Fiber1 gene-based quantitative fluorescence detection method for fowl aviadenoviruses

Abstract

The present invention relates to a Fiber1 gene-based quantitative fluorescence detection method for fowl aviadenoviruses, belonging to the technical field of animal medical tests. The method uses designed primers and probes in combination with a specific reaction system and reaction conditions to detect fowl aviadenoviruses. The method is fast and effective, and has excellent detection sensitivity. By means of the method, fowl aviadenovirus detection and monitoring can be performed periodically, which helps to formulate effective prevention, control and eradication measures in a timely manner, so as to block large-area outbreaks of Angara disease in fowls, thus laying a foundation for prevention, control and eradication of Angara disease in fowls.

Description

A quantitative fluorescence detection method for avian Ankara virus based on the Fiber1 gene Technical Field

[0001] This invention belongs to the field of animal medical testing technology, specifically relating to a quantitative fluorescence detection method for avian Ankara virus based on the Fiber1 gene. Background Technology

[0002] Avian Ankara disease, also known as pericardial effusion syndrome or pericardial effusion-hepatitis syndrome, is an acute infectious disease caused by infection with virulent serotype 4 of group I avian adenovirus (FAdV). The typical lesion is the accumulation of pale yellow, transparent or gelatinous exudate in the pericardium, accompanied by a certain degree of hepatitis.

[0003] Ankara disease was first reported in the Ankara region of Pakistan in March 1987, and subsequently spread to the United States, India, South Africa, Canada, South Korea, New Zealand, Mexico, Iraq, Chile, Japan, Russia, and European countries. From 2012 to 2014, reports of the disease began in my country, but because it did not cause a large-scale epidemic at that time, it did not attract much attention from the Chinese poultry industry. However, since the summer of 2015, more than twenty provinces and municipalities, including Jiangsu, Anhui, Henan, Hebei, Hubei, Hunan, Jiangxi, Beijing, Heilongjiang, Liaoning, Jilin, Zhejiang, Shandong, Inner Mongolia, Shanxi, Guangdong, Guangxi, Yunnan, Sichuan, Hainan, Guizhou, and Chongqing, have successively reported the occurrence of the disease, showing an explosive epidemic trend. Young chickens, ducks, and geese aged 3-10 weeks are most susceptible, with mortality rates reaching 10%-80%. In breeding poultry and laying hens, it can cause a 10%-30% decrease in egg production. However, there is currently no effective treatment for avian Ankara virus infection. Therefore, establishing a method for rapid and accurate detection of avian Ankara virus is of great significance for the prevention and eradication of avian Ankara disease.

[0004] Avian Ankara disease can be diagnosed through microbiological and viral isolation methods, but these methods are time-consuming, complex, and require highly skilled personnel. Serological and PCR methods are currently the most common viral detection methods. Serological methods are relatively simple, but cross-reactivity is a common problem. Compared to serological methods, PCR methods are more sensitive and specific, making them more suitable for detecting avian Ankara virus. However, conventional open PCR testing can lead to environmental contamination and false positives. Quantitative real-time PCR (qPCR) can overcome this drawback, offering simple operation and higher sensitivity and specificity. Therefore, establishing a qPCR method for avian Ankara virus detection is most suitable for the clinical diagnosis of avian Ankara disease.

[0005] Wang Guokang et al. established a real-time quantitative PCR detection method based on the hexazosome gene of avian Ankara virus. This method used the chicken embryo half-maximal infectious dose (EID50) of FAdV-4HB1510 strain as a standard for sensitivity analysis. Therefore, it could not achieve absolute quantification to reflect the method's sensitivity and the intensity of infection in the sample. [1] .

[0006] In addition, other quantitative real-time PCR detection methods based on hexazosinogen genes, although their sensitivity has been quantitatively analyzed, have sensitivity ranges of 2.03 copies / μL to 7.1 × 10⁻⁶. 2 The copy number / μL is inconsistent, making it impossible to detect lower concentrations. [2-7] .

[0007] Zhang Yundan's method had a sensitivity of 2.03 copies / μL, but in its clinical applicability evaluation trial, the samples used were suspected cases of avian Ankara virus infection (only showing clinical symptoms, not confirmed cases), and the detection rate was only 80%. Therefore, it could not accurately reflect the detection rate of the method. [2] .

