Primer set and probe set for detecting chicken circular virus agv2 and gyv7 double real-time fluorescence quantitative PCR

Abstract

The present application relates to a kind of primer set and probe set for chicken circle virus AGV2 and GyV7 dual real-time fluorescent quantitative PCR detection, the sequence of the primer set and probe set is as shown in SEQ ID NO.1-6, the present application provides primer set and probe set and kit for chicken circle virus AGV2 and GyV7 dual real-time fluorescent quantitative PCR detection, and establishes the detection method capable of simultaneously detecting chicken circle virus AGV2 and GyV7, with the advantages of simultaneous detection, rapid detection, high efficiency, quantitative accuracy, high sensitivity, strong specificity, good repeatability, for chicken population to carry out new chicken circle virus AGV2 and GyV7 molecular epidemiology survey and subsequent scientific prevention and control related disease lay foundation, with very important research significance.

Description

Technical Field

[0001] The invention belongs to the field of veterinary medicine, and in particular relates to a primer set and a probe set for dual real-time fluorescence quantitative PCR detection of chicken circoviruses AGV2 and GyV7. Background Art

[0002] Gyroviruses (GyVs) are single-stranded circular DNA viruses. This genus previously had only one member, chicken infectious anemia virus (CIAV). Since 2011, researchers have isolated nine viruses with genetic similarities to CIAV. Gyroviruses have a wide range of reservoirs, including chickens, humans, and wild birds, and some can infect both chickens and humans. This genus was previously classified as belonging to the Circoviridae family. However, due to their structural and genetic similarity to members of the Anelloviridae family, the latest viral classification approved by the International Committee on Taxonomy of Viruses in 2016 reassigned GyVs to the Anelloviridae family. The genus Gyrovirus can be divided into three clades: Clade A, which includes CIAV, HGyV / AGV2, GyV3, GyV6, GyV7, and GyV9; Clade B, which includes GyV4 and GyV5; and Clade C, which includes GyV8. The circovirus genome is approximately 2.2 to 2.4 kb and consists of a 5' non-coding region and three overlapping open reading frames (ORFs), which encode the structural protein VP1, the cytoskeletal protein VP2, and the apoptotic protein VP3, respectively.

[0003] In 2011, researchers isolated human GyVs (HGyV) from the skin of healthy French adults. That same year, a GyV strain, Avian GyV2 (AGV2), was isolated from the serum of sick chickens in Brazil that presented with depression and weight loss. This virus shared only 40% identity with CIAV, with its VP1, VP2, and VP3 amino acid sequences sharing 38.8%, 40.3%, and 32.2% homologies with the corresponding CIAV proteins, respectively. In 2012, researchers detected HGyV in the feces of patients with diarrhea, with a detection rate of 1-9.3%. This study indicated that HGyV is closely related to AGV2, with only 3-7% difference in the VP1-3 protein sequences between HGyV and AGV2, indicating that HGyV and AGV2 belong to the same species. Recently, GyV7 was detected in infected chickens. Its 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] Real-time quantitative PCR (RT-PCR) monitors the progress of PCR amplification in real time using fluorescent signals. During the exponential phase of PCR amplification, the linear relationship between the template Ct value and the initial copy number of that template exists, providing the basis for quantification. Unlike conventional PCR, which is cumbersome and lacks quantitative analysis, real-time qPCR has rapidly developed due to its ease of use, high sensitivity, and excellent reproducibility. It has been applied to various areas of life science research, such as differential gene expression analysis, SNP detection, allele detection, drug development, clinical diagnostics, and transgenic research. Currently, real-time qPCR technology primarily encompasses two categories, fluorescent dyes and fluorescent probes, depending on the fluorescent chemical used. Commonly mentioned are TaqMan probes, FRET hybridization probes (fluorescence resonance energy transfer probes), and molecular beacons. The TaqMan probe method involves adding a specific fluorescent probe to a pair of primers during PCR amplification. This probe binds specifically to the template, with its binding site located between the two primers. The 5' end of the probe is labeled with a fluorescent reporter group, such as FAM, VIC, ROX, or JOE, and the 3' end is labeled with a fluorescent quencher group, such as Eclipse or TAMRA. Real-time fluorescence quantitative PCR technology can not only qualitatively detect the presence of pathogens but also quantitatively analyze viral levels, making it widely used in pathogen nucleic acid detection. Multiplex real-time fluorescence quantitative PCR is a specialized form of fluorescence quantitative PCR. Its distinguishing feature is that a single PCR run can simultaneously detect multiple pathogens, making it highly effective for identifying pathogens with complex pathogens or those with multiple genotypes.

