CN111218526B - PCR primer pair and probe for rapidly identifying I subgroup serum 11 type avian adenovirus and application thereof - Google Patents
PCR primer pair and probe for rapidly identifying I subgroup serum 11 type avian adenovirus and application thereof Download PDFInfo
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Abstract
The invention provides a PCR primer pair and a probe for rapidly identifying and detecting I subgroup serum 11 type avian adenovirus, wherein the sequence of the PCR primer pair is shown as SEQ ID NO. 1 and SEQ ID NO. 2, and the sequence of the probe is shown as SEQ ID NO. 3. The detection method comprises the following steps: taking the DNA of the avian adenovirus sample of the subgroup I serum 11 to be detected as a template, carrying out fluorescent quantitative PCR amplification by using the PCR primer pair and the probe, and collecting fluorescent signals. The lowest detectable template concentration of the invention is 31 copies/. Mu.L, which is about 100 times higher than that of the conventional PCR method, other common poultry viruses have no specific amplification, no cross reaction is found, and the variation coefficient in batch and batch is less than 1%.
Description
Technical Field
The invention belongs to the technical field of virus nucleic acid detection, and particularly relates to a PCR primer pair, a probe and a detection method for rapidly identifying and detecting I subgroup 11 avian adenovirus.
Background
Avian adenovirus (FAdV) is a double-stranded linear DNA virus without a capsular structure, belonging to the family adenoviridae, genus avian adenovirus. Avian adenoviruses are divided into 3 subgroups, wherein subgroup i FAdV is divided into 5 virulent species and 12 serotypes. The chicken flock infected with FAdV can cause various diseases such as inclusion body hepatitis, pericardial effusion-hepatitis syndrome, myogastric erosion and the like, and causes serious economic loss for poultry industry. FAdV can be transmitted in various modes such as horizontal transmission, vertical transmission, semen transmission and the like, and mainly invades liver cells of chickens, and causes degeneration necrosis of the liver cells and nuclear inclusion bodies.
In recent years, most of the regions in China have been reported to contain hepatitis, and most of the contained hepatitis is mainly caused by FAdV-D (FAdV-11 is the main factor) and FAdV-E (FAdV-8 is the main factor). For example, chang et al showed 65.1% FAdV-11 isolate in our country between 2007-2014, indicating that the major epidemic strain in our country between 2007-2014 was FAdV-11 (Changjin L, haiying L, dongdong W, et al Charaking of fowl adenoviruses isolated between 2007 and 2014 in China[J ]. Vet Microbiol,2016, 197:62-67). Although the breeding hens are infected with FAdV-11 without obvious clinical symptoms, FAdV-11 can infect offspring chicks in a vertical transmission mode, thereby causing the chicks to develop serious symptoms. Meanwhile, FAdV-11 can also infect chicken flocks in a horizontal transmission mode, and the chicken flocks can become lifelong carriers after infection and can intermittently expel toxin. This severely jeopardizes the healthy development of the poultry industry. As no safe and effective FAdV-11 vaccine is developed at present, the phenomenon that FAdV-11 infects chicken flocks still occurs, and meanwhile FAdV-11 can be mixed with FAdV-4, FAdV-8 and other serotypes of adenoviruses, which provides great challenges for effective prevention and control of adenoviruses (Niu Y, sun Q, zhang G, et al, epidemic investigation of outbreaks of fowl adenovirus infections in commercial chickens in China [ J ]. Transbound Emerg Dis,2018,65 (1): e121-e 126).
At present, FAdV-11 infection is detected mainly by traditional methods such as SPF chicken embryo inoculation, common PCR amplification and the like. Because the SPF chick embryo inoculated with the disease has long virus reproduction period and complex operation, and meanwhile, after the target strip is amplified by common PCR, the amplified target strip is detected by utilizing agarose gel electrophoresis technology, then the nucleic acid strip is observed in a DNA electrophoresis pattern observer and photographed, whether the disease sample is positive is judged artificially, and the disease sample with very low virus content is difficult to monitor. Furthermore, although foreign scholars refer to SYBR Green I dye method fluorescence quantitative PCR method established by Gunes, A et al, can detect 12 serotypes in Sup>A broad spectrum (Gunes, A., mark, A., grafl, B., berger, E., hess, M.,2012.real-time PCR assay for universal detection and quantitation of all five species of fowl adenoviruses (FAdV-A to FAdV-E). J.Virol.methods, vol.183, pp.147-153), the method is limited to detecting 12 serotypes in Sup>A broad spectrum, namely, can only identify avian adenoviruses and other viruses, and can not identify and detect avian adenoviruses of FAdV-11 and other 11 serotypes. Therefore, it is highly desirable to establish a discrimination detection technique which is simple and rapid to operate, high in sensitivity and good in specificity.