[0008] FAdV-4's spike protein 1 (Fiber1) is located on the surface of the viral particle and has a specific antigenic determinant. Furthermore, studies have shown that adenovirus fiber proteins are highly expressed in the early stages of viral infection in the host, making them an important target for clinical diagnosis.

[0009] Based on the above problems, there is an urgent need to establish a real-time quantitative PCR detection method based on the Fiber1 gene of avian Ankara virus, which can lay an important foundation for the prevention and eradication of avian Ankara disease, and also provide support for the epidemiological investigation of clinical avian Ankara disease.

[0010] References

[0011] [1] Wang Guokang, Lu Qin, Jia Miaomiao, et al. Establishment and preliminary evaluation of a real-time quantitative PCR detection method for avian adenovirus type 4 [J]. Chinese Journal of Veterinary Medicine, 2022, 58(02):22-26.

[0012] [2] Zhang Yundan, Yang Yuan, Wang Jun, et al. Establishment and application of real-time fluorescence quantitative PCR detection method for type 4 avian adenovirus serum using TaqMan probe [J]. Northwest Agriculture Journal, 2019, 28(06): 868-876.

[0013] [3] Wu Shuang, Zhang Cong, Yuan Huisha, et al. Establishment and application of real-time fluorescence quantitative PCR detection method for avian adenovirus type 4 using TaqMan probe [J]. Jiangsu Journal of Agricultural Sciences, 2023, 39(01):134-141.

[0014] [4] Lu Qingxia, Jin Qianyue, Feng Lili, et al. Establishment and application of SYBR Green I real-time quantitative PCR detection method for avian adenovirus serological type 4 [J]. Henan Agricultural Sciences, 2022, 51(12):131-138.

[0015] [5] Zhao Lichan, Wang Zhanxin, Luo Yangyang, et al. Establishment and preliminary application of TaqMan real-time PCR method for avian adenovirus type 4 [J]. Guangdong Animal Husbandry and Veterinary Science and Technology, 2020, 45(05):48-52.

[0016] [6] Liu Lin, He Chunhui, Wang Sainan, et al. Establishment of TaqMan real-time quantitative PCR detection method for avian adenovirus type 4 [J]. Chinese Journal of Veterinary Medicine, 2020, 40(05): 928-932.

[0017] [7] Luo Yangyang, Li Qunhui, Lin Limiao, et al. Establishment and application of TaqMan real-time PCR detection method for avian adenovirus serological type 4 [J]. Chinese Journal of Veterinary Medicine, 2020, 56(04):39-42+47+143.

[0018] Summary of the Invention

[0019] The F1F1 / F1R1 primer sequence and FIP probe sequence designed based on the Fiber1 gene have high specificity, specifically amplifying only avian Ankara virus, while showing no specific amplification for common avian infectors such as Escherichia coli and Salmonella; DNA viruses such as avian leukosis virus; and parasites such as Histomoniasis and Eimeria tenella. The avian Ankara virus fluorescence quantitative PCR detection method established in this invention has high sensitivity, with a minimum detection limit of 1.9 copies / μL for plasmid standards. The detection rate of commercial samples containing avian Ankara virus using this method is 100%.

[0020] This application provides a primer designed based on the Fiber1 gene, including primer 1;

[0021] The upstream sequence number of primer 1 is SEQ ID NO:7, and the downstream sequence number is SEQ ID NO:8.

[0022] In some preferred embodiments, the probe of primer 1 is F1R; the sequence number is SEQ ID NO:25.

[0023] This application also provides a primer probe designed based on the Fiber1 gene, including probe F1R; the sequence number is SEQ ID NO:25.

[0024] In addition, this application also provides a method for detecting avian Ankara virus, utilizing the aforementioned primers and probes designed based on the Fiber1 gene, comprising the following steps:

[0025] 1) Prepare the solution to be tested;

[0026] 2) Preparation of standards: The nucleic acid sequences between upstream and downstream primers designed using the Fiber1 gene of avian Ankara virus were chemically synthesized and ligated into the pUC-18T vector as plasmid standards;

[0027] 3) Preparation of standard solutions: Dilute the synthesized plasmid standard and prepare a series of standard solutions with gradient concentrations;

[0028] 4) qPCR detection: Primers and probes are added to the genomic DNA of a series of standard solution samples and the sample to be tested for qPCR detection;

[0029] 5) Using a series of standard solutions to obtain a series of plasmid standard samples, a CT value-concentration standard curve was obtained; using the CT value of the sample to be tested, the concentration of the Fiber1 gene in the solution to be tested was quantitatively detected.

