CN107267635B - Kit for detecting resistance of pig to be detected to porcine reproductive and respiratory syndrome virus - Google Patents
Kit for detecting resistance of pig to be detected to porcine reproductive and respiratory syndrome virus Download PDFInfo
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Abstract
The invention discloses a kit for detecting the resistance of a pig to be detected to porcine reproductive and respiratory syndrome virus. The invention provides a kit for detecting the resistance of a pig to be detected to porcine reproductive and respiratory syndrome virus, which comprises a substance for detecting specific SNP; the specific SNP is the 125 th nucleotide of a target sequence of a specific primer pair in the pig genome DNA; the specific primer pair consists of a primer F and a primer R; the primer F is a single-stranded DNA molecule shown in a sequence 1 of a sequence table; the primer R is a single-stranded DNA molecule shown in a sequence 2 of a sequence table. Based on the specific SNP, the resistance of the pig to be detected with the AA genotype to the porcine reproductive and respiratory syndrome virus is higher than that of the pig to be detected with the GG genotype. The method provided by the invention is used for breeding pigs, and pigs to be selected can be screened in early stage, so that the problem of porcine reproductive and respiratory syndrome in actual production is effectively relieved, and the economic benefit is improved. The invention will play a great role in the breeding work of pigs.
Description
Technical Field
The invention relates to a kit for detecting resistance of a pig to be detected to porcine reproductive and respiratory syndrome virus.
Background
The porcine reproductive and respiratory syndrome is an important infectious disease of pigs and is mainly characterized by reproductive disorders of pregnant sows and respiratory symptoms of pigs at all ages. Porcine reproductive respiratory syndrome virus causes porcine reproductive respiratory syndrome. Sick pigs and pigs with viruses are important sources of infection, and the main transmission routes are contact infection, air transmission and sperm transmission, and can also be vertically transmitted through placenta. The susceptible pigs can be infected with viruses through various ways such as oral inoculation, nasal inoculation, intramuscular inoculation, intraperitoneal inoculation, intravenous inoculation and intrauterine inoculation, and the viruses can be transmitted to other susceptible pigs by contacting 2-14 weeks after the pigs are infected with the viruses. The virus can be detected from nasal cavity, feces and urine of sick pig.
Porcine reproductive and respiratory syndrome was first reported in 1987 in the United states, and was subsequently reported in Canada, Germany, Spain, and the United kingdom. In 1995, the disease was discovered in continental China and started to prevail in several provinces. The porcine reproductive and respiratory syndrome is one of the important reasons for harming the pig production in China.
Disclosure of Invention
The invention aims to provide a kit for detecting the resistance of a pig to be detected to porcine reproductive and respiratory syndrome virus.
The invention provides a kit for detecting the resistance of a pig to be detected to porcine reproductive and respiratory syndrome virus, which comprises a substance for detecting specific SNP; the specific SNP is the 125 th nucleotide of a target sequence of a specific primer pair in the pig genome DNA; the specific primer pair consists of a primer F and a primer R;
the primer F is (a1) or (a 2):
(a1) a single-stranded DNA molecule shown in sequence 1 of the sequence table;
(a2) DNA molecule which is obtained by substituting one or more nucleotides in the sequence 1 and has the same function as the sequence 1;
the primer R is (b1) or (b 2):
(b1) a single-stranded DNA molecule shown in a sequence 2 of a sequence table;
(b2) and (b) a DNA molecule which is obtained by substituting the sequence 2 by one or more nucleotides and has the same function as the sequence 2.
The specific SNP is G/A polymorphism.
In the kit, the substance for detecting the specific SNP is the specific primer pair.
The kit also comprises a carrier recorded with the following judgment standards: based on the specific SNP, the resistance of the pig to be detected with the AA genotype to the porcine reproductive and respiratory syndrome virus is higher than that of the pig to be detected with the GG genotype.
The kit also comprises a vector which is recorded with the method for detecting the resistance of the pig to be detected to the porcine reproductive and respiratory syndrome virus.
The pig to be detected is a big white pig.
The invention also protects the application of the kit, which is (c1) or (c 2):
(c1) detecting the application of the resistance of the pig to the porcine reproductive and respiratory syndrome virus;
(c2) and (5) breeding pigs.
The pig to be detected is a big white pig. The pig is a big white pig.
