CN110527748B - PCR-RFLP primer for distinguishing avian adenovirus type C and duck adenovirus type 3 and method thereof - Google Patents
PCR-RFLP primer for distinguishing avian adenovirus type C and duck adenovirus type 3 and method thereof Download PDFInfo
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Abstract
The invention relates to a PCR-RFLP primer for distinguishing avian adenovirus type C and duck adenovirus type 3 and a method thereof, wherein the sequence of the primer is as follows: an upstream primer HF: 5 '-GAAAYTGGGGSCTGAAATAC-3'; a downstream primer HR: 5 '-TTGTAGTTYGTGGCCATCTG-3', the primer designed by the invention has strong specificity, can effectively amplify the avian adenovirus type C and the duck adenovirus type 3, and can not amplify other common poultry pathogens; according to the invention, the rapid identification of the C type of the avian adenovirus and the 3 type of the duck adenovirus can be achieved only by using a group of primers of an upstream primer HF and a downstream primer HR and combining the conventional XhoI restriction endonuclease digestion, and the blank of research in related fields at home and abroad is filled.
Description
Technical Field
The invention belongs to the technical field of veterinary infectious disease rapid diagnosis, and particularly relates to a PCR-RFLP primer for distinguishing avian adenovirus type C and duck adenovirus type 3 and a method thereof.
Background
According to the International Committee for viral Classification (ICTV) classification (https:// talk. ictvon. org/taxonomy /), 5 genera (Genus) were set under the family Adenoviridae (Adenovirdae): avian adenovirus (aviadovorus), AT-rich adenovirus (Atadenovirus), sialidase adenovirus (Siadenovirus), mammalian adenovirus (massadefovir), and acipenser meridionus (ichtdenovorus). Adenoviruses are double-stranded DNA non-enveloped viruses with icosahedral symmetry, and the capsid is composed of 252 capsomeres, 240 of which are hexons (hexons) and 12 of which are penton bases (pentons), each of which binds 1 to 2 fibers. The genome sizes of different adenoviruses vary, typically between 26-45 kb. The genome of adenovirus can be divided into early transcribed gene (E) and late transcribed gene (L) according to the initiation time of replication of adenovirus DNA. Early transcriptional genes, which contain four regions, El (E1A and E1B), E2(E2A and E2B), E3 and E4, are mainly responsible for regulating and controlling the proliferative replication of viruses. Late transcribed genes include five regions, all of Ll, L2, L3, L4 and L5, and are primarily functional in being responsible for encoding structural proteins of the virus.
Both avian adenovirus type C (Fowl Aviadenovirus C, FAdV-C) and Duck adenovirus type 3 (Duck adenovirus 3, DAdV-3) belong to the family of Adenoviridae (Adenoviridae) and the genus avian adenovirus (Aviadenovirus). Since 2015, the avian adenovirus type C is widely prevalent in China and is frequently acute, which mainly causes inclusion body hepatitis and pericarditis syndrome of chickens and causes huge economic loss to the chicken industry. In recent years, the avian adenovirus type C host is expanded, not only chickens but also ducks are infected by the avian adenovirus type C host, and the infected ducks are subjected to cesarean section to become pericardial effusion and liver necrosis. The duck adenovirus type 3 is also a duck adenovirus newly discovered in recent years, the duck liver swelling, bleeding and necrosis are mainly caused by the virus infection, and the phenomenon that duck groups are infected with the virus is increasingly common in recent years, so that the healthy development of the duck breeding industry is seriously influenced. Both the avian adenovirus type C and the duck adenovirus type 3 can infect ducks to cause liver necrosis, the clinical judgment is not easy to be accurate, and a molecular biological method for identifying the avian adenovirus type C and the duck adenovirus type 3 is not reported in the field at present.
Disclosure of Invention
The invention aims to provide a PCR-RFLP primer which has high sensitivity and high specificity and can effectively distinguish the C type of avian adenovirus and the 3 type of duck adenovirus and a method thereof.
The purpose of the invention is realized by the following technical scheme: a PCR-RFLP primer for distinguishing C types of avian adenoviruses and 3 types of duck adenoviruses is disclosed, and the sequence of the primer is as follows:
an upstream primer HF: 5 '-GAAAYTGGGGSCTGAAATAC-3';
a downstream primer HR: 5 '-TTGTAGTTYGTGGCCATCTG-3'.