[0030] 4. The detection method according to claim 3, wherein the qPCR detection method in step 4) is:

[0031] i) Prepare the solution for the qPCR reaction system;

[0032] ii) The solution is subjected to pre-denaturation and cyclic reaction.

[0033] In some preferred embodiments, the primer concentration in the qPCR reaction system solution in step i) is 0.1 μM to 0.4 μM, preferably 0.2 μM.

[0034] In some preferred embodiments, the probe concentration in the qPCR reaction system solution in step i) is 0.05 μM to 0.15 μM, preferably 0.1 μM.

[0035] In some preferred embodiments, the annealing temperature of the primer 1 reaction system is 58–60°C, preferably 60°C.

[0036] In some preferred embodiments, the annealing time is 20 to 40 seconds, preferably 30 seconds.

[0037] In some preferred embodiments, the cyclic reaction is repeated 40 to 50 times, preferably 45 times.

[0038] In some preferred embodiments, the pre-denaturation conditions are 93–97°C for 25–35 s, with one cycle; preferably 95°C for 30 s, with one cycle.

[0039] In some preferred embodiments, the cyclic reaction process is 93-97℃ for 8-12s, 58-62℃ for 25-35s; the number of cycles is 40-50; preferably 95℃ for 10s, 60℃ for 30s; the number of cycles is 45. Beneficial effects:

[0040] This application provides a real-time quantitative PCR diagnostic method specific to avian Ankara virus. The method is rapid and effective (with an effectiveness rate of up to 100%). Regular detection and monitoring of avian Ankara virus in cloacal samples from poultry flocks using this method helps to formulate effective prevention and control measures in a timely manner, prevent large-scale outbreaks of avian Ankara disease, and lay the foundation for the prevention and control of avian Ankara disease.

[0041] 1. Identify conserved and highly specific fragments in the avian Ankara virus fiber gene sequence;

[0042] 2. Optimal reaction conditions for the invention of a real-time fluorescence quantitative PCR diagnostic method for avian Ankara virus.

[0043] 3. The method has good sensitivity and can accurately detect even lower gene copy numbers: the lowest detectable gene copy number is 1.9 copies / μL;

[0044] 4. The method has a high detection rate: it can detect all commercially available biological samples containing avian Ankara virus, with a detection rate of up to 100%. Attached Figure Description

[0045] Figure 1. Identification of primer amplification effect;

[0046] Wherein, M: molecular weight marker; 1: PCR amplification result of FADV-4 genome using primers F1F-1 / F1R-1; 2: PCR amplification result of FADV-4 genome using primers F1F-2 / F1R-2; 3: PCR amplification result of FADV-4 genome using primers F1F-3 / F1R-3; 4: PCR amplification result of FADV-4 genome using primers F1F-4 / F1R-4; 5: PCR amplification result of FADV-4 genome using primers F1F-5 / F1R-5; 6: PCR amplification result of FADV-4 genome using primers F1F-6 / F1R-6.

[0047] Figure 2. Identification of the synthesized plasmid standard;

[0048] Where M: molecular weight marker; 1: primer pair F1F-1 / F1R-1, 1.9 × 10⁻⁶ 10 PCR amplification results of copy / μL plasmid standard.

[0049] Figure 3. Identification of optimal reaction conditions for qPCR;