The invention also protects the application of the substance for detecting the specific SNP in the preparation of the kit; the specific SNP is the 125 th nucleotide of a target sequence of a specific primer pair in the pig genome DNA; the specific primer pair consists of a primer F and a primer R;
the primer F is (a1) or (a 2):
(a1) a single-stranded DNA molecule shown in sequence 1 of the sequence table;
(a2) DNA molecule which is obtained by substituting one or more nucleotides in the sequence 1 and has the same function as the sequence 1;
the primer R is (b1) or (b 2):
(b1) a single-stranded DNA molecule shown in a sequence 2 of a sequence table;
(b2) DNA molecule which is obtained by substituting one or more nucleotides in the sequence 2 and has the same function as the sequence 2;
the kit has the function of detecting the resistance of the pigs to be detected to the porcine reproductive and respiratory syndrome virus.
In the application, the substance for detecting the specific SNP is the specific primer pair.
The pig to be detected is a big white pig.
The invention also protects a specific primer pair, which consists of a primer F and a primer R;
the primer F is (a1) or (a 2):
(a1) a single-stranded DNA molecule shown in sequence 1 of the sequence table;
(a2) DNA molecule which is obtained by substituting one or more nucleotides in the sequence 1 and has the same function as the sequence 1;
the primer R is (b1) or (b 2):
(b1) a single-stranded DNA molecule shown in a sequence 2 of a sequence table;
(b2) and (b) a DNA molecule which is obtained by substituting the sequence 2 by one or more nucleotides and has the same function as the sequence 2.
The invention also protects the application of the specific primer pair in the preparation of the kit; the kit has the function of detecting the resistance of the pigs to be detected to the porcine reproductive and respiratory syndrome virus. The pig to be detected is a big white pig.
The invention also protects the application of the specific primer pair, which is (c1) or (c 2):
(c1) detecting the application of the resistance of the pig to the porcine reproductive and respiratory syndrome virus;
(c2) and (5) breeding pigs.
The pig to be detected is a big white pig. The pig is a big white pig.
The invention also provides a method for detecting the resistance of the pig to the porcine reproductive and respiratory syndrome virus, which comprises the following steps:
taking the genome DNA of a pig to be detected as a template, carrying out PCR amplification by adopting the specific primer pair, and then sequencing the PCR amplification product;
if only one PCR amplification product is obtained and the 125 th nucleotide is G, the genotype of the pig to be detected is GG; if only one PCR amplification product is obtained and the 125 th nucleotide is A, the genotype of the pig to be detected is AA; if the PCR amplification products have two types, wherein one 125 th nucleotide is G, the other 125 th nucleotide is A, and the genotype of the pig to be detected is GA;
the resistance of the pig to be detected with the AA genotype to the porcine reproductive and respiratory syndrome virus is higher than that of the pig to be detected with the GG genotype.
The pig to be detected is a big white pig.
Any of the porcine reproductive and respiratory syndrome viruses described above may specifically be the ATCC VR-2385 strain.
The method provided by the invention is used for breeding pigs, and pigs to be selected can be screened in early stage, so that the problem of porcine reproductive and respiratory syndrome in actual production is effectively relieved, and the economic benefit is improved. The detection method of the invention has simple operation, low cost and high accuracy, and can realize automatic direct detection. The invention will play a great role in the breeding work of pigs.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
In the examples, the kit for detecting antibodies against porcine reproductive and respiratory syndrome was a porcine reproductive and respiratory syndrome antibody (PRRS-Ab) ELISA kit (Jianglai organism) purchased from Philadelphia dealbata and having a product number of JL 21926-48T.
In the examples, the Porcine Reproductive Respiratory Syndrome Virus (PRRSV) used for challenge was ATCC VR-2385.
The onset of porcine reproductive and respiratory syndrome is characterized by: decreased food intake, preference for lying and no desire to move, body temperature of about 40.5 deg.C, eye dropsy, ear cyanosis, eyelid edema, conjunctivitis, cough, and asthma.
Examples of the following,
First, obtaining of test population
The method comprises the following steps of (1) taking blood of 40-day large white piglet (with the weight of 11 +/-2 kg), detecting a porcine reproductive and respiratory syndrome antibody (antibody detection), collecting a nasal swab to detect the porcine reproductive and respiratory syndrome virus (virus detection), screening piglets with positive antibody detection and/or positive virus detection, and forming a test population by piglets with negative antibody detection and negative virus detection.
Second, genotype detection
The following operations were performed for each piglet of the experimental group:
1. blood was collected and genomic DNA was extracted.
2. And (3) performing PCR amplification by using the genomic DNA extracted in the step (1) as a template and adopting a primer pair consisting of F and R.
F (sequence 1 of the sequence table): 5'-TGAGAGGGGCCCTCCAGGTGA-3', respectively;
r (sequence 2 of the sequence table): 5'-TGTGATGTGTGGAGCACTTCC-3' are provided.
