CN109136407B

CN109136407B - PCR method for rapidly detecting avian adenovirus-I group

Info

Publication number: CN109136407B
Application number: CN201811105544.9A
Authority: CN
Inventors: 王建琳; 尹燕博; 栾庆东; 江之瑶
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2018-09-21
Filing date: 2018-09-21
Publication date: 2021-11-16
Anticipated expiration: 2038-09-21
Also published as: CN109136407A

Abstract

The invention discloses a PCR method for rapidly detecting avian adenovirus-I group. The invention adopts the hands-free nucleic acid method to obtain nucleic acid for PCR amplification, compared with the prior art, the invention does not need to carry out fussy treatment on the sample, thus obviously shortening the detection time; the concentration of the obtained nucleic acid is 4-14 times higher, and the purity of the nucleic acid is not influenced; the sensitivity of the detection on the same sample is 4 times and 10 times higher. The hands-free nucleic acid PCR method has the advantages of short time, strong specificity, high sensitivity and good stability, and can quickly, sensitively and specifically detect the avian adenovirus-I group.

Description

PCR method for rapidly detecting avian adenovirus-I group

Technical Field

The invention relates to a PCR method for rapidly detecting avian adenovirus-I group.

Background

The avian adenovirus belongs to the genus avian adenovirus of the family adenoviridae, and can be divided into 3 groups according to different antigenicity; based on serocross-neutralization reactivity, the avian adenovirus-group I can be divided into 12 serotypes (serotypes 1-7, 8a, 8b, 9-11). The avian adenovirus-group I is widely distributed in the world, and recently, researches report that chicken hepatitis-pericardial effusion syndrome, inclusion body hepatitis and the like caused by infection of different serotype strains of the avian adenovirus-group I are widely prevalent in China, particularly, the chicken hepatitis-pericardial effusion syndrome caused by the avian adenovirus of serotype 4 causes a great amount of death of broiler chickens, and causes serious economic loss to the chicken industry in China. In order to effectively control the infection of the avian adenovirus-I group, a rapid and accurate diagnosis method needs to be established.

The diagnosis method of the avian adenovirus-I group comprises the traditional pathogen separation identification, serological method and molecular biological technology. The diagnosis of avian adenovirus-I group infection is carried out by observing the nuclear inclusion body of the hepatocyte through histopathology and observing the virus particle shape through an electron microscope, and then virus separation is carried out by using chick embryo, chick embryo kidney cell, chick embryo hepatocyte, Vero cell and the like. Then, the antibody is detected by serological methods such as agar gel precipitation test, indirect hemagglutination test, spot immune combination test, enzyme-linked immunosorbent assay, immunoperoxidase test, virus neutralization test of tissue culture and chick embryo culture and the like for diagnosis, but the antibody exists in infected chickens and immune chickens, and the detection result is difficult to explain. The molecular biology technology detects the avian adenovirus-I group instead of detecting an antibody, the diagnosis can be carried out at the early stage of virus infection, the PCR diagnosis for the avian adenovirus-I group Hexon gene has higher sensitivity and specificity, in addition, the Hexon gene amplified by the PCR can be used as a probe for spot hybridization, and the amplified Hexon is subjected to restriction fragment length polymorphism analysis after restriction enzyme digestion to carry out the serotyping on the avian adenovirus-I group. Therefore, the existing PCR diagnosis method is a sensitive and specific diagnosis method for detecting the infection of the avian adenovirus-I group, but the PCR method has more steps and longer time consumption, and the problem of how to shorten the time and improve the efficiency on the basis of the original PCR is solved by the existing rapid diagnosis.

Disclosure of Invention

The invention aims to provide a PCR method for rapidly detecting avian adenovirus group I.

The invention firstly provides a method for obtaining virus nucleic acid, which comprises the following steps: processing the sample by Buffer to obtain virus nucleic acid; the Buffer is an aqueous solution containing 45-50mM Tris-HCl, 5.8-6.0M urea and 1ml/100ml Tween 20.

The Buffer can be specifically an aqueous solution containing 50mM Tris-HCl, 6M urea and 1ml/100ml Tween 20.

The processing method comprises the following steps: mixing the sample with the Buffer, standing and reacting at 98 ℃ for 8-10min, and then standing and reacting at 4 ℃ for 10-15 min.