[0050] Wherein, M: molecular weight marker; 1-6: qPCR results of primers F1F-1 and F1R-1 at a concentration of 0.1 μM, and probes at concentrations of 0.05 μM (1, 2), 0.1 μM (3, 4), and 0.15 μM (5, 6), respectively, performed at annealing temperatures of 58℃ (1, 3, 5) and 60℃ (2, 4, 6), with agarose gel electrophoresis results; 7-12: qPCR results of primers F1F-1 and F1R-1 at a concentration of 0.2 μM, and probes at concentrations of 0.05 μM (7, 8), 0.1 μM (9, 10), and 0.15 μM (11, 12), respectively, performed at annealing temperatures of 58℃ (1, 3, 5) and 60℃ (2, 4, 6), with agarose gel electrophoresis results. qPCR was performed at annealing temperatures of 58℃ (7, 9, 11) and 60℃ (8, 10, 12), and the agarose gel electrophoresis results of the amplified products were obtained; 13-18: qPCR was performed with primers F1F-1 and F1R-1 at a concentration of 0.3 μM and probes at concentrations of 0.05 μM (13, 14), 0.1 μM (15, 16), and 0.15 μM (17, 18) at annealing temperatures of 58℃ (13, 15, 17) and 60℃ (14, 16, 18), and the agarose gel electrophoresis results of the amplified products were obtained; 19-24: qPCR was performed with primers F1F-1 and F1R-1 at annealing temperatures of 0.3 μM and 0.05 μM (13, 14), 0.1 μM (15, 16), and 0.15 μM (17, 18), respectively, and the agarose gel electrophoresis results of the amplified products were obtained; At a concentration of 0.4 μM, qPCR was performed with probes at concentrations of 0.05 μM (19, 20), 0.1 μM (21, 22), and 0.15 μM (23, 24), respectively, at annealing temperatures of 58 °C (19, 21, 23) and 60 °C (20, 22, 24). The agarose gel electrophoresis results of the amplified products were obtained.

[0051] Figure 4. Results of the fluorescence quantitative PCR sensitivity test;

[0052] Among them, 1-8: respectively with 1.9×10 7 The amplification curves of qPCR were obtained using plasmid standards ranging from 1.9 copies / μL to 1.9 copies / μL as templates.

[0053] Figure 5. Standard curve for quantitative PCR;

[0054] 1.9×10 7 Using plasmid standards ranging from 1.9 copies / μL to 1.9 copies / μL as templates, qPCR was performed, and a standard curve was plotted with template concentration on the x-axis and Ct value on the y-axis.

[0055] Figure 6. Agarose gel electrophoresis to verify the results of the sensitivity test;

[0056] Where M: molecular weight indicator; 1-8: 1.9 × 10⁻⁸ respectively 7 The results of agarose gel electrophoresis of the products amplified by qPCR from plasmid standards ranging from 1.9 copies / μL to 1.9 copies / μL.

[0057] Figure 7. Results of the specificity assay for quantitative real-time PCR;

[0058] Among them, 1: with 1.9×10 6 Amplification curves of qPCR using copy / μL plasmid standards as templates; others: amplification curves of qPCR using the genomes of Eimeria coccidia, Histomonia solani, Escherichia coli, Salmonella, and avian leukosis virus as templates.

[0059] Figure 8. Results of the specificity test verified by agarose gel electrophoresis;

[0060] Where M: molecular weight indicator; 1-6: molecular weight indicators respectively, with a molecular weight of 1.9 × 10⁻⁶. 6 The results of qPCR were obtained using plasmid standards (copy / μL), Eimeria coli, Histomonia spp., Escherichia coli, Salmonella, and avian leukosis virus genomes as templates, and the amplified products were subjected to agarose gel electrophoresis.

[0061] Figure 9. Results of the repeatability test of quantitative real-time PCR;

[0062] Among them, 1-2: with 1.9×10 10 Amplification curve of qPCR using copies / μL plasmid standard as template.

[0063] Figure 10. Detection results of biological products containing avian Ankara virus by real-time quantitative PCR;

[0064] Among them, 1-4: respectively with 1.9×10 7 Using genomic DNA extracted from plasmid standards (copy / μL), He Yibang triple inactivated mange vaccine, Wohua triple inactivated mange vaccine, and Harbin Pharmaceutical Group triple inactivated mange vaccine as templates, qPCR amplification curves were obtained.

[0065] Figure 11. Agarose gel electrophoresis verifies the detection results of qPCR for biological products;

[0066] Where M: molecular weight indicator; 1-4: molecular weight indicators respectively, with a molecular weight of 1.9 × 10⁻⁴. 7 The results of qPCR were obtained using genomic DNA extracted from plasmid standards (copy / μL), He Yibang triple inactivated mange vaccine, Wohua triple inactivated mange vaccine, and Harbin Pharmaceutical Group triple inactivated mange vaccine as templates. The amplified products were then subjected to agarose gel electrophoresis.

