CN112048570A - PCR primer for detecting duck adenovirus type 4 and detection method and application thereof - Google Patents
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Abstract
The invention discloses a PCR primer for detecting duck adenovirus type 4, a detection method and application thereof. The invention establishes a primer capable of rapidly detecting the duck adenovirus type 4 and a PCR detection method thereof for the first time, and has the advantages of simple operation, low cost and short time; moreover, the PCR detection method for detecting duck adenovirus type 4 has high accuracy, good specificity and high sensitivity, and the lowest detection limit is 6 multiplied by 101The copies is beneficial to popularization and application in clinic, and provides technical support for developing adenovirus type 4 molecular epidemic virology investigation and related detection work in duck groups.
Description
Technical Field
The invention belongs to the field of molecular biology, and relates to a PCR primer for detecting duck adenovirus type 4, a detection method and application thereof.
Background
In recent years, along with the rapid development of waterfowl industry, the infection of adenovirus in duck group is increasing, and the duck adenovirus infection is mainly characterized by inclusion body hepatitis. According to the survey, the duck adenovirus is divided into 4 genotypes, including duck adenovirus type 1 (duck adenovirus 1, DAdV-1), duck adenovirus type 2 (duck adenovirus 2, DAdV-2), duck adenovirus type 3 (duck adenovirus 3, DAdV-3) and duck adenovirus type 4 (duck adenovirus 4, DAdV-4). Duck adenovirus type 4 was first discovered in 2019 and was isolated in the laboratory by researchers from egg laying sheldrake disease clinically with salpingitis, and according to the phylogenetic tree analysis, the novel adenovirus was found to belong to avian adenovirus genus (Aviadenovirus), named Duck adenovirus type 4.
During replication of an adenovirus in a host cell, the virion will first enter the nucleus of the host, transcribe and translate viral proteins in the nucleus, and then assemble into new virions. Duck adenovirus type 4 virions are icosahedral in symmetry, with a capsid composed of 252 capsomeres, comprising 240 hexons (hexon), 12 vertex pentons (penton), each of which also has a trimeric spike protein (fiber) protruding from it. Hexon, penton and spike proteins are the major structural proteins with immunogenicity, in which the spike protein is non-covalently bound to the penton. Like duck adenovirus type 3, duck adenovirus type 4 also has two fiber protein genes (fiber-1 and fiber-2), while duck adenovirus type 2 has only one fiber protein gene.
The existing method for detecting duck adenovirus pathogeny mainly comprises virus separation and identification, agar diffusion test, common PCR and the like. However, no detection method for duck adenovirus type 4 exists. Based on the fact that researchers find that the duck adenovirus type 4 positivity rate reaches about 15%, the trend of continuous rising is possible, so that the effective PCR primer (amplification primer) for detecting duck adenovirus type 4 is provided, and the establishment of a set of rapid detection method and system has important significance clinically.
Disclosure of Invention
The invention aims to provide a PCR primer for detecting duck adenovirus type 4;
the invention also aims to provide a PCR kit for detecting duck adenovirus type 4;
the invention also aims to provide a PCR method for detecting duck adenovirus type 4;
the invention also aims to provide the application of the PCR primer in the preparation of a duck adenovirus type 4 detection reagent;
the invention also aims to provide application of the PCR method in diagnosing avian duck adenovirus type 4 infection.
The technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided:
a PCR primer for detecting duck adenovirus type 4 has the following nucleotide sequence:
and (3) primer F: 5'-GGGCAAATGACCAACCTCTA-3' (SEQ ID NO. 1);
and (3) primer R: 5'-TGTCCCTGAACCCGATGTAA-3' (SEQ ID NO. 2).
The inventor finds that different serotypes of duck adenovirus have different hexon sizes, the total length of duck adenovirus type 4 hexon gene has 2832 nucleotides, and the evolutionary tree analysis is carried out according to the total length amino acid sequence of duck adenovirus type 4 hexon, so that duck adenovirus type 4 belongs to a new branch, and the genetic distance between the duck adenovirus type 4 and turkey is closer than the genetic distance between the duck adenovirus type 2 and duck adenovirus type 3.
The invention designs a primer according to a duck adenovirus type 4 gene sequence (GenBank accession number: MN 733730). The duck adenovirus type 4 specific region (nucleotide sequence of the conserved region of the Hexon gene) is selected as a site for primer design, so that the specificity of the detection method is ensured. Then, after screening, the primer F/R with high sensitivity and strong specificity is screened out.
