CN114540512A - Primer for detecting chicken APOA1 gene and detection method and kit thereof - Google Patents
Primer for detecting chicken APOA1 gene and detection method and kit thereof Download PDFInfo
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Abstract
The invention discloses a primer for detecting chicken APOA1 gene, a detection method and a kit thereof. The primer sequence is shown as SEQ ID NO.1 and SEQ ID NO.2, and specifically comprises the following components: APOA 1-F: TCCTTCTGGCAGCACGATG; (SEQ ID NO.1) APOA 1-R: CCACAGCAGAGGACTCGAAC (SEQ ID NO. 2). The primer sequence and the corresponding PCR detection method designed by the invention can quickly detect the content of the chicken APOA1, and have high detection sensitivity and good specificity. The energy and protein content of the corresponding daily ration can be adjusted and evaluated according to the APOA1 content of the chicken.
Description
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to a primer for detecting chicken APOA1 gene, a detection method and a kit thereof.
Background
APOA1 is called Apolipoprotein a-I, also called Apolipoprotein a1, is encoded by the APOA1 gene, has a molecular weight of about 28kDa, is synthesized in the liver, constitutes a major High-density Lipoprotein (HDL) component in the body, and plays an important role in the reverse cholesterol transport process, in which cholesterol is transported from peripheral tissues to the liver for metabolism, as a major structural component of the HDL (about 70%). Apolipoproteins are proteins that bind to water-insoluble lipids and are used as vehicles to shuttle and circulate continuously in the systemic circulation and lymphatic system, playing an important role in transporting lipid substances. In addition, the protein also plays a significant role in the virus infection process. For example, through clinical sample investigation, APOA1 protein levels significantly different from those of healthy subjects were also detected in the blood of patients infected with the new coronavirus, while the levels of serum high density lipoprotein and APOA1 protein were too high, and the risk of infection with the new coronavirus was greater. Therefore, the change of the protein level can be used as an important early warning signal for the new coronavirus infection, and the fact that the APOA1 protein level in blood is possibly a functional protein for resisting the virus infection is also revealed, but the change condition of chicken APOA1 protein in the avian virus infection process and whether the function of resisting the virus infection is exerted are unknown, and the change condition is worthy of further exploration of a plurality of researchers.
The real-time fluorescent quantitative PCR technology was introduced by Applied Biosystems in 1996, and not only does the technology realize qualitative to quantitative leap of PCR, but also compared with conventional PCR, the technology has the characteristics of stronger specificity, effective solution of PCR pollution problem, high automation degree and the like. The method is characterized in that a PCR product is labeled and tracked through a fluorescent dye or a specific probe labeled by fluorescence, the reaction process is monitored on line in real time, the product can be analyzed by combining with corresponding software, and the initial concentration of a sample template to be detected is calculated, so that the detection sensitivity and specificity can be effectively improved by using a fluorescent PCR method to detect the chicken APOA 1.
However, few literature reports currently exist about a method for detecting chicken APOA1, which is few and single, only comprises a common PCR method and a Coomassie brilliant blue staining method, and has low detection sensitivity and specificity.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the primer for detecting the chicken APOA1 gene, the detection method and the kit thereof, which can quickly detect the content of chicken APOA1, and have high detection sensitivity and good specificity.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
a primer for detecting chicken APOA1 gene has the sequence shown in SEQ ID NO.1 and SEQ ID NO. 2. The method comprises the following specific steps:
APOA1-F：TCCTTCTGGCAGCACGATG；(SEQ ID NO.1)
APOA1-R：CCACAGCAGAGGACTCGAAC。(SEQ ID NO.2)
further, the primer sequence was a sequence complementary to a sequence in the chicken APOA1 gene.
A method for detecting chicken APOA1 gene uses sample DNA as template, and adopts the above-mentioned primer to make PCR amplification, and the amplified product is undergone the process of enzyme digestion electrophoresis to judge result.
