Universal primers for simultaneous detection of chicken circovirus AGV2, GyV3 and GyV7

Abstract

The present invention relates to universal primers for simultaneously detecting chicken circovirus AGV2, GyV3 and GyV7, the sequences of the universal primers are shown in SEQ ID NO.1-2, and the kit for simultaneously detecting chicken circovirus AGV2, GyV3 and GyV7 comprises the universal primers. The present invention analyzes the genome structural characteristics of AGV2, GyV3 and GyV7, comprehensively utilizes a variety of bioinformatics tools, and cleverly designs primers to obtain primers for simultaneously detecting AGV2, GyV3 and GyV7. The method is simple and rapid, and can simultaneously detect AGV2, GyV3 and GyV7; it has strong specificity and has no crossover to common chicken infectious diseases (egg drop syndrome virus, avian adenovirus type 4, avian influenza virus H9, etc.); the optimization procedure is simple, and conventionally 6 primers need to be designed for 3 pathogens, while the present invention only needs to design 2 primers, which is convenient for condition optimization.

Description

Technical Field

[0001] The invention belongs to the field of avian disease, and in particular relates to a universal primer for simultaneously detecting chicken circovirus AGV2, GyV3 and GyV7. Background Art

[0002] Gyrovirus (GyVs) is a single-stranded circular DNA virus. In the past, this genus had only one member, chicken infectious anemia virus (CIAV). Since 2011, researchers have isolated nine viruses with similar genetic structures to CIAV. Gyroviruses have a wide range of hosts, including chickens, humans, and wild birds. Some Gyroviruses can infect both chickens and humans. This genus used to belong to the Circoviridae family. Since Gyroviruses are more similar to members of the Anelloviridae family in terms of structure and genetic information, GyVs was reassigned to the Anelloviridae family in the latest virus classification approved by the International Committee on Taxonomy of Viruses in 2016. The genus Gyrovirus can be divided into three branches: Branch A, which includes CIAV, HGyV / AGV2, GyV3, GyV6, GyV7, and GyV9; Branch B, which includes GyV4 and GyV5; and Branch C, which includes GyV8. The circovirus genome is about 2.2-2.4 kb, consisting of a 5' non-coding region and three overlapping open reading frames (ORFs). The three overlapping ORFs encode the structural protein VP1, the skeleton protein VP2, and the apoptotic protein VP3, respectively.

[0003] In 2011, researchers isolated human GyVs (HGyV) from the skin surface of healthy French adults; in the same year, a GyV was also isolated from the serum of a group of sick chickens in Brazil that showed depression and weight loss, namely chicken GyVs2 (Avian GyV2, AGV2). This virus has only 40% homology with CIAV, and the amino acid sequences of its VP1, VP2 and VP3 proteins are 38.8%, 40.3% and 32.2% homologous to the corresponding proteins of CIAV, respectively. In 2012, researchers detected HGyV in the feces of diarrhea patients, and the detection rate was 1-9.3%. The study pointed out that HGyV is closely related to AGV2, and the difference in the VP1-3 protein sequences of HGyV and AGV2 is only 3-7%, indicating that HGyV and AGV2 belong to the same virus. In 2012, researchers first isolated and identified human GyV3 from the feces of children with acute gastroenteritis in Chile and Hong Kong, China. In 2018, Chinese scholars first identified GyV3 from the glandular stomach of sick chickens with typical infectious glandular gastritis in chickens. Another study confirmed through pathogenicity tests in chickens and mice that GyV3 can cause infectious glandular gastritis and anemia in chickens, and can cross-species infect mice, causing gastroenteritis and anemia in mice. The genome of GyV3 contains approximately 2356 base sequences and has a similar genomic structure to other GyVs. The homology of its VP1 and VP2 proteins with the amino acid sequences of the corresponding proteins of HGyV / AGV2 are 62% and 63%, respectively, and it has 57% similarity with the amino acid sequences of other known GyVs VP3. Recently, GyV7 was detected in infected chickens. The genome is 2439bp, with a non-coding region of 514bp. The amino acid sequences of its VP1, VP2 and VP3 proteins have the highest homology with the corresponding proteins of GyV7 at 49%, 53% and 63%, respectively.

