PCR (Polymerase Chain Reaction) primer group for detecting and distinguishing avian reovirus and application of PCR primer group

Abstract

The invention discloses a PCR primer group for detecting and distinguishing avian reoviruses and application of the PCR primer group, and belongs to the technical field of molecular biology. The invention provides a PCR primer for detecting and / or distinguishing the avian reovirus, the primer group has good specificity on the avian reovirus and has no cross reaction with other major poultry diseases, a sensitivity test result shows that the sensitivity is about 10 times higher than that of a conventional PCR method, other common poultry viruses have no specific amplification, and the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result is about 10 times higher than that of the conventional PCR method. No cross reaction is found, intra-batch and inter-batch variation coefficients are both smaller than 2%, and the detection method has the advantages of being high in sensitivity, high in specificity, good in repeatability and the like.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a PCR primer set for detecting and distinguishing avian reovirus and an application thereof. Background Art

[0002] Avian Reovirus (ARV) can cause viral arthritis, intestinal diseases and stunting syndrome, and lead to immunosuppression, increasing the susceptibility of chickens to other secondary diseases. In recent years, the prevalence of the disease in my country has been on the rise, resulting in low feed conversion rate, increased mortality and culling of chickens, reduced production efficiency, and serious economic losses to the poultry industry. Due to the complexity of ARV multi-segment gene fragments, it is more likely to produce gene rearrangement and recombination within gene fragments, leading to the generation of ARV mutant strains. Currently, the widely used commercial vaccines (such as S1133, ZJS, etc.) are all genotype I, but multiple genotypes are prevalent in my country, resulting in unsatisfactory clinical immune protection effects, which poses a huge challenge to the prevention and control of the disease.

[0003] ARV virus particles are spherical and are non-enveloped double-stranded RNA viruses. The viral genome consists of 10 segmented double-stranded RNAs, which are divided into three groups according to their size: L (L1, L2, L3), M (M1, M2, M3), and S (S1, S2, S3, S4). Among them, the σC protein encoded by the S1 gene is a highly variable protein that can induce the body to produce type-specific neutralizing antibodies, which is related to the pathogenicity and infectivity of the virus and is the main basis for the typing of ARV epidemic strains. ARV is prone to genetic mutation or recombination to produce new strains. Based on the results of genetic evolution analysis of the σC gene, ARV can be divided into 6 genotypes, and there are large differences in immunogenicity and pathogenicity between different genotypes. Epidemiological surveys in recent years have found that genotype I, genotype I subtype, genotype II, and genotype VI are the dominant genotypes of ARV prevalence in my country. Therefore, it is of great significance to establish an efficient, sensitive, and specific differential diagnosis method for the prevention and control of viral arthritis.

[0004] At present, the methods for detecting ARV pathogens include virus isolation and identification, serology, ordinary PCR, and universal fluorescent quantitative PCR, but these methods have low clinical detection efficiency and are time-consuming and labor-intensive. Patent 202210151323.5 discloses a multiplex PCR kit and its application for detecting or distinguishing four different genotypes of avian reovirus (genotype I, genotype I subtype, genotype II, and genotype V). However, the multiplex PCR detection method involved in this technical solution has the problems of high detection limit and low sensitivity. Summary of the invention

[0005] [Technical issues] The technical problem to be solved by the present invention is to provide a PCR primer set with reduced detection limit and improved sensitivity, which can effectively detect and / or distinguish avian reovirus. Technical Solution

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions: In a first aspect, the present invention provides a PCR primer set for detecting and / or distinguishing avian reovirus, the primer set comprising at least one of the following (a)-(d): (a) The first primer set includes: The upstream primer ARV1-qF1 having a nucleotide sequence as shown in SEQ ID NO.1; Downstream primer ARV1-qR1 having a nucleotide sequence as shown in SEQ ID NO.2; The first probe; (b) The second primer set includes: The upstream primer ARV1-sub-qF1 whose nucleotide sequence is shown in SEQ ID NO.12; Downstream primer ARV1-sub-qR1 having a nucleotide sequence as shown in SEQ ID NO.13; The second probe; (c) The third primer set includes: The upstream primer ARV2-qF1 having a nucleotide sequence as shown in SEQ ID NO.23; Downstream primer ARV2-qR1 having a nucleotide sequence as shown in SEQ ID NO.24; The third probe; (d) The fourth primer set includes: The upstream primer ARV6-qF1 whose nucleotide sequence is shown in SEQ ID NO.34; Downstream primer ARV6-qR1 having a nucleotide sequence as shown in SEQ ID NO.35; The fourth probe.

[0007] In one embodiment, the first probe, the second probe, the third probe and the fourth probe are nucleic acid probes for detecting target nucleic acid sequences; the first probe, the second probe, the third probe and the fourth probe are connected with a fluorescent group and a quenching group.

[0008] In one embodiment, the fluorescent group includes: FAM, HEX, TET, CY3, CY5, CY7, ROX, JOE, Texas Red, Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 647, VIC, NED, TAMRA, AMCA or fluorescein amide.

[0009] In one embodiment, the quencher group includes: BHQ1, BHQ2, BHQ3, DABCYL, Iowa BlackFQ, Iowa Black RQ, TQ3 or Eclipse.

[0010] In one embodiment, the fluorescent group is attached to the 5' end of the probe, and the quencher group is attached to the 3' end of the probe.

[0011] In one embodiment, the fluorescent groups attached to the first probe, the second probe, the third probe and the fourth probe are different.

[0012] In one embodiment, the first probe is: 5'-VIC-CCGCGTGCGCAGAGGATSTGTT-MGB-3'; And / or, the second probe is: 5'-FAM-CTTCTGCTCRCAACGAGTCTCYTTGA-BHQ1-3'; And / or, the third probe is: 5'-CY5-CTGAAAMCCGGCSCGAGGCAC-BHQ3-3'; And / or, the fourth probe is: 5'-Texas Red-CCGGRAAHGTCGCCTGCTGRAA-BHQ2-3'.

