CN112522447A - Absolute fluorescence quantitative PCR detection method for chicken infectious anemia virus - Google Patents
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Abstract
The invention relates to an absolute fluorescent quantitative PCR detection method of chicken infectious anemia virus, which scientifically designs a primer for specifically amplifying a VP1 gene fragment of the chicken infectious anemia virus, and then establishes the absolute fluorescent quantitative PCR method by using a TB Green (deoxyribonucleic acid) Premix Ex Taq II reagent of TaKaRa company. According to the highly conserved region of the chicken infectious anemia virus VP1 gene, an upstream primer and a downstream primer with the amplification fragment size of 140bp are scientifically designed and synthesized, and the primer sequences are as follows: the upstream primer is VP 1-F: 5'-GCCCCGGTACGTATAGTGTG-3', respectively; the downstream primer is VP 1-R: 5'-CCCGTACATGTGGTCTGCAT-3', respectively; the method can be used for the rapid and specific detection of the infectious chicken anemia virus vaccine contamination residues and low-content infectious chicken anemia viruses in clinical samples by enterprises; the content of the infectious chicken anemia virus can be quantitatively detected, so that the change rule and the like of the infectious chicken anemia virus in vivo can be known, and an effective means is provided for further researching the molecular biological characteristics of the infectious chicken anemia virus and formulating an effective prevention and control strategy.
Description
Technical Field
The invention relates to an absolute fluorescent quantitative PCR (polymerase chain reaction) detection method for chicken infectious anemia virus, belonging to the technical field of biology.
Background
Infectious Chicken Anemia Virus (CIAV) infection mainly causes chick aplastic Anemia and systemic lymphoid tissue atrophy; after the adult chicken is infected with the infectious chicken anemia virus, the infection is often subclinical and toxic, so that the production performance and the immune function are reduced. The infectious chicken anemia virus can be spread horizontally and vertically. Since the first report in 1979, infectious chicken anemia virus has spread around the world. In 1992, infectious anemia viruses of chicken are first isolated in Heilongjiang province in China, and then are sequentially isolated from chicken flocks in Henan, Shandong, Jiangsu, Liaoning, Jilin and the like. In recent years, due to the large-scale and intensive development of the chicken raising industry in China and the lack of an effective prevention and control strategy, the prevalence range of the disease in China is continuously expanded, and serious economic loss is brought to the chicken raising industry. At present, serological detection methods (virus neutralization test, indirect fluorescent antibody test, ELISA test, etc.) are commonly used clinically to confirm the diagnosis of cases suspected to be infected with the infectious chicken anemia virus. With the continuous progress of detection technology, nucleic acid probe technology, PCR technology, real-time fluorescence quantitative PCR technology and the like are gradually applied to the specific detection of the infectious chicken anemia virus. At the present stage, the detection accuracy of the infectious chicken anemia virus vaccine contamination residues and low-content infectious chicken anemia viruses in clinical samples in production practice is low, and a sensitive method for quantitatively detecting the infectious chicken anemia viruses specifically is lacked. Therefore, the invention is beneficial to perfecting the detection method of the infectious chicken anemia virus and provides a technical support for further researching the molecular biological characteristics of the infectious chicken anemia virus and formulating an effective prevention and control strategy.
Disclosure of Invention
The invention aims to provide an absolute fluorescent quantitative PCR detection method for chicken infectious anemia virus aiming at the defects of the existing problems.
