A PCR primer set for detecting and differentiating avian reovirus and its application

Abstract

The present invention discloses a PCR primer set for detecting and differentiating avian reovirus and its application, belonging to the technical field of molecular biology. The present invention provides PCR primers for detecting and / or differentiating avian reovirus. The primer set has good specificity for chicken reovirus and no cross-reaction with several other major poultry diseases. The sensitivity test results show that it is about 10 times higher than the conventional PCR method. There is no specific amplification for other common poultry viruses, and no cross-reaction is found. The within-batch and between-batch coefficient of variation are both less than 2%. The detection method of the present invention has the advantages of high sensitivity, strong specificity, and good repeatability.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and particularly relates to a PCR primer set for detecting and differentiating avian reovirus and its application. Background Art

[0002] Avian Reovirus (ARV) can cause viral arthritis, intestinal diseases and growth retardation syndrome, and lead to immunosuppression, enhancing the susceptibility of chicken flocks to secondary diseases. In recent years, the prevalence of this disease has shown an upward trend in China, resulting in low feed conversion rate, increased dead, culled and disabled chickens, reduced production efficiency, and causing serious economic losses to the poultry farming industry. Due to the complexity of the multi-segment gene fragments of ARV, it is easier to generate gene rearrangement and intragenic recombination phenomena, thus leading to the emergence of ARV variant strains. Currently, the widely used commercial vaccines (such as S1133, ZJS, etc.) are all genotype I, but multiple genotype strains are prevalent in China, resulting in unsatisfactory clinical immunoprotective effects and bringing huge challenges to the prevention and control of this disease.

[0003] The ARV virion is spherical, an envelopeless double-stranded RNA virus, and its viral genome consists of 10 segments of double-stranded RNA, which are divided into 3 groups according to their sizes: L (L1, L2, L3), M (M1, M2, M3), S (S1, S2, S3, S4). Among them, the σC protein encoded by the S1 gene is a highly variable protein, which can induce the body to produce type-specific neutralizing antibodies, is related to the virulence and infectivity of the virus, and is the main basis for typing ARV epidemic strains. ARV is prone to gene mutation or recombination to produce new strains. Based on the genetic evolutionary analysis results of the σC gene, ARV can be divided into 6 genotypes, and there are significant differences in immunogenicity and virulence among different genotype strains. In recent years, epidemiological investigations have found that genotype I, genotype I subtype, genotype II and genotype VI are the dominant genotypes of ARV prevalence in China. Therefore, establishing an efficient, highly sensitive and specific differential diagnosis method is of great significance for the prevention and control of viral arthritis diseases.

[0004] Currently, the methods for detecting ARV pathogens include virus isolation and identification, serology, ordinary PCR, and universal fluorescence quantitative PCR, etc., but these methods have low clinical detection efficiency and are time-consuming and laborious. The patent of 202210151323.5 discloses a multiplex PCR kit for detecting or differentiating 4 different genotypes of avian reovirus (genotype I, genotype I subtype, genotype II and genotype V) and its application. However, the multiplex PCR detection method involved in this technical solution has problems of high detection limit and low sensitivity. Summary of the Invention

[0005] [Technical Problem]

[0006] The technical problem to be solved by the present invention is to provide a PCR primer set with reduced detection limit and increased sensitivity, which can effectively detect and / or distinguish avian reovirus.

[0007] Technical solution

[0008] To solve the above technical problem, the present invention provides the following technical solution:

[0009] 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):

[0010] (a) The first primer set includes:

[0011] The upstream primer ARV1-qF1 with the nucleotide sequence shown in SEQ ID NO.1;

[0012] The downstream primer ARV1-qR1 with the nucleotide sequence shown in SEQ ID NO.2;

[0013] The first probe;

[0014] (b) The second primer set includes:

[0015] The upstream primer ARV1-sub-qF1 with the nucleotide sequence shown in SEQ ID NO.12;

[0016] The downstream primer ARV1-sub-qR1 with the nucleotide sequence shown in SEQ ID NO.13;

[0017] The second probe;

[0018] (c) The third primer set includes:

[0019] The upstream primer ARV2-qF1 with the nucleotide sequence shown in SEQ ID NO.23;

[0020] The downstream primer ARV2-qR1 with the nucleotide sequence shown in SEQ ID NO.24;

[0021] The third probe;

[0022] (d) The fourth primer set includes:

[0023] The upstream primer ARV6-qF1 with the nucleotide sequence shown in SEQ ID NO.34;

[0024] The downstream primer ARV6-qR1 with the nucleotide sequence shown in SEQ ID NO.35;

[0025] The fourth probe.

[0026] In one embodiment, the first probe, the second probe, the third probe, and the fourth probe are nucleic acid probes for detecting a target nucleic acid sequence; a fluorophore and a quencher are attached to the first probe, the second probe, the third probe, and the fourth probe.

[0027] In one embodiment, the fluorophore 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.

[0028] In one embodiment, the quencher includes: BHQ1, BHQ2, BHQ3, DABCYL, Iowa Black FQ, Iowa Black RQ, TQ3, or Eclipse.