[0067] Figure 12 Results of sensitivity detection by real-time PCR for bioproduct validation

[0068] Among them, 1-2: genomic DNA of the Yibangxin triple inactivated influenza vaccine (with a copy number of 1.43 copies / μL of the avian Ankara virus Fiber1 gene) diluted 100-fold, respectively. 2 Using copies / μL as a template, the amplification curve of qPCR was obtained.

[0069] Figure 13. Agarose gel electrophoresis results of the sensitivity of quantitative real-time PCR for bioproduct validation.

[0070] Wherein, M: molecular weight marker; 1: the result of agarose gel electrophoresis detection of the product after qPCR amplification using 100-fold diluted Yibangxin triple inactivated avian influenza vaccine genomic DNA (avian Ankara virus Fiber1 gene copy number of 1.43 copies / μL) as a template; 2: the result of agarose gel electrophoresis detection of the product using Yibangxin triple inactivated avian influenza vaccine genomic DNA (avian Ankara virus Fiber1 gene copy number of 1.43 × 10⁻⁶). 2 The product of qPCR amplification using copies / μL as template was detected by agarose gel electrophoresis. Detailed Implementation

[0071] Example 1

[0072] 1. Design and synthesis of primers and probes

[0073] The Fiber1 gene (MG148335.1:30459-31754; see Table 1, SEQ ID NO:1) of the CH / AHMC / 2015 FAdV-4 virus strain isolated in our laboratory was compared with multiple FAdV-4 virus nucleotide sequences (see Table 1, SEQ ID NO:2-6) downloaded from the GenBank database using DNAMan biological software to determine conserved nucleotide regions. Fiber1 gene-specific primers were designed using Primer Premier 5.0 software. After primer amplification effect analysis, the best amplification primers were selected, and a specific TaqMan probe was designed within the amplified sequence, labeled with FAM at the 5' end and MGB at the 3' end. Both primers and probes were synthesized by General Biotechnology (Anhui) Co., Ltd., and the sequences are shown in Table 2.

[0074] Table 1. Fiber1 gene sequence of FAdV-4 virus

[0075] As mentioned above, a number of conserved nucleotide regions were screened for avian Ankara virus, and they were located in different positions. We designed the following 6 pairs of primers and probes for the sequences of the above-mentioned conserved regions.

[0076] Table 2 Primer and probe sequences

[0077] Table2 Primer and probe sequence

[0078] 2. Primer amplification effect analysis

[0079] Five 9-day-old SPF chicken embryos were inoculated with the avian Ankara virus CH / AHMC / 2015 strain, isolated from the Anhui Provincial Key Laboratory of Poultry Disease Prevention and Monitoring at Anhui University of Science and Technology, via the allantoic cavity route, at a dose of 0.1 mL per embryo. Liver tissues from dead embryos were collected after 24 hours of incubation, observed, and homogenized. The liver tissue was centrifuged at 16000×g for 5 min, and the supernatant was used to extract viral genomic DNA according to the EZNATM Viral DNA Kit instructions. The nucleic acid concentration was then determined.

[0080] Genomic DNA of avian Ankara virus strain CH / AHMC / 2015 was amplified using primers 1-6.

[0081] Table 3: PCR reaction system (25 μL system)

[0082] PCR amplification reaction procedure:

[0083] Table 4: PCR reaction conditions

[0084] The PCR amplification products of the genomic DNA of avian Ankara virus strain CH / AHMC / 2015 were subjected to agarose gel electrophoresis using six pairs of primers. The results showed that the amplification products of the avian Ankara virus Fiber1 gene by the first pair of primers, F1F-1 and F1R-1, were brighter than those by the other primers (Figure 1).

[0085] Table 5: Gray-scale analysis of electrophoretic bands of amplification products from each primer pair

[0086] Gray-scale analysis of each band showed that, under the same conditions, the agarose gel electrophoresis band of primer F1F-1 / F1R-1 had the highest gray-scale value, at 47007.66. The gray-scale values ​​of primers 2–6 were all distributed between 43000 and 44000. Primer 1 was significantly better than primers 2–6, showing a significant increase in gray-scale value and good specificity and accuracy. Therefore, F1F-1 / F1R-1 was determined to be the optimal primer for establishing subsequent experimental methods (Table 5).