Further, the PCR reaction system included 2 XPro TaqHS Probe Premix 12.5. mu.L, 1. mu.L each of the upstream and downstream primers 10 pmol/. mu.L, 2. mu.L of the template DNA, and finally 25. mu.L with sterile water.
Further, the PCR procedure was: 15min at 55 ℃; pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 10 s; annealing at 60 ℃ for 30 s; the last two steps were performed for 39 cycles.
A kit for detecting chicken APOA1 gene comprises the primer.
Further, the kit may also include one or more enzymes/reagents for the PCR reaction, and optionally other components and means.
The invention has the beneficial effects that:
the primer sequence and the corresponding PCR detection method designed by the invention can quickly detect the content of the chicken APOA1, and have high detection sensitivity and good specificity. The energy and protein content of the corresponding daily ration can be adjusted and evaluated according to the APOA1 content of the chicken.
Drawings
FIG. 1 is a gene detection map;
FIG. 2 shows the result of PCR electrophoresis;
FIG. 3 shows the results of fluorescence detection.
Detailed Description
The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.
Example 1 detection of the chicken APOA1 Gene
1. Material
The chicken APOA1 gene is synthesized by Ongko biotechnology, Inc., and a virus genome DNA extraction kit, a common agarose gel DNA recovery kit and a common plasmid mini-extraction kit are purchased from Tiangen Biotechnology limited; 2 XTaq PCR Master Mix, DL2000 DNA Marker, etc. were purchased from Bao bioengineering (Dalian) Co., Ltd.
2. Detection method
(1) Design of primers
Primers specific to the APOA1 gene were designed on-line using Primer-BLAST according to the NCBI reference nucleotide sequence chicken APOA1(GenBank accession No.: NM-205525.4), and were synthesized by Shanghai Biotechnology engineering, Inc. The length of the target fragment gene is 127bp, and is positioned at nucleotide position 143-169 of APOA1 Chicken. The detailed information of the primers is shown in the following table.
TABLE 1 primer APOA1
(2) Extracting gene of sample to be detected
Taking tissues of a control group (a proper amount of healthy SPF (specific pathogen free) chickens) and a test group (virus infected chickens) and grinding the tissues into fine powder by liquid nitrogen, adding a proper amount of normal saline, uniformly grinding the fine powder (in a tissue homogenate state) and centrifuging the fine powder at 3000r/min for 3min, taking 200 mu L of supernate into a clean centrifugal tube, and then extracting nucleic acid by using an RNA extraction kit.
(3) PCR detection
Adopting 25 muL reaction system, using 2 muL synthetic gene with same concentration as template, the total volume of the fluorescent quantitative PCR reaction system is 25 muL, which includes:
2 XPro TaqHS Probe Premix 12.5. mu.L, upstream and downstream primers (10 pmol/. mu.L) each 1. mu.L, template DNA 2. mu.L, and finally 25. mu.L with sterile water.
The Bio-Rad CFX connect fluorescent quantitative PCR instrument amplification program is as follows: 15min at 55 ℃; 30s at 95 ℃; 10s at 95 ℃; 30s at 60 ℃; the last two steps were performed for 39 cycles.
Both the housekeeping gene (ACTIN) and the gene of interest need to be arranged in 3 parallel repeats.
3. The result of the detection
(1) Firstly, observing whether the difference of CT values of three repeated tests is large (generally not exceeding 0.5), and then respectively calculating the average value of the CT values of the three parallel repeated tests;
(2) subtracting the average value of housekeeping genes from the average value of the target genes to obtain a delta ct value;
(3) calculating the mean value of the two delta values of the control group and recording the mean value as A;
(4) and respectively subtracting A from the four delta values of the test group and the control group to obtain a delta-delta ct value.
(5) And calculating 2-delta ct, namely performing significance analysis on the calculation result by using GraphPad Prism software, and obtaining the result shown in figure 1.