[0029] In one embodiment, the fluorophore is attached to the 5'-end of the probe, and the quencher is attached to the 3'-end of the probe.

[0030] In one embodiment, the fluorophores attached to the first probe, the second probe, the third probe, and the fourth probe are different.

[0031] In one embodiment, the first probe is: 5'-VIC-CCGCGTGCGCAGAGGATSTGTT-MGB-3';

[0032] and / or, the second probe is: 5'-FAM-CTTCTGCTCRCAACGAGTCTCYTTGA-BHQ1-3';

[0033] and / or, the third probe is: 5'-CY5-CTGAAAMCCGGCSCGAGGCAC-BHQ3-3';

[0034] and / or, the fourth probe is: 5'-Texas Red-CCGGRAAHGTCGCCTGCTGRAA-BHQ2-3'.

[0035] 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.

[0036] In one embodiment, the first set of primer sets is used to detect and / or distinguish avian reovirus (ARV) genotype I, the second set of primer sets is used to detect and / or distinguish avian reovirus (ARV) genotype I subtypes, the third set of primer sets is used to detect and / or distinguish avian reovirus (ARV) genotype II, and the fourth set of primer sets is used to detect and / or distinguish avian reovirus (ARV) genotype VI.

[0037] In a second aspect, the present invention also provides a reagent for detecting and / or distinguishing avian reovirus, and the reagent contains the primer sets described in the first aspect.

[0038] In a third aspect, the present invention also provides the use of the primer sets described in the first aspect in the preparation of a reagent for detecting and / or distinguishing avian reovirus.

[0039] In a fourth aspect, the present invention provides a method for non-diagnostically detecting and / or distinguishing avian reovirus, and the method includes the following steps:

[0040] S1. Extract the genomic RNA of avian reovirus;

[0041] S2. Mix the primer sets described in the first aspect and the genomic RNA of avian reovirus in step S1, and perform fluorescence quantitative PCR;

[0042] S3. Detect and / or distinguish avian reovirus according to the fluorescence signal obtained from the fluorescence quantitative PCR in step S2.

[0043] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] (1) According to the sigma C gene sequence, specific amplification primers and probes were designed in the conserved regions of four different genotypes of chicken reovirus (genotype I, genotype I subtype, genotype II, genotype VI), and a multiplex fluorescence quantitative PCR detection method was established for the differential detection of four different genotypes of chicken reovirus (genotype I, genotype I subtype, genotype II, and genotype VI). Compared with other primers and probes, the probe combinations 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) have good specificity for different genotypes of chicken reovirus, showing fluorescence only for the target genotype and Ct values ≤ 40 in the detection of genotype I, genotype I subtype, genotype II, and genotype VI of chicken reovirus.

[0046] (2) The results of the specificity test 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, genotype III strain of ARV, genotype IV strain of ARV, genotype V strain of ARV).

[0047] (3) The results of the sensitivity test showed that the lowest detectable template of the detection method of the present invention was 10 copies, which was about 10 times higher than the conventional PCR method. There was no specific amplification for other common poultry viruses, and no cross-reaction was found. The coefficient of variation within and between batches was less than 2%, indicating that the detection method of the present invention has the advantages of high sensitivity, strong specificity, and good repeatability, and has broad application prospects.

[0048] (4) The real-time fluorescence quantitative PCR detection method of the present invention directly monitors the dynamic changes of fluorescence signals during the PCR amplification process through an optoelectronic conduction system, so as to achieve accurate quantitative analysis. Compared with the prior art, this method has the following significant advantages: ① Precise quantification: It can perform real-time quantitative analysis on nucleic acid templates to ensure the accuracy and reliability of the results. ② Closed reaction system: The whole process is designed with a completely closed reaction, without subsequent PCR processing steps, effectively reducing the risk of contamination and the false positive rate. ③ Automated operation: It realizes the automatic detection and recording of fluorescence signals throughout the process, and the results are intuitively displayed, avoiding errors caused by subjective human judgment. ④ High efficiency and high throughput: It has the ability to quickly detect and can meet the needs of high-throughput sample detection, significantly improving work efficiency. Through these optimized features, this method provides an efficient, accurate, and low-pollution solution for nucleic acid detection. Description of the Drawings

[0049] Figure 1 It is the genetic evolution analysis results of 15 type I ARV gene strains, 15 type I subtype ARV gene strains, 15 type II gene strains, 15 type VI gene strains, 1 type III gene strain, 1 type IV gene strain, and 1 type V gene strain.

[0050] Figure 2 It is the quadruple fluorescence quantitative detection results of ARV positive cultures of type I S1133 strain, type I subtype GD / SB / 202301 strain, type II HN / WLK / 202305 strain, and type VI FJ / ZYQ / 202402 strain using the first set of primer and probe combinations.

[0051] Figure 3 It is the quadruple fluorescence quantitative detection results of ARV positive cultures of type I S1133 strain, type I subtype GD / SB / 202301 strain, type II HN / WLK / 202305 strain, and type VI FJ / ZYQ / 202402 strain using the second set of primer and probe combinations.