Wherein, S is G/C, and Y is C/T.
Analysis of nucleotide sequences of avian adenovirus type C and duck adenovirus type 3 hexon genes revealed that the cleavage sites of these two adenoviruses were different at 1783-1788, the cleavage site of avian adenovirus type C was CTCGAG and could be recognized by XhoI restriction endonuclease, while the sequence of duck adenovirus type 3 was CTAGAG (as shown in FIG. 1) and could not be recognized by XhoI restriction endonuclease. According to the invention, specific primers are designed and enzyme cutting sites are selected according to the difference of enzyme cutting sites of avian adenovirus type C and duck adenovirus type 3 hexon gene sequences at 1783-1788. Only one primer group is needed, nucleotide sequence characteristics of two adenovirus hexon genes are combined, and the avian adenovirus type C and the duck adenovirus type 3 can be specifically identified by utilizing XhoI restriction endonuclease for enzyme digestion analysis.
The primers designed by the invention can amplify both avian adenovirus type C and duck adenovirus type 3, and the amplified bands are 716bp in size. The PCR product of the C-type amplification of the avian adenovirus is cut into 2 fragments after being cut by XhoI restriction endonuclease, wherein the size of one fragment is 446bp, the size of the other fragment is 270bp, while the PCR product of the 3-type amplification of the duck adenovirus is not changed after being cut by the XhoI restriction endonuclease, and is still 716 bp.
The PCR-RFLP method for distinguishing the avian adenovirus type C and the duck adenovirus type 3 by using the primer comprises the following steps:
(1) extracting DNA of the virus from the test sample.
(2) PCR amplification reaction: using the upstream primer HF: 5 '-GAAAYTGGGGSCTGAAATAC-3' and downstream primer HR: 5 '-TTGTAGTTYGTGGCCATCTG-3', and carrying out PCR amplification reaction by using the DNA obtained in the step (1) as a template; the primers can amplify DNA of avian adenovirus type C and duck adenovirus type 3, and the sizes of PCR products are 716 bp;
wherein, the optimization system of the PCR amplification reaction in the step (2) is as follows: the following components were contained in a 50. mu.L system:
the reaction conditions of the PCR amplification reaction in the step (2) are as follows: pre-denaturation at 94 deg.C for 5 min; 30s at 94 ℃, 30s at 53.5 ℃ and 46s at 72 ℃ for 35 cycles; 7min at 72 ℃; hold at 10 ℃.
(3) And (3) enzyme digestion of a PCR product: carrying out XhoI enzyme digestion reaction on the PCR product obtained by amplification in the step (2);
wherein the enzyme digestion reaction system in the step (3) is as follows: the following components were contained in a 30. mu.L system:
the reaction conditions of the enzyme digestion reaction in the step (3) are as follows: reacting in water bath at 37 ℃ for 15 min.
(4) Agarose gel electrophoresis: carrying out agarose gel electrophoresis on the product obtained by enzyme digestion in the step (3); observing agarose gel electrophoresis, cutting a PCR product amplified by DNA of the avian adenovirus type C into 2 fragments after the digestion of XhoI restriction endonuclease, wherein the sizes of the 2 fragments are 446bp and 270bp respectively; the size of the fragment of the PCR product of duck adenovirus type 3 DNA amplification is unchanged after the restriction enzyme digestion by XhoI restriction enzyme, and is still 716 bp; and observing the number of fragments of the enzyme digestion product according to agarose gel electrophoresis, and specifically identifying the avian adenovirus type C and the duck adenovirus type 3.
The primer is applied to the preparation of a kit for detecting avian adenovirus type C and duck adenovirus type 3.
Compared with the prior art, the invention has the advantages that:
1. the primer designed by the invention has strong specificity, can effectively amplify the C type of the avian adenovirus and the 3 type of the duck adenovirus, but can not amplify other common poultry pathogens such as MDPV, GPV, DPV, EDSV, E.coli, RA, PM and the like; in addition, the primer designed by the invention also has the advantage of high sensitivity; the primer can be applied to a kit for detecting the avian adenovirus type C and the duck adenovirus type 3.