[0004] AGV2, GyV3 and GyV7 are newly discovered viruses in chickens in recent years, and co-infection of these three viruses is relatively common in chickens. However, there are currently no research reports on the simultaneous PCR detection of the three newly emerging chicken circle viruses AGV2, GyV3 and GyV7. The establishment of the present invention can fill the gaps in related fields at home and abroad. The PCR detection method established by the present invention can simultaneously detect the prevalent AGV2, GyV3 and GyV7 in chickens, laying the foundation for epidemiological surveys of newly emerging AGV2, GyV3 and GyV7 viruses in chickens and subsequent scientific prevention and control of related diseases, which has very important research significance. Summary of the invention

[0005] The purpose of the present invention is to provide universal primers for simultaneously detecting chicken circovirus AGV2, GyV3 and GyV7, which can be combined with DNA sequencing to simultaneously perform scientific identification and detection of the infection of chicken circovirus AGV2, GyV3 and GyV7.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A universal primer for simultaneously detecting chicken circle virus AGV2, GyV3 and GyV7, the universal primer is as follows:

[0008] GyVs-F: 5'-GGGCAGTGAATTGCCGYTTAG-3' (SEQ ID NO. 1);

[0009] GyVs-R: 5'-CCGGGGVCKTGGGTTGAAKA-3' (SEQ ID NO. 2).

[0010] Among them, Y=C / T, V=G / A / C, K=G / T.

[0011] The universal primer is used in preparing a kit for simultaneously detecting chicken circovirus AGV2, GyV3 and GyV7.

[0012] A kit for simultaneously detecting chicken circovirus AGV2, GyV3 and GyV7, the kit comprising the universal primers.

[0013] A detection method for simultaneously detecting chicken circovirus AGV2, GyV3 and GyV7 using the universal primers, the method is not for the purpose of diagnosis, and the PCR reaction system is: the following components are contained in a 50 μL system, wherein 2×Premix Taq PCR amplification reaction solution 25 μL, 20 μM upstream primer GyVs-F 1 μL, 20 μM downstream primer GyVs-R 1 μL, prepared DNA template 2 μL, sterile water is added to a final volume of 50 μL, and PCR amplification is performed after mixing;

[0014] The PCR amplification conditions were as follows: pre-denaturation at 94°C for 3 min before entering the cycle, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 45 s. After 35 cycles, extension at 72°C for 10 min was performed. After the reaction, identification was performed by conventional agarose gel electrophoresis.

[0015] The PCR products are as follows: AGV2: 629bp; GyV3: 653bp; GyV7: 649bp; combined with DNA sequencing, chicken circovirus AGV2, GyV3 and GyV7 were identified and detected simultaneously.

[0016] The universal primers designed by the present invention can specifically amplify AGV2, GyV3 and GyV7 at the same time. Therefore, AGV2, GyV3 and GyV7 can be detected simultaneously based on only one pair of primers, and the problem of cross interference due to too many primers will not occur. Moreover, the universal primers cannot amplify other common chicken infectious diseases (egg drop syndrome virus, avian adenovirus type 4, avian influenza virus H9, Newcastle disease virus, J subgroup avian leukosis virus, etc.), so it has good specificity.

[0017] Compared with the prior art, the advantages of the present invention are: the present invention analyzes the structural characteristics of the genomes of AGV2, GyV3 and GyV7, comprehensively utilizes a variety of bioinformatics tools, and cleverly designs primers to obtain primers for simultaneous detection of AGV2, GyV3 and GyV7. The method is simple and rapid, and can detect AGV2, GyV3 and GyV7 at the same time; it has strong specificity and has no crossover to common chicken infectious diseases (egg drop syndrome virus, avian adenovirus type 4, avian influenza virus H9, chicken Newcastle disease virus, J subgroup avian leukosis virus, etc.); the optimization procedure is simple (conventionally, 6 primers need to be designed for 3 pathogens, while the present invention only needs to design 2 primers, which is convenient for condition optimization); combined with DNA sequencing, the infection of chicken circovirus AGV2, GyV3 and GyV7 can be scientifically identified and detected at the same time. The establishment of the present invention can fill the gaps in related fields at home and abroad. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a graph of the electrophoresis results of the PCR method of the present invention, wherein M: DNA molecular weight DL2000; 1: AGV2; 2: GyV3; 3: GyV7; 4: AGV2 and GyV3 co-infection; 5: AGV2 and GyV7 co-infection; 6: GyV3 and GyV7 co-infection; 7: AGV2, GyV3 and GyV7 co-infection; 8: EDSV; 9: FAdV-4; 10: AIV H9; 11: NDV; 12: ALV-J.