[0013] In one embodiment, the avian reovirus includes at least one of ARV genotype I, ARV genotype I subtype, ARV genotype II, and ARV genotype VI.

[0014] In one embodiment, the first primer group is used to detect and / or distinguish avian reovirus ARV genotype I, the second primer group is used to detect and / or distinguish avian reovirus ARV genotype I subtypes, the third primer group is used to detect and / or distinguish avian reovirus ARV genotype II, and the fourth primer group is used to detect and / or distinguish avian reovirus ARV genotype VI.

[0015] In a second aspect, the present invention further provides a reagent for detecting and / or distinguishing avian reovirus, wherein the reagent contains the primer set described in the first aspect.

[0016] In a third aspect, the present invention further provides use of the primer set described in the first aspect in preparing a reagent for detecting and / or distinguishing avian reovirus.

[0017] In a fourth aspect, the present invention provides a method for detecting and / or distinguishing avian reovirus for non-diagnostic purposes, the method comprising the following steps: S1. Extract avian reovirus genomic RNA; S2, mixing the primer set described in the first aspect and the avian reovirus genomic RNA described in step S1, and performing fluorescent quantitative PCR; S3. Detecting and / or distinguishing avian reovirus according to the fluorescent signal obtained by the fluorescent quantitative PCR in step S2.

[0018] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) Based on the sigma C gene sequence, the present invention designed specific amplification primers and probes in the conserved regions of four different genotypes of chicken reovirus (genotype I, genotype I subtype, genotype II, and genotype VI), and established a multiplex fluorescence quantitative PCR detection method that can be used to identify and detect four different genotypes of chicken reovirus (genotype I, genotype I subtype, genotype II, and genotype VI). Compared with other primers and probes, the probe combination designed in the present invention (ARV1-qF1, ARV1-qR1 and ARV1-qP1; ARV1-sub-qF1, ARV1- sub-qR1 and ARV1-sub-qP1; ARV2-qF1, ARV2-qR1 and ARV2-qP1; ARV6-qF1, ARV6-qR1 and ARV6-qP1) has good specificity for different genotypes of chicken reovirus, and only shows fluorescence for the target genotype in the detection of chicken reovirus genotype I, genotype I subtype, genotype II, and genotype VI, and the Ct values ​​are all ≤40.

[0021] (2) The results of specificity tests showed that the primers and probes of the present invention had no cross-reaction with several other major poultry diseases (MDV, ILTV, FAdV, CIAV, IBDV, NDV, AIV, IBV, REV, ALV, ARV genotype III strain, ARV genotype IV strain, ARV genotype V strain).

[0022] (3) The results of the sensitivity test showed that the detection method of the present invention can detect a minimum of 10 copies of the template, which is about 10 times higher than the conventional PCR method. Other common poultry viruses were not specifically amplified and no cross-reaction was found. The intra-batch and inter-batch coefficients of variation were both less than 2%, indicating that the detection method of the present invention has the advantages of high sensitivity, strong specificity, good repeatability, etc., and has broad application prospects.

[0023] (4) The real-time fluorescence quantitative PCR detection method of the present invention directly monitors the dynamic changes of the fluorescence signal during the PCR amplification process through a photoelectric transmission system, thereby realizing accurate quantitative analysis. Compared with the prior art, this method has the following significant advantages: ① Accurate quantification: It can perform real-time quantitative analysis of nucleic acid templates to ensure the accuracy and reliability of the results. ② Closed reaction system: It adopts a fully closed reaction design, and does not require subsequent PCR processing steps, effectively reducing the risk of contamination and false positive rate. ③ Automated operation: It realizes automatic detection and recording of the fluorescence signal throughout the process, and the results are intuitively displayed to avoid errors caused by human subjective judgment. ④ High efficiency and high throughput: It has rapid detection capabilities, can meet the needs of high-throughput sample detection, and significantly improves work efficiency. Through these optimized characteristics, this method provides an efficient, accurate, and low-pollution solution for nucleic acid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 These are the results of genetic evolution analysis of 15 ARV genotype I strains, 15 ARV genotype I subtype strains, 15 genotype II strains, 15 genotype VI strains, 1 genotype III strain, 1 genotype IV strain, and 1 genotype V strain.

[0025] Figure 2 It is the result of quadruple fluorescence quantitative detection of ARV-positive cultures of genotype I S1133 strain, genotype I subtype GD / SB / 202301 strain, genotype II HN / WLK / 202305 strain, and genotype VI FJ / ZYQ / 202402 strain using the first set of primers and probe combinations.

[0026] Figure 3 It is the result of quadruple fluorescence quantitative detection of ARV-positive cultures of genotype I S1133 strain, genotype I subtype GD / SB / 202301 strain, genotype II HN / WLK / 202305 strain, and genotype VI FJ / ZYQ / 202402 strain using the second set of primers and probe combinations.

[0027] Figure 4It is the result of quadruple fluorescence quantitative detection of ARV-positive cultures of genotype I S1133 strain, genotype I subtype GD / SB / 202301 strain, genotype II HN / WLK / 202305 strain, and genotype VI FJ / ZYQ / 202402 strain using the third set of primers and probe combinations.

[0028] Figure 5 This is a graph showing the results of PCR amplification of four recombinant standard plasmids, where M: DNA molecular weight standard (DL2000); 1: standard plasmid for genotype I strains; 2: standard plasmid for genotype I subtype strains; 3: standard plasmid for genotype II strains; 4: standard plasmid for genotype VI strains; 5-8: negative controls.

[0029] Figure 6 It is a quadruple fluorescence quantitative PCR standard curve containing standard plasmids of Sigma C genes of genotype I strains, genotype I subtype strains, genotype II strains, and genotype VI strains.