2. The method for distinguishing the avian adenovirus type C from the duck adenovirus type 3 is simple, rapid and high in accuracy: the method comprises the steps of detecting 58 clinically collected dead duck disease materials with liver necrosis, extracting DNA in a detection sample, carrying out PCR amplification, carrying out XhoI enzyme digestion on a PCR product obtained by amplification, carrying out agarose gel electrophoresis on the enzyme digestion product, observing the agarose gel electrophoresis, and showing that the avian adenovirus type C is 2 parts positive, the positivity is 3.4%, the duck adenovirus type 3 is 6 parts positive, the positivity is 10.3%, and no sample with the avian adenovirus type C and the duck adenovirus type 3 being co-positive is detected.
3. According to the invention, the rapid identification of the C type of the avian adenovirus and the 3 type of the duck adenovirus can be achieved only by using a group of primers of an upstream primer HF and a downstream primer HR and combining the conventional XhoI restriction endonuclease digestion, and the blank of research in related fields at home and abroad is filled.
4. The identification method is simple, economic, convenient, efficient and high in accuracy, and can be used for quickly identifying the C type of the avian adenovirus and the 3 type of the duck adenovirus.
Drawings
FIG. 1 is a diagram comparing the cleavage sites of avian adenovirus type C and duck adenovirus type 3.
FIG. 2 is the electrophoresis diagram of the PCR-RFLP method for distinguishing the avian adenovirus type C from the duck adenovirus type 3.
FIG. 3 is an electrophoretogram of a specificity assay.
Detailed Description
The invention is described in detail below with reference to the drawings and examples of the specification:
the first embodiment is as follows:
1. material
1.1 instanceTesting reagents and consumables: 200 μ L PCR tubes were purchased from Corning; the Viral nucleic acid extraction Kit EasyPure Viral DNA/RNA Kit and the bacterial genome extraction Kit EasyPure bacteriological DNA Kit are purchased from Beijing Quanyujin biotechnology, Inc.; premix TaqTM(TaKaRa TaqTMVersion 2.0plus dye) PCR kit and QuickcutYMThe Spe I restriction enzyme was purchased from Takara Bio Inc.
1.2 strains: avian adenovirus type C (strain FJ 1674), duck adenovirus type 3 (strain AH 06), Muscovy Duck Parvovirus (MDPV), Goose Parvovirus (GPV), Duck Plague Virus (DPV), Egg Drop Syndrome Virus (EDSV), duck-derived escherichia coli (e.coli), Riemerella Anatipestifer (RA), and duck-derived avian Pasteurella Multocida (PM) were all preserved by the livestock veterinary institute of the academy of agricultural sciences of fujian province.
2. Gene sequence analysis, enzyme cutting site selection and primer design of avian adenovirus type C and duck adenovirus type 3 hexon
2.1 Gene sequence analysis and restriction site selection
According to the difference of nucleotide sequences of avian adenovirus type C and duck adenovirus type 3 hexon genes, the restriction sites of the two adenoviruses are different at 1783-1788, as shown in figure 1, the restriction site of avian adenovirus type C is CTCGAG and can be recognized by XhoI restriction endonuclease, while the sequence of duck adenovirus type 3 is CTAGAG and can not be recognized by XhoI restriction endonuclease. Therefore, the site is selected as a specific enzyme cutting site for distinguishing the avian adenovirus type C and the duck adenovirus type 3.
2.2 primer design
Primers were designed based on the hexon genes of avian adenovirus type C (GenBank accession No.: NC 015323) and duck adenovirus type 3 (GenBank accession No.: KR135164) and had the following sequences:
an upstream primer HF: 5 '-GAAAYTGGGGSCTGAAATAC-3'
A downstream primer HR: 5 '-TTGTAGTTYGTGGCCATCTG-3'
The primer needs to satisfy the following characteristics: the primer can amplify both avian adenovirus type C and duck adenovirus type 3, and the amplified bands are 716bp in size. Secondly, after the PCR product of the C-type amplification of the avian adenovirus is cut by XhoI restriction endonuclease, the PCR product can be cut into 2 fragments, wherein the size of one fragment is 446bp, the size of the other fragment is 270bp, and the PCR product of the 3-type amplification of the duck adenovirus is not changed after being cut by the XhoI restriction endonuclease and is also the fragment of the original PCR product. ③ after the PCR product of the mixed amplification of the avian adenovirus type C and the duck adenovirus type 3 is cut by XhoI restriction endonuclease, 3 bands can be displayed by electrophoresis, wherein 1 band with the size of 716bp is the product which is not cut by the XhoI enzyme after the PCR amplification of the duck adenovirus type 3, and the other 2 segments (1 band with the size of 446bp and the other 1 band with the size of 270bp) are the product which is obtained after the PCR product of the C amplification of the avian adenovirus type is cut by the XhoI enzyme. According to agarose gel electrophoresis observation, the enzyme digestion product has 1 band (716bp), 2 bands (446bp and 270bp) or 3 bands (716bp, 446bp and 270bp), and the avian adenovirus type C and duck adenovirus type 3 can be specifically identified.