[0019] Figure 2 This is the result of AGV2 sensitivity analysis, where M: DNA molecular weight DL2000; 1: 3.62×10 5 pg;2:3.62×10 4 pg;3:3.62×10 3 pg;4:3.62×10 2 pg;5:3.62×10 1 pg;6:3.62×10 0 pg;7: negative control for the experiment.

[0020] Figure 3This is the result of GyV3 sensitivity analysis, where M is DNA molecular weight DL2000; 1 is 5.91×10 5 pg;2:5.91×10 4 pg;3:5.91×10 3 pg;4:5.91×10 2 pg;5:5.91×10 1 pg;6:5.91×10 0 pg;7: negative control for the experiment.

[0021] Figure 4 This is the result of GyV7 sensitivity analysis, where M is DNA molecular weight DL2000; 1 is 6.16×10 5 pg;2:6.16×10 4 pg;3:6.16×10 3 pg;4:6.16×10 2 pg;5:6.16×10 1 pg;6:6.16×10 0 pg;7: negative control for the experiment. DETAILED DESCRIPTION

[0022] The present invention is described in detail below with reference to the accompanying drawings and embodiments:

[0023] Embodiment 1:

[0024] 1. Materials and methods

[0025] 1.1 strains

[0026] The experimental pathogens, including chicken circovirus type 2 (AGV2), chicken circovirus type 7 (GyV7), egg drop syndrome virus (EDSV), avian adenovirus type 4 (FAdV-4), avian influenza virus H9 (AIV H9), Newcastle disease virus (NDV), and avian leukosis virus subgroup J (ALV-J), were all identified and preserved by the Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences.

[0027] 1.2 Primer design

[0028] According to the genome characteristics of chicken circovirus AGV2, GyV3 and GyV7 in the National Center of Biotechnology Information (NCBI) database GenBank, a variety of biological software were used to cleverly design primer sets for identifying AGV2, GyV3 and GyV7. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The primer information is as follows:

[0029] GyVs-F: 5'-GGGCAGTGAATTGCCGYTTAG-3';

[0030] GyVs-R: 5'-CCGGGGVCKTGGGTTGAAKA-3'.

[0031] PCR product sizes: AGV2: 629 bp; GyV3: 653 bp; GyV7: 649 bp.

[0032] 1.3 Preparation of viral nucleic acid

[0033] The DNA of AGV2, GyV3, GyV7, EDSV, FAdV-4 and the RNA of AIV H9, NDV and ALV-J were extracted using the EasyPure Viral DNA / RNA Kit. One-Step gDNA Removal and cDNA Synthesis SuperMix) was used to reverse transcribe the extracted RNA into cDNA for later use.

[0034] 1.4 PCR method establishment

[0035] According to the PCR kit Premix Taq TM (TaKaRa Taq TM The 50 μL system recommended by Version 2.0plus dye was used for amplification, including 25 μL of 2×Premix Taq PCR amplification reaction solution, 1 μL of upstream primer GyVs-F (20 μM), 1 μL of downstream primer GyVs-R (20 μM), 2 μL of prepared DNA template, and sterile water was added to the final volume of 50 μL, and PCR amplification was performed after mixing.

[0036] The optimized amplification conditions were as follows: pre-denaturation at 94°C for 3 min before entering the cycle, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 45 s. After 35 cycles, extension was performed at 72°C for 10 min. After the reaction was completed, identification was performed by conventional agarose gel electrophoresis.

[0037] Results Figure 1: The target fragment size of AGV2 is 629bp (lane 1); the target fragment size of GyV3 is 653bp (lane 2); the target fragment size of GyV7 is 649bp (lane 3); the target fragment size of AGV2 and GyV3 double positive can only see one, which cannot be distinguished by the naked eye (lane 4); the target fragment size of AGV2 and GyV7 double positive can only see one, which cannot be distinguished by the naked eye (lane 5); the target fragment size of GyV3 and GyV7 double positive can only see one, which cannot be distinguished by the naked eye (lane 6); the target fragment size of AGV2, GyV3 and GyV7 co-positive can only see one, which cannot be distinguished by the naked eye (lane 7). Combined with DNA sequencing, the chicken circle virus AGV2, GyV3 and GyV7 can be further scientifically identified and detected.

[0038] 1.5 Specificity of multiplex PCR methods

[0039] According to the optimized PCR conditions, the cDNA templates of EDSV, FAdV-4 DNA and AIV H9, NDV and ALV-J RNA reverse transcribed were amplified. The results are shown in Figure 1 No band amplification was observed for EDSV (lane 8), FAdV-4 (lane 9), AIV H9 (lane 10), NDV (lane 11), and ALV-J (lane 12), indicating that the method established in the present invention has strong specificity.