The technical scheme of the invention is as follows: the absolute fluorescent quantitative PCR detection method for the chicken infectious anemia virus is characterized by comprising the following steps of:
(1) extracting chicken infectious anemia virus genome DNA: extraction of infectious chicken anemia virus genomic DNA Using AxyPrep DNA miniprep kit from AxygenTM(iii) a multiple source Genomic DNA finish;
(2) design and synthesis of primers: according to the highly conserved region of the chicken infectious anemia virus VP1 gene, an upstream primer and a downstream primer with the amplification fragment size of 140bp are designed and synthesized, and the primer sequences are as follows: the upstream primer is VP 1-F: 5'-GCCCCGGTACGTATAGTGTG-3', respectively; the downstream primer is VP 1-R: 5'-CCCGTACATGTGGTCTGCAT-3', respectively; before the primer is used, dissolving the primer by using ultrapure water, and storing the primer at the temperature of minus 20 ℃ for later use, wherein the use concentration of the primer is 10 pmol;
(3) establishment of an absolute fluorescent quantitative PCR method: the establishment of the absolute fluorescent quantitative PCR method was carried out by using TB Green-box Premix Ex Taq-II reagent of TaKaRa, 20. mu.L: solution A: 2 mu L of template and 6 mu L of double distilled water; and B, liquid B: TB Green Premix Ex Taq II (2X) 10. mu.L, upstream and downstream primers 0.8. mu.L each, ROX Reference Dye II (50X) 0.4. mu.L; when the sample is added, the solution B is added into the eight-tube reaction tube, then the solution A is added, and centrifugal treatment is carried out before the machine is arranged; the reaction procedure was as follows: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s, and annealing at 60 ℃ for 34 s; the reaction program had 40 cycles;
(4) drawing a standard curve: the pcDNA3.1-VP1 plasmid successfully prepared in the laboratory is used as a positive standard substance for standby; converting the positive standard plasmid into copy number, and continuously diluting by 10 timesFrom 1: 10 to 1: 109Setting 9 concentration gradients in total, setting 3 repetitions for each gradient, amplifying in an ABI 7500 type fluorescence quantitative PCR instrument, and setting deionized water as a negative control; collecting data after amplification is finished, obtaining an optimal detection area according to an amplification curve graph and the CT value condition, and drawing a standard curve;
(5) sensitivity and specificity test of the absolute fluorescent quantitative PCR method: the standard curve is drawn to show that the chicken infectious anemia virus with the virus DNA copy number of only 10 can be detected, and the sensitivity of the absolute fluorescent quantitative PCR method is reflected; extracting corresponding nucleic acid from the existing fowl adenovirus FAdV, avian leukosis virus ALV and reticuloendotheliosis virus REV in a laboratory, and operating the program according to an amplification system; meanwhile, a positive control of the infectious chicken anemia virus and a negative control of deionized water are set, and the detection result shows the specificity of the absolute fluorescent quantitative PCR method.
The principle and the most central key technology of the invention are to scientifically design a primer for specifically amplifying the gene segment of the chicken infectious anemia virus VP1, and then establish the absolute fluorescent quantitative PCR method by using TB Green ™ Premix Ex Taq II reagent of TaKaRa company. According to the highly conserved region of the chicken infectious anemia virus VP1 gene, an upstream primer and a downstream primer with the amplification fragment size of 140bp are scientifically designed and synthesized, and the primer sequences are as follows: the upstream primer is VP 1-F: 5'-GCCCCGGTACGTATAGTGTG-3', respectively; the downstream primer is VP 1-R: 5'-CCCGTACATGTGGTCTGCAT-3', respectively; the invention discloses a primer sequence for PCR amplification of chicken infectious anemia virus VP1 gene.
The scheme of the invention is as follows:
(1) extracting chicken infectious anemia virus genome DNA: extraction of infectious chicken anemia virus genomic DNA Using AxyPrep DNA miniprep kit from AxygenTMMultiple source Genomic DNA.
(2) Design and synthesis of primers: according to the highly conserved region of the chicken infectious anemia virus VP1 gene, an upstream primer and a downstream primer with the amplification fragment size of 140bp are designed and synthesized, and the primer sequences are as follows: the upstream primer is VP 1-F: 5'-GCCCCGGTACGTATAGTGTG-3', respectively; the downstream primer is VP 1-R: 5'-CCCGTACATGTGGTCTGCAT-3', respectively; before use, the primers were dissolved in ultrapure water and stored at-20 ℃ at a concentration of 10 pmol.
