CN108866240B - Primers and enzyme for identifying DAdV-3 and DAdV-A and application thereof - Google Patents
Primers and enzyme for identifying DAdV-3 and DAdV-A and application thereof Download PDFInfo
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Abstract
The invention provides a primer and an enzyme for identifying DAdV-3 and DAdV-A and application thereof, belonging to the field of duck pathology research, wherein the primer shown as SEQ ID NO.1-2 and SalI enzyme are adopted to establish a method for identifying and diagnosing two duck adenoviruses (DAdV-3 and DAdV-A) appearing in duck groups in China, the method has the sensitivity equivalent to PCR, and only needs to carry out enzyme digestion on a PCR product for additional 10min and then carry out conventional agarose gel electrophoresis, so that the method is simple, practical, convenient and quick, provides support for developing adenovirus molecular epidemic virology investigation and scientific prevention and control of related diseases in the duck groups, and has very important research significance.
Description
Technical Field
The invention belongs to the field of duck pathology research, and particularly relates to primers and enzymes for identifying DAdV-3 and DAdV-A and application thereof.
Background
The adenovirus particles are non-envelope, nucleocapsid is icosahedral symmetric, linear, double-stranded DNA virus, and the whole length of genome is about 33 kd. Among them, Hexon protein (Hexon) is the most major structural protein of viruses of various genera in the family adenoviridae, and each Hexon protein carries species-specific antigenic determinants of the major genus of viruses on the surface, is a target of virus neutralizing antibodies, contains major protective antigenic gene clusters of viruses and is closely related to the pathogenicity of the viruses.
According to the latest classification of the International Committee for viral classifications (https:// talk. ictvon. org /), the Family adenoviridae (Family:Adenoviridae) The lower 5 Genera (Genera) were assigned, respectively AT-rich adenovirus (Genus: Atadenovirus), avian adenovirus (Genus: Aviadenovirus), piscine adenovirus (Genus: Ichtadenovirus), mammalian adenovirus (Genus: Mastadenovirus) and sialidase virus (Genus: Siadenovirus).
It was found that there are many adenoviral infections in birds, of which 5 are adenovirus infections in chickens: avian adenovirus type A (Fowl aviadenovirus type A, FAdV-A), avian adenovirus type B (Fowl aviadenovirus type B, FAdV-B), avian adenovirus type C (Fowl aviadenovirus type C, FAdV-C), avian adenovirus type D (Fowl aviadenovirus type D, FAdV-D), avian adenovirus type E (Fowl aviadenovirus type E, FAdV-E), all belong to the genus of avian adenovirus. There are 4 kinds of adenoviruses infecting turkeys (Turkey), which belong to two genera of the family adenoviridae, respectively, wherein Turkey adenovirus type B (Turkey avidenovirus B), Turkey adenovirus type C (Turkey avidenovirus C) and Turkey adenovirus type D (Turkey avidenovirus D) belong to the genus avian adenovirus; while Turkey sialidase virus type A (Turkey siadenovirus A) belongs to the genus sialidase virus. There are 2 kinds of adenoviruses infecting parrots (Psittacine), respectively belonging to two genera of the family adenoviridae, wherein the Psittacine adenoviridae type A (Psittacine atadenovirus A) belongs to the AT-rich adenoviridae genus; and the Psittacine adenovirus type B (Psittacine avidenovirus B) belongs to the avian adenovirus genus.