In a second aspect of the present invention, there is provided:
a PCR kit for detecting duck adenovirus type 4 comprises the PCR primer.
Further, the kit also comprises a positive standard substance.
The positive standard substance is nucleic acid of duck adenovirus type 4 positive sample
In a third aspect of the present invention, there is provided:
a PCR method for detecting duck adenovirus type 4 comprises the following steps:
(1) extracting DNA of a sample to be detected;
(2) using the DNA of a sample to be detected as a template, and amplifying by using the PCR primer to obtain an amplification product;
(3) and (3) performing electrophoretic analysis, and comparing the electrophoretic band of the amplification product obtained in the step (2) with the electrophoretic band of the positive standard substance to judge whether the sample to be detected is duck adenovirus type 4.
The electrophoresis analysis in the step (3) includes electrophoresis using 1% agarose gel, and of course, other conventional electrophoresis in the art can be reasonably used as an alternative according to actual requirements.
Further, the determining of the sample to be detected in the step (3) includes the following conditions:
(a) if the electrophoresis strip of the amplification product obtained in the step (2) and the electrophoresis strip of the positive standard product both have a strip with the size of 442bp, judging that the sample to be detected is duck adenovirus type 4;
(b) and (3) if the electrophoresis band of the amplification product obtained in the step (2) does not have any band, judging that the sample to be detected is negative and does not contain duck adenovirus type 4.
Further, the reaction system for PCR primer amplification in the step (2) is as follows:
further, the conditions for PCR primer amplification in the step (2) are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 20s, annealing at 55 ℃ for 20s, and extension at 72 ℃ for 30 s; circulating for 35 times; final extension at 72 ℃ for 10 min.
In a fourth aspect of the present invention, there is provided:
the PCR primer is applied to the preparation of a duck adenovirus type 4 detection reagent.
In a fifth aspect of the present invention, there is provided:
the application of the PCR method in diagnosing the adenovirus type 4 infection of the poultry ducks.
The invention has the beneficial effects that:
1. the invention establishes a PCR primer and a method for quickly detecting duck adenovirus type 4 for the first time, and the method optimally designs a specific primer of duck adenovirus type 4 based on a nucleotide sequence of a conserved region of a Hexon gene of duck adenovirus type 4, and finally realizes the quick detection of duck adenovirus type 4;
2. the method disclosed by the invention is simple to operate, can be used for quickly detecting the duck adenovirus type 4, has the characteristics of strong specificity and high sensitivity, provides technical support for developing adenovirus type 4 molecular epidemic virology research in duck groups, and has very important clinical significance.
Drawings
FIG. 1 is a gel electrophoresis diagram established for the duck adenovirus type 4 PCR detection method; wherein DAdV-4 represents a duck adenovirus type 4 positive sample, and water is used as a negative control;
FIG. 2 is a specific gel electrophoresis diagram of duck adenovirus type 4 PCR detection method; wherein, Lane 1 is duck parvovirus (MPV), Lane 2 is Goose Parvovirus (GPV), Lane 3 is duck adenovirus type 1 (DAdV-1), Lane 4 is duck adenovirus type 3 (DAdV-3), Lane 5 is DAdV-4 as positive control, and water as negative control;
FIG. 3 is a graph showing the sensitivity test of duck adenovirus type 4 nucleic acid, lanes 1 to 6 show the concentrations of 6X 105～6×100Six gradient concentrations of copies/. mu.l;
FIG. 4 is a gel electrophoresis chart of clinical samples, wherein 1-9 are clinical samples, and 10 is DAdV-4;
FIG. 5 is a Hexon gene evolutionary tree analysis diagram of duck adenovirus type 4 clinical samples, wherein S5 and S6 are clinical test positive samples 5 and 6, respectively.
Detailed Description
In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.
Primer design
Designing primers according to a duck adenovirus type 4 Hexon gene sequence, and selecting primers F and R with high sensitivity and strong specificity after a large amount of comparison and screening, wherein the base sequences are as follows:
and (3) primer F: 5'-GGGCAAATGACCAACCTCTA-3' (SEQ ID NO. 1);
and (3) primer R: 5'-TGTCCCTGAACCCGATGTAA-3' (SEQ ID NO. 2).
Establishment of duck adenovirus type 4 PCR detection method
The PCR method for detecting the duck adenovirus type 4 comprises the following steps:
(1) taking DNA of the extracted duck adenovirus type 4 positive sample as a template, and performing PCR amplification reaction and gel electrophoresis analysis; water was used as a negative control.