Disclosure of Invention
In view of the foregoing, it is desirable to provide a real-time fluorescent PCR method for rapidly identifying and detecting subgroup I serotype 11 avian adenoviruses, which addresses the problems associated with the prior art.
The technical scheme of the invention is as follows:
in a first aspect, the invention provides a PCR primer pair and probe for rapid differential detection of subgroup I serogroup 11 avian adenovirus; the sequences of the PCR primer pairs are shown as SEQ ID NO. 1 and SEQ ID NO. 2, and the sequences of the probes are shown as SEQ ID NO. 3.
Further, FAM is bound to the 5 '-end of the probe, and TAMRA is bound to the 3' -end of the probe.
In a second aspect, the invention provides a kit for rapid differential detection of subgroup I sero-11 avian adenovirus, comprising said PCR primer pair and said probe.
Further, the kit also comprises a positive recombinant plasmid standard containing FAdV-11hexon genes.
In a third aspect, the invention also provides application of the PCR primer pair and the probe in preparation of a preparation for detecting or screening I subgroup 11 avian adenovirus.
In a fourth aspect, the present invention provides a method for rapidly identifying subgroup I serotype 11 avian adenoviruses in a sample for identifying and separating subgroup I serotype 11 avian adenoviruses from a complex-component sample or a complex-infected sample; the method comprises the following steps: taking the DNA of the avian adenovirus sample of the subgroup I serum 11 to be detected as a template, carrying out fluorescent quantitative PCR amplification by using the PCR primer pair and the probe, and collecting fluorescent signals.
Further, in the reaction system for fluorescent quantitative PCR amplification, 6pmol of the primer shown as SEQ ID NO. 1, 6pmol of the primer shown as SEQ ID NO. 2, and 4pmol of the probe were contained per 20. Mu.L.
Further, the reaction system for fluorescent quantitative PCR amplification is as follows: 2 XMaster Mix (Probe qPCR) 10. Mu.L, rox reference dye (50X) 0.04. Mu.L, DNA template 2. Mu.L, SEQ ID NO:1 primer (6 pmol) 0.2. Mu.L, SEQ ID NO:2 primer (6 pmol) 0.2. Mu.L, SEQ ID NO:3 Probe (4 pmol) 0.1. Mu.L, and 20. Mu.L was supplemented with sterile double distilled water.
Further, the reaction conditions of the fluorescent quantitative PCR amplification are as follows: pre-denaturation at 95 ℃ for 20sec; denaturation at 95℃for 3sec, annealing/extension at 60℃for 30sec for 40 cycles.
The invention has the advantages and beneficial effects that:
according to the invention, a specific amplification primer and a probe are designed in a conservation area according to the hexon gene sequence of FAdV-11, and a specific method for detecting FAdV-11 is established.
The specificity test results show that the primers and the probes of the invention have no cross reaction with other poultry main diseases (FAdV-4, FAdV-8, H9 subtype AIV, NDV, IBV and ILTV).
The sensitivity test result shows that the lowest detectable template concentration of the detection method is 31 copies/mu L, which is about 100 times higher than that of the conventional PCR method, other common poultry viruses have no specific amplification, no cross reaction is found, and the variation coefficients in and among batches are less than 1%, so that the detection method has the advantages of high sensitivity, strong specificity, good repeatability and the like, and has wide application prospect.
The real-time fluorescence quantitative PCR detection method can directly detect the change of a fluorescence signal in the PCR amplification process through the photoelectric conduction system to obtain a quantitative result, overcomes a plurality of defects in the prior art, and has the following advantages: (1) the template can be quantified; (2) the sealing reaction is carried out, the post-treatment of PCR is not needed, the pollution is less, and the false positive rate is low; (3) the observation and recording are automatic, the result is visual, and errors caused by artificial judgment are avoided; (4) the working efficiency is high, and the high-flux detection is facilitated.