In recent years, with the continuous expansion of the feeding scale of waterfowls (especially ducks) in China and the development of breeding modes, new epidemic diseases in the waterfowls are more and more complicated. There are also reports of adenovirus infection in duck groups of different adenoviruses belonging to the family adenoviridae, and there are duck adenovirus type a (duck adenovirus a, abbreviated as DAdV-a) belonging to the genus AT-rich adenovirus. In 2014, with the development of high throughput technology, 1 duck adenovirus (GR strain, GenBank accession NC 024486) was sequenced in the whole genome by Marek A et al and named duck adenovirus type 2 (DAdV-2) and classified as members of the genus avian adenovirus based on genomic characteristics and genetic evolutionary characteristics (Marek A, Kaj n GL, Kosio C, et al complex genome sequences of pig adenovirus type 1 and duck adenovirus type 2 expanded the number of animals with the gene in the genus Aviangio [ J ] Virology, 2014, 462-. Subsequently, since 2014, a new duck adenovirus (CH-GD-2014 strain, GenBank accession number KR 135164) appears in Muscovy ducks in China, and the virus and the GR strain of the DAdV-2 isolate have the following obvious differences: the nucleotide homology between the whole genome sequence and GR strain is only 92.3 percent; ② the nucleotide homology of the Hexon genes of the CH-GD-2014 strain and the GR strain is only 76.6 percent and is lower than 80.0 percent; ③ CH-GD-2014 strain has 2 spike proteins (Fiber 1, Fiber 2), while GR strain has only 1 spike protein (Fiber). Based on the characteristics, a new duck adenovirus is popular in duck groups in China and is named as duck adenovirus type 3 (DAdV-3) (Zhou Zhehai duck adenovirus type 3 genome sequence analysis and pathogenicity research [ D ]. Master thesis of southern China agriculture university, 2016.). This brings practical demands to DAdV-3 and DAdV-A, and generally, for differential diagnosis of two pathogens, primers are designed for 2 pathogens respectively (i.e., 4 primers are required in total) to establish a differential diagnosis method. Based on the nucleotide sequence characteristics of the conserved regions of the Hexon genes of DAdV-3 and DAdV-A, the research establishes a method which can simultaneously amplify DAdV-3 and DAdV-A by only 2 primers, but has SalI enzyme polymorphism difference in an amplification region (namely SalI enzyme cutting site difference in a PCR amplification region), and can effectively distinguish enzyme cutting products by carrying out conventional agarose gel electrophoresis. The sensitivity of the method is equivalent to that of PCR, electrophoresis is carried out after the PCR product is subjected to enzyme digestion for only 10min (the product is not required to be subjected to gel recovery), the method is simple, practical, convenient and quick, particularly, mixed infection of DAdV-3 and DAdV-A can be accurately distinguished, no primer and enzyme report for differential diagnosis of DAdV-3 and DAdV-A based on similar principles is seen at present, and the research can fill the blank in the related research field.
Disclosure of Invention
The invention aims to provide primers and enzymes for identifying DAdV-3 and DAdV-A and application thereof, wherein DAdV-3 and DAdV-A can be amplified simultaneously by only 2 primers, but SalI enzyme polymorphism difference exists in an amplification region (namely SalI enzyme digestion site difference exists in a PCR amplification region), and enzyme digestion products can be effectively distinguished by performing conventional agarose gel electrophoresis.
The following technical scheme is adopted for achieving the purpose:
a set of primers and enzymes for identifying DAdV-3 and DAdV-A, said primers being as follows:
DAdV-3A-F1：5’-CATACMGYGGMACAGCTTACAATC-3’，
DAdV-3A-R1：5’-ATCSCKAAAACCWATGTAGTTA-3’；
the enzyme is SalI enzyme.
Another object of the present invention is to provide a kit for detecting DAdV-3 and DAdV-A, comprising the primer and the enzyme.
Amplification was performed according to the 50. mu.L system recommended by the DreamTaq Green PCR Master Mix (2X) kit, wherein 25. mu.L of the 2 XPCR Master Mix amplification reaction solution, 1.0. mu.L of each of 10. mu.M primers DAdV-3A-F1 and DAdV-3A-R1, 1.0. mu.L of each of the extracted nucleic acid templates (DAdV-3, DAdV-A), and sterile deionized water was added to a final volume of 50. mu.L, and PCR amplification was performed after mixing.