[0005] Currently, there are no reports of simultaneous dual TaqMan real-time fluorescence quantitative PCR detection of the newly discovered chicken circoviruses AGV2 and GyV7. The development of this invention fills a gap in related fields both domestically and internationally. The TaqMan real-time fluorescence quantitative PCR detection method established in this invention can detect and accurately quantify the chicken circoviruses AGV2 and GyV7 prevalent in chicken flocks, laying the foundation for molecular epidemiological surveys of the newly discovered chicken circoviruses AGV2 and GyV7 in chicken flocks and subsequent scientific prevention and control of related diseases, thus having extremely important research significance. Summary of the Invention

[0006] The purpose of the present invention is to provide a primer set and a probe set for dual real-time fluorescence quantitative PCR detection of chicken circovirus AGV2 and GyV7, to establish a detection method that can simultaneously detect chicken circovirus AGV2 and GyV7, to simplify the operation procedure and save costs.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A primer set and probe set for dual real-time fluorescence quantitative PCR detection of chicken circovirus AGV2 and GyV7, the primer set comprising:

[0009] Primer sequences for AGV2:

[0010] AGV2-qT-F: 5'-CCACGGGCAAGACACTAAAT-3' (SEQ ID NO.1),

[0011] AGV2-qT-R: 5'-GGATGCTCGTATGCCGTTAATA-3' (SEQ ID NO. 2),

[0012] Primer sequences for GyV7:

[0013] GyV7-qT-F: 5'-TGCAACCCAACTCCTCATATAC-3' (SEQ ID NO.3),

[0014] GyV7-qT-R: 5'-TCCATCGTCCTCGGTAGAAA-3' (SEQ ID NO. 4);

[0015] The probe set includes:

[0016] Probe sequence for AGV2:

[0017] AGV2-qT-probe: 5'-TCTCGCCGACAAGCAACAACTACT-3' (SEQ ID NO.5);

[0018] Probe sequence for GyV7:

[0019] GyV7-qT-probe: 5'-AAGATGGCAAGACGAGCAAGACGA-3' (SEQ ID NO. 6);

[0020] Among them, the 5'-end of AGV2-qT-probe is labeled with the fluorescent reporter group VIC, and the 3'-end is labeled with Eclipse;

[0021] The 5'-end of GyV7-qT-probe is labeled with the fluorescent reporter group Texas Red, and the 3'-end is labeled with Eclipse.

[0022] The dual real-time fluorescence quantitative PCR detection method for detecting chicken circovirus AGV2 and GyV7 using the primer set and probe set is not for diagnosis. The TaqMan real-time fluorescence quantitative PCR reaction system is as follows: the system contains the following components in 20 μL: TaqMan qPCR Mix 10 μL, primer concentrations of 10 μmol·L -1 AGV2-qT-F and AGV2-qT-R, 0.2 μL each, with a concentration of 10 μmol·L -1 AGV2-qT-probe 0.4 μL, primer concentrations of 10 μmol·L -1 0.2 μL each of GyV7-qT-F and GyV7-qT-R, with a concentration of 10 μmol·L -1 Add 0.4 μL of GyV7-qT-probe, 1 μL each of AGV2 and GyV7 nucleic acid DNA templates or 2 μL of nucleic acid template to be tested, and add sterile deionized water to a final volume of 20 μL;

[0023] The reaction conditions of real-time fluorescence quantitative PCR were as follows: pre-denaturation at 95°C for 2 min; 40 cycles of 95°C for 10 s and 60°C for 30 s.

[0024] After the real-time fluorescence quantitative PCR reaction is completed, the test results are analyzed. When only the VIC channel shows a positive amplification signal, the result is judged as AGV2 positive; when only the Texas Red channel shows a positive amplification signal, the result is judged as GyV7 positive; if positive amplification signals are seen in both the VIC channel and the Texas Red channel, the result is judged as AGV2 and GyV7 co-infection.