Drawings
FIG. 1 is a graph showing the PCR amplification results of the standard plasmid of example 1 of the present invention, wherein M: DNA molecular weight standard (DL 2000); 1-3: a standard plasmid; 4: a positive control; 5: negative control.
FIG. 2 is a graph showing the results of ordinary PCR amplification in example 1 of the present invention, wherein 1 to 10: plasmid concentrations were 1.0X10 respectively 9 copies/μL、1.0×10 8 copies/μL、1.0×10 7 copies/μL、1.0×10 6 copies/μL、1.0×10 5 copies/μL、1.0×10 4 copies/μL、1.0×10 3 copies/μL、1.0×10 2 copies/μL、1.0×10 1 COPIES/. Mu.L, 1.0X100 COPIES/. Mu.L; 11: negative control.
FIG. 3 is a standard chart of FAdV-11 TaqMan fluorescent quantitative PCR in example 1 of the present invention.
FIG. 4 is a graph of FAdV-11 TaqMan fluorescent quantitative PCR kinetics for example 1 of the present invention, wherein 1-10: standard plasmid concentrations were 3.10X10 respectively 9 copies/μL、3.10×10 8 copies/μL、3.10×10 7 copies/μL、3.10×10 6 copies/μL、3.10×10 5 copies/μL、3.10×10 4 copies/μL、3.10×10 3 copies/μL、3.10×10 2 copies/μL、3.10×10 1 copies/μL、3.10×10 0 cobies/. Mu.L; 11: negative control.
FIG. 5 is a graph showing the specific amplification of FAdV-11 TaqMan fluorescent quantitative PCR method of example 2 of the present invention.
FIG. 6 is a survival curve of experimental chickens after FAdV-11 challenge in example 3 of the present invention.
FIG. 7 is a graph showing the results of the test chicken detoxification cases after FAdV-11 detoxification in example 3 of the present invention.
FIG. 8 is a graph showing the results of the viral load of each tissue of the test chicken after FAdV-11 challenge in example 3 of the present invention.
Detailed Description
In order to better illustrate the problems addressed by the present invention, the technical solutions employed and the effects achieved, reference will now be made further to specific embodiments and related data. It should be noted that the present disclosure includes, but is not limited to, the following examples and their combination embodiments.
The embodiments of the present invention are not to be construed as specific techniques or conditions, according to techniques or conditions described in the literature in this field or according to the product specifications. The reagents or instruments used are conventional products available in commercial and other ways, and the manufacturers are not identified.
Example 1
Establishment of FAdV-11 TaqMan fluorescent quantitative PCR detection method
1. Material
1.1 strains
The avian adenovirus type 11 (FAdV-11), the avian adenovirus type 8 (FAdV-8), the avian adenovirus type 4 (FAdV-4), the avian influenza virus type H9 (AIV H9), the Newcastle Disease Virus (NDV), the Marek's Disease Virus (MDV), the Infectious Bronchitis Virus (IBV) and the infectious laryngotracheitis virus (ILTV) are all obtained by the commercial route.
1.2 major reagents and instruments
Humoral virus DNA/RNA miniprep kit was purchased from Axygen, pMD-19T vector and PrimeScript TM The RT Master Mix (Perfect Real Time) kit was purchased from Takara,probe qPCR Mix was purchased from TOYOBO company, applied Biosystems 7500 FAST type real-time fluorescent quantitative PCR instrument was purchased from ABI company.