[0052] Figure 4 It is the quadruple fluorescence quantitative detection results of ARV positive cultures of type I S1133 strain, type I subtype GD / SB / 202301 strain, type II HN / WLK / 202305 strain, and type VI FJ / ZYQ / 202402 strain using the third set of primer and probe combinations.

[0053] Figure 5 It is the PCR amplification result diagram of four recombinant standard plasmids, where M: DNA molecular weight standard (DL2000); 1: standard plasmid of type I strain; 2: standard plasmid of type I subtype strain; 3: standard plasmid of type II strain; 4: standard plasmid of type VI strain; 5 - 8: negative control.

[0054] Figure 6 It is the quadruple fluorescence quantitative PCR standard curve of standard quality plasmids containing the Sigma C gene of type I strain, type I subtype strain, type II strain, and type VI strain.

[0055] Figure 7 It is the sensitivity test results of the VIC channel of ARV positive plasmids containing the 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 Copy / Reaction; 11: Negative control.

[0056] Figure 8 are the results of the sensitivity test of the FAM channel of the ARV positive plasmid containing the Sigma C gene of different concentrations of genotype I subtype strains. 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 Copy / Reaction; 11: Negative control.

[0057] Figure 9 are the results of the sensitivity test of the CY5 channel of the ARV positive plasmid containing the Sigma C gene of different concentrations of genotype II strains. 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 Copy / Reaction; 11: Negative control.

[0058] Figure 10 are the results of the sensitivity test of the Texas Red channel of the ARV positive plasmid containing the Sigma C gene of different concentrations of genotype VI strains. 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 , 1.0×101 、1.0×10 0 Copy / reaction; 11: Negative control.

[0059] Figure 11 These are the specific test results of the identification and detection method of the present invention. Detailed implementation manners

[0060] The present invention will be further described in detail below in conjunction with the specific implementation manners. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not limit the present invention in any way.

[0061] In the present invention, the term "about" or "approximately" should be understood to include all values within the allowable measurement error range.

[0062] The experimental methods in the following embodiments are all conventional methods, unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources, unless otherwise specified.

[0063] Sources of reagents used in the following embodiments:

[0064] (1) Cells and viruses

[0065] Liver cancer cells of chicken embryos (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 / 202306 (GenBank: PQ629506.1), ARV genotype IV strain: 4-A5B4 (GenBank: PQ498471.1), ARV genotype V strain: GX / LSQ / 202403 (GenBank: PQ629507.1), avian 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 disease virus (IBDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), Marek's disease virus (MDV) were all obtained through commercial channels.

[0066] (2)Virus nucleic acid extraction kit

[0067] The nucleic acid extraction kit used in the present invention: Virus nucleic acid extraction kit (magnetic bead method) (product number: CPWG003) of Guangdong Biaoyun Biotechnology Co., Ltd.

[0068] Example:

[0069] Example 1 Establishment of a fluorescence quantitative PCR method for the differential detection of type I, type I subtype, type II, and type VI strains of avian reovirus genes

[0070] I. Design and synthesis of primers and probes

[0071] (I) Sequence alignment

[0072] By consulting the literature and downloading the complete genome sequences of 15 type I strains of ARV genes, 15 type I subtype strains of ARV genes, 15 type II strains of genes, and 15 type VI strains of genes in the GenBank database, a total of 60 strains. Genetic evolution analysis was carried out using MEGA software. The results showed that the 60 reference sequences were respectively in 4 different branches of type I gene (small dotted line), type I subtype gene (solid line), type II gene (bold solid line), and type VI gene (large dotted line) ( Figure 1 ). Sequence alignment analysis was carried out using SnapGene software. The results showed that according to the type I strains of ARV genes, type I subtype strains of genes, type II strains of genes, and type VI strains of genes, the conserved sequence segments of the common Sigma C gene among different genotype strains were the same and could be used to design four groups of primer pairs and probes respectively. The information of the above 60 strains is as follows.

[0073] The information of the above 60 strains is as follows:

[0074] 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: 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).

[0075] 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).

[0076] 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).

[0077] 4. ARV genotype 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 (GenBank: ON262189.1), GF20_3-4_a (GenBank: LC604647.1), SD-201913077 (GenBank: ON262191.1), REO / Broiler / SDWF04 / 22 (GenBank: OR836594.1), REO / Broiler / 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).

[0078] (II) Primer and Probe Design

[0079] Based on the above sequence alignment results, the common fragment of the Sigma C gene of ARV genotype I strains, ARV genotype I subtype strains, genotype II strains, and genotype VI strains was selected. Primer and probe design software was used to design primers and probes 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 quality plasmids for the 4 genotype strains. At the same time, factors such as the annealing temperature, length, CG content of the primers and probes, the selection of the fluorescent group and quenching group of the probe, and the mutual interference between the primers and probes should be fully considered. Finally, the designed primers and probes were subjected to sequence alignment in the GenBank database to ensure their specificity and amplification efficiency.