The reaction system of the PCR amplification reaction in step (1) is shown in Table 1.
TABLE 1 reaction System for PCR amplification reaction
The reaction procedure of the PCR amplification reaction in the step (1) is as follows:
pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 20s, annealing at 55 ℃ for 20s, and extension at 72 ℃ for 30 s; circulating for 35 times; final extension at 72 ℃ for 10 min.
(2) Analyzing the result of the PCR amplification product of the duck adenovirus type 4 positive sample.
The analysis result of the PCR amplification product of the duck adenovirus type 4 positive sample in 1% agarose gel electrophoresis is shown in FIG. 1, and the result shows that the duck adenovirus type 4 positive sample in lane 1 of 1% agarose gel electrophoresis shows a band with the size of 442bp, which is consistent with the size of the expected amplification fragment, and the control (water) in lane 2 does not show any band, which indicates that the method is feasible.
Based on the test, the feasibility of the method is verified, and the effect of the detection method is further detected.
Specificity test
To test the specificity of the primers for amplifying duck adenovirus type 4 of the present invention, the following tests were performed:
the nucleic acids of duck adenovirus type 1 (DAdV-1), duck adenovirus type 3 (DAdV-3), duck parvovirus (MPV) and Goose Parvovirus (GPV) and DAdV-4 are respectively extracted, the nucleic acids of DAdV-1, DAdV-3, MPV, GPV and DAdV-4 are used as PCR templates, water is used as a negative control, and the PCR method for detecting duck adenovirus type 4 by PCR is used for detection and analysis, and the result is shown in figure 2.
As can be seen from FIG. 2, the detection method of the invention can only specifically amplify duck adenovirus type 4, a band with the size of 442bp appears only in an electrophoretogram of duck adenovirus type 4, and no band with the size of 442bp appears in the electrophoretogram of DAdV-1, DAdV-3, MPV and GPV nucleic acids, which indicates that the DAdV-1, DAdV-3, MPV and GPV nucleic acids are not amplified, and indicates that primers F and R designed by the invention have good specificity and can only be used for PCR detection of duck adenovirus type 4.
Sensitivity test
To test the sensitivity of the primers for amplifying duck adenovirus type 4 of the present invention, the following tests were performed:
1. preparation of duck adenovirus type 4 positive plasmid
The preparation steps of the duck adenovirus type 4 positive plasmid p-DAdV4 in the embodiment are as follows:
extracting nucleic acid of the duck adenovirus type 4 positive sample, and performing PCR amplification by using the primer F and the primer R as amplification primers. And respectively recovering purified and amplified products, cloning to a PMD18T-Vector plasmid, sending the plasmid with a positive PCR result to Shanghai biotechnological engineering Limited company for sequencing, and confirming that the fragment is correctly inserted into the Vector, namely, the fragment is used as a duck adenovirus type 4 positive standard sample.
2. Determination of duck adenovirus type 4 positive plasmid sensitivity
As can be seen from FIG. 3, the brightness of the amplified band of duck adenovirus type 4 positive plasmid p-DAdV4 in the detection method of the invention decreases with the decrease of plasmid concentration, and the minimum detection limit of the method for p-DAdV4 is 6X 101copies, which demonstrates the high sensitivity of the method of the invention.
Clinical sample testing
In order to verify the practical use effect of the primers for amplifying duck adenovirus type 4 and the method thereof according to the present invention, the following experiments were performed:
(1) extracting nucleic acid from 9 parts of suspected duck adenovirus type 4 duck liver;
(2) the extracted sample DNA is used as a template, the primers F and R designed by the invention are used for amplification, meanwhile, the duck adenovirus type 4 positive sample is used as a positive control, and water is used as a negative control. The reaction system and the reaction program are described in the PCR method for detecting the duck adenovirus type 4 by the PCR.
The results of the detection are shown in FIG. 4.
As can be seen from the PCR amplification chart of clinical samples of duck adenovirus shown in FIG. 4, 2 clinical samples (positive sample 5 and positive sample 6) all showed 442bp bands, which are consistent with the DAdV-4 band, and were determined as duck adenovirus type 4.
The PCR products of 2 clinical samples (positive sample 5 and positive sample 6) which were detected to be positive were subjected to clone sequencing, and the obtained Hexon gene sequence after sequencing was analyzed by genetic evolutionary tree analysis using N-J method (FIG. 5).