The treatment method specifically comprises the following steps: mixing the sample with the Buffer, standing and reacting at 98 ℃ for 8min, and then standing and reacting at 4 ℃ for 15 min.

The Buffer was mixed with the sample in equal volume. And when the sample to be detected is a tissue, grinding the tissue, and centrifuging to obtain a supernatant.

The invention also provides a method for detecting the avian adenovirus-I group, which comprises the following steps:

(1) obtaining a virus to be detected or virus nucleic acid of a sample to be detected by adopting any one of the methods;

(2) and (2) detecting whether the specific fragment of the avian adenovirus-I group exists in the virus nucleic acid obtained in the step (1), wherein if the specific fragment of the avian adenovirus-I group exists in the virus nucleic acid, the virus to be detected is the avian adenovirus group I or the avian adenovirus-I group exists in the sample to be detected.

The method for detecting whether the specific fragment of the avian adenovirus-I group exists in the virus nucleic acid obtained in the step (1) comprises the following steps: performing PCR amplification on the virus nucleic acid obtained in the step (1) by using a specific primer pair, wherein if a specific band of 850-910bp exists in an amplification product, a specific fragment of the avian adenovirus-I group exists in the amplification product;

the specific primer pair consists of a primer F and a primer R;

the primer F is (a1) or (a 2):

(a1) a single-stranded DNA molecule shown in sequence 1 of the sequence table;

(a2) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 1 and have the same functions as the sequence 1;

the primer R is (a3) or (a 4):

(a3) a single-stranded DNA molecule shown in a sequence 2 of a sequence table;

(a4) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 2 and has the same function as the sequence 2.

The reaction system for PCR amplification specifically may be: nucleic acid 2. mu.L, 10 XPCRBufer 3. mu.L, 25mM Mgcl₂mu.L, 2.5. mu.L of 10mM dNTP, 0.5. mu.L (25pmol/w) of each of the forward primer F and the reverse primer R, 0.5. mu.L of Taq enzyme, and sterilized ultrapure water to 30. mu.L.

The reaction procedure of the PCR amplification can be specifically as follows: denaturation at 94 deg.C for 5 min; 1min at 94 ℃, l min at 55 ℃, 90s at 72 ℃ and 30 cycles; extension at 72 ℃ for 10 min.

Any one of the specific fragments can be specifically shown as a sequence 3 in a sequence table.

The invention also protects the application of any one of the above buffers in obtaining viral nucleic acid.

The invention also protects the application of any of the above buffers in the detection of the avian adenovirus-I group.

The invention also provides a kit for detecting the avian adenovirus-I group, which comprises any one of the Buffer and the specific primer pair.

Any one of the viruses to be detected can be a 12-strain standard serotype strain (FAV-1, FAV-2, FAV-3, FAV-4, FAV-5, FAV-6, FAV-7, FAV-8, FAV-9, FAV-10, FAV-11 and FAV-12) of the avian adenovirus-I group, a serotype 4 avian adenovirus isolate (FAV-QDLX-140421-B), a serotype 8a avian adenovirus isolate (FAV-SDWF-140620-B), a serotype 8B avian adenovirus isolate (FAV-QDLX-111025-B) or a serotype 11 avian adenovirus isolate (FAV-SDLQ-140704-G).

Any of the above samples may be animal tissue, such as liver tissue.

Compared with the prior art, the invention adopts a hands-free nucleic acid method to obtain nucleic acid for PCR amplification, does not need to carry out fussy treatment on a sample, and obviously shortens the detection time; the concentration of the obtained nucleic acid is 4-14 times higher, and the purity of the nucleic acid is not influenced; the sensitivity of the detection on the same sample is 4 times and 10 times higher. The hands-free nucleic acid PCR method has the advantages of short time, strong specificity, high sensitivity and good stability, and can quickly, sensitively and specifically detect the avian adenovirus-I group.

Drawings

FIG. 1 shows PCR amplification results of nucleic acids extracted by different methods. 1: negative control; m: marker; 2: traditional Trizol nucleic acid extraction PCR method; 3: hands-free nucleic acid PCR method; 4: takara kit nucleic acid extraction PCR method.

FIG. 2 shows the result of detecting 12 standard serotype strains of avian adenovirus group I by hands-free nucleic acid PCR method. M: marker; 1-12: avian adenovirus serotype 1-12.