3. Sequencing the PCR amplification product obtained in the step 2, and obtaining the genotype of each piglet according to the sequencing result.
The PCR amplification product of each piglet is 240 bp. According to the sequencing result of the PCR amplification product, all the pigs to be tested in the test population belong to 3 genotypes respectively.
If only one PCR amplification product exists and the 125 th nucleotide is G, the genotype of the piglet is GG. If only one PCR amplification product exists and the 125 th nucleotide is A, the genotype of the piglet is AA. If there are two PCR amplification products, wherein one 125 th nucleotide is G and the other 125 th nucleotide is A, the genotype of the piglet is GA.
From the test population, 21 individuals with the gene type GG were randomly selected to form a group GG (all individuals were labeled with GG) and 21 individuals with the gene type AA were randomly selected to form a group AA (all individuals were labeled with AA).
Thirdly, raising
The group GG and the group AA were mixed and raised in an environment isolated from the outside. The mixed feeding is that the GG group individuals and the AA group individuals can freely contact. The environment isolated from the outside is the environment without the spread of the porcine reproductive and respiratory syndrome virus from the outside.
Obtaining diseased pigs
When piglets in the third step are raised to 9 months of age, 1 pig is randomly extracted from the GG group and the AA group and taken out, the porcine reproductive and respiratory syndrome virus is used for virus attack, and the two pigs have the incidence characteristics of the porcine reproductive and respiratory syndrome and can detect the porcine reproductive and respiratory syndrome virus by the nasal swab under continuous observation.
The diseased pig extracted from the GG group and subjected to the steps is named diseased pig-GG. The diseased pig extracted from the AA group and obtained by the steps is named as a diseased pig-AA.
Fifth, infection simulation
And (3) when the piglets in the third step are raised to 10 months of age, the sick pigs obtained in the fourth step are taken as infection sources to simulate infection: the sick pig-GG and the sick pig-AA are put back to the original population on the same day.
Sixthly, counting the incidence rate
Counting days from the time when the sick pigs are placed in the step five, and counting the number of the sick pigs in each group after 40 days, namely counting the number of the pigs with the porcine reproductive and respiratory syndrome in each group (counting the number of the pigs without the sick pigs-GG and the sick pigs-AA).
The above procedure was repeated three times.
The number of sick pigs in three repeated experiments of the GG group is respectively as follows: 10, 10 and 12.
The number of sick pigs in three repeated experiments of the AA group is respectively as follows: 4, 5 and 6.
The results show that the resistance of the AA genotype pig to the porcine reproductive and respiratory syndrome virus is obviously higher than that of the GG genotype pig.
SEQUENCE LISTING
<110> Beijing academy of agricultural profession
<120> kit for detecting resistance of pig to porcine reproductive and respiratory syndrome virus
<130>GNCYX171349
<160>2
<170>PatentIn version 3.5
<210>1
<211>21
<212>DNA
<213> Artificial sequence
<400>1
tgagaggggc cctccaggtg a 21
<210>2
<211>21
<212>DNA
<213> Artificial sequence
<400>2
tgtgatgtgt ggagcacttc c 21
Claims (1)
1. The application of the specific primer pair for detecting the specific SNP in the preparation of a kit for detecting the resistance of the white pig to the porcine reproductive and respiratory syndrome virus;
the specific primer pair consists of a primer F and a primer R; the primer F is a single-stranded DNA molecule shown in a sequence 1 of a sequence table; the primer R is a single-stranded DNA molecule shown in a sequence 2 of a sequence table;
the specific SNP is 125 th nucleotide of a target sequence amplified by the specific primer pair in pig genome DNA, and is G/A polymorphism.
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| CN201710607206.4A CN107267635B (en) | 2017-07-24 | 2017-07-24 | Kit for detecting resistance of pig to be detected to porcine reproductive and respiratory syndrome virus |
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| CN201710607206.4A CN107267635B (en) | 2017-07-24 | 2017-07-24 | Kit for detecting resistance of pig to be detected to porcine reproductive and respiratory syndrome virus |
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| CN107267635A CN107267635A (en) | 2017-10-20 |
| CN107267635B true CN107267635B (en) | 2020-08-21 |
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Families Citing this family (1)
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| CN114774468B (en) * | 2022-04-20 | 2022-12-20 | 温氏食品集团股份有限公司 | Allele molecular marker and anti-blue-ear-disease pig group construction method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008052335A1 (en) * | 2006-10-31 | 2008-