[0025] A kit for dual real-time fluorescence quantitative PCR detection of chicken circovirus AGV2 and GyV7, comprising the primer set and probe set.

[0026] The present invention provides a primer set, a probe set, and a kit for dual real-time fluorescence quantitative PCR detection of chicken circovirus AGV2 and GyV7, and establishes a detection method capable of simultaneously detecting chicken circovirus AGV2 and GyV7, which has the following advantages and effects:

[0027] 1. Simultaneous detection, rapid detection, and high efficiency: The dual TaqMan real-time fluorescence quantitative PCR detection method established by the present invention using the primer set and probe set can simultaneously detect, differentially diagnose, and accurately quantify AGV2 and GyV7 in chickens, simplifying the operating procedures and saving costs. At the same time, this detection method does not require conventional agarose gel electrophoresis detection. After the reaction is completed, the result can be determined by the program provided by the real-time fluorescence quantitative PCR machine.

[0028] 2. Accurate quantification: By preparing standards and drawing standard curves, the infection of AGV2 and GyV7 in the samples to be tested can be directly and accurately quantified based on the Ct values ​​of AGV2 and GyV7.

[0029] 4. High sensitivity: The minimum detection limit of AGV2 is 53.1 copies / μL; the minimum detection limit of GyV7 is 42.7 copies / μL.

[0030] 5. Strong specificity: No positive amplification fluorescence signal was observed for other common chicken pathogens (such as EDSV, FAdV-4, AIV H9, NDV and ALV-J). Amplification signals were only detected for AGV2 (VIC channel) and GyV7 (Texas Red channel).

[0031] 6. Good repeatability: The intra-group coefficient of variation for AGV2 detection of the established real-time fluorescence quantitative PCR detection method was 0.49-1.38%, and the inter-group coefficient of variation was 0.69-1.74%. The intra-group coefficient of variation for GyV7 detection was 0.72-1.19%, and the inter-group coefficient of variation was 0.86-1.66%, indicating that the TaqMan real-time fluorescence quantitative PCR method established by the present invention has good repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the standard curve for detecting AGV2 by the dual real-time fluorescence quantitative PCR method.

[0033] Figure 2 This is the standard curve for detecting GyV7 using the dual real-time fluorescence quantitative PCR method.

[0034] Figure 3 This is the specific detection result diagram of the dual real-time fluorescence quantitative PCR method; among them, 1: AGV2; 2: GyV7; experimental controls: EDSV, FAdV-4, AIV H9, NDV and ALV-J, which cannot be effectively distinguished by the naked eye. DETAILED DESCRIPTION

[0035] The present invention is described in detail below with reference to the accompanying drawings and embodiments:

[0036] Example 1:

[0037] 1. Materials and Methods

[0038] 1.1 strains

[0039] The experimental pathogens 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.

[0040] 1.2 Primer and probe design

[0041] Based on the genomic characteristics of chicken circovirus AGV2 and GyV7 in the National Center of Biotechnology Information (NCBI) database GenBank, specific primers and probes for AGV2 and GyV7 were designed, respectively. The primers and probes were synthesized at Sangon Biotech (Shanghai) Co., Ltd.

[0042] The primer sequences for AGV2 are:

[0043] AGV2-qT-F: 5'-CCACGGGCAAGACACTAAAT-3'

[0044] AGV2-qT-R: 5'-GGATGCTCGTATGCCGTTAATA-3'

[0045] The probe sequence for AGV2 is:

[0046] AGV2-qT-probe: 5'(VIC)-TCTCGCCGACAAGCAACAACTACT-3'(Eclipse);

[0047] The primer sequences for GyV7 are:

[0048] GyV7-qT-F: 5'-TGCAACCCAACTCCTCATATAC-3'

[0049] GyV7-qT-R: 5'-TCCATCGTCCTCGGTAGAAA-3'

[0050] The probe sequence for GyV7 is:

[0051] GyV7-qT-probe: 5'(Texas Red)-AAGATGGCAAGACGAGCAAGACGA-3'(Eclipse);

[0052] The 5'-end of AGV2-qT-probe is labeled with the fluorescent reporter group VIC, and the 3'-end is labeled with Eclipse.

[0053] wherein the 5'-end of GyV7-qT-probe is labeled with a fluorescent reporter group Texas Red and the 3'-end is labeled with Eclipse.