[0030] Figure 7 The results of the VIC channel sensitivity test of ARV positive plasmids containing Sigma C gene of type I strain at different concentrations. The template amounts of 1-10 are 1.0×10 9 , 1.0×10 8 , 1.0×10 7 , 1.0×10 6 , 1.0×10 5 , 1.0×10 4 , 1.0×10 3 , 1.0×10 2 , 1.0×10 1 , 1.0×10 0 copies / reaction; 11: negative control.

[0031] Figure 8 The results of the FAM channel sensitivity test of ARV positive plasmids containing the Sigma C gene of the genotype I subtype strain at different concentrations. The template amounts of 1-10 are 1.0×10 9 , 1.0×10 8 , 1.0×10 7 , 1.0×10 6 , 1.0×10 5 , 1.0×10 4 , 1.0×10 3 , 1.0×10 2 , 1.0×10 1 , 1.0×10 0 copies / reaction; 11: negative control.

[0032] Fig. 9 The results of the sensitivity test of the ARV positive plasmid CY5 channel containing the Sigma C gene of the genotype II strain at different concentrations. The template amounts of 1-10 are 1.0×10 9 , 1.0×10 8 , 1.0×10 7 , 1.0×10 6 , 1.0×10 5 , 1.0×10 4 , 1.0×10 3 , 1.0×10 2 , 1.0×10 1 , 1.0×10 0 copies / reaction; 11: negative control.

[0033] Fig.10 The results of the Texas Red channel sensitivity test of ARV positive plasmids containing the Sigma C gene of the genotype VI strain at different concentrations. The template amounts of 1-10 are 1.0×10 9 , 1.0×10 8 , 1.0×10 7 , 1.0×10 6 , 1.0×10 5 , 1.0×10 4 , 1.0×10 3 , 1.0×10 2 , 1.0×10 1 , 1.0×10 0 copies / reaction; 11: negative control.

[0034] Fig.11 It is the specific test result of the identification detection method of the present invention. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0036] In the present invention, the term "about" or "approximately" should be understood to include all numerical values ​​within the allowed measurement error range.

[0037] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0038] The sources of the reagents used in the following examples are: (1) Cells and viruses Chicken embryo hepatocellular carcinoma cells (LMH); ARV genotype I subtype strain: GD / SB / 202301 (GenBank: PQ490793.1), ARV genotype II strain: HN / WLK / 202305 (GenBank: PQ490783.1); ARV genotype VI strain FJ / ZYQ / 202402 (GenBank: PQ490773.1), ARV genotype III strain: FJ / WCJ / 20230 6 (GenBank: PQ629506.1), ARV genotype IV strain: 4-A5B4 (GenBank: PQ498471.1), ARV genotype V strain: GX / LSQ / 202403 (GenBank: PQ629507.1), fowl adenovirus (FAdV), chicken infectious anemia virus (CIAV), reticuloendotheliosis virus (REV), avian leukosis virus (ALV) were all identified and preserved by South China Agricultural University. Genotype I S1133 strain, H9 subtype avian influenza virus (AIV H9), Newcastle disease virus (NDV), infectious bursal virus (IBDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), Marek's disease virus (MDV) were all obtained from commercial sources.

[0039] (2) Viral nucleic acid extraction kit The nucleic acid extraction kit used in the present invention is a virus nucleic acid extraction kit (magnetic bead method) produced by Guangdong Biaoyun Biotechnology Co., Ltd. (Cat. No.: CPWG003).

[0040] Example:

[0041] Example 1 Establishment of a fluorescent quantitative PCR method for distinguishing and detecting chicken reovirus genotype I, type I subtype, type II and type VI strains

[0042] 1. Design and synthesis of primers and probes 1. Sequence alignment By consulting the literature and downloading the whole genome sequences of 60 strains from the GenBank database, including 15 ARV genotype I strains, 15 ARV genotype I subtype strains, 15 genotype II strains, and 15 genotype VI strains, the results showed that the 60 reference sequences were in four different branches: genotype I (small dotted line), genotype I subtype (solid line), genotype II (bold solid line), and genotype VI (large dotted line). Figure 1). Sequence alignment analysis was performed using SnapGene software, and the results showed that the conserved sequence segments of the Sigma C gene shared by different genotype strains were the same among ARV genotype I strains, genotype I subtype strains, genotype II strains, and genotype VI strains, and could be used to design four sets of primer pairs and probes, respectively. The information of the aforementioned 60 strains is as follows.

[0043] The information of the above 60 strains is as follows: 1. ARV genotype I strains: S1133 (GenBank: OR612119.1), 1733 (GenBank: KF741712.1), 2048 (GenBank: AF204945.1), LN09-1 (GenBank: KP288843.1), GX110116 (GenBank: KF741752.1), SD-2010-0085 (GenBank: KC963039.1), HB10-1 (GenBank: KP288833.1), SD09-1 (GenBank: KP288843.1). GenBank: KP288853.1), BJ10-1 (GenBank: KP288867.1), GX / 2010 / 1 (GenBank: KJ476705.1), GuangxiR1 (GenBank: KC183744.1 ), LN10-1 (GenBank: KP288870.1), HB06 (GenBank: EU526387.1), 141045 (GenBank: OR612109.1), B-98 (GenBank: DQ643974.1).

[0044] 2. ARV genotype I subtype strains: SDJN01 (GenBank: ON807302.1), GDLWA23 (GenBank: PP505765.1), HZJY / 170308 (GenBank: MK189474.1), REO / Broiler / SDWF01 / 22 (GenBank: OR836582.1), GDLJW23 (GenBank: PP505762.1), FJCQF23 (GenBank: PP505750.1), REO / Broiler / SDRZ01 / 21 (GenBank: OR836581.1), FJCFF23 (GenBank: PP505749.1), GDHWZ23 (GenBank: PP505757.1), Reo / Broiler / YTLY / 161021 (GenBank: MK189465.1), Reo / Breeder / BZHM / 150814 (GenBank: MK189483.1); FJCYB23 (GenBank: PP505751.1); GDBC8A23 (GenBank: PP505763.1), JXTDY23 (GenBank: PP505761.1), GD / SB / 202301 (GenBank: PQ490793.1).