Example 2 repeatability test
The nucleic acids of the control and test groups were subjected to relative quantitative PCR amplification under the same conditions using the synthesized gene as a detection template, and 3 replicates were set for each nucleic acid, and the results are shown in fig. 2, fig. 3, and table 2.
In fig. 2, M1: the 600bp is sequentially from bottom to top: 100bp, 200bp, 300bp, 400bp, 500bp and 600 bp; m2: 2000bp is from bottom to top: 100bp, 250bp, 500bp, 750bp, 1000bp and 2000 bp; n: negative control; p: a positive control; s: and (5) detecting the sample.
TABLE 2 test results
| Name of test sample | Positive control | Sample 1 | |
|
Sample No. 4 | Sample No. 5 | Sample No. 6 |
| CT value of detection result | 19.97 | 26.87 | 30.09 | N | N | N | N |
Sample 1 and sample 2 in table 2 are APOA1 test samples; samples 3 to 6 are samples for verifying the specificity.
According to the detection results of fig. 2, fig. 3 and table 2, when the sample is subjected to multiple repeatability tests, the detection results have no significant difference, and thus, the scheme of the application has good repeatability.
Sequence listing
<110> Chengdubi-Mi-Tech detection technology service Co., Ltd
<120> primer for detecting chicken APOA1 gene, detection method and kit thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tccttctggc agcacgatg 19
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Claims (7)
1. A primer for detecting chicken APOA1 gene is characterized in that the sequence of the primer is shown as SEQ ID NO.1 and SEQ ID NO. 2.
2. The primer of claim 1, wherein the primer sequence is a sequence complementary to a sequence in chicken APOA1 gene.
3. A method for detecting chicken APOA1 gene is characterized in that sample DNA is used as a template, the primer of claim 1 is used for PCR amplification, and the result is judged by enzyme digestion electrophoresis of the amplified product.
4. The method of claim 3, wherein the PCR reaction system comprises 2 XPro TaqHS Probe Premix 12.5. mu.L, 1. mu.L each of the upstream and downstream primers 10 pmol/. mu.L, 2. mu.L of template DNA, and finally 25. mu.L of sterile water.
5. The method of claim 3, wherein the PCR program is: 15min at 55 ℃; pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 10 s; annealing at 60 ℃ for 30 s; the last two steps were performed for 39 cycles.
6. A kit for detecting chicken APOA1 gene, which is characterized by comprising the primer of claim 1.
7. The kit of claim 6, further comprising one or more enzymes/reagents for PCR reaction, and optionally other components and means.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106573970A (en) * | 2014-05-15 | 2017-04-19 | 克利夫兰心脏实验室公司 | Compositions and methods for purification and detection of HDL and APOA1 |
| CN107462729A (en) * | 2017-08-10 | 2017-12-12 | 迈克生物股份有限公司 | A kind of Apolipoprotein A1 detection kit and detection method |
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2022
- 2022-03-21 CN CN202210320805.9A patent/CN114540512A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106573970A (en) * | 2014-05-15 | 2017-04-19 | 克利夫兰心脏实验室公司 | Compositions and methods for purification and detection of HDL and APOA1 |
| CN107462729A (en) * | 2017-08-10 | 2017-12-12 | 迈克生物股份有限公司 | A kind of Apolipoprotein A1 detection kit and detection method |
Non-Patent Citations (3)
| Title |
|---|
| 夏安琪: "江西不同地方品种鸡apoA-I、apoB基因多态性与脂肪代谢的关联性分析", 中国知网学位论文数据库, no. 02, pages 13 - 15 * |
| 李亚等: "ABCA1基因多态性与血浆载脂蛋白A1水平的关系", 第四军医大学学报, vol. 29, no. 11, pages 1018 - 1020 * |
| 汪晓燕等: "载脂蛋白A1、B基因多态性对非创伤性股骨头坏死发生的影响", 中国骨伤, vol. 21, no. 02, pages 99 - 102 * |
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