[0054] 1.3 Preparation of nucleic acids

[0055] DNA of AGV2, GyV7, EDSV, FAdV-4 and RNA of AIV H9, NDV and ALV-J were extracted according to the viral nucleic acid extraction kit (EasyPure Viral DNA / RNA Kit). The extracted RNA was reverse transcribed into cDNA for standby by using reverse transcription kit (One-Step gDNA Removal and cDNA Synthesis SuperMix).

[0056] 1.4 Construction of positive standards

[0057] 1.4.1 Construction of AGV2 positive standard

[0058] AGV2-VP2 was cloned into pUC57-KANA vector after full gene synthesis, which was used as the standard (P-AGV2) of this study. After determining its concentration by spectrophotometer, the corresponding copy number was calculated as 5.31 x 1010copies / μL. After linearization enzyme digestion, continuous 10-fold dilution was performed, and the obtained concentration of 5.31 x 1010copies / μL to 5.31 x 1011copies / μL were all frozen at -20°C for standby. 8 7 0

[0059] 1.4.2 Construction of GyV7 positive standard

[0060] GyV7-VP2 was cloned into pUC57-KANA vector after full gene synthesis, which was used as the standard (P-GyV7) of this study. After determining its concentration by spectrophotometer, the corresponding copy number was calculated as 4.27 x 1010copies / μL. After linearization enzyme digestion, continuous 10-fold dilution was performed, and the obtained concentration of 4.27 x 1010copies / μL to 4.27 x 1011copies / μL were all frozen at -20°C for standby. 8 7 0

[0061] 1.5 Optimization of double TaqMan real-time fluorescent quantitative PCR reaction conditions

[0062] ​​​​​​​A 20 μL real-time fluorescence quantitative PCR reaction system was prepared according to the TaqMan instructions. Different primer final concentrations and different reaction conditions were optimized to determine the optimal reaction conditions for the established real-time fluorescence quantitative PCR method.

[0063] The optimized TaqMan real-time fluorescence quantitative PCR optimal reaction system (20 μL) is as follows: TaqMan qPCRMix 10 μL, AGV2-qT-F (primer concentration is 10 μmol·L -1 ) and AGV2-qT-R (primer concentration was 10 μmol·L -1 ) 0.2 μL each, AGV2-qT-probe (probe concentration was 10 μmol·L -1 )0.4μL, GyV7-qT-F (primer concentration was 10μmol·L -1 ) and GyV7-qT-R (primer concentration was 10 μmol·L -1 ) 0.2 μL each, GyV7-qT-probe (probe concentration was 10 μmol·L -1 ) 0.4 μL, 1 μL each of AGV2 and GyV7 nucleic acid DNA templates (2.0 μL of the cDNA template to be tested is added for clinical testing), and sterile deionized water is added to a final volume of 20 μL. The optimized reaction conditions for real-time fluorescence quantitative PCR are: pre-denaturation at 95°C for 2 minutes, followed by 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds.

[0064] 1.6 Establishment of standard curve

[0065] 1.6.1 Establishment of AGV2 Standard Curve

[0066] The optimal reaction conditions of the optimized dual TaqMan real-time fluorescence quantitative PCR method were used to analyze the expression of different plasmid concentrations (5.31×10 6 ~5.31×10 3 The positive standard (P-AGV2) with 5.31×10 copies / μL was used as a template to perform real-time fluorescence quantitative PCR amplification reaction and obtain the corresponding amplification kinetic curve. From the amplification kinetic curves of plasmids with different concentrations, it can be seen that the established real-time fluorescence quantitative PCR method has a high sensitivity and specificity at 5.31×10 6 ~5.31×10 3 The results showed a good linear relationship within the reaction range of 100 copies / μL (P-AGV2 plasmid). The correlation coefficient for AGV2 was 0.999. The standard curve for the real-time fluorescence quantitative PCR method for detecting AGV2 was obtained by taking the common logarithm (logC) of the copy number in each concentration standard template as the horizontal axis and the cycle threshold (Ct value) as the vertical axis (see Figure 1)’s linear equation Y=-3.48X+39.37.