FIG. 3 shows the specificity of detection of avian adenovirus group I by hands-free nucleic acid PCR method. M: marker; 1: a serum type 4 avian adenovirus isolate; 2: an avian adenovirus type 8a isolate; 3: an avian adenovirus type 8b isolate; 4: avian adenovirus type 11 isolate; 5: fowlpox virus; 6: newcastle disease virus; 7: H9N2 subtype avian influenza virus; 8: chicken infectious anemia virus; 9: infectious bronchitis virus; 10: and (5) negative control.

FIG. 4 shows the results of sensitivity detection by the hands-free PCR method and the conventional Trizol-extracted PCR method. M: marker; 1-9: the results of hands-free nucleic acid PCR are respectively original poison, diluted 10, 100, 1000, 2000, 5000, 10000, 20000 and 40000 times; 10-18: the traditional Trizol nucleic acid extraction PCR result is respectively 10 times, 100 times, 1000 times, 2000 times, 5000 times, 10000 times, 20000 times and 40000 times of original toxicity and dilution.

FIG. 5 shows the result of PCR-based sensitivity detection of nucleic acids extracted by Takara kit. M: marker; 1-10: original poison, 10 times diluted, 100, 1000, 2000, 5000, 10000, 20000, 40000 and negative control.

FIG. 6 shows the results of detection of avian adenovirus-I group clinical disease by hands-free PCR and traditional Trizol nucleic acid extraction PCR. A: detecting the result of the poultry adenovirus-I group clinical disease material by a traditional Trizol nucleic acid extraction PCR method; b: the result of detecting the poultry adenovirus-I group clinical disease material by a hands-free nucleic acid PCR method.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

12 standard serotype strains of avian adenovirus group I (FAV-1, FAV-2, FAV-3, FAV-4, FAV-5, FAV-6, FAV-7, FAV-8, FAV-9, FAV-10, FAV-11 and FAV-12) were purchased from Chinese veterinary drug inspection.

Avian Adenovirus serotype 4 isolate (FAV-QDLX-140421-B), Avian Adenovirus serotype 8a isolate (FAV-SDWF-140620-B), Avian Adenovirus serotype 8B isolate (FAV-QDLX-111025-B), Avian Adenovirus type 11 isolate (FAV-SDLQ-140704-G) are described in the documents Wang J, Wang S, Zou K, Zhang Y, Xu S, YinY. variant serogroups of wlfo Adenoviral Isolated from Commercial Poultwen and 2017in SoRegions of China, Avian Dis,2018,62 (2). 171- -176, publicly available from the university of agricultural, Qingdao.

Fowlpox virus (fowlpox virus quail attenuated vaccine strain CVCC AV1003 strain) is described in the literature: the construction of recombinant fowlpox virus expressing HA protein of H5 subtype avian influenza virus and its immune potency were first assessed [ D ] chinese academy of agricultural sciences, 2012.

Newcastle disease virus (newcastle disease attenuated vaccine strain LaSota strain) is described in the literature: the study of the effects of drugs on different strains of newcastle disease virus [ D ]. north river agricultural university, 2008. the public is available from Qingdao agricultural university.

Subtype H9N2 avian influenza virus (subtype H9N2 avian influenza virus strain, strain C/SD/196/11) is described in the literature: the different pathogenic H9N2 subtype avian influenza viruses induce the difference of mRNA transcription levels of TLR-7 and Mx genes of chickens, and the veterinary science of animal husbandry, 2017,48(5): 907-.

Chicken infectious anemia virus is described in literature: li Changjin, Li Haiying, Wang Dongdong, Wang Jinggjin, Wang Youding, Wang Shouchun, Li Jida, Liu Ping, Wang Jianlin, Xu Shouzhen, Cui Shangjin, Zhang Yi, Yin Yanbo, Characterisation of how infected between Ween and 2014in China, Veterinary Microbiology,2016,19762-67.

Infectious bronchitis virus (infectious bronchitis attenuated vaccine strain M41) is described in the literature: cloning and sequencing of genes of infectious bronchitis virus M41 and vaccine strains H52 and H120S1 in Xiujun, Zheng Haizhou, Zuli, et al [ J ]. proceedings of northern university of Hebei (Nature science edition), 2004,28(2): 181-.