(3) Establishment of an absolute fluorescent quantitative PCR method: the invention establishes an absolute fluorescent quantitative PCR method by using TB Green-box Premix Ex Taq-II reagent of TaKaRa company, wherein the reaction system is 20 mu L: solution A: 2 mu L of template and 6 mu L of double distilled water; and B, liquid B: TB Green Premix Ex Taq II (2X) 10. mu.L, upstream and downstream primers 0.8. mu.L each, and ROX Reference Dye II (50X) 0.4. mu.L. When adding sample, firstly adding the solution B into the eight-tube reaction tube, then adding the solution A, and centrifuging before loading. The reaction procedure was as follows: the reaction sequence was 40 cycles of pre-denaturation at 95 ℃ for 30s, followed by denaturation at 95 ℃ for 5s and final annealing at 60 ℃ for 34 s.
(4) Drawing a standard curve: the pcDNA3.1-VP1 plasmid successfully prepared in the laboratory was used as a positive plasmid standard for future use. Converting the positive standard plasmid into copy number, and continuously diluting 10 times at a ratio of 1: 10 to 1: 109 A total of 9 concentration gradients were set up, 3 replicates for each gradient, and amplification was performed in an ABI 7500 model fluorescent quantitative PCR instrument. While deionized water was set as a negative control. And collecting data after the amplification is finished, obtaining an optimal detection area according to the amplification curve graph and the CT value condition, and drawing a standard curve.
(5) Sensitivity and specificity test of the absolute fluorescent quantitative PCR method: after the amplification is finished, the amplification curve (see fig. 1), the standard curve (see fig. 2) and the melting curve (see fig. 3) are comprehensively analyzed. The copy number and the CT value in the standard curve are in a linear inverse proportion relationship, the lowest copy number of the detection sensitivity of the invention is 10, and the established method is shown to have high sensitivity. The regression equation for the gene fragment is: y = -3.061X +33.796, standard curve correlation coefficient R2The amplification efficiency is 112.2 percent and is more than 0.999, which meets the requirement of the test and can be used for the quantitative analysis of the subsequent VP1 gene segment. Melting curve analysis showsOnly a specific single peak appears, and no primer dimer peak and non-specific amplification products appear, which indicates that the established method has high specificity. Meanwhile, the method is used for detecting the existing poultry adenovirus FAdV, avian leukemia virus ALV and reticuloendotheliosis virus REV in a laboratory, and the results are all negative.
Advantageous effects
The invention establishes an absolute fluorescent quantitative PCR detection method for the infectious chicken anemia virus, which can be used for the rapid and specific detection of the infectious chicken anemia virus vaccine contamination residue and the low content of the infectious chicken anemia virus in clinical samples by enterprises; in addition, the content of the infectious chicken anemia viruses can be quantitatively detected, so that the change rule and the like of the infectious chicken anemia viruses in vivo can be known, and an effective means is provided for further researching the molecular biological characteristics of the infectious chicken anemia viruses and formulating an effective prevention and control strategy. Therefore, the invention has certain production practice application value.
Drawings
FIG. 1 is a graph showing the amplification of pcDNA3.1-VP1 recombinant plasmid.
FIG. 2 is a standard curve of pcDNA3.1-VP1 recombinant plasmid.
FIG. 3 is a melting curve of pcDNA3.1-VP1 recombinant plasmid.
FIG. 4 is a sample of tissue infected with chicken infectious anemia virus detected by PCR;
lane M, Super DNA Marker;
lanes 3-5, liver tissue of 3 chickens on day 63 were CIAV mono-infected;
lanes 6-8, spleen tissue from 3 chickens on day 63 were CIAV mono-infected;
lanes 9-11, kidney tissue of 3 chickens on day 63 were CIAV mono-infected;
lanes 12-14, liver tissue of 3 chickens co-infected at day 63 with CIAV and ALV-J;
lanes 15-17, spleen tissue of 3 chickens on day 63 co-infected with CIAV and ALV-J;
lanes 18-20, CIAV and ALV-J Co-infected renal tissue of 3 chickens on day 63.
FIG. 5 is a graph of copy number in absolute fluorescent quantitative PCR detection of tissue samples infected with chicken infectious anemia virus. (absolute fluorescent quantitative PCR detects the DNA copy number of the infectious chicken anemia virus in the organ tissues (liver, spleen and kidney) on the 63 th day after the single infection of the infectious chicken anemia virus, the co-infection of the infectious chicken anemia virus and the subgroup J avian leukosis virus; CIAV is the single infection of the infectious chicken anemia virus, and CIAV + ALV-J is the co-infection of the infectious chicken anemia virus and the subgroup J avian leukosis virus).