[0045] 3. ARV genotype II strains: REO / Broiler / SDYT06 / 23 (GenBank: OR836587.1), REO / Broiler / SDYT03 / 23 (GenBank: OR836585.1), REO / Broiler / SDYT04 / 23 (GenBank: OR836586.1), REO / Broiler / SDWF02 / 21 (GenBank: OR836583.1), PHC-2020-0545 (GenBank: MW174790.1), GF20_4-1_a (GenBank: LC604645.1), JS-202011786 (GenBank: ON262177.1), FJ-202010797 (GenBank: ON262174.1), SD-201911535 (GenBank: ON262173.1), SD-202011003 (GenBank: ON262176.1), ISR / 5050 / 2022 (GenBank: OQ939603.1), ARV / ck / Israel / 406276 / 2021 (GenBank: OR233084.1), ISR / 4313 / 2021 (GenBank: OR019796.1), FJ-202010829 (GenBank: ON262175.1), HN / WLK / 202305 (GenBank: PQ490783.1).

[0046] 4. ARV gene type VI strains: GF20_3-7_b (GenBank: LC604648.1), GF20_4-7_b (GenBank: LC604649.1), LINA019-S1 / China / 2023 (GenBank: PQ249300.1), SD-20207277-4 (G enBank: ON262189.1), GF20_3-4_a (GenBank: LC604647.1), SD-201913077 (GenBank: ON262191.1), REO / Broiler / SDWF04 / 22 (GenBank: OR836594.1), REO / Bro iler / SDWF05 / 22 (GenBank: OR836595.1), SD-20209560 (GenBank: ON262190.1), 918 (GenBank: AF297215.1), 1017-1 (GenBank: AF297216.1), D3386 / 1 / 5 / 16SA (GenBank: OP816635.1), ISR / 7993 / 2015 (GenBank: OQ939623.1), Reo / PA / Broiler / 03200 / 12 (GenBank: KP727785.1), FJ / ZYQ / 202402 (GenBank: PQ490773.1).

[0047] 2. Primer and probe design Based on the above sequence alignment results, the common fragments of the Sigma C gene of ARV genotype I strains, ARV genotype I subtype strains, genotype II strains and genotype VI strains were selected. Primers and probes were designed for the Sigma C gene of ARV genotype I strains, genotype I subtype strains, genotype II strains and genotype VI strains, as well as standard plasmids of the four genotype strains using primer and probe design software. At the same time, the annealing temperature, length, CG content of primers and probes, the selection of fluorescent groups and quenching groups of probes, and the mutual interference between primers and probes should be fully considered. Finally, the designed primers and probes were sequenced in the GenBank database to ensure their specificity and amplification efficiency.

[0048] The information of primers and probes is shown in Table 1, which were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The primers used for ARV genotype I strains are as follows: The primer pair ARV1-qF1 and ARV1-qR1 can be used to amplify all ARV genotype I strains used for sequence alignment, and the probe ARV1-qP1 emits VIC fluorescence; The primer pair ARV1-qF2 and ARV1-qR2 can be used to amplify all ARV genotype I strains used for sequence alignment, and the probe ARV1-qP2 emits VIC fluorescence; The primer pair ARV1-qF3 and ARV1-qR3 can be used to amplify all ARV genotype I strains used for sequence alignment, and emit VIC fluorescence through the probe ARV1-qP3.

[0049] The primers and probes used for ARV genotype I subtype strains are as follows: The primer pair ARV1-sub-qF1 and ARV1-sub-qR1 can be used to amplify all ARV genotype I subtype strains used for sequence alignment, and the probe ARV1-sub-qP1 emits FAM fluorescence; The primer pair ARV1-sub-qF2 and ARV1-sub-qR2 can be used to amplify all ARV genotype I subtype strains used for sequence alignment, and the probe ARV1-sub-qP2 emits FAM fluorescence; The primer pair ARV1-sub-qF3 and ARV1-sub-qR3 can be used to amplify all ARV genotype I subtype strains used for sequence alignment, and the probe ARV1-sub-qP3 emits FAM fluorescence.

[0050] The primers and probes used for ARV genotype II strains are as follows: The primer pair ARV2-qF1 and ARV2-qR1 can be used to amplify all ARV genotype II strains used for sequence alignment, emitting CY5 fluorescence through the probe ARV2-qP1; The primer pair ARV2-qF2 and ARV2-qR2 can be used to amplify all ARV genotype II strains used for sequence alignment, emitting CY5 fluorescence through the probe ARV2-qP2; The primer pair ARV2-qF3 and ARV2-qR3 can be used to amplify all ARV genotype II strains used for sequence alignment, and the probe ARV2-qP3 emits CY5 fluorescence.

[0051] The primers and probes used for ARV genotype VI strains are as follows: The primer pair ARV6-qF1 and ARV6-qR1 can be used to amplify all ARV genotype VI strains used for sequence alignment, and emits Texas Red fluorescence through the probe ARV6-qP1; The primer pair ARV6-qF2 and ARV6-qR2 can be used to amplify all ARV genotype VI strains used for sequence alignment, and emits Texas Red fluorescence through the probe ARV6-qP2; The primer pair ARV6-qF3 and ARV6-qR3 can be used to amplify all ARV genotype VI strains used for sequence alignment, and emits Texas Red fluorescence through the probe ARV6-qP3.

[0052] Through experimental screening and verification, primers and probes for the identification and detection of ARV genotype I strains, ARV genotype I subtype strains, genotype II strains and genotype VI strains were finally obtained.

[0053] Table 1 Primer and probe sequence information

[0054] Note: “N” stands for no data (no fluorescent molecules).