Example 1 comparison of methods for obtaining viral nucleic acids

One, hands-free nucleic acid method for obtaining virus nucleic acid

Hands-free Buffer: an aqueous solution containing 50mM Tris-HCl (pH 7.5), 6M urea and 1% (by volume) Tween 20.

Mixing 20 mu L of the hands-free Buffer with a sample to be detected (when the sample to be detected is a tissue, grinding the tissue and centrifuging to take a supernatant), standing and reacting at 98 ℃ for 8min after mixing according to the volume of 1:1, and then standing and reacting at 4 ℃ for 15 min.

Second, traditional Trizol nucleic acid extraction method to obtain virus nucleic acid

1. 200 μ L of sample to be tested was taken, mixed with 700 μ L of Trizol reagent (Beijing Optimalaceae, New Biotechnology Co., Ltd., product number: 15596026) by shaking thoroughly, and allowed to stand at 4 ℃ for 10 minutes.

2. After completion of step 1, 150. mu.L of chloroform was added, followed by vigorous shaking for 5 seconds, standing at 4 ℃ for 3 minutes, centrifugation at 12000r/min for 15 minutes, and the supernatant was collected.

3. Adding 500 μ L isopropanol into the supernatant obtained in step 2, reversing, standing at 4 deg.C for 10min, centrifuging at 12000r/min for 15min, and discarding the supernatant.

4. Washing the precipitate obtained in step 3 with 1mL of 75% (volume percentage) ethanol aqueous solution for three times, centrifuging at 7500r/min for 5min, collecting the precipitate, drying, dissolving the nucleic acid precipitate with 20 μ L of DEPC water, and storing in a refrigerator at-80 deg.C.

Third, TAKARA nucleic acid extraction kit method for obtaining virus nucleic acid

Viral nucleic acid was extracted according to the TAKARA kit (Takara Bio Inc., cat # 9766).

Effect comparison of four and three methods

1. The results of nucleic acid extraction and nucleic acid concentration measurement of the same serum type 4 avian adenovirus isolate by the three methods described in the first, second, and third above are shown in table 1. The results show that the concentration of nucleic acid extracted by the hands-free nucleic acid method is much higher than that of the other two methods.

TABLE 1 comparison of nucleic acid concentrations extracted by different nucleic acid extraction methods

2. Extracting nucleic acid from the same serum type 4 avian adenovirus isolate by the three methods of the first, second and third, performing PCR amplification on the extracted nucleic acid, and performing gel electrophoresis detection on the amplified product.

And (3) PCR reaction system: nucleic acid 2. mu.L, 10 XPCRRbuffer 3. mu.LL，25mM MgCl₂mu.L, 10mM dNTP 2.5. mu.L, upstream primer F and downstream primer R each 0.5. mu.L (25pmol/L), Taq enzyme 0.5. mu.L (Dalibao bioengineering Co., Ltd.), and sterilized ultrapure water to 30. mu.L. A negative control using DEPC water instead of nucleic acid was set up.

PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; 1min at 94 ℃, l min at 55 ℃, 90s at 72 ℃ and 30 cycles; extension at 72 ℃ for 10 min.

F:5 '-CAARTTCAGRCAGACGGT-3' (SEQ ID NO: 1);

5'-AAGAGGCCCGGGCAATGC-3' (SEQ ID NO: 2).

The results are shown in FIG. 1. The results show that after the nucleic acid extracted by the three methods is amplified by PCR, the product is 877bp (sequence 3 in the sequence table), and the electrophoresis bands are all free of impurity bands.

The results show that the nucleic acid extracted by the hands-free nucleic acid method has high concentration, simple and convenient operation and less time consumption, and the purity of the extracted nucleic acid can meet the detection requirement.

Example 2 detection of avian adenovirus group I by hands-free nucleic acid PCR

Detection of Standard Strain

The strain to be tested: 12 standard serotype strains of avian adenovirus group-I.

And (2) extracting nucleic acid of a strain to be detected by adopting the hands-free nucleic acid method established in the step one of the embodiment 1, carrying out PCR amplification on the extracted nucleic acid, and carrying out gel electrophoresis detection on an amplification product.

And (3) PCR reaction system: nucleic acid 2. mu.L, 10 XPCR buffer 3. mu.L, 25mM MgCl₂mu.L, 10mM dNTP 2.5. mu.L, upstream primer F and downstream primer R each 0.5. mu.L (25pmol/L), Taq enzyme 0.5. mu.L (Dalibao bioengineering Co., Ltd.), and sterilized ultrapure water to 30. mu.L.