[0080] The primer and probe information is shown in Table 1 and was synthesized by Sangon Biotech (Shanghai) Co., Ltd. Among them:

[0081] The primers for ARV genotype I strains are as follows:

[0082] The primer pair ARV1-qF1 and ARV1-qR1 can be used to amplify all type I strains of the ARV gene for sequence alignment, and emit VIC fluorescence through the probe ARV1-qP1;

[0083] The primer pair ARV1-qF2 and ARV1-qR2 can be used to amplify all type I strains of the ARV gene for sequence alignment, and emit VIC fluorescence through the probe ARV1-qP2;

[0084] The primer pair ARV1-qF3 and ARV1-qR3 can be used to amplify all type I strains of the ARV gene for sequence alignment, and emit VIC fluorescence through the probe ARV1-qP3.

[0085] The primers and probes for type I subtype strains of the ARV gene are as follows:

[0086] The primer pair ARV1-sub-qF1 and ARV1-sub-qR1 can be used to amplify all type I subtype strains of the ARV gene for sequence alignment, and emit FAM fluorescence through the probe ARV1-sub-qP1;

[0087] The primer pair ARV1-sub-qF2 and ARV1-sub-qR2 can be used to amplify all type I subtype strains of the ARV gene for sequence alignment, and emit FAM fluorescence through the probe ARV1-sub-qP2;

[0088] The primer pair ARV1-sub-qF3 and ARV1-sub-qR3 can be used to amplify all type I subtype strains of the ARV gene for sequence alignment, and emit FAM fluorescence through the probe ARV1-sub-qP3.

[0089] The primers and probes for type II strains of the ARV gene are as follows:

[0090] The primer pair ARV2-qF1 and ARV2-qR1 can be used to amplify all type II strains of the ARV gene for sequence alignment, and emit CY5 fluorescence through the probe ARV2-qP1;

[0091] The primer pair ARV2-qF2 and ARV2-qR2 can be used to amplify all type II strains of the ARV gene for sequence alignment, and emit CY5 fluorescence through the probe ARV2-qP2;

[0092] The primer pair ARV2-qF3 and ARV2-qR3 can be used to amplify all type II strains of the ARV gene for sequence alignment, and emit CY5 fluorescence through the probe ARV2-qP3.

[0093] The primers and probes for type VI strains of the ARV gene are as follows:

[0094] The primer pair ARV6-qF1 and ARV6-qR1 can be used to amplify all type VI strains of the ARV gene for sequence alignment, and emit Texas Red fluorescence through the probe ARV6-qP1;

[0095] The primer pair ARV6-qF2 and ARV6-qR2 can be used to amplify all type VI strains of the ARV gene for sequence alignment, and emit Texas Red fluorescence through the probe ARV6-qP2;

[0096] The primer pair ARV6-qF3 and ARV6-qR3 can be used to amplify all type VI strains of the ARV gene for sequence alignment, and emit Texas Red fluorescence through the probe ARV6-qP3.

[0097] Through experimental screening and verification, primers and probes for the differential detection of type I strains, type I subtype strains, type II strains, and type VI strains of the ARV gene were finally obtained.

[0098] Table 1 Primer and probe sequence information

[0099]

[0100] Note: "N" represents no data (no fluorescent molecules).

[0101] (III) Establishment of a fluorescence quantitative PCR method for the differential detection of type I, type I subtype, type II, and type VI strains of the ARV gene

[0102] 1. Sample treatment and genomic RNA extraction

[0103] Biological materials such as cell cultures, tissue organs, and anal swabs containing ARV genotype I, genotype I subtypes, genotype II, and genotype VI strains were pretreated for different types of samples according to the instructions of the virus nucleic acid extraction kit (magnetic bead method). For tissue samples, an appropriate amount of normal saline was added and mixed well, ground thoroughly, centrifuged at 12,000 x g for 5 - 10 min, and the supernatant was taken for extraction. For swab samples, an appropriate amount of sample preservation solution or normal saline was added to ensure that the solution could immerse the swab head. After soaking, it was vortexed vigorously for 1 min, and the supernatant was taken for extraction. The nucleic acid extraction method was referred to the instructions of the virus nucleic acid extraction kit (magnetic bead method) of Guangdong Biaoyun Biotechnology Co., Ltd., and the nucleic acid was extracted using the Bori NPA-32 series of fully automatic nucleic acid extraction and purification instruments. The specific experimental steps were as follows: First, take out the pre-packaged reagent plate and invert it several times to resuspend the magnetic beads. By hand shaking or using a 96-well plate centrifuge for a short time, the reagents and magnetic beads were concentrated at the bottom of the well plate. Then carefully tear off the aluminum foil film of the reagent plate, avoiding vibration to prevent the reagents from splashing out, and confirm the direction of the reagent plate. Next, add 400 μL of the sample to the sample wells of the reagent plate. Subsequently, load the reagent plate into the fully automatic nucleic acid extraction instrument and install the magnetic rod sheath. Perform the automated extraction experiment according to the instrument's recommended program. After the automated program ended, transfer the elution product in the nucleic acid collection well to a nuclease-free centrifuge tube and store it at -20°C or below.