[0055] 3. Establishment of a fluorescence quantitative PCR method for the identification and detection of ARV genotype I, genotype I subtypes, genotype II, and genotype VI strains 1. Sample processing and genomic RNA extraction Cell culture, tissue organs, anal swabs and other biological materials containing ARV genotype I, genotype I subtype, genotype II and genotype VI strains were pre-treated according to the instructions of the viral nucleic acid extraction kit (magnetic bead method). For tissue samples, it is necessary to add an appropriate amount of physiological saline to mix, fully grind, centrifuge at 12000 xg for 5-10 min, and take the supernatant for extraction. For swab samples, it is necessary to add an appropriate amount of sample preservation solution or physiological saline to ensure that the solution can immerse the swab brush head, vortex vigorously for 1 min after soaking, and take the supernatant for extraction. The nucleic acid extraction method refers to the instructions of the Virus Nucleic Acid Extraction Kit (Magnetic Bead Method) of Guangdong Biaoyun Biotechnology Co., Ltd., and the nucleic acid extraction is performed using the Biori NPA-32 series fully automatic nucleic acid extraction and purification instrument. The specific experimental steps include: First, take out the pre-packed reagent plate, invert and mix several times to resuspend the magnetic beads. The reagents and magnetic beads are concentrated to the bottom of the well plate by shaking by hand or using a 96-well plate centrifuge for short centrifugation. Then carefully tear off the aluminum foil of the reagent plate, avoid vibration, prevent the reagent from splashing, and confirm the direction of the reagent plate. Then add 400 μL of sample to the sample well of the reagent plate. Then load the reagent plate into the fully automatic nucleic acid extraction and purification instrument and install the magnetic rod sheath. Perform the automated extraction experiment according to the instrument's recommended procedure. After the automated procedure is completed, transfer the eluted product in the nucleic acid mobile phone well to a nuclease-free centrifuge tube and store it at -20°C or below.

[0056] 2. Quadruple fluorescence quantitative PCR reaction system and conditions Take the HifairV C58P2 Multiplex One Step RT-qPCR Probe Kit (UDGPlus) of Yisheng Biotechnology as an example. The reaction system is shown in Table 2 and is prepared at 20 μL / tube, including 16 μL of amplification reagents containing primers, probes, and qPCR enzymes, and 4 μL of template.

[0057] Table 2 Fluorescence quantitative PCR reaction system

[0058] The reaction program was set on a fluorescence quantitative PCR instrument (ABI 7500Fast) according to Table 3; the fluorescence detection channels were selected as FAM, CY5, VIC and Texas Red. The specific detection channel settings can be operated according to the instruction manual of each instrument.

[0059] Table 3 Fluorescence quantitative PCR reaction program setting parameters

[0060] Example 2 Validation of the Fluorescence Quantitative PCR Method for Identifying and Detecting ARV Genotype I, Genotype I Subtypes, Genotype II, and Genotype VI Strains

[0061] ARV genotype I strain (S1133), genotype I subtype strain (GD / SB / 202301), genotype II strain (HN / WLK / 202305) and genotype VI strain (FJ / ZYQ / 202402) were infected with LMH cell culture, and genomic RNA and negative control samples (nuclease-free water) were extracted. Quadruple qPCR detection was performed using the method of Example 1 to verify the effectiveness of the positive control.

[0062] When the quadruple qPCR assay was performed using primer and probe combinations of ARV1-qF1, ARV1-qR1, ARV1-qP1, ARV1-sub-qF1, ARV1-sub-qR1, ARV1-sub-qP1, ARV2-qF1, ARV2-qR1, ARV2-qP1, ARV6-qF1, ARV6-qR1, and ARV6-qP1, the test results were as follows Figure 2As shown, the Ct value of the VIC fluorescence channel of the ARV genotype I strain S1133 sample is 16.69, and there is an obvious amplification curve; while the FAM, CY5, and Texas Red fluorescence channels have no Ct values. The Ct value of the FAM fluorescence channel of the genotype I subtype strain GD / SB / 202301 sample is 23.49, and there is an obvious amplification curve; while the VIC, CY5, and Texas Red fluorescence channels have no Ct values. The Ct value of the CY5 fluorescence channel of the genotype II strain HN / WLK / 202305 sample is 11.85, and there is an obvious amplification curve; while the FAM, VIC, and Texas Red fluorescence channels have no Ct values. The Ct value of the Texas Red fluorescence channel of the genotype VI strain FJ / ZYQ / 202402 sample is 15.11, and there is an obvious amplification curve; while the FAM, VIC, and CY5 fluorescence channels have no Ct values. There were no Ct values ​​in the four fluorescence channels of VIC, FAM, CY5 and Texas Red for ARV genotype III strain FJ / WCJ / 202306, ARV genotype IV strain 4-A5B4, ARV genotype V strain GX / LSQ / 202403 and negative control samples.

[0063] When the quadruple qPCR assay was performed using primer and probe combinations of ARV1-qF2, ARV1-qR2, ARV1-qP2, ARV1-sub-qF2, ARV1-sub-qR2, ARV1-sub-qP2, ARV2-qF2, ARV2-qR2, ARV2-qP2, ARV6-qF2, ARV6-qR2, and ARV6-qP2, the test results were as follows Figure 3As shown, the Ct value of the VIC fluorescence channel of the ARV genotype I strain S1133 sample was 19.91, the Ct value of the FAM fluorescence channel was 35.83, and there was an obvious amplification curve; while there were no Ct values ​​for the CY5 and Texas Red fluorescence channels. The Ct value of the FAM fluorescence channel of the ARV genotype I subtype strain GD / SB / 202301 sample was 26.2, the Ct value of the CY5 fluorescence channel was 36.87, and there was an obvious amplification curve; while there were no Ct values ​​for the VIC and Texas Red fluorescence channels. The Ct value of the CY5 fluorescence channel of the ARV genotype II strain HN / WLK / 202305 sample was 19.09, the Ct value of the VIC fluorescence channel was 33.51, and there was an obvious amplification curve; while there were no Ct values ​​for the FAM and Texas Red fluorescence channels. The Ct value of the Texas Red fluorescence channel of the ARV gene type VI strain FJ / ZYQ / 202402 sample was 9.03, the Ct value of the VIC fluorescence channel was 28.92, and the Ct value of the CY5 fluorescence channel was 22.49, and there was an obvious amplification curve; while there was no Ct value in the FAM fluorescence channel. The Ct value of the Texas Red fluorescence channel of the ARV gene type IV strain 4-A5B4 sample was 34.26, and the Ct value of the CY5 fluorescence channel of the ARV gene type V strain GX / LSQ / 202403 sample was 36.35, while the VIC, FAM, CY5 and Texas Red fluorescence channels of the ARV gene type III strain FJ / WCJ / 202306 sample and the negative control sample had no Ct value.