The amplification conditions are pre-denaturation at 95 ℃ for 3 min, then circulation is carried out, denaturation at 95 ℃ for 30 s, annealing at delta T (52 ℃ -62 ℃) for 30 s, extension at 72 ℃ for 35s, final extension at 72 ℃ for 10min is carried out after 40 cycles are finished, and identification is carried out according to conventional agarose gel electrophoresis after reaction is finished. Δ T (52 ℃ -62 ℃) indicates that the annealing temperature is optimized in this interval. The optimized optimal annealing temperature is 56 ℃.
The invention has the advantages that:
based on the nucleotide sequence characteristics of the conserved regions of the Hexon genes of DAdV-3 and DAdV-A, the invention establishes a method which can simultaneously amplify DAdV-3 and DAdV-A by only 2 primers, but has SalI enzyme polymorphism difference in an amplification region (namely SalI enzyme cutting site difference in a PCR amplification region), and can effectively distinguish enzyme cutting products by carrying out conventional agarose gel electrophoresis. The sensitivity of the method is equivalent to that of PCR, electrophoresis is carried out only after the PCR product is subjected to enzyme digestion for additional 10min, the method is simple, practical, convenient and quick, particularly, mixed infection of DAdV-3 and DAdV-A can be accurately distinguished, reports of primers and enzymes for differential diagnosis of DAdV-3 and DAdV-A based on similar principles are not seen at present, and the research can fill up the blank of related research fields.
Drawings
FIG. 1 is a region of upstream primer design.
FIG. 2 downstream primer design region.
FIG. 3 shows a comparison of nucleotide sequences at positions 459-465 of DAdV-3 and DAdV-A.
FIG. 4 electrophoresis of PCR products, wherein M: DL2000 molecular weight standard; 1: DAdV-3; 2: DAdV-A.
FIG. 5 specificity assay; m: DL2000 molecular weight standard; 1: DAdV-3; 2: DAdV-A; 3: a DEV; 4: MDPV; 5: DuCV; 6: GPV; 7: E. coli; 8: R.A.; 9: P.M.; 10: DAdV-2; 11: and (5) sterilizing the deionized water.
FIG. 6 electrophoresis of the products after Sal I enzymatic cleavage identification, wherein M1: DL500 molecular weight standard; m2: DL2000 molecular weight standard; 1: carrying out enzyme digestion electrophoresis on the DAdV-3 product; 2: carrying out enzyme digestion electrophoresis on the DAdV-A product; 3: the products of the co-infection of DAdV-3 and DAdV-A are subjected to enzyme digestion electrophoresis.
Detailed Description
Example 1
1. Material
1.1 strains and strains
The pathogens DAdV-3 (strain W1FJ, GenBank accession No. MH 349774) and DAdV-A (strain FJ12025, GenBank accession No. KF 286430) for the test were maintained at the animal husbandry and veterinary institute of the academy of agricultural sciences, Fujian province.
Control strains Duck Enteritis Virus (DEV), Muscovy Duck Parvovirus (MDPV), duck circovirus (DuCV) and duck Goose Parvovirus (GPV) for tests; the control strains duck escherichia coli (E. coli), riemerella anatipestifer (R.A.) and duck pasteurella multocida (P.M.) for the test were all preserved by the animal husbandry and veterinary institute of agricultural and scientific institute of Fujian province. The Hexon gene (GR strain, GenBank accession NC 024486) of Duck adenovirus type 2 (DAdV-2) was synthesized in its entire sequence by Biotechnology engineering (Shanghai) GmbH.
1.2 primer design
According to the representative strain of the Adenoviridae DAdV-3 in GenBank of the National Center of Biotechnology Information (NCBI) database: CH-GD-2014 strain (KR 135164), W1GD strain (MH 349773), W1FJ strain (MH 349774), W1AH strain (MH 349775) and representative DAdV-A strains: the Hexon genes of E05 strain (EF 532411), FJ12025 strain (KF 286430), D11-JW-012 strain (KJ 452170) and D11-JW-017 strain (KJ 452171) were aligned by nucleotide series analysis using Lasergene DNASAR MegAlign to determine the DAdV-3 and DAdV-A gene conserved regions as the upstream primer design region (FIG. 1) and the downstream primer design region (FIG. 2), respectively.