F:5 '-CAARTTCAGRCAGACGGT-3' (SEQ ID NO: 1);

5'-AAGAGGCCCGGGCAATGC-3' (SEQ ID NO: 2).

The results are shown in FIG. 2. The result shows that the hands-free nucleic acid PCR method can be used for detecting 12 serotype strains of the avian adenovirus-I group.

II, specificity

And (3) virus to be detected: four serotype avian adenovirus-group I (avian adenovirus serotype 4 isolate, avian adenovirus serotype 8a isolate, avian adenovirus serotype 8b isolate, avian adenovirus serotype 11 isolate), fowl pox virus, Newcastle disease virus, avian influenza virus subtype H9N2, infectious anemia virus of chicken, infectious bronchitis virus.

The hands-free nucleic acid method established in the first step of the embodiment 1 is adopted to extract the virus nucleic acid to be detected, the extracted nucleic acid is subjected to PCR amplification, and the amplification product is subjected to gel electrophoresis detection.

And (3) PCR reaction system: nucleic acid 2. mu.L, 10 XPCR buffer 3. mu.L, 25mM MgCl₂mu.L, 10mM dNTP 2.5. mu.L, upstream primer F and downstream primer R each 0.5. mu.L (25pmol/L), Taq enzyme 0.5. mu.L (Dalibao bioengineering Co., Ltd.), and sterilized ultrapure water to 30. mu.L. A negative control using DEPC water instead of nucleic acid was set up.

PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; 1min at 94 ℃, lmin at 55 ℃, 90s at 72 ℃ and 30 cycles; extension at 72 ℃ for 10 min.

F:5 '-CAARTTCAGRCAGACGGT-3' (SEQ ID NO: 1);

5'-AAGAGGCCCGGGCAATGC-3' (SEQ ID NO: 2).

The results are shown in FIG. 3. The result shows that except the four serotype avian adenovirus-I groups, the other viruses are all negative, and the method for detecting the avian adenovirus-I group has good specificity.

Third, sensitivity

And (3) virus to be detected: avian adenovirus serotype 4 isolate.

Taking the original virus to be detected and samples diluted by 10 times, 100 times, 1000 times, 2000 times, 5000 times, 10000 times, 20000 times and 40000 times of the original virus, extracting nucleic acid by adopting the three methods of the first, second and third of the exemption example 1, carrying out PCR amplification on the extracted nucleic acid, and carrying out gel electrophoresis detection on the amplified product.

And (3) PCR reaction system: nucleic acid 2. mu.L, 10 XPCR buffer 3. mu.L, 25mM MgCl₂4μL，10mM dNTP 2.5μL, upstream primer F and downstream primer R each 0.5. mu.L (25pmol/w), Taq enzyme 0.5. mu.L (Dalianbao bioengineering Co., Ltd.), and sterilized ultrapure water was added to 30. mu.L. A negative control using DEPC water instead of nucleic acid was set up.

F:5 '-CAARTTCAGRCAGACGGT-3' (SEQ ID NO: 1);

5'-AAGAGGCCCGGGCAATGC-3' (SEQ ID NO: 2).

The results are shown in FIGS. 4 and 5. The hands-free nucleic acid PCR method can detect the PCR detection result of extracting nucleic acid after 20000 times of original virus dilution at the lowest; the PCR method for extracting nucleic acid by using Trizol can detect the PCR detection result of extracting nucleic acid after the original virus is diluted by 5000 times at least; the PCR method for extracting nucleic acid by using the Takara kit can detect the original virus with the dilution of 2000 times at least. Compared with the traditional Trizol nucleic acid extraction PCR method and the Takara kit nucleic acid extraction PCR method, the sensitivity of the hands-free nucleic acid PCR method is higher, and is respectively 4 times and 10 times higher than that of the two methods.

Fourth, clinical sample detection

Clinical samples: chicken liver 23 parts.

And (3) extracting nucleic acid by adopting three methods described in the first and second exemptions of example 1, carrying out PCR amplification on the extracted nucleic acid, and carrying out gel electrophoresis detection on an amplification product.