[0064] When the quadruple qPCR assay was performed using primer and probe combinations of ARV1-qF3, ARV1-qR3, ARV1-qP3, ARV1-sub-qF3, ARV1-sub-qR3, ARV1-sub-qP3, ARV2-qF3, ARV2-qR3, ARV2-qP3, ARV6-qF3, ARV6-qR3, and ARV6-qP3, the test results were as follows Figure 4As shown, the Ct value of the VIC fluorescence channel of the ARV genotype I strain S1133 sample was 24.51, the Ct value of the FAM fluorescence channel was 29.33, and there was an obvious amplification curve; while there were no Ct values ​​for the CY5 and Texas Red fluorescence channels. The Ct value of the FAM fluorescence channel of the ARV genotype I subtype strain GD / SB / 202301 sample was 24.82, the Ct value of the CY5 fluorescence channel was 38.23, and there was an obvious amplification curve; while there were no Ct values ​​for the VIC and Texas Red fluorescence channels. The Ct value of the CY5 fluorescence channel of the ARV genotype II strain HN / WLK / 202305 sample was 18.89, the Ct value of the VIC fluorescence channel was 33.06, and there was an obvious amplification curve; while there were no Ct values ​​for the FAM and Texas Red fluorescence channels. The Ct value of the Texas Red fluorescence channel of the ARV gene type VI strain FJ / ZYQ / 202402 sample was 15.82, the Ct value of the FAM fluorescence channel was 37.93, and there was an obvious amplification curve; while the VIC and CY5 fluorescence channels had no Ct values. The Ct value of the Texas Red fluorescence channel of the ARV gene type V strain GX / LSQ / 202403 sample was 30.83; while the VIC, FAM, CY5 and Texas Red fluorescence channels of the ARV gene type III strain FJ / WCJ / 202306, the ARV gene type IV strain 4-A5B4 and the negative control sample had no Ct values.

[0065] It can be seen that when the ARV1-qF1, ARV1-qR1, ARV1-qP1, ARV1-sub-qF1, ARV1-sub-qR1, ARV1-sub-qP1, ARV2-qF1, ARV2-qR1, ARV2-qP1, ARV6-qF1, ARV6-qR1, ARV6-qP1 primer and probe combination is used for quadruple qPCR detection, for ARV genotype I strain samples: the Ct value of VIC is ≤40, while the three fluorescence channels of FAM, CY5, and TexasRed have no Ct value; for ARV genotype I subtype strain samples: the Ct value of FAM is ≤40, while the three fluorescence channels of VIC, CY5, and TexasRed have no Ct value; for ARV genotype II strain samples: the Ct value of CY5 is ≤40, while the three fluorescence channels of VIC, FAM, and TexasRed have no Ct value. There were no Ct values ​​in the three fluorescence channels of Red; the Ct values ​​of Texas Red of ARV genotype VI strain samples were all ≤40, while there were no Ct values ​​in the three fluorescence channels of VIC, FAM, and CY5; the Ct values ​​of VIC, FAM, CY5, and Texas Red of ARV genotype III strain FJ / WCJ / 202306, ARV genotype IV strain 4-A5B4, ARV genotype V strain GX / LSQ / 202403, and negative control samples were all ≤40. Red has no Ct value; this is in line with the judgment criteria for differential diagnosis of ARV genotype I, genotype I subtype, genotype II and genotype VI strains, and is fully capable of distinguishing the four ARV genotype strains. The method of Example 1 and the primer and probe combinations of ARV1-qF1, ARV1-qR1, ARV1-qP1, ARV1-sub-qF1, ARV1-sub-qR1, ARV1-sub-qP1, ARV2-qF1, ARV2-qR1, ARV2-qP1, ARV6-qF1, ARV6-qR1, ARV6-qP1 can be used to identify and detect other ARV genotype I, genotype I subtype, genotype II and genotype VI strains.

[0066] Example 3 Sensitivity test

[0067] 1. Preparation of recombinant plasmid standards (1) Screening the conserved region of the Sigma C gene of ARV genotype I strains, designing the standard plasmid primer pair of the Sigma C gene of ARV-1, named ARV1-F (SEQ ID NO.10) and ARV1-R (SEQ ID NO.11) as shown in Table 1. The RNA of ARV genotype I strains was extracted, and the fragment size of RT-PCR amplification was 563 bp ( Figure 5), recover the positive amplification product, clone the positive amplification product into the puc57 vector, screen the positive recombinant plasmid and send it to Shanghai Biotech Co., Ltd. for sequencing identification. The positive recombinant plasmid with correct sequencing was named puc57-ARV1. The OD260 / OD280 ratio of puc57-ARV1 was 1.89 and the nucleic acid concentration was 120 ng / μL using an ultraviolet spectrophotometer. Referring to the calculation method of plasmid DNA copy number, the concentration of puc57-ARV1 plasmid DNA solution was calculated to be 4.04×10 10 copies / μL.