[0104] 2. Quadruple fluorescence quantitative PCR reaction system and conditions

[0105] Taking the HifairV C58P2 Multiplex One Step RT-qPCR Probe Kit (UDGPlus) of Yeasen Biotech Co., Ltd. as an example. The reaction system was prepared as shown in Table 2 at 20 μL / tube, including a total of 16 μL of amplification reagents containing primers, probes, and qPCR enzymes, and 4 μL of the template amount.

[0106] Table 2 Fluorescence quantitative PCR reaction system

[0107]

[0108] Set the reaction program on the fluorescence quantitative PCR instrument (ABI 7500Fast) according to Table 3; for the fluorescence detection channel selection: FAM, CY5, VIC, and Texas Red, the specific detection channel settings can be operated with reference to the instruction manuals of each instrument.

[0109] Table 3 Fluorescence quantitative PCR reaction program setting parameters

[0110]

[0111] Verification of Fluorescent Quantitative PCR Method for Identification and Detection of Type I, Subtype I, Type II, and Type VI Strains of ARV Genes

[0112] After infecting LMH cell cultures with type I ARV gene strain (S1133), type I subtype ARV gene strain (GD / SB / 202301), type II ARV gene strain (HN / WLK / 202305), and type VI ARV gene strain (FJ / ZYQ / 202402), genomic RNA was extracted, as well as a negative control sample (nuclease-free water). The quadruple qPCR detection was performed using the method of Example 1 to verify the effectiveness of the positive control.

[0113] When using 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 for quadruple qPCR detection, the test results are as Figure 2 shown. For the sample of type I ARV gene strain S1133, the Ct value in the VIC fluorescence channel was 16.69, and there was an obvious amplification curve; while there was no Ct value in the FAM, CY5, and Texas Red fluorescence channels. For the sample of type I subtype ARV gene strain GD / SB / 202301, the Ct value in the FAM fluorescence channel was 23.49, and there was an obvious amplification curve; while there was no Ct value in the VIC, CY5, and Texas Red fluorescence channels. For the sample of type II ARV gene strain HN / WLK / 202305, the Ct value in the CY5 fluorescence channel was 11.85, and there was an obvious amplification curve; while there was no Ct value in the FAM, VIC, and Texas Red fluorescence channels. For the sample of type VI ARV gene strain FJ / ZYQ / 202402, the Ct value in the Texas Red fluorescence channel was 15.11, and there was an obvious amplification curve; while there was no Ct value in the FAM, VIC, and CY5 fluorescence channels. For type III ARV gene strain FJ / WCJ / 202306, type IV ARV gene strain 4-A5B4, type V ARV gene strain GX / LSQ / 202403, and the negative control sample, there was no Ct value in the VIC, FAM, CY5, and Texas Red fluorescence channels.

[0114] When using the 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, ARV6-qP2 for quadruple qPCR detection, the test results are as follows Figure 3 shown. For the sample of ARV genotype I strain S1133, the Ct value in the VIC fluorescence channel is 19.91, and the Ct value in the FAM fluorescence channel is 35.83, and there is an obvious amplification curve; while there is no Ct value in the CY5 and Texas Red fluorescence channels. For the sample of ARV genotype I subtype strain GD / SB / 202301, the Ct value in the FAM fluorescence channel is 26.2, and the Ct value in the CY5 fluorescence channel is 36.87, and there is an obvious amplification curve; while there is no Ct value in the VIC and Texas Red fluorescence channels. For the sample of ARV genotype II strain HN / WLK / 202305, the Ct value in the CY5 fluorescence channel is 19.09, and the Ct value in the VIC fluorescence channel is 33.51, and there is an obvious amplification curve; while there is no Ct value in the FAM and Texas Red fluorescence channels. For the sample of ARV genotype VI strain FJ / ZYQ / 202402, the Ct value in the Texas Red fluorescence channel is 9.03, the Ct value in the VIC fluorescence channel is 28.92, and the Ct value in the CY5 fluorescence channel is 22.49, and there is an obvious amplification curve; while there is no Ct value in the FAM fluorescence channel. For the sample of ARV genotype IV strain 4-A5B4, the Ct value in the Texas Red fluorescence channel is 34.26, and for the sample of ARV genotype V strain GX / LSQ / 202403, the Ct value in the CY5 fluorescence channel is 36.35, while there is no Ct value in the VIC, FAM, CY5 and Texas Red fluorescence channels for the sample of ARV genotype III strain FJ / WCJ / 202306 and the negative control sample.