[0067] 1.6.2 Establishment of GyV7 Standard Curve

[0068] The optimal reaction conditions of the optimized dual TaqMan real-time fluorescence quantitative PCR method were used to analyze the expression of different concentrations (4.27×10 6 ~4.27×10 3 The positive standard (P-GyV7) with a concentration of 4.27×10 6 ~4.27×10 3 There is a good linear relationship in the range of copies / μL. Among them, the correlation coefficient for GyV7 is 0.996. The common logarithm of the copy number in each concentration standard template (logC) is used as the horizontal axis, and the cycle threshold value (Ct value) is used as the vertical axis to obtain the standard curve based on the real-time fluorescence quantitative PCR method for detecting GyV7 (see Figure 2 )’s linear equation Y=-3.34X+38.31.

[0069] 1.7 Sensitivity test

[0070] The optimized TaqMan real-time fluorescence quantitative PCR method was used to detect different concentrations of plasmids (P-AGV2 plasmid and P-GyV7 plasmid). The results showed that the minimum detection limits of AGV2 and GyV7 were 53.1 copies / μL and 42.7 copies / μL, respectively.

[0071] 1.8 Specificity test

[0072] The optimized dual TaqMan real-time fluorescence quantitative PCR method was used to amplify common chicken pathogens such as EDSV, FAdV-4, AIV H9, NDV and ALV-J using AGV2 and GyV7 as positive controls to evaluate the specificity of the established real-time fluorescence quantitative PCR method.

[0073] From the amplification curve (see Figure 3)It was found that the established double TaqMan real-time fluorescent quantitative PCR method only had AGV2 amplification signal in VIC channel; only GyV7 amplification signal in Texas Red channel; if the sample was AGV2 and GyV7 co-infection, amplification signal was found in both VIC channel and Texas Red channel. No positive amplification fluorescence signal was found for other common chicken pathogens (such as EDSV, FAdV-4, AIV H9, NDV and ALV-J), indicating that the established real-time fluorescent quantitative PCR method had strong specificity.

[0074] 1.9 repeatability test

[0075] 1.9.1 AGV2 repeatability

[0076] The optimized double TaqMan real-time fluorescent quantitative PCR method was used to detect different plasmid concentrations (5.31 x 10 6 copies / μL, 5.31 x 10 4 copies / μL, 5.31 x 10 2 copies / μL) of positive standard (P-AGV2) as template. Each standard contained 3 repeated contents, and the intra-group coefficient of variation was calculated. The above standard was divided and stored at -20℃, and was taken out every 7 days for detection by the optimized real-time fluorescent quantitative PCR method, a total of 3 times, and the inter-group coefficient of variation was calculated.

[0077] The results of intra-group and inter-group repeatability test of different dilutions of standard showed (see Table 1) that the intra-group coefficient of variation was 0.49-1.38%, and the inter-group coefficient of variation was 0.69-1.74%, indicating that the TaqMan real-time fluorescent quantitative PCR method established in this study had good repeatability.

[0078] Table 1 Intra-group and inter-group coefficient of variation of real-time fluorescent quantitative PCR

[0079]

[0080] 1.9.2 GyV7 repeatability

[0081] The optimized double TaqMan real-time fluorescent quantitative PCR method was used to detect different plasmid concentrations (4.27 x 10 6 copies / μL, 4.27 x 10 4 copies / μL, 4.27 x 10 2The detection was performed with the positive standard (P-GyV7) as template, which was diluted to 1.0 x 105copies / μL. Each standard was repeated 3 times, and the intra-group coefficient of variation was calculated. The above standard was divided and stored at -20℃, and was taken out every 7 days, and was detected by the optimized real-time fluorescent quantitative PCR method, and was detected 3 times, and the inter-group coefficient of variation was calculated.

[0082] The results of the intra-group and inter-group repeatability test of the different dilution standards showed (see Table 2) that the intra-group coefficient of variation was 0.72-1.19%, and the inter-group coefficient of variation was 0.86-1.66%, indicating that the TaqMan real-time fluorescent quantitative PCR method established in the present study was good in repeatability.