3, establishment of PCR and XhoI restriction enzyme digestion method
3.1 extraction of viral DNA from viral samples
Using Beijing Algorithm Biotech Limited
The Viral DNA/RNAKit nucleic acid extraction kit extracts nucleic acid DNA of avian adenovirus type C (FJ1674 strain) and duck adenovirus type 3 (AH06 strain) according to the operation method of the instruction.
3.2 PCR amplification reaction and optimization of conditions:
the upstream primer HF designed in the step 2.2 is utilized: 5 '-GAAAYTGGGGSCTGAAATAC-3' and downstream primer HR: 5 '-TTGTAGTTYGTGGCCATCTG-3', using the DNA obtained in step 3.1 as a template to perform PCR amplification reaction, detecting the PCR product by using conventional agarose gel electrophoresis, and performing gel imaging, wherein the result shows that the avian adenovirus type C and the duck adenovirus type 3 can be amplified, and the amplified band sizes are 716 bp.
Wherein, during PCR amplification reaction, the used Dream Taq Green PCR Master Mix (2x) kit is purchased from Thermo Scientific company, PCR reaction liquid is optimized on the basis of combining the reaction system configuration recommended by the kit, the specific configuration method of the optimized 50 muL optimal reaction system is shown in Table 1, and the optimal reaction conditions are as follows: pre-denaturation at 94 deg.C for 5 min; 30s at 94 ℃, 30s at 53.5 ℃ and 46s at 72 ℃ for 35 cycles; 7min at 72 ℃; hold at 10 ℃.
TABLE 1 preparation of PCR reaction solution
3.3 cleavage of the PCR product
Carrying out XhoI enzyme digestion reaction on the PCR product obtained by amplification in the step 3.2: quickcut by Bao bioengineering (Dalian) Co., LtdTMThe reaction solution was prepared from the enzyme digestion reaction system (30. mu.L system, see Table 2 for specific configuration) recommended by XhoI kit. Reaction conditions are as follows: reacting in water bath at 37 ℃ for 15 min.
TABLE 2 preparation of enzyme digestion reaction solution
3.4 agarose gel electrophoresis: the cleavage product obtained in step 3.3 was subjected to agarose gel electrophoresis, and the results are shown in FIG. 2. Wherein, M in FIG. 2 is DL2000 molecular weight standard; 1 is an avian adenovirus C type PCR amplification product; 2 is duck adenovirus type 3 PCR amplification product; 3 is a mixed PCR amplification product of avian adenovirus type C and duck adenovirus type 3; 4 is the product of poultry adenovirus C type PCR amplification product after enzyme digestion; 5 is the product of duck adenovirus type 3 PCR amplification product after enzyme digestion; 6 is the product of poultry adenovirus type C and duck adenovirus type 3 mixed PCR amplification product after enzyme digestion; 7 is negative control;
from the results shown in fig. 2, it can be seen that: the primers designed by the invention can amplify both avian adenovirus type C and duck adenovirus type 3, and the amplified bands are 716bp in size. The PCR product of the C-type amplification of the avian adenovirus is cut into 2 fragments after being cut by XhoI restriction endonuclease, wherein the size of one fragment is 446bp, the size of the other fragment is 270bp, and the PCR product of the 3-type amplification of the duck adenovirus is not changed after being cut by the XhoI restriction endonuclease and is also the fragment of the original PCR product. After the PCR product of poultry adenovirus type C and duck adenovirus type 3 mixed amplification is subjected to enzyme digestion by XhoI restriction endonuclease, electrophoresis can show 3 bands, wherein 1 band with the size of 716bp is a product which is not subjected to enzyme digestion by XhoI after duck adenovirus type 3 PCR amplification, and the other 2 segments (1 band with the size of 446bp and the other 1 band with the size of 270bp) are products which are subjected to enzyme digestion by XhoI enzyme and obtained from the PCR product of poultry adenovirus type C amplification.