[0087] The amplification product sequence (SEQ ID NO:9) of the first pair of primers: F1F-1 and F1R-1 is as follows:

[0088] The amplification product sequence (SEQ ID NO:12) of the second pair of primers: F1F-2 and F1R-2 is as follows:

[0089] The amplification product sequence (SEQ ID NO:15) of the third pair of primers: F1F-3 and F1R-3 is as follows:

[0090] The amplification product sequence (SEQ ID NO:18) of the fourth pair of primers: F1F-4 and F1R-4 is as follows:

[0091] The amplification product sequence (SEQ ID NO:21) of the fifth pair of primers: F1F-5 and F1R-5 is as follows:

[0092] The amplification product sequence (SEQ ID NO:24) of the sixth pair of primers: F1F-6 and F1R-6 is as follows:

[0093] 3. Preparation of recombinant plasmid standards

[0094] The nucleic acid sequence of the Fiber1 gene between the first pair of primers, F1F-1 and F1R-1, was entrusted to General Biotechnology (Anhui) Co., Ltd. to be chemically synthesized and ligated into the pUC-18T vector.

[0095] Plasmid concentration is determined according to the formula:

[0096] Copy number = plasmid concentration (ng / μL) × 10 -9 ×6.02×10 23 / (660×total plasmid length), calculate the plasmid copy number.

[0097] The final calculated copy number of the plasmid standard was 1.9 × 10⁻⁶. 10 Copy / μL. Using the reaction system in Table 3 and the reaction procedure in Table 4 for 1.9 × 10⁻⁶ μL. 10 PCR amplification was performed using a standard plasmid of 1 copy / μL. The results showed that, using the plasmid standard as a template, a fragment of the same size as the target fragment could be amplified (Figure 2), indicating that the recombinant plasmid standard was successfully constructed.

[0098] The plasmid standard was serially diluted 10-fold using ddH2O to obtain plasmids of 1.9 × 10⁻⁶ ppm. 9 Copies / μL, 1.9 × 10 8Copies / μL, 1.9 × 10 7 Copies / μL, 1.9 × 10 6 Copies / μL, 1.9 × 10 5 Copies / μL, 1.9 × 10 4 Copies / μL, 1.9 × 10 3 Copies / μL, 1.9 × 10 2 Copies / μL, 1.9 × 10 1 Plasmids of 1.9 copies / μL and 1.9 copies / μL.

[0099] 4. Optimization of conditions for quantitative real-time PCR (i.e., qPCR)

[0100] Using the primer and probe concentrations in Table 6, prepare the qPCR reaction system. If the volume is less than 20 μL, make up the difference with ddH2O. Perform the qPCR reaction under the reaction conditions shown in Table 7.

[0101] Table 6: qPCR reaction system (20 μL system)

[0102] qPCR amplification reaction procedure:

[0103] Table 7: qPCR reaction conditions

[0104] 4.1 Concentration of upstream and downstream primers

[0105] Based on Table 6, the final concentrations of primers in the reaction system are changed as shown in the table below, while other processes remain the same as described above.

[0106] Table 8: Concentration of upstream and downstream primers

[0107] 4.2 Probe concentration

[0108] Based on Table 6, the final concentration of the probe in the reaction system is changed as shown in the table below, while other processes remain the same as described above.

[0109] Table 9: Probe Concentration

[0110] 4.3 qPCR reaction conditions

[0111] The reaction systems in Tables 8 and 9 were subjected to qPCR reactions under the conditions in Table 10.

[0112] Table 10: qPCR reaction conditions

[0113] The qPCR reaction products were subjected to agarose gel electrophoresis. Figure 3 and Table 11 show that when the primer concentration in the qPCR reaction system was 0.2 μmol / L and the probe concentration was 0.1 μmol / L, the agarose gel electrophoresis bands of the qPCR amplification products were the brightest and had the highest gray value at an annealing temperature of 60℃, indicating the best amplification effect.

[0114] Table 11: Analysis of optimal reaction conditions for qPCR

[0115] When the primer concentration is 0.2 μmol / L, the gray value of the electrophoresis result is distributed in the range of 9400 to 12000. The gray value is the most obvious, the electrophoretic band of the product is the best, and the amplification effect is good and significant.