The primer design software Oligo 7.0 is used for designing a set of primers as follows:
DAdV-3A-F1：5’-CATACMGYGGMACAGCTTACAATC-3’，
DAdV-3A-R1: 5 '-ATCSCKAAAACCWATGTAGTTA-3'. The primers were synthesized by Biotechnology engineering (Shanghai) Inc.
Note that when DAdV-3 and DAdV-A were amplified using DAdV-3A-F1/DAdV-3A-R1, the band sizes of the PCR products of DAdV-3 and DAdV-A were 524bp and 521bp, respectively (conventional agarose electrophoresis, no significant distinction with the naked eye). However, there was a characteristic SalI enzyme cleavage site difference in the amplified regions of DAdV-3 and DAdV-A, wherein the nucleotide sequence of DAdV-3 at position 459-465 was GTCGAC (recognized by SalI enzyme), while DAdV-A had no position recognized by SalI enzyme in the amplified region, and the results of the sequence alignment are shown in FIG. 3.
1.3 Primary reagents
DreamTaq Green PCR Master Mix (2X) was purchased from Thermoscientific, EasyPure Genomic DNA Kit, EasyPure Bacteria Genomic DNA Kit, all from Beijing Quanjin Biotechnology, Inc.; DNA molecular weight Standard DL500, DNA molecular weight Standard DL2000 and Quickcut SalI enzymes were purchased from Baori physician technical (Beijing) Ltd.
Establishment of test methods
2.1 extraction of genomic DNA
DAdV-3, DAdV-A, DEV, MDPV, DuCV, GPV the corresponding Genomic DNA was extracted according to the method of the easy pure Genomic DNA Kit and frozen at-80 ℃ for future use.
E. coli, R.A. and P.M. the corresponding Genomic DNA was extracted according to the method of the EasyPure Bacteria Genomic DNA Kit and frozen at-80 ℃ for future use.
2.2 configuration of reaction solution and optimization of annealing temperature
Amplification was performed according to the 50. mu.L system recommended by the DreamTaq Green PCR Master Mix (2X) kit, wherein 25. mu.L of the 2 XPCR Master Mix amplification reaction solution, 1.0. mu.L of each of the primers DAdV-3A-F1 and DAdV-3A-R1 (10. mu.M), 1.0. mu.L of each of the extracted nucleic acid templates (DAdV-3, DAdV-A), and sterile deionized water was added to a final volume of 50. mu.L, and PCR amplification was performed after mixing.
The amplification conditions are pre-denaturation at 95 ℃ for 3 min, then circulation is carried out, denaturation at 95 ℃ for 30 s, annealing at delta T (52 ℃ -62 ℃) for 30 s, extension at 72 ℃ for 35s, final extension at 72 ℃ for 10min is carried out after 40 cycles are finished, and identification is carried out according to conventional agarose gel electrophoresis after reaction is finished. Delta T (52 ℃ -62 ℃) indicates that the annealing temperature is optimized in the interval, and the optimized optimal annealing temperature is 56 ℃.
As a result, it was found (FIG. 4) that a band of 524bp in size appeared when DAdV-3 was added to the template alone (lane 1); when DAdV-A alone was added to the template, a band of 521bp in size (lane 2) appeared, which was indistinguishable to the naked eye by conventional agarose electrophoresis.
2.3 specificity test
And amplifying DEV, MDPV, DuCV, GPV, E.coli, R.A. and P.M. and sterilized deionized water in the optimized PCR system and amplification conditions in a contrast way, wherein no amplification band is seen. The results are shown in FIG. 5, DEV (lane 3), MDPV (lane 4), DuCV (lane 5), GPV (lane 6), E. coli (lane 7), R.A. (lane 8) and P.M. (lane 9), DAdV-2 (lane 10) and sterilized deionized water (lane 11), indicating that the established method is highly specific and has no cross-reaction to common waterfowl pathogens.