As shown in FIG. 5, the Hexon genetic evolutionary trees of the positive samples 4 and 5 show that 2 positive samples are in the same cluster with the registered DAdV-4 on the NCBI and are far away from the DAdV-1, the DAdV-2 and the DAdV-3, so that the positive samples 5 and 6 can be determined to be duck adenovirus type 4, and the results are consistent with the results of the PCR method for rapidly detecting duck adenovirus type 4 in the research. But compared with the PCR method in the invention, the sequencing method in the prior art has longer time consumption, more complex detection procedure and higher cost, so the PCR method in the invention has more competitive power, and can be widely applied to various aspects such as preparation of duck adenovirus type 4 detection reagent, diagnosis of avian duck adenovirus type 4 infection or epidemiological investigation and the like on the basis of the detection.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> institute of animal health of academy of agricultural sciences of Guangdong province
<120> PCR primer for detecting duck adenovirus type 4, detection method and application thereof
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gggcaaatga ccaacctcta 20
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Claims (9)
1. A PCR primer for detecting duck adenovirus type 4 is characterized in that the nucleotide sequence of the PCR primer is as follows:
and (3) primer F: 5'-GGGCAAATGACCAACCTCTA-3', respectively;
and (3) primer R: 5'-TGTCCCTGAACCCGATGTAA-3' are provided.
2. A PCR kit for detecting duck adenovirus type 4, which comprises the PCR primer as claimed in claim 1.
3. The PCR kit of claim 2, wherein the kit further comprises a positive standard.
4. A PCR method for detecting duck adenovirus type 4 is characterized by comprising the following steps:
(1) extracting DNA of a sample to be detected;
(2) amplifying by using the PCR primer of claim 1 by using a sample DNA to be detected as a template to obtain an amplification product;
(3) and (3) performing electrophoretic analysis, and comparing the electrophoretic band of the amplification product obtained in the step (2) with the electrophoretic band of the positive standard substance to judge whether the sample to be detected is duck adenovirus type 4.
5. The PCR method according to claim 4, wherein the determining the sample to be tested in step (3) includes the following steps:
(a) if the electrophoresis strip of the amplification product obtained in the step (2) and the electrophoresis strip of the positive standard product both have a strip with the size of 442bp, judging that the sample to be detected is duck adenovirus type 4;
(b) and (3) if the electrophoresis band of the amplification product obtained in the step (2) does not have any band, judging that the sample to be detected is negative and does not contain duck adenovirus type 4.
6. The PCR method according to claim 4, wherein the reaction system for PCR primer amplification in step (2) is:
7. the PCR method according to claim 4, wherein the conditions for the PCR primer amplification in step (2) are: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 20s, annealing at 55 ℃ for 20s, and extension at 72 ℃ for 30 s; circulating for 35 times; final extension at 72 ℃ for 10 min.
8. The use of the PCR primer of claim 1 in the preparation of a duck adenovirus type 4 detection reagent.
9. Use of the PCR method according to any one of claims 4 to 7 in an avian duck adenovirus type 4 epidemiological survey.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112575122A (en) * | 2020-12-29 | 2021-03-30 | 商丘美兰生物工程有限公司 | Dual PCR primer group for rapidly detecting duck type 2 adenovirus and duck circovirus as well as detection method and application thereof |
| CN112626279A (en) * | 2021-01-21 | 2021-04-09 | 福建省农业科学院畜牧兽医研究所 | Real-time fluorescent quantitative PCR (polymerase chain reaction) primer and kit for detecting new duck type 4 adenovirus |
| CN112646933A (en) * | 2021-01-21 | 2021-04-13 | 福建省农业科学院畜牧兽医研究所 | Real-time fluorescent quantitative PCR (polymerase chain reaction) detection primer, probe and kit for duck type 4 adenovirus |
| CN112725533A (en) * | 2021-01-21 | 2021-04-30 | 福建省农业科学院畜牧兽医研究所 | Primer group for real-time fluorescent quantitative PCR identification of duck type 1 adenovirus and duck type 4 adenovirus |
| CN112725532A (en) * | 2021-01-21 | 2021-04-30 | 福建省农业科学院畜牧兽医研究所 | Real-time fluorescent quantitative PCR (polymerase chain reaction) detection primer for identifying FAdV-4 and DAdV-4 and kit thereof |
| CN114196786A (en) * | 2021-11-11 | 2022-03-18 | 佛山科学技术学院 | Poultry adenovirus type 4 and 8 dual fluorescent quantitative PCR rapid detection kit and method |
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