[0116] Based on this, the concentration of the probe was changed to 0.05–0.15 μmol / L, and the gray value was distributed in the range of 9400–12000. Among them, the effect was best when the concentration of the probe was 0.1 μmol / L; the second best was 0.15 μmol / L, and the last best was 0.05 μmol / L.

[0117] When the primer concentration is 0.4 μmol / L, the gray values ​​of the electrophoresis results are distributed between 7400 and 8400, with the gray values ​​showing a decreasing trend.

[0118] When the primer concentration was 0.1 or 0.3 μmol / L, the gray values ​​of the electrophoresis results ranged from approximately 3000 to 8000, a significant decrease compared to the gray values ​​obtained with a primer concentration of 0.2 μmol / L. Furthermore, when the primer concentration was 0.3 μmol / L, the order of effectiveness for changing the probe concentration to 0.1 μmol / L, 0.15 μmol / L, and 0.05 μmol / L was 0.15 μmol / L > 0.1 μmol / L > 0.05 μmol / L. These results further demonstrate that the optimal conditions for primer and probe concentrations are uncertain.

[0119] Therefore, the optimal reaction conditions for quantitative real-time PCR were determined as follows:

[0120] Table 12: qPCR reaction system (20 μL system)

[0121] Table 13: qPCR reaction conditions

[0122] Example 2: Establishment of a standard curve for quantitative real-time PCR (i.e., qPCR)

[0123] Fiber1 gene plasmid standards diluted 10-fold (1.9 × 10⁻⁶)7 Using plasmid concentrations ranging from 1.9 copies / μL to 1.9 copies / μL as templates, quantitative real-time PCR amplification was performed under optimized conditions to obtain amplification curves (Figure 4). A standard curve was established with plasmid concentration on the x-axis and Ct value of qPCR on the y-axis (Figure 5).

[0124] The standard curve equation is Y = -(1.414)ln(x) + 36.252, and the correlation coefficient is R. 2 =0.991, indicating that the particle size of this standard is 1.9×10 7 It exhibits good linearity in the concentration range of 1.9 copies / μL to 1.9 copies / μL.

[0125] Example 3

[0126] 1. Sensitivity test

[0127] 1.9×10 7 Using plasmid standards ranging from 1.9 copies / μL to 1.9 copies / μL as templates, quantitative real-time PCR amplification was performed under optimized conditions (Figures 4 and 6).

[0128] Curves 1-8 in Figure 4 show 1.9 × 10 7 Plasmid standards with concentrations ranging from 1.9 copies / μL to 1.9 copies / μL can be detected; and the agarose gel electrophoresis results in Figure 6 show that the size of the amplified fragment is consistent with the target fragment, indicating that this method has good sensitivity for detecting plasmid standards as low as 1.9 copies / μL.

[0129] 2. Specificity test

[0130] The optimal real-time PCR reaction conditions and reaction system of Example 1 were used to detect the genomic DNA of major parasites infecting birds: Eimeria virulence and Histomonia spp.; bacteria: Escherichia coli and Salmonella; and DNA viruses: avian leukosis virus and 1.9 × 10⁻⁶ genomic DNA. 6 The specificity of the method was verified by detecting plasmid standards at a concentration of 1 copy / μL.

[0131] The results in Figures 7 and 8 show that this method can only amplify recombinant standard plasmids, and no amplification curves were found for other common parasites, viruses, and bacteria infecting birds. The method has good and significant specificity.

[0132] 3. Repeatability experiment

[0133] The optimal real-time PCR method described in Example 1 was used to analyze 1.9 × 10⁻⁶ PCR samples. 10 Repeatability tests were conducted using recombinant plasmid standards at a concentration of 1 copy / μL to evaluate the reproducibility of this method. The results showed that the amplification curves of this quantitative real-time PCR method were essentially consistent during repeated operations, indicating good reproducibility (Figure 9).