[0068] (2) Screening the conserved region of the Sigma C gene of ARV genotype I subtype strains, designing the standard plasmid primer pair of the Sigma C gene of ARV-1-sub, named ARV1-sub-F (SEQ ID NO.21) and ARV1-sub-R (SEQ ID NO.22) as shown in Table 1. The RNA of ARV genotype I subtype strains was extracted, and the fragment size of RT-PCR amplification was 531 bp ( Figure 5 ), recover the positive amplification product, clone the positive amplification product into the puc57 vector, screen the positive recombinant plasmid and send it to Shanghai Biotech Co., Ltd. for sequencing identification. The positive recombinant plasmid with correct sequencing was named puc57-ARV1-sub. The OD260 / OD 280 ratio of puc57-ARV1-sub was 1.92 and the nucleic acid concentration was 109 ng / μL using an ultraviolet spectrophotometer. Referring to the calculation method of plasmid DNA copy number, the concentration of puc57-ARV1-sub plasmid DNA solution was calculated to be 3.07×10 10 copies / μL.

[0069] (3) Screening the conserved region of the Sigma C gene of ARV genotype II strains, designing the standard plasmid primer pair of the Sigma C gene of ARV-2, named ARV2-F (SEQ ID NO.32) and ARV2-R (SEQ ID NO.33) as shown in Table 1. The RNA of ARV genotype II strains was extracted, and the fragment size of RT-PCR amplification was 521 bp ( Figure 5 ), recover the positive amplification product, clone the positive amplification product into the puc57 vector, select the positive recombinant plasmid and send it to Shanghai Biotech Co., Ltd. for sequencing identification. The positive recombinant plasmid with correct sequencing was named puc57-ARV2. The OD260 / OD280 ratio of puc57-ARV2 was 1.89 and the nucleic acid concentration was 113 ng / μL using an ultraviolet spectrophotometer. Referring to the calculation method of plasmid DNA copy number, the concentration of puc57-ARV2 plasmid DNA solution was calculated to be 3.8×1010 copies / μL.

[0070] (4) The conserved region of the Sigma C gene of ARV genotype VI strains was screened, and the standard plasmid primer pairs of the Sigma C gene of ARV-6 were designed, which were named ARV6-F (SEQ ID NO.43) and ARV6-R (SEQ ID NO.44) as shown in Table 1. RNA of ARV genotype VI strains was extracted, and the fragment size of RT-PCR amplification was 626 bp ( Figure 5 ), recover the positive amplification product, clone the positive amplification product into the puc57 vector, screen the positive recombinant plasmid and send it to Shanghai Biotech Co., Ltd. for sequencing identification. The positive recombinant plasmid with correct sequencing was named puc57-ARV6. The OD260 / OD280 ratio of puc57-ARV6 was 1.95 and the nucleic acid concentration was 113 ng / μL using an ultraviolet spectrophotometer. Referring to the calculation method of plasmid DNA copy number, the concentration of puc57-ARV6 plasmid DNA solution was calculated to be 3.8×10 10 copies / μL.

[0071] 2. Fluorescence quantitative PCR standard curve and sensitivity detection First, the four recombinant standard plasmids (puc57-ARV1, puc57-ARV1-sub, puc57-ARV2, and puc57-ARV6) were diluted with nuclease-free water so that the copy number of each plasmid was 10 9 The number of copies / 4 μL was then diluted 10-fold with nuclease-free water, with 10 copies per 4 μL. 0 , 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 and 10 8 For the template sample with the same copy number, three replicates were set for each dilution, and a negative control (nuclease-free water) was set. The program was set in the fluorescence quantitative PCR instrument according to the reaction conditions of the method established in Example 1 for detection.

[0072] The standard curve of the quadruple fluorescence quantitative PCR was established as follows Figure 6 As shown, the standard curve of fluorescent quantitative PCR for genotype I is y = -3.5257x + 40.942 (R 2 =0.9987, and the standard curve of fluorescent quantitative PCR for genotype I subtype was y = -3.5812x + 41.326 (R 2=0.9982, the standard curve of genotype II fluorescence quantitative PCR is y = -3.4012x +40.411 (R 2 = 0.9975), the standard curve of fluorescent quantitative PCR for genotype VI is y = -3.4668x + 40.388 (R 2 = 0.9977). In summary, the correlation coefficients (R 2 ) were between 0.9975 and 0.9987, and the amplification efficiency (E) was between 90% and 110%, indicating that the method had a good linear relationship.

[0073] The sensitivity test results of the four recombinant standard plasmids (puc57-ARV1, puc57-ARV1-sub, puc57-ARV2, and puc57-ARV6) are as follows: Figure 7 , 8 , 9, and 10 show that for standards containing 9 different concentrations of 4 recombinant standard plasmids, the corresponding Ct values ​​of the four fluorescence channels of VIC, FAM, CY5, and Texas Red were all ≤40, and the results were all positive with obvious amplification curves; the minimum template concentration that can be detected for the 4 genotype strains is 10 viral copies. The minimum detection limit of mPCR for ARV-I, ARV-I-sub, and ARV-II in the Chinese patent application number 202210151323.5 is 10 2 , 10 2 and 10 1 Virus copies, which shows that the present invention has higher sensitivity.

[0074] Example 4: Specificity test

[0075] The cDNA of 6 different genotypes of ARV (ARV-I, ARV-I-sub, ARV-Ⅱ, ARV-III, ARV-IV, ARV-V and ARV-VI) and other avian virus genomes (DNA of Marek's disease virus (MDV), infectious laryngotracheitis virus (ILTV), fowl adenovirus (FAdV), chicken infectious anemia virus (CIAV) or cDNA of infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), avian influenza virus (AIV), infectious bronchitis virus (IBV), reticuloendotheliosis virus (REV), avian leukosis virus (ALV)) were used as templates to perform quadruple fluorescence quantitative PCR amplification under the above reaction conditions to detect the specificity of the method. Each sample was repeated 3 times, and a negative control was set up at the same time.

[0076] The results are as follows Fig.11As shown, the method of the present invention can only detect 4 ARV genotypes (genotype I, genotype I subtype, genotype II, genotype VI) strains, and has no cross reaction with other viruses, and has good specificity.

