muscovy duck reovirus, goose reovirus and novel duck reovirus using the triple RT-PCR primer set of claim 1, wherein the method is not for diagnosis, and is characterized by: The sample to be tested is used as a template, and the primer set is used to perform a triple RT-PCR reaction; then, the amplified product is detected by agarose gel electrophoresis; wherein the addition amount of each primer pair sequence in the primer set is: The primer pair sequence for amplifying MDRV virus: the primer pair sequence for amplifying GRV virus: the primer pair sequence for amplifying NDRV virus = 0.55:0.3:0.

4.

7. The triple RT-PCR detection method according to claim 6, characterized in that: The total volume of the reaction system of the PCR reaction is 20 μL, including: 10 μL of 2×Dream Taq Green PCR Master Mix, 0.55 μL of MDRV-F and MDRV- R mixed primers with a concentration of 10 μmol / L; 0.3 μL of GRV-F and GRV- R mixed primers with a concentration of 10 μmol / L; 0.4 μL of NDRV-F and NDRV- R mixed primers with a concentration of 10 μmol / L; 3 μL of pMD18-MDRV, pMD18-GRV and pMD18-NDRV plasmid mixed template, added with double distilled water to 20 μL; The PCR reaction process was as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 57.7°C for 30 s, extension at 72°C for 1 min, for a total of 39 cycles, and extension at 72°C for 5 min.

8. The triple RT-PCR detection method according to claim 6, characterized in that: The length of the amplified product fragment of the triple RT-PCR reaction for MDRV virus is 708 bp, the length of the amplified product fragment of GRV virus is 324 bp, and the length of the amplified product fragment of NDRV virus is 198 bp.

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