[0134] Example 4: Clinical Sample Testing

[0135] The genomes of three commercially available chicken new meningitis triple inactivated vaccines (Ebang new meningitis triple inactivated vaccine, Wohua new meningitis triple inactivated vaccine, and Harbin Pharmaceutical Group chicken new meningitis triple inactivated vaccine) were extracted according to the EZNATM Viral DNA Kit instructions;

[0136] Qualitative results: The real-time quantitative PCR detection method obtained in Example 1 was used to detect the genomes of three commercial vaccines to verify the detection effect of the method. The verification analysis was repeated three times. The results showed that the avian Ankara real-time quantitative PCR detection method established in this study can detect commercial biological products containing avian Ankara virus, with a detection rate of 100% (Figures 10 and 11).

[0137] Quantitative results: Based on the standard curve obtained in Example 2, the copy number of the avian Ankara virus Fiber1 gene in the genomes of the extracted Yibang New Menopausal Triple Inactivated Vaccine, Wohua New Menopausal Triple Inactivated Vaccine, and Harbin Pharmaceutical Group Chicken New Menopausal Triple Inactivated Vaccine was calculated to be 1.43 × 10⁻⁶. 2 Copies / μL, 2.21 × 10 2 Copy / μL and 3.27×10 6 Copy / μL.

[0138] Sensitivity verification: Genomic DNA from the Yibangxin triple inactivated influenza vaccine was serially diluted to obtain a Fiber1 gene copy number concentration of 1.43 × 10⁻⁶. 1 Biological samples with a genomic DNA concentration of 1.43 copies / μL and a Fiber1 gene concentration of 1.43 copies / μL were detected using the real-time quantitative PCR method obtained in Example 1. The results showed that the avian Ankara real-time quantitative PCR method established in this study could detect biological samples containing a Fiber1 gene copy number concentration of 1.43 copies / μL (Figures 12 and 13), consistent with the results of Example 3, further demonstrating the high sensitivity of this method.

Claims

1. A primer designed based on the Fiber1 gene, comprising primer 1; The upstream sequence number of primer 1 is SEQ ID NO:7, and the downstream sequence number is SEQ ID NO:

8.

2. A probe designed based on the Fiber1 gene, comprising probe F1R; sequence number SEQ ID NO:

25.

3. A method for detecting avian Ankara virus, utilizing the primers designed based on the Fiber1 gene as described in claim 1 and the probe designed based on the Fiber1 gene as described in claim 2, comprising the following steps: 1) Prepare the solution to be tested; 2) Preparation of standards: The nucleic acid sequences between upstream and downstream primers designed using the Fiber1 gene of avian Ankara virus were chemically synthesized and ligated into the pUC-18T vector as plasmid standards; 3) Preparation of standard solutions: Dilute the synthesized plasmid standard and prepare a series of standard solutions with gradient concentrations; 4) qPCR detection: Primers and probes are added to the genomic DNA of a series of standard solution samples and the sample to be tested for qPCR detection; 5) Using a series of standard solutions to obtain a series of plasmid standard samples, a CT value-concentration standard curve was obtained; using the CT value of the sample to be tested, the concentration of the Fiber1 gene in the solution to be tested was quantitatively detected.

4. The detection method according to claim 3, wherein, The qPCR detection method in step 4) is as follows: i) Prepare the solution for the qPCR reaction system; ii) The solution is subjected to pre-denaturation and cyclic reaction.

5. The detection method according to claim 3, wherein, The primer concentration in the qPCR reaction system solution in step i) is 0.1 μM to 0.4 μM, preferably 0.2 μM.

6. The detection method according to claim 3, wherein, The probe concentration in the qPCR reaction system solution in step i) is 0.05 μM to 0.15 μM, preferably 0.1 μM.

7. The detection method according to claim 3, wherein, The annealing temperature of the primer 1 reaction system is 58–60°C, preferably 60°C.

8. The detection method according to claim 3, wherein, The annealing time is 20-40 seconds, preferably 30 seconds.

9. The detection method according to claim 3, wherein, The cyclic reaction is repeated 40 to 50 times, preferably 45 times.

10. The detection method according to claim 3, wherein, The pre-denaturation conditions are 93–97°C for 25–35 seconds, with one cycle; preferably 95°C for 30 seconds, with one cycle.

11. The detection method according to claim 3, wherein, The cyclic reaction process is 93-97℃ for 8-12s, 58-62℃ for 25-35s; the number of cycles is 40-50; preferably 95℃ for 10s, 60℃ for 30s; the number of cycles is 45.

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