[0040] 1.6 Sensitivity of PCR

[0041] According to the optimized PCR conditions, the DNA extracted from AGV2, GyV3 and GyV7 was diluted 10 times (the concentration of AGV2 was 3.62×10 5 pg-3.62×10 0 The concentrations of pg and GyV3 were 5.91×10 5 pg-5.91×10 0 The concentrations of pg and GyV7 were 6.16×10 5 pg-6.16×10 0 pg) was reverse transcribed and then amplified. The results were as follows Figure 2 , 3 , as shown in Figure 4.

[0042] The results show that the minimum detection limit of AGV2 is 3.62×10 1 pg (i.e. 36.2pg) Figure 2 Lane 5); the minimum detection limit of GyV3 is 5.91×10 1 pg (i.e. 59.1pg) Figure 3Lane 5); the minimum detection limit of GyV7 is 6.16×10 1 pg (i.e. 61.6pg) Figure 4 Lane 5)

[0043] 1.7 Clinical Application

[0044] After 196 samples of chicken diseased materials sent for clinical inspection were processed according to conventional methods, the corresponding nucleic acid DNA was extracted using the virus nucleic acid extraction kit EasyPure Viral DNA / RNA Kit, and then the AGV2, GyV3 and GyV7 infection were detected using the established PCR method. The results showed that 61 samples were positive for chicken circovirus (AGV2, GyV3 and GyV7), with a positive rate of 31.12%. The positive PCR products were cloned and sequenced, and the results showed that 11 samples were positive for AGV2 single infection, with a positive rate of 5.61%; 24 samples were positive for GyV3 single infection, with a positive rate of 12.24%; 5 samples were positive for GyV7 single infection, with a positive rate of 2.55%; 9 samples were positive for AGV2 and GyV3 co-infection, with a positive rate of 4.59%; 4 samples were positive for AGV2 and GyV7 co-infection, with a positive rate of 2.04%; 6 samples were positive for GyV3 and GyV7 co-infection, with a positive rate of 3.06%; 2 samples were positive for AGV2, GyV3 and GyV7 co-infection, with a positive rate of 1.02%.

[0045] The above-mentioned embodiments are intended to illustrate that the present invention can be implemented or used by professionals in the field. Modifications to the above-mentioned embodiments will be obvious to professionals in the field. Therefore, the present invention includes but is not limited to the above-mentioned embodiments. Any method, process, or product that complies with the claims or description of the specification and complies with the principles and novelty and creative features disclosed herein falls within the scope of protection of the present invention.

Claims

1. A universal primer for simultaneously detecting chicken circovirus AGV2, GyV3 and GyV7, characterized in that: The universal primers are as follows: GyVs-F: 5'- GGGCAGTGAATTGCCGYTTAG -3'; GyVs-R: 5'-CCGGGGVCKTGGGTTGAAKA-3'.

2. Use of the universal primer as claimed in claim 1 in preparing a kit for simultaneously detecting chicken circovirus AGV2, GyV3 and GyV7.

3. A kit for simultaneously detecting chicken circle viruses AGV2, GyV3 and GyV7, characterized in that: The kit comprises the universal primer as claimed in claim 1.

4. A method for detecting chicken circovirus AGV2, GyV3 and GyV7 simultaneously using the universal primers described in claim 1, the method not being aimed at diagnosis, characterized in that: The PCR reaction system is as follows: the following components are contained in a 50 μL system, including 25 μL of 2×Premix Taq PCR amplification reaction solution, 1 μL of 20 μM upstream primer GyVs-F, 1 μL of 20 μM downstream primer GyVs-R, 2 μL of prepared DNA template, and sterile water is added to a final volume of 50 μL, and PCR amplification is performed after mixing; The PCR amplification conditions were as follows: pre-denaturation at 94 °C for 3 min before entering the cycle, denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 45 s. After 35 cycles, extension was performed at 72 °C for 10 min. After the reaction was completed, identification was performed by conventional agarose gel electrophoresis. Combined with DNA sequencing, chicken circovirus AGV2, GyV3 and GyV7 were further identified and detected.

5. The detection method according to claim 4, characterized in that: The PCR products are as follows: AGV2: 629 bp; GyV3: 653 bp; GyV7: 649 bp; combined with DNA sequencing, chicken circovirus AGV2, GyV3 and GyV7 were identified and detected simultaneously.

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