2.4 enzyme digestion identification
Carrying out enzyme digestion identification on the PCR product by using SalI enzyme, wherein the enzyme digestion system is a 30 mu L system: of these, 20. mu.L of 10 XQuickCut Green Buffer 3. mu. L, QuickCut ™ Sal I enzyme 3. mu. L, PCR product was supplemented with sterile deionized water to a final volume of 30. mu.L. Mixing the mixture gently, centrifuging the mixture instantly, and reacting the mixture in water bath at 37 ℃ for 10 min. The results are shown in FIG. 6, where two DAdV-3 products were obtained, with sizes of 101bp and 423bp, respectively (lane 1); the DAdV-A product remained 521bp in size (lane 2); there are three DAdV-3 and DAdV-A products, 521bp, 423bp and 101bp, respectively (lane 3).
Clinical application
After 49 clinical inspection duck-origin disease materials are ground according to a conventional method, the EasyPure Genomic DNA Kit is used for extracting corresponding nucleic acid DNA, and the optimized method is used for detecting DAdV-3 and DAdV-A infection. After the PCR product is subjected to SalI enzyme digestion, conventional agarose gel electrophoresis is carried out, and the result shows that 9 parts of DAdV-3 is detected to be positive in infection, wherein the positive rate is 18.37%; 5 parts of DAdV-A is detected to be positive, and the positive rate is 10.20%; 2 portions of DAdV-3 and DAdV-A were detected to be positive by coinfection, with a positive rate of 4.08%.
The positive target fragments after the PCR reaction were cut and recovered with an agarose gel recovery kit (DP 209, Tiangen Biochemical technology, Beijing, Ltd.). According to the description of pEASY-T1 Simple Cloning Kit Cloning ligation Kit (CT 111-01, Beijing Quanjin Biotechnology Co., Ltd.)HexonCloning the gene fragment to a pEASY-T1 vector, randomly selecting 8 single colonies, culturing the single colonies in an ampicillin (the content is 100 mu g/mL) resistant LB liquid culture medium for 14 h, and extracting corresponding plasmids by using a rapid plasmid miniextraction kit (DP 105, Tiangen Biochemical technology (Beijing) Co., Ltd.). When PCR amplification is usedThe extracted plasmids are respectively subjected to PCR identification by the primers and conditions, and the screened positive recombinant plasmids are sent to the doctor of medical science and technology (Beijing) Co. The sequencing results were verified by BLAST analysis at NCBI for Hexon gene fragments of DAdV-3 and DAdV-A, wherein the homology of the DAdV-3 positive fragment and the Hexon gene of DAdV-3 (strain W1FJ, GenBank accession No. MH 349774) was above 99.0%; the homology between the DAdV-A positive fragment and the Hexon gene of DAdV-A (FJ 12025 strain, GenBank accession KF 286430) is more than 99.0%. The coincidence rate of the sequencing result and the PCR detection result is 100 percent.
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> primers and enzymes for identifying DAdV-3 and DAdV-A and uses thereof
<130> 2
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 24
<212> DNA
<213> Artificial sequence
<400> 1
catacmgygg macagcttac aatc 24
<210> 2
<211> 22
<212> DNA
<213> Artificial sequence
<400> 2
atcsckaaaa ccwatgtagt ta 22
Claims (2)
1. A set of primers and enzymes for identifying DAdV-3 and DAdV-A, characterized by: the primers are as follows:
DAdV-3A-F1：5’-CATACMGYGGMACAGCTTACAATC-3’，
DAdV-3A-R1: 5 '-ATCSCKAAAACCWATGTAGTTA-3'; the enzyme is SalI enzyme.
2. A kit for detecting DAdV-3 and DAdV-a, comprising the primer of claim 1 and an enzyme.
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| CN108842005A (en) * | 2018-07-28 | 2018-11-20 | 福建省农业科学院畜牧兽医研究所 | The MGB probe and its mating primer and method of Ana 1 aviadenovirus c-type and Ana 1 aviadenovirus Type B are detected simultaneously |
| 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 |
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