Detailed description of the invention
For better understanding of the content of the present invention, the following embodiments are combined to demonstrate the effect of the absolute fluorescent quantitative PCR detection method for infectious chicken anemia virus of the present invention.
Examples
(1) Extracting chicken infectious anemia virus genome DNA: extraction of infectious chicken anemia virus genomic DNA Using AxyPrep DNA miniprep kit from AxygenTMMultiple source Genomic DNA.
(2) PCR detection of tissue samples infected with infectious chicken anemia Virus: the primers are a pair of primers which are synthesized in a laboratory and aim at the gene VP3 of the chicken infectious anemia virus, the size of an amplified fragment is 366bp, and the sequences of the primers are as follows: the upstream primer is VP 3F: 5'-ATGAACGCTCTCCAAGAAGATAC-3', respectively; the downstream primer is VP 3R: 5'-TTACAGTCTTATACGCCTTTTTGCG-3', respectively; the experiment was performed by PCR amplification using 2 x Taq Master Mix PCR enzyme from tokyo kezan biotechnology ltd, reaction system 25 μ L: 2 xTaq Master Mix 12.5. mu.L, template 2. mu.L, upstream primer and downstream primer each 1. mu.L, sterilized ddH2O8.5. mu.L. The reaction procedure is as follows: 5min at 95 ℃; 35 cycles of 95 ℃ for 30s, 56 ℃ for 30s and 72 ℃ for 30 s; the reaction was stopped at 72 ℃ for 10min and 4 ℃. The PCR product was electrophoresed through a 1% agarose gel and then observed on a gel imager (see FIG. 4).
(3) Detecting a tissue sample infected with the chicken infectious anemia virus by absolute fluorescent quantitative PCR: the absolute fluorescent quantitative PCR method is established by using TB Green-box Premix Ex Taq-II reagent of TaKaRa company, and the reaction system is 20 mu L: solution A: 2 mu L of template and 6 mu L of double distilled water; and B, liquid B: TB Green Premix Ex Taq II (2X) 10. mu.L, upstream and downstream primers 0.8. mu.L each, and ROX Reference Dye II (50X) 0.4. mu.L. When adding sample, firstly adding the solution B into the eight-tube reaction tube, then adding the solution A, and centrifuging before loading. The reaction procedure was as follows: the reaction sequence was 40 cycles of pre-denaturation at 95 ℃ for 30s, followed by denaturation at 95 ℃ for 5s and final annealing at 60 ℃ for 34 s. According to the standard curve and the CT value, the copy number of the chicken infectious anemia virus in the tissue sample is calculated, the content of the chicken infectious anemia virus in different organ tissues is compared (see figure 5), and the result shows that the content of the chicken infectious anemia virus in the spleen is the highest and is far higher than that in the liver and the kidney.
(4) Comparing the PCR detection method with the absolute fluorescent quantitative PCR detection method for the infectious chicken anemia virus: by comparing the tissue sample (see figure 4) of the infectious chicken anemia virus detected by PCR with the tissue sample (see figure 5) of the infectious chicken anemia virus detected by absolute fluorescent quantitative PCR, the conventional PCR can detect a certain content of the infectious chicken anemia virus and the reaction content can be only approximately determined by the intensity of the strip, while the absolute fluorescent quantitative PCR can detect less content of the infectious chicken anemia virus (10 copies), and can accurately detect the content of the infectious chicken anemia virus in different organ tissues (liver, spleen and kidney).