2. Method of
2.1 primer design and Synthesis
The Hexon gene contains avian adenovirus type specificity, group specificity and subgroup specificity antigenic determinants, is a main parting gene and virulence gene, and is screened out a highly conserved specific region of FAdV-11Hexon genes by largely comparing DNA sequences of I subgroup serum 11 type avian adenovirus Hexon genes in GenBank, and a specific primer pair and a TaqMan probe for detecting the FAdV-11Hexon genes are designed. The specific primers were designated FAdV-11-F (SEQ ID NO: 1) and FAdV-11-R (SEQ ID NO: 2) respectively, and the Probe was designated Probe (SEQ ID NO: 3). The primer pair and probe sequences designed are as follows:
FAdV-11-F(SEQ ID NO:1)：5'-gaggaccttcttttaaacc-3'
FAdV-11-R(SEQ ID NO:2)：5'-gtagggagttgtcatctg-3'
Probe(SEQ ID NO:3)：5'-FAM-tcttgctcgcctctgtgtca-TAMRA-3'
2.2 nucleic acid extraction of samples
Nucleic acid extraction was performed on tissue sample milling solutions (see example 4) and virus solutions, and the extraction method was referred to the instructions of the Axygen company humoral viral DNA/RNA miniprep kit. The specific experimental steps comprise: collecting 200 mu L of supernatant (or virus liquid) after tissue homogenate centrifugation, and transferring the supernatant into a 1.5mL sterile centrifuge tube; adding 200 mu L Buffer V-L, vortex shaking and mixing uniformly, centrifuging at 12,000rpm for 1min, standing at room temperature for 5min to fully crack the sample; adding 75 μl Buffer V-N, vortex shaking, mixing to denature protein, and centrifuging at 12,000rpm for 5min; the supernatant was transferred to a new sterile 2mL centrifuge tube, 250. Mu.L of isopropanol (1% glacial acetic acid) was added, inverted 6-8 times, and mixed well. Placing the preparation tube into a new 2mL centrifuge tube, transferring the mixed solution into the preparation tube, centrifuging at 6,000rpm for 1min, and discarding the filtrate; 500. Mu.L Buffer W1A (added with absolute ethanol with a specified volume) is added into a preparation tube, and the mixture is left standing at room temperature for 1min, centrifuged at 12,000rpm for 1min, and the liquid in the collection tube is discarded; then adding 800 mu L Buffer W2 into a collecting pipe, centrifuging at 12,000rpm for 1min, and discarding liquid in the collecting pipe; the column was then returned to a 2mL centrifuge tube and centrifuged at 12,000rpm for 1min; transferring the preparation tube into a brand new 1.5mL sterile centrifuge tube; finally, 50 mu L of TE buffer is added in the center of the prepared tube membrane, the tube membrane is stood still at room temperature for 1min, and the DNA/RNA is eluted by centrifugation at 12,000rpm for 1min; the nucleic acid sample was placed in a-20℃refrigerator for further use.
2.3 reverse transcription
According to PrimeScript TM Reverse transcription reactions were performed using RT Master Mix (Perfect Real Time) kit instructions. Preparing 20. Mu.L of the reaction system on ice, namely taking 4. Mu.L of 5X PrimeScript RT Master Mix (Perfect Real Time), adding RNase Free dH into Total RNA template (less than or equal to 1. Mu.g) 2 O to 20. Mu.L; slightly and evenly mixed, and then subjected to reverse transcription reaction, and stripsThe parts are as follows: 37℃for 15min (reverse transcription reaction), 85℃for 5sec (reverse transcriptase inactivation reaction), and 4 ℃.
2.4 Standard plasmid construction
Screening the conserved region of FAdV-11hexon gene, designing standard plasmid primer pair of FAdV-11hexon gene, named hexon-F (SEQ ID NO. 4) and hexon-R (SEQ ID NO. 5), respectively. The designed standard plasmid primer sequences were as follows:
hexon-F(SEQ ID NO.4)：5'-GGCGTGCTAGACACTAGACA-3'
hexon-R(SEQ ID NO.5)：5'-TGTCTAGTGTCTAGCACGCC-3'
extracting FAdV-11 strain DNA, recovering positive amplified products after PCR amplification fragment size is 550bp (figure 1), cloning the positive amplified products into a pMD-19T vector, screening positive recombinant plasmid and sending the positive recombinant plasmid to Shanghai biological company for sequencing and identification. The correctly sequenced positive recombinant plasmid was designated pMD-19T-hexaon, the OD 260/OD 280 ratio of pMD-19T-hexaon was 1.88, and the nucleic acid concentration was 110ng/uL using an ultraviolet spectrophotometer. It was diluted 10-fold, a total of 8 gradients (10 -1 -10 -8 ) Then, PCR amplification was performed using FAdV-11-F (SEQ ID NO: 1) and FAdV-11-R (SEQ ID NO: 2) as primers. Reference plasmid DNA copy number calculation method, the concentration of pMD-19T-hexaon plasmid DNA solution was calculated to be 3.10X10 10 copies/μL。
2.5 qPCR reaction system and conditions
The FAdV-11 TaqMan fluorescent quantitative PCR amplification reaction system is as follows: 2 XMaster Mix (Probe qPCR) 10. Mu.L, rox reference dye (50X) 0.04. Mu.L, DNA template 2. Mu.L, SEQ ID NO:1 primer (6 pmol) 0.5. Mu.L, SEQ ID NO:2 primer (6 pmol) 0.5. Mu.L, SEQ ID NO:3 Probe (4 pmol) 0.5. Mu.L and 20. Mu.L supplemented with sterile double distilled water. The optimized reaction conditions are as follows: pre-denaturation at 95 ℃ for 20sec; denaturation at 95℃for 3sec, annealing/extension at 60℃for 30sec for 40 cycles.