[0077] Example 5: Repeatability test

[0078] According to the method of diluting the standard plasmids in Example 3, the four standard plasmids (puc57-ARV1, puc57-ARV1-sub, puc57-ARV2, puc57-ARV6) were diluted with 10 6 and 10 4 Two concentrations of copy number / 4 μL were used as templates, and the two dilutions were tested three times at different times. The same template was tested three times at the same time each time, and three replicate wells were set. According to the reaction system and reaction procedure of Example 1, quadruple fluorescence quantitative PCR test was carried out to analyze the stability of the method.

[0079] The results are shown in Table 4. The intra-assay and inter-assay coefficients of variation were both less than 2%, indicating that the quadruple fluorescence quantitative PCR method established in the present invention has good repeatability and stability, and the results are stable and reliable (Table 4).

[0080] Table 4 Repeatability results of the quadruple fluorescence quantitative PCR method

[0081] Example 6: Detection of clinical samples

[0082] Twenty-five 28-day-old SPF chickens were randomly divided into 5 groups, 5 in each group, and ARV genotype I strain (S1133), genotype I subtype strain (GD / SB / 202301), genotype II strain (HN / WLK / 202305) and genotype VI strain (FJ / ZYQ / 202402) were respectively injected at 10 6 TCID 50 The dose was injected into SPF chickens through the foot pad, and a blank control group without virus challenge was set up. Ten days after the virus challenge, three samples of anal swabs, tendons and cecal tonsils were collected from each group of SPF chickens, as well as LMH cell culture samples of vaccine strain S1133 and isolated strains GD / SB / 202301, HN / WLK / 202305, and FJ / ZYQ / 202402, a total of 79 samples, and viral genomic RNA was extracted according to the instructions of the nucleic acid extraction kit.

[0083] The test results are shown in Table 5. For each type of sample after ARV genotype I strain, genotype I subtype strain, genotype II strain and genotype VI strain infected SPF chicken, the quadruple fluorescence quantitative PCR method established by the present invention can effectively identify and detect. The method of the present invention can sensitively, quickly, accurately and specifically identify and detect ARV genotype I strain, genotype I subtype strain, genotype II strain and genotype VI strain in samples such as cell culture, anal swabs and tissues.

[0084] Table 5 Test results of clinical samples

[0085] Note: “-” represents no Ct value or Ct value > 40.

[0086] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A PCR primer set for detecting and / or distinguishing avian reovirus, characterized in that: The primer set includes at least one of the following (a)-(d): (a) The first primer set includes: The upstream primer ARV1-qF1 having a nucleotide sequence as shown in SEQ ID NO.1; Downstream primer ARV1-qR1 having a nucleotide sequence as shown in SEQ ID NO.2; The first probe; (b) The second primer set includes: The upstream primer ARV1-sub-qF1 having a nucleotide sequence as shown in SEQ ID NO.12; Downstream primer ARV1-sub-qR1 having a nucleotide sequence as shown in SEQ ID NO.13; The second probe; (c) The third primer set includes: The upstream primer ARV2-qF1 having a nucleotide sequence as shown in SEQ ID NO.23; Downstream primer ARV2-qR1 having a nucleotide sequence as shown in SEQ ID NO.24; The third probe; (d) The fourth primer set includes: The upstream primer ARV6-qF1 whose nucleotide sequence is shown in SEQ ID NO.34; Downstream primer ARV6-qR1 having a nucleotide sequence as shown in SEQ ID NO.35; The fourth probe.

2. The PCR primer set according to claim 1, characterized in that: The avian reovirus includes at least one of ARV genotype I, ARV genotype I subtype, ARV genotype II, and ARV genotype VI.

3. The PCR primer set according to claim 2, characterized in that: The first primer group is used to detect and / or distinguish avian reovirus ARV genotype I, the second primer group is used to detect and / or distinguish avian reovirus ARV genotype I subtypes, the third primer group is used to detect and / or distinguish avian reovirus ARV genotype II, and the fourth primer group is used to detect and / or distinguish avian reovirus ARV genotype VI.

4. The PCR primer set according to claim 1, characterized in that: The first probe, the second probe, the third probe and the fourth probe are nucleic acid probes for detecting target nucleic acid sequences; the first probe, the second probe, the third probe and the fourth probe are connected with fluorescent groups and quenching groups; the fluorescent groups connected with the first probe, the second probe, the third probe and the fourth probe are different.

5. The PCR primer set according to claim 4, characterized in that: The fluorescent groups include: FAM, HEX, TET, CY3, CY5, CY7, ROX, JOE, Texas Red, Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 647, VIC, NED, TAMRA, AMCA or fluorescein amide.

6. The PCR primer set according to claim 4, characterized in that: The quenching groups include: BHQ1, BHQ2, BHQ3, DABCYL, Iowa Black FQ, Iowa Black RQ, TQ3 or Eclipse.

7. The PCR primer set according to claim 4, characterized in that: The first probe is: 5'-VIC-CCGCGTGCGCAGAGGATSTGTT-MGB-3'; And / or, the second probe is: 5'-FAM-CTTCTGCTCRCAACGAGTCTCYTTGA-BHQ1-3'; And / or, the third probe is: 5'-CY5-CTGAAAMCCGGCSCGAGGCAC-BHQ3-3'; And / or, the fourth probe is: 5'-Texas Red-CCGGRAAHGTCGCCTGCTGRAA-BHQ2-3'.

8. A reagent for detecting and / or distinguishing avian reovirus, characterized in that: The reagent contains the PCR primer set according to any one of claims 1 to 7.

9. Use of the PCR primer set according to any one of claims 1 to 7 in the preparation of a reagent for detecting and / or distinguishing avian reovirus.

10. A method for detecting and / or distinguishing avian reovirus for non-diagnostic purposes, characterized in that: The following steps are involved: S1. Extract avian reovirus genomic RNA; S2, mixing the PCR primer set according to any one of claims 1 to 7 and the avian reovirus genomic RNA described in step S1, and performing fluorescent quantitative PCR; S3. Detecting and / or distinguishing avian reovirus according to the fluorescent signal obtained by the fluorescent quantitative PCR in step S2.

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