Sequence listing
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Claims (1)
1. The absolute fluorescent quantitative PCR detection method for the chicken infectious anemia virus is characterized by comprising the following steps of:
(1) extracting chicken infectious anemia virus genome DNA: extraction of infectious chicken anemia virus genomic DNA Using AxyPrep DNA miniprep kit from AxygenTM(iii) a multiple source Genomic DNA finish;
(2) design and synthesis of primers: according to the highly conserved region of the chicken infectious anemia virus VP1 gene, an upstream primer and a downstream primer with the amplification fragment size of 140bp are designed and synthesized, and the primer sequences are as follows: the upstream primer is VP 1-F: 5'-GCCCCGGTACGTATAGTGTG-3', respectively; the downstream primer is VP 1-R: 5'-CCCGTACATGTGGTCTGCAT-3', respectively; before the primer is used, dissolving the primer by using ultrapure water, and storing the primer at the temperature of minus 20 ℃ for later use, wherein the use concentration of the primer is 10 pmol;
(3) establishment of an absolute fluorescent quantitative PCR method: the establishment of the absolute fluorescent quantitative PCR method was carried out by using TB Green-box Premix Ex Taq-II reagent of TaKaRa, 20. mu.L: solution A: 2 mu L of template and 6 mu L of double distilled water; and B, liquid B: TB Green Premix Ex Taq II (2X) 10. mu.L, upstream and downstream primers 0.8. mu.L each, ROX Reference Dye II (50X) 0.4. mu.L; when the sample is added, the solution B is added into the eight-tube reaction tube, then the solution A is added, and centrifugal treatment is carried out before the machine is arranged; the reaction procedure was as follows: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s, and annealing at 60 ℃ for 34s, wherein the reaction program is 40 cycles;
(4) drawing a standard curve: the pcDNA3.1-VP1 plasmid successfully prepared in the laboratory is used as a positive standard substance for standby; converting the positive standard plasmid into copy number, and continuously diluting at 10 times ratio from 1: 10 to 1: 109Setting 9 concentration gradients in total, setting 3 times for each gradient, and performing in ABI 7500 type fluorescence quantitative PCR instrumentAmplifying, and setting deionized water as a negative control; collecting data after amplification is finished, obtaining an optimal detection area according to an amplification curve graph and the CT value condition, and drawing a standard curve;
(5) sensitivity and specificity test of the absolute fluorescent quantitative PCR method: the standard curve is drawn to show that the chicken infectious anemia virus with the virus DNA copy number of only 10 can be detected, and the sensitivity of the absolute fluorescent quantitative PCR method is reflected; extracting corresponding nucleic acid from the existing fowl adenovirus FAdV, avian leukosis virus ALV and reticuloendotheliosis virus REV in a laboratory, and operating the program according to an amplification system; meanwhile, a positive control of the infectious chicken anemia virus and a negative control of deionized water are set, and the detection result shows the specificity of the absolute fluorescent quantitative PCR method.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112877478A (en) * | 2021-04-12 | 2021-06-01 | 广东省农业科学院动物卫生研究所 | Real-time fluorescence quantitative PCR detection reagent for glandular stomach necrosis virus and application thereof |
| CN113214365A (en) * | 2021-05-12 | 2021-08-06 | 扬州大学 | ELISA kit for detecting chicken infectious anemia virus antibody based on polypeptide |
| CN113897356A (en) * | 2021-10-20 | 2022-01-07 | 佛山科学技术学院 | Fluorescent quantitative PCR kit and primers for detecting chicken infectious anemia virus |
| CN114959120A (en) * | 2022-06-30 | 2022-08-30 | 华南农业大学 | Primer probe set and kit for detecting chicken infectious anemia virus by RAA fluorescence method and application of primer probe set and kit |
| CN116463297A (en) * | 2023-03-09 | 2023-07-21 | 扬州大学 | Recombinant serum type 4 avian adenovirus expressing chicken infectious anemia virus VP1 protein and preparation method thereof |
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| CN113214365A (en) * | 2021-05-12 | 2021-08-06 | 扬州大学 | ELISA kit for detecting chicken infectious anemia virus antibody based on polypeptide |
| CN113897356A (en) * | 2021-10-20 | 2022-01-07 | 佛山科学技术学院 | Fluorescent quantitative PCR kit and primers for detecting chicken infectious anemia virus |
| CN113897356B (en) * | 2021-10-20 | 2024-04-30 | 佛山科学技术学院 | Fluorescent quantitative PCR (polymerase chain reaction) kit and primer for detecting chicken infectious anemia virus |
| CN114959120A (en) * | 2022-06-30 | 2022-08-30 | 华南农业大学 | Primer probe set and kit for detecting chicken infectious anemia virus by RAA fluorescence method and application of primer probe set and kit |
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