3. Results
3.1 Construction of pMD-19T-hexon Standard plasmid and PCR results
FIG. 1 provides a map of the PCR amplification results of the pMD-19T-hexon standard plasmid, and it can be seen that the PCR amplified fragment size is 550bp. The result of the common PCR amplification is shown in FIG. 2, and the PCR is the most suitable3.10X10 were detected at low energy 3 COPies/. Mu.L. The common PCR uses FAdV-11-F (SEQ ID NO: 1) and FAdV-11-R (SEQ ID NO: 2) as primers, the standard plasmid is diluted by 10 times as a template, the lowest detection concentration of the common PCR amplification is detected, and the quantitative PCR can be protruded from the side surface, so that the PCR has high sensitivity.
3.2 qPCR standard curve
The standard plasmid pMD-19T-hexon was subjected to multiple dilution at a ratio of 10-fold, and a total of 9 gradients were diluted (10 -1 -10 -9 ) qPCR standard curves were drawn using the gradient diluted plasmid as template (FIG. 3). The results show that: y= -3.5876x+41.17 (R 2 = 0.9986), qPCR amplification results showed good linearity between different gradient log values and Ct values.
Example 2
Sensitivity and repeatability detection
The standard plasmid pMD-19T-hexan after gradient dilution was used as a template according to 3.10X10 9 ～3.10×10 0 Fluorescence quantitative PCR amplification was performed on the copies/uL respectively, and Ct values greater than 36 were regarded as negative. qPCR standard curves were plotted for different concentrations. As shown in FIG. 4, the method has high sensitivity, and the lowest detectable template concentration is 3.10X10 1 COPies/. Mu.L, compared with ordinary PCR (3.10X10) 3 cobies/. Mu.L) sensitivity was 100 times higher.
At 3.10X10 5 Copies/. Mu.L and 3.10X10 6 Two concentrations of the standard plasmid pMD-19T-hexan, copies/. Mu.L, were used as templates, the two dilutions were assayed 3 times in duplicate at different time periods, 3 replicates were performed simultaneously on the same template each time, and 3 duplicate wells were set up for real-time fluorescent quantitative PCR according to the amplification system and conditions of example 1. The results show that the variation coefficient of the FAdV-11 TaqMan fluorescence quantitative PCR method is smaller than 1% in the batch and between the batch, which shows that the established FAdV-11 TaqMan fluorescence quantitative PCR method has better repeatability and stable and reliable results (Table 1).
TABLE 1 repeatability of FAdV-11 TaqMan fluorescent quantitative PCR method
Example 3
Specific experiment of FAdV-11 TaqMan fluorescent quantitative PCR detection method
Common viruses that have been collected in the laboratory and cause poultry to become ill include serogroup 8 avian adenovirus (FAdV-8), serogroup 4 avian adenovirus (FAdV-4), H9 subtype avian influenza virus (AIV H9), newcastle Disease Virus (NDV), marek's Disease Virus (MDV), infectious Bronchitis Virus (IBV), infectious laryngotracheitis virus (ILTV), and were used to detect the specificity of the real-time fluorescent quantitative PCR method of serogroup 11 avian adenovirus according to the present invention, and the reaction system and amplification reaction conditions were amplified in reference example 1. As shown in FIG. 5, FAdV-8, FAdV-4, MDV, AIV H9, NDV, IBV, ILTV and sterile double distilled water all had no amplification curve, and were negative, only FAdV-11 samples were able to amplify positive curves. The fluorescent quantitative PCR method has high specificity.