And (3) PCR reaction system: nucleic acid 2. mu.L, 10 XPCR buffer 3. mu.L, 25mM MgCl₂mu.L, 10mM dNTP 2.5. mu.L, upstream primer F and downstream primer R each 0.5. mu.L (25pmol/w), Taq enzyme 0.5. mu.L (Dalibao bioengineering Co., Ltd.), and sterilized ultrapure water to 30. mu.L. A negative control using DEPC water instead of nucleic acid was set up.

F:5 '-CAARTTCAGRCAGACGGT-3' (SEQ ID NO: 1);

5'-AAGAGGCCCGGGCAATGC-3' (SEQ ID NO: 2).

The results are shown in FIG. 5. The result shows that the detection results of the hands-free nucleic acid PCR method and the traditional Trizol nucleic acid extraction PCR method are consistent, the detection results of 4 diseased materials are positive, and the detection rate of the avian adenovirus-I group is 17.39%.

Sequence listing

<110> Qingdao agricultural university

<120> PCR method for rapidly detecting avian adenovirus-I group

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caarttcagr cagacggt 18

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aagaggcccg ggcaatgc 18

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<400> 3

aaattcaggc agacggtcgt agctcccact cgcaatgtca ccaccgaaaa ggcacaacgt 60

ctgcagatca gattctaccc gatccagacg gatgacacgc caaacagcta tcgcgtgcgc 120

tacagcgtca acgttgggga cagctgggtg ttggacatgg gggcgaccta tttcgacatc 180

aagggaatcc tagaccgagg gccgtccttc aagccctact gcggcacggc ttacaacccg 240

ctggctccca aggagtccat gtttaacaac tggtcggaga cggcacccgg gcagaacgtg 300

tccgcctccg gtcagctgtc caatgtctat accaacacga gcaccaccaa agacacgacg 360

gcggcgcagg tgacgaagat ttccggcgtc tttcccaacc ccaaccagga cccggaacga 420

atcctctgcg cagtagaaac gcacaccgcg tgctcggtcg cttcgccaag tctcagtaca 480

attacgctta cggtgctacg tcaagcccgt cgccgccgac ggttcccagt ccctcacgca 540

gaccccctac tggatcatga acaacgcggg caccgaatac ctgggggcgg tagccgtcga 600

ggactacacc aacagcctct cgtacccaga taccatgatc gtgccgcctc ccgaggatta 660

cgacgattac aacataggca ccacgcgtgc gctcagtccc aactacatcg gtttcagaga 720

caacttcatc aacctgctgt atcacgactc cggcgtgtgc tcgggcaccc tcaactcgga 780

gcgttcgggc atgaacgtgg tggtcgagct gcccgaccgg aataccgagc tcagctacca 840

gtacatgctg gccgacatga tgtcccgcca tcactat 877

Claims

1. A method of obtaining viral nucleic acid comprising the steps of: processing the sample by Buffer to obtain virus nucleic acid; the Buffer is an aqueous solution consisting of 50mM Tris-HCl, 6M urea and 1% Tween 20 by volume percentage;

the processing method comprises the following steps: mixing the sample with the Buffer, standing and reacting at 98 ℃ for 8min, and then standing and reacting at 4 ℃ for 15 min.

2. A method for detecting avian adenovirus-group I, comprising the steps of:

(1) obtaining a test virus or viral nucleic acid from a test sample using the method of claim 1;

(2) detecting whether a specific fragment of the avian adenovirus-I group exists in the viral nucleic acid obtained in the step (1), wherein if the specific fragment of the avian adenovirus-I group exists in the viral nucleic acid, the virus to be detected is the avian adenovirus-I group or the avian adenovirus-I group exists in the sample to be detected;

the methods are methods for non-disease diagnostic and therapeutic purposes.

3. The application of a Buffer in obtaining viral nucleic acid, wherein the Buffer is an aqueous solution consisting of 50mM Tris-HCl, 6M urea and 1% Tween 20 by volume percentage;

mixing the sample with the Buffer, standing and reacting at 98 ℃ for 8min, and then standing and reacting at 4 ℃ for 15 min.

4. The application of Buffer in the detection of avian adenovirus-I group is characterized in that the Buffer is an aqueous solution consisting of 50mM Tris-HCl, 6M urea and 1% Tween 20 by volume percentage; the use is a non-disease diagnostic and therapeutic use;