[0115] When using the 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, ARV6-qP3 for quadruple qPCR detection, the test results are as follows Figure 4As shown, the Ct value of the ARV gene type I strain S1133 sample in the VIC fluorescence channel was 24.51, and the Ct value in the FAM fluorescence channel was 29.33, and there was an obvious amplification curve; while there was no Ct value in the CY5 and Texas Red fluorescence channels. The Ct value of the ARV gene type I subtype strain GD / SB / 202301 sample in the FAM fluorescence channel was 24.82, and the Ct value in the CY5 fluorescence channel was 38.23, and there was an obvious amplification curve; while there was no Ct value in the VIC and Texas Red fluorescence channels. The Ct value of the ARV gene type II strain HN / WLK / 202305 sample in the CY5 fluorescence channel was 18.89, and the Ct value in the VIC fluorescence channel was 33.06, and there was an obvious amplification curve; while there was no Ct value in the FAM and Texas Red fluorescence channels. The Ct value of the ARV gene type VI strain FJ / ZYQ / 202402 sample in the Texas Red fluorescence channel was 15.82, and the Ct value in the FAM fluorescence channel was 37.93, and there was an obvious amplification curve; while there was no Ct value in the VIC and CY5 fluorescence channels. The Ct value of the ARV gene type V strain GX / LSQ / 202403 sample in the Texas Red fluorescence channel was 30.83; while there was no Ct value in 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.

[0116] It can be seen from this that when using 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, and ARV6-qP1 for quadruple qPCR detection, for samples of ARV genotype I strains: the Ct values of VIC are all ≤ 40, while there are no Ct values in the three fluorescence channels of FAM, CY5, and TexasRed; for samples of ARV genotype I subtype strains: the Ct values of FAM are all ≤ 40, while there are no Ct values in the three fluorescence channels of VIC, CY5, and TexasRed; for samples of ARV genotype II strains: the Ct values of CY5 are all ≤ 40, while there are no Ct values in the three fluorescence channels of VIC, FAM, and TexasRed; for samples of ARV genotype VI strains: the Ct values of TexasRed are all ≤ 40, while there are no Ct values in the three fluorescence channels of VIC, FAM, and CY5; for ARV genotype III strain FJ / WCJ / 202306, ARV genotype IV strain 4-A5B4, ARV genotype V strain GX / LSQ / 202403, and the negative control sample, there are no Ct values in VIC, FAM, CY5, and TexasRed; this meets the determination criteria for the differential diagnosis of ARV genotype I, genotype I subtype, genotype II, and genotype VI strains, and can completely distinguish 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, and ARV6-qP1 can be used for the differential detection of other ARV genotype I, genotype I subtype, genotype II, and genotype VI strains.

[0117] Example 3 Sensitivity Test

[0118] 1. Preparation of Recombinant Plasmid Standard

[0119] (1) Screen the conserved region of the Sigma C gene of ARV genotype I strain, design the standard plasmid primer pair for the Sigma C gene of ARV-1, and name them ARV1-F (SEQ ID NO.10) and ARV1-R (SEQ ID NO.11) as shown in Table 1. Extract the RNA of ARV genotype I strain, and amplify the fragment with a size of 563 bp by RT-PCR ( Figure 5), the positive amplification products were recovered, cloned into the puc57 vector, and the positive recombinant plasmids were screened and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing and identification. The correctly sequenced positive recombinant plasmid was named puc57-ARV1. The OD260 / OD280 ratio of puc57-ARV1 was measured to be 1.89 using a UV spectrophotometer, and the nucleic acid concentration was 120 ng / μL. Referring to the calculation method of plasmid DNA copy number, the concentration of the puc57-ARV1 plasmid DNA solution was calculated to be 4.04×10 10 copies / μL.

[0120] (2) The conserved region of the Sigma C gene of the ARV gene type I subtype strain was screened, and a pair of standard plasmid primers for the Sigma C gene of ARV-1-sub was designed, named ARV1-sub-F (SEQ ID NO.21) and ARV1-sub-R (SEQ ID NO.22) as shown in Table 1. The RNA of the ARV gene type I subtype strain was extracted, and the amplified fragment size by RT-PCR was 531 bp ( Figure 5 ), the positive amplification products were recovered, cloned into the puc57 vector, and the positive recombinant plasmids were screened and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing and identification. The correctly sequenced positive recombinant plasmid was named puc57-ARV1-sub. The OD260 / OD280 ratio of puc57-ARV1-sub was measured to be 1.92 using a UV spectrophotometer, and the nucleic acid concentration was 109 ng / μL. Referring to the calculation method of plasmid DNA copy number, the concentration of the puc57-ARV1-sub plasmid DNA solution was calculated to be 3.07×10 10 copies / μL.

[0121] (3) The conserved region of the Sigma C gene of the ARV gene type II strain was screened, and a pair of standard plasmid primers for the Sigma C gene of ARV-2 was designed, named ARV2-F (SEQ ID NO.32) and ARV2-R (SEQ ID NO.33) as shown in Table 1. The RNA of the ARV gene type II strain was extracted, and the amplified fragment size by RT-PCR was 521 bp ( Figure 5 ), the positive amplification products were recovered, cloned into the puc57 vector, and the positive recombinant plasmids were screened and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing and identification. The correctly sequenced positive recombinant plasmid was named puc57-ARV2. The OD260 / OD280 ratio of puc57-ARV2 was measured to be 1.89 using a UV spectrophotometer, and the nucleic acid concentration was 113 ng / μL. Referring to the calculation method of plasmid DNA copy number, the concentration of the puc57-ARV2 plasmid DNA solution was calculated to be 3.8×1010 copies / μL.