Specificity test of PCR
4.1 extraction of DNA from viral and bacterial samples
Using Beijing-Authentic Biotechnology Ltd
The Viral DNA/RNA Kit nucleic acid extraction Kit is used for extracting nucleic acid of avian adenovirus type C (FJ1674 strain), duck adenovirus type 3 (AH06 strain), Muscovy Duck Parvovirus (MDPV), Goose Parvovirus (GPV), Duck Plague Virus (DPV) and Egg Drop Syndrome Virus (EDSV) according to the operation method of the instruction book.
The nucleic acid of duck-origin escherichia coli (e.coli), Riemerella Anatipestifer (RA) and duck-origin Pasteurella Multocida (PM) was extracted using the bacterial genome extraction kit EasyPure bacteriological DNAKit of beijing holothurian limited company according to the method of the specification.
4.2 PCR amplification reaction:
and (3) carrying out PCR amplification reaction by using the DNA extracted in the step 4.1 as a template and using the optimized conditions in the step 3.2.
4.3 cleavage of PCR product: and (3) carrying out XhoI enzyme digestion on the PCR product obtained by amplification in the step 4.2.
4.4 agarose gel electrophoresis: the result of agarose gel electrophoresis of the enzyme digestion product is shown in FIG. 3, wherein M in FIG. 3 is DL2000 molecular weight standard; 1 is an avian adenovirus C type PCR amplification product; 2 is duck adenovirus type 3 PCR amplification product; 3 is a mixed PCR amplification product of avian adenovirus type C and duck adenovirus type 3; 4 is the product of poultry adenovirus C type PCR amplification product after enzyme digestion; 5 is the product of duck adenovirus type 3 PCR amplification product after enzyme digestion; 6 is the product of poultry adenovirus type C and duck adenovirus type 3 mixed PCR amplification product after enzyme digestion; 7 is negative control; 8 is Muscovy Duck Parvovirus (MDPV); 9 is Goose Parvovirus (GPV); 10 is Duck Plague Virus (DPV); egg Drop Syndrome Virus (EDSV) 11; 12 is duck origin escherichia coli (e.coli); 13 is Riemerella Anatipestifer (RA); 14 is duck source fowl Pasteurella Multocida (PM).
As can be seen from fig. 3: both the avian adenovirus type C and the duck adenovirus type 3 can be amplified, and the sizes of the amplified bands are 716 bp. Common pathogens (MDPV, GPV, DPV, EDSV, E.coli, RA and PM) of the poultry are detected, and no band is amplified, so that the primer disclosed by the invention is good in specificity.
5. Clinical testing
The method is used for detecting 58 clinically collected dead duck-origin disease samples with liver necrosis, and the detection result shows that the avian adenovirus type C is 2 parts positive, the positive rate is 3.4%, the duck adenovirus type 3 is 6 parts positive, the positive rate is 10.3%, and the samples with the avian adenovirus type C and the duck adenovirus type 3 which are jointly positive are not detected.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Sequence listing
<110> animal husbandry and veterinary institute of agricultural academy of sciences of Fujian province
<120> PCR-RFLP primer for distinguishing avian adenovirus type C and duck adenovirus type 3 and method thereof
<160> 2
<170> SIPOSequenceListing 1.0
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<212> DNA
<213> Artificial sequence (Artificial sequence)
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gaaaytgggg sctgaaatac 20
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<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
ttgtagttyg tggccatctg 20
Claims (7)
1. A PCR-RFLP primer for distinguishing C types of avian adenovirus and 3 types of duck adenovirus is characterized in that: the sequences of the primers are as follows:
an upstream primer HF: 5 '-GAAAYTGGGGSCTGAAATAC-3';
a downstream primer HR: 5 '-TTGTAGTTYGTGGCCATCTG-3'.
2. Use of the primers of claim 1 in the preparation of kits for detecting avian adenovirus type C and duck adenovirus type 3, which is not a method for disease diagnosis and treatment.