[0083] Table 2 Intra-group and inter-group coefficient of variation of real-time fluorescent quantitative PCR

[0084]

[0085]

[0086] 2Clinical sample detection

[0087] According to the conventional method, 165 clinical samples were treated, and the corresponding nucleic acid DNA was extracted by using the viral nucleic acid extraction kit EasyPure Viral DNA / RNA Kit, and the AGV2 and GyV7 infection was detected by using the optimized double TaqMan real-time fluorescent quantitative PCR method. The results showed that 23 AGV2 infection positives were detected, and the positive rate was 13.94%; 17 GyV7 infection positives were detected, and the positive rate was 10.30%; 4 AGV2 and GyV7 co-infection positives were detected, and the positive rate was 2.42%, indicating that the method established could be used for the molecular epidemiological investigation of AGV2 and GyV7 and the subsequent research on the pathogenic mechanism.

[0088] The above embodiments are intended to exemplify the present application which can be implemented or used by the skilled in the art, and it will be obvious to those skilled in the art that modifications can be made to the above embodiments, and therefore the present application includes but is not limited to the above embodiments, and any method, process, product which meets the present claims or the description, which meets the principles and novelty, and the creative features disclosed herein, falls within the protection scope of the present application.

Claims

1. A primer-probe combination for dual real-time fluorescence quantitative PCR detection of chicken circovirus AGV2 and GyV7, characterized by: including a primer pair targeting AGV2, a primer pair targeting GyV7, a probe targeting AGV2, and a probe targeting GyV7; The nucleotide sequence of the primer pair for AGV2 is: AGV2-qT-F: 5'-CCACGGGCAAGACACTAAAT-3', AGV2-qT-R: 5'-GGATGCTCGTATGCCGTTAATA-3'; The nucleotide sequence of the primer pair for GyV7 is: GyV7-qT-F: 5'-TGCAACCCAACTCCTCATATAC-3', GyV7-qT-R: 5'-TCCATCGTCCTCGGTAGAAA-3'; The nucleotide sequence of the probe for AGV2 is: AGV2-qT-probe: 5'-TCTCGCCGACAAGCAACAACTACT-3'; The nucleotide sequence of the probe for GyV7 is: GyV7-qT-probe: 5'-AAGATGGCAAGACGAGCAAGACGA-3'; Among them, the 5' end of AGV2-qT-probe is labeled with the fluorescent reporter group VIC, and the 3' end is labeled with Eclipse; The 5' end of GyV7-qT-probe is labeled with the fluorescent reporter group Texas Red, and the 3' end is labeled with Eclipse.

2. A dual real-time fluorescence quantitative PCR method for detecting chicken circovirus AGV2 and GyV7 using the primer-probe combination of claim 1, wherein the method is not intended for disease diagnosis and is characterized by: Dual real-time fluorescence quantitative PCR reaction system: The 20 μL system contains the following components: TaqMan qPCR Mix 10 μL, primer concentrations of 10 μmol·L -1 AGV2-qT-F and AGV2-qT-R, 0.2 μL each, with a concentration of 10 μmol·L -1 AGV2-qT-probe 0.4 μL, primer concentrations of 10 μmol·L -1 0.2 μL each of GyV7-qT-F and GyV7-qT-R, with a concentration of 10 μmol·L -1 0.4 μL of GyV7-qT-probe, 2 μL of nucleic acid template to be tested, and add sterile deionized water to a final volume of 20 μL; The reaction conditions of dual real-time fluorescence quantitative PCR were as follows: pre-denaturation at 95°C for 2 min; 40 cycles of 95°C for 10 s and 60°C for 30 s.

3. A kit for dual real-time fluorescence quantitative PCR detection of chicken circovirus AGV2 and GyV7, characterized by: The kit comprises the primer-probe combination according to claim 1.

4. The kit according to claim 3, wherein: The PCR reaction system is as follows: 20 μL system contains the following components: TaqMan qPCR Mix 10 μL, primer concentration is 10 μmol·L -1 AGV2-qT-F and AGV2-qT-R, 0.2 μL each, with a concentration of 10 μmol·L -1 AGV2-qT-probe 0.4 μL, primer concentrations of 10 μmol·L -1 0.2 μL each of GyV7-qT-F and GyV7-qT-R, with a concentration of 10 μmol·L -1 0.4 μL of GyV7-qT-probe, 2 μL of nucleic acid template to be tested, and add sterile deionized water to a final volume of 20 μL; The PCR reaction conditions were as follows: pre-denaturation at 95°C for 2 min, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s.

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