[0122] (4) Screen the conserved region of the Sigma C gene of avian reovirus (ARV) genotype VI strains, design standard plasmid primer pairs for the Sigma C gene of ARV-6, and name them ARV6-F (SEQ ID NO.43) and ARV6-R (SEQ ID NO.44) as shown in Table 1. Extract the RNA of ARV genotype VI strains, and amplify the fragment with a size of 626 bp by RT-PCR ( Figure 5 ). Recover the positive amplification products, clone the positive amplification products into the puc57 vector, and screen the positive recombinant plasmids for sequencing and identification by Sangon Biotech (Shanghai) Co., Ltd. The correctly sequenced positive recombinant plasmid was named puc57-ARV6. Use a UV spectrophotometer to measure the OD260 / OD280 ratio of puc57-ARV6 to be 1.95, and the nucleic acid concentration was 113 ng / μL. Refer to the calculation method of plasmid DNA copy number to calculate the concentration of the puc57-ARV6 plasmid DNA solution to be 3.8×10 10 copies / μL.

[0123] 2. Fluorescent quantitative PCR standard curve and sensitivity detection

[0124] First, dilute the 4 recombinant standard plasmids (puc57-ARV1, puc57-ARV1-sub, puc57-ARV2, puc57-ARV6) with nuclease-free water so that the copy number of each plasmid is 10 9 copies / 4 μL, and then perform 10-fold serial dilutions with nuclease-free water. Take the template samples diluted to contain 10 0 , 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 and 10 8 copies, set 3 replicates for each dilution, and set a negative control (nuclease-free water) at the same time. Set the program on a fluorescent quantitative PCR instrument according to the reaction conditions of the method established in Example 1 for detection.

[0125] The established quadruple fluorescent quantitative PCR standard curve is as Figure 6 shown. The fluorescent quantitative PCR standard curve for genotype I is y = -3.5257x + 40.942 (R 2 = 0.9987), and the fluorescent quantitative PCR standard curve for genotype I subtype is y = -3.5812x + 41.326 (R2 = 0.9982), the standard curve of fluorescence quantitative PCR for genotype II was y = -3.4012x + 40.411 (R 2 = 0.9975), and the standard curve of fluorescence quantitative PCR for genotype VI was y = -3.4668x + 40.388

[0126] (R 2 = 0.9977). In summary, the correlation coefficients (R 2 ) of the four standard curves were between 0.9975 and 0.9987, and the amplification efficiencies (E) were all between 90% and 110%, indicating that this method had a good linear relationship.

[0127] The sensitivity test results of 4 recombinant standard plasmids (puc57 - ARV1, puc57 - ARV1 - sub, puc57 - ARV2, puc57 - ARV6) were as shown in Figure 7 , 8 , 9, and 10: For the standards with 9 different concentrations containing 4 recombinant standard plasmids, the Ct values of the four fluorescence channels of VIC, FAM, CY5, and Texas Red corresponding to them were all ≤ 40, the results were all positive, and there were obvious amplification curves; the lowest template concentration that could be detected for 4 genotype strains was 10 virus copies. While in the Chinese patent with the application number 202210151323.5, the lowest detection limits of mPCR for ARV - I, ARV - I - sub, and ARV - II were 10 2 , 10 2 and 10 1 virus copies respectively. Thus, it can be seen that the sensitivity of the present invention is higher.

[0128] Example 4: Specificity test

[0129] Using the cDNA of 6 different genotype strains of ARV (ARV - I, ARV - I - sub, ARV - Ⅱ, ARV - III, ARV - IV, ARV - V, and ARV - VI) and the genomic DNA of other avian viruses (Marek's disease virus (MDV), infectious laryngotracheitis virus of chicken (ILTV), fowl adenovirus (FAdV), DNA of chicken infectious anemia virus (CIAV) or cDNA of infectious bursal disease virus of chicken (IBDV), Newcastle disease virus (NDV), avian influenza virus (AIV), infectious bronchitis virus (IBV), reticuloendotheliosis virus (REV), avian leukosis virus (ALV)) as templates, quadruple fluorescence quantitative PCR amplification was carried out under the above reaction conditions to detect the specificity of this method. Each sample was replicated 3 times, and a negative control was set simultaneously.

[0130] The results were as shown in Figure 11As shown, the method of the present invention can only detect 4 genotypes of ARV (genotype I, genotype I subtype, genotype II, genotype VI) strains, and has no cross-reaction with other viruses, showing good specificity.

[0131] Example 5: Repeatability detection

[0132] According to the method of diluting the standard quality plasmid in Example 3, the 4 standard quality plasmids (puc57-ARV1, puc57-ARV1-sub, puc57-ARV2, puc57-ARV6) were respectively used as templates at two concentrations of 10 6 and 10 4 copies / 4 μL. Three repeated experiments were carried out on these two dilutions at different times. Each time, three repeated determinations were carried out on the same template simultaneously, and 3 repeated wells were set. The quadruple fluorescence quantitative PCR test was carried out according to the "reaction system and reaction program of Example 1" to analyze the stability of the method.