3. Use according to claim 2, characterized in that: the kit for detecting the avian adenovirus type C and the duck adenovirus type 3 comprises the following steps:
(1) extracting DNA of the virus from a test sample;
(2) PCR amplification reaction: using the upstream primer HF: 5 '-GAAAYTGGGGSCTGAAATAC-3' and downstream primer HR: 5 '-TTGTAGTTYGTGGCCATCTG-3', and carrying out PCR amplification reaction by using the DNA obtained in the step (1) as a template; the primers can amplify DNA of avian adenovirus type C and duck adenovirus type 3, and the sizes of PCR products are 716bp after gel recovery;
(3) and (3) enzyme digestion of a PCR product: carrying out XhoI enzyme digestion reaction on the PCR product obtained by amplification in the step (2);
(4) agarose gel electrophoresis: carrying out agarose gel electrophoresis on the product obtained by enzyme digestion in the step (3); observing agarose gel electrophoresis, cutting a PCR product amplified by DNA of the avian adenovirus type C into 2 fragments after the digestion of XhoI restriction endonuclease, wherein the sizes of the 2 fragments are 446bp and 270bp respectively; the size of the fragment of the PCR product of duck adenovirus type 3 DNA amplification is unchanged after the restriction enzyme digestion by XhoI restriction enzyme, and is still 716 bp; and observing the fragment quantity of the enzyme digestion product according to agarose gel electrophoresis, and specifically identifying the avian adenovirus type C and the duck adenovirus type 3.
4. Use according to claim 3, characterized in that: the optimization system of the PCR amplification reaction in the step (2) is as follows: the following components were contained in a 50. mu.L system:
。
5. Use according to claim 3, characterized in that: the reaction conditions of the PCR amplification reaction in the step (2) are as follows: pre-denaturation at 94 deg.C for 5 min; 30s at 94 ℃, 30s at 53.5 ℃ and 46s at 72 ℃ for 35 cycles; 7min at 72 ℃; hold at 10 ℃.
6. Use according to claim 3, characterized in that: the system of the enzyme digestion reaction in the step (3) is as follows: the following components were contained in a 30. mu.L system:
。
7. Use according to claim 3, characterized in that: the reaction conditions of the enzyme digestion reaction in the step (3) are as follows: reacting in water bath at 37 ℃ for 15 min.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107043831A (en) * | 2017-06-13 | 2017-08-15 | 福建省农业科学院畜牧兽医研究所 | Ana 1 aviadenovirus A types and 2 type Real time PCR detection primers, probe and kit |
| CN108842004A (en) * | 2018-07-28 | 2018-11-20 | 福建省农业科学院畜牧兽医研究所 | Distinguish real-time fluorescence quantitative PCR primer and its differentiating method and the application of Ana 1 aviadenovirus c-type and Ana 1 aviadenovirus Type B |
| CN108950070A (en) * | 2018-07-28 | 2018-12-07 | 福建省农业科学院畜牧兽医研究所 | Identify the PCR primer and its discrimination method and application of Ana 1 aviadenovirus c-type and Ana 1 aviadenovirus Type B |
| CN109182597A (en) * | 2018-09-03 | 2019-01-11 | 广州维伯鑫生物科技有限公司 | It is a kind of to detect I, II, III groups of multiple fluorescence quantitative PCR kits of aviadenovirus and its detection method simultaneously |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107586886B (en) * | 2017-10-30 | 2020-12-01 | 嘉兴安宇生物科技有限公司 | Reagent and method for rapidly detecting porcine adenovirus |
-
2019
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107043831A (en) * | 2017-06-13 | 2017-08-15 | 福建省农业科学院畜牧兽医研究所 | Ana 1 aviadenovirus A types and 2 type Real time PCR detection primers, probe and kit |
| CN108842004A (en) * | 2018-07-28 | 2018-11-20 | 福建省农业科学院畜牧兽医研究所 | Distinguish real-time fluorescence quantitative PCR primer and its differentiating method and the application of Ana 1 aviadenovirus c-type and Ana 1 aviadenovirus Type B |
| CN108950070A (en) * | 2018-07-28 | 2018-12-07 | 福建省农业科学院畜牧兽医研究所 | Identify the PCR primer and its discrimination method and application of Ana 1 aviadenovirus c-type and Ana 1 aviadenovirus Type B |
| CN109182597A (en) * | 2018-09-03 | 2019-01-11 | 广州维伯鑫生物科技有限公司 | It is a kind of to detect I, II, III groups of multiple fluorescence quantitative PCR kits of aviadenovirus and its detection method simultaneously |
Non-Patent Citations (1)
| Title |
|---|
| 鸭腺病毒3型Hexon基因的克隆及SYBR GreenⅠ实时荧光定量PCR方法的建立;陈翠腾等;《中国兽医学报》;20190615(第06期);全文 * |
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