Example 4
Clinical sample detection is carried out by adopting the FAdV-11 TaqMan fluorescent quantitative PCR method established by the invention
Dilution of serotype 11 avian adenovirus to 2X 10 6 TCID 50 Per 0.1mL, 10 SPF chickens of 3 days of age were infected by intramuscular injection. Anal swabs of test chickens are collected before and on the 2 nd and 4 th days after the detoxification, 1mL of sterile PBS is added, and after shaking and mixing, the supernatant is collected by centrifugation. In addition, on the 4 th day after the toxicity attack, tissue samples of heart, liver, spleen, lung, kidney, bursa of Fabricius, pancreas, small intestine, adenosis stomach, myostomach and the like of 3 test chickens are collected, and a tissue grinding fluid is prepared. DNA is extracted by referring to a humoral virus nucleic acid extraction kit, and the FAdV-11 proliferation and detoxification condition in chickens is detected by using an established FAdV-11 TaqMan real-time fluorescent quantitative PCR method.
The results show that the test chickens infected by FAdV-11 have clinical symptoms such as mental depression, disharmony and yellow green feces and the like on the 3 rd day after the toxicity attack, and begin to die on the 3 rd day after the toxicity attack, and the death rate reaches 100 percent (figure 6). The detoxification of test chickens following FAdV-11 infection suggests that fecal port transmission in production is an important transmission mode for FAdV-11 (FIG. 7). Meanwhile, the virus content of the collected organs of each tissue is detected, and the virus can be replicated and proliferated in the organs of each tissue of the test chicken, wherein the virus content in the liver is highest, and the pancreas and small intestine and the heart are the lowest (figure 8).
In conclusion, the invention designs a specific amplification primer and a probe according to the hexon gene sequence of FAdV-11, and establishes a specific method for detecting FAdV-11, and the detection method has the advantages of high sensitivity, strong specificity, good repeatability and the like for the serum 11 type avian adenovirus of subgroup I, the lowest detectable template concentration is 31 copies/mu L, which is about 100 times higher than that of the conventional PCR method, other common poultry viruses have no specific amplification, no cross reaction is found, and the intra-batch and inter-batch variation coefficients are less than 1%.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Sequence listing
<110> Wenshi food group Co., ltd
<120> PCR primer pair and probe for rapidly identifying I subgroup serum 11 type avian adenovirus and application thereof
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Claims (9)
1. A PCR primer pair and a probe for rapidly identifying I subgroup serum 11 type avian adenovirus are characterized in that the sequences of the PCR primer pair are shown as SEQ ID NO. 1 and SEQ ID NO. 2; the sequence of the probe is shown as SEQ ID NO. 3.
2. The PCR primer pair and probe for rapid identification of subgroup I sero-11 avian adenovirus as claimed in claim 1, wherein the probe has FAM bound at the 5 'end and TAMRA bound at the 3' end.
3. The use of a PCR primer pair and probe as claimed in claim 1 in the preparation of a formulation for detecting or screening subgroup I sero 11 avian adenovirus.
4. A kit for rapid identification of subgroup I sero-11 avian adenovirus, said kit comprising a PCR primer pair and a probe as described in claim 1.
5. The kit for rapid identification of subgroup I sero-11 avian adenovirus as claimed in claim 4, further comprising a positive recombinant plasmid standard comprising the FAdV-11hexon gene.
6. A method for rapidly identifying subgroup I serogroup 11 avian adenoviruses, said method being for non-diagnostic purposes, said method comprising: taking the DNA of the avian adenovirus sample of the serum 11 type of the subgroup I to be detected as a template, carrying out fluorescent quantitative PCR amplification by using the PCR primer pair and the probe as set forth in claim 1, and collecting fluorescent signals.
7. The method for rapid identification of subgroup I serogroup 11 avian adenovirus as claimed in claim 6, wherein the reaction system composition used for PCR amplification comprises: each 20. Mu.L of the reaction system contained 6pmol of the primer of claim 1 and 4pmol of the probe.
8. The method for rapid identification of subgroup I sero-11 avian adenovirus as claimed in claim 6, wherein each 20 μl of the reaction system for PCR amplification comprises: 2 XMaster Mix 10. Mu.L, 50X Rox reference dye 0.04.04. Mu.L, DNA template 2. Mu.L, 6pmol of the primer shown in SEQ ID NO:1, 6pmol of the primer shown in SEQ ID NO:2, 4pmol of the probe shown in SEQ ID NO:3, and sterilized double distilled water was added to 20. Mu.L.
9. The method for rapid identification of subgroup I sero-11 avian adenovirus as claimed in claim 6, wherein the reaction conditions for PCR amplification are: pre-denaturation at 95 ℃ for 20 seconds; denaturation at 95℃for 3sec, annealing/extension at 60℃for 30sec for 40 cycles.
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