[0133] The results are shown in Table 4. The within-batch and between-batch coefficients of variation are both less than 2%, indicating that the quadruple fluorescence quantitative PCR method established by the present invention has good repeatability and stability, and the results are stable and reliable (Table 4).

[0134] Table 4 Repeatability results of the quadruple fluorescence quantitative PCR method

[0135]

[0136] Example 6: Detection of clinical samples

[0137] Twenty-five 28-day-old SPF chickens were randomly divided into 5 groups, with 5 chickens in each group. The 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 inoculated into SPF chickens by footpad injection at a dose of 10 6 TCID 50 . At the same time, a non-challenged blank control group was set. At 10 days after challenge, 3 samples of anal swabs, tendons and cecal tonsils of SPF chickens in each group were collected respectively, as well as LMH cell culture samples of the vaccine strain S1133 and the isolated strains GD / SB / 202301, HN / WLK / 202305, FJ / ZYQ / 202402, a total of 79 samples. The viral genomic RNA was extracted respectively according to the instructions of the nucleic acid extraction kit.

[0138] The detection results are shown in Table 5. For various types of samples after SPF chickens are infected with ARV genotype I strains, genotype I subtype strains, genotype II strains, and genotype VI strains, the quadruple fluorescence quantitative PCR method established by the present invention can effectively identify and detect them. The method of the present invention can sensitively, rapidly, accurately, and specifically identify and detect the fluorescence quantitative PCR detection method of ARV genotype I strains, genotype I subtype strains, genotype II strains, and genotype VI strains in samples such as cell cultures, anal swabs, and tissues.

[0139] Table 5 Detection Results of Clinical Samples

[0140]

[0141] Note: "-" represents no Ct value or Ct value > 40.

[0142] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A PCR primer set for detecting and / or differentiating avian reovirus, characterized in that, The primer set includes the primer sets of (a)-(d) below: (a) The first primer set includes: The upstream primer ARV1-qF1 with the nucleotide sequence shown in SEQ ID NO.1; The downstream primer ARV1-qR1 with the nucleotide sequence shown in SEQ ID NO.2; The first probe; (b) The second primer set includes: The upstream primer ARV1-sub-qF1 with the nucleotide sequence shown in SEQ ID NO.12; The downstream primer ARV1-sub-qR1 with the nucleotide sequence shown in SEQ ID NO.13; The second probe; (c) The third primer set includes: The upstream primer ARV2-qF1 with the nucleotide sequence shown in SEQ ID NO.23; The downstream primer ARV2-qR1 with the nucleotide sequence shown in SEQ ID NO.24; The third probe; (d) The fourth primer set includes: The upstream primer ARV6-qF1 with the nucleotide sequence shown in SEQ ID NO.34; The downstream primer ARV6-qR1 with the nucleotide sequence shown in SEQ ID NO.35; The fourth probe; The avian reovirus includes avian reovirus gene type I, avian reovirus gene type I subtype, avian reovirus gene type II, and avian reovirus gene type VI The first probe is: 5’-VIC-CCGCGTGCGCAGAGGATSTGTT-MGB-3’; The second probe is: 5’-FAM-CTTCTGCTCRCAACGAGTCTCYTTGA-BHQ1-3’; The third probe is: 5’-CY5-CTGAAAMCCGGCSCGAGGCAC-BHQ3-3’; The fourth probe is: 5’-Texas Red-CCGGRAAHGTCGCCTGCTGRAA-BHQ2-3’.

2. The PCR primer set according to claim 1, characterized in that, The first primer set is used for detecting and / or differentiating avian reovirus gene type I, the second primer set is used for detecting and / or differentiating avian reovirus gene type I subtype, the third primer set is used for detecting and / or differentiating avian reovirus gene type II, and the fourth primer set is used for detecting and / or differentiating avian reovirus gene type VI.

3. The PCR primer set according to claim 1, wherein The first probe, the second probe, the third probe, and the fourth probe are nucleic acid probes for detecting the target nucleic acid sequence; a fluorophore and a quencher are linked to the first probe, the second probe, the third probe, and the fourth probe; the fluorophores linked to the first probe, the second probe, the third probe, and the fourth probe are different.

4. The PCR primer set according to claim 3, characterized in that The fluorophores include: FAM, HEX, TET, CY3, CY5, CY7, ROX, JOE, Texas Red, Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor647, VIC, NED, TAMRA, AMCA, or fluorescein amide.

5. The PCR primer set according to claim 3, characterized in that, The quenching groups include: BHQ1, BHQ2, BHQ3, DABCYL, Iowa Black FQ, Iowa Black RQ, TQ3 or Eclipse.

6. A reagent for detecting and / or differentiating avian reovirus, characterized in that, The reagent contains the PCR primer set according to any one of claims 1-5.

7. Use of the PCR primer set according to any one of claims 1-5 in the preparation of a reagent for detecting and / or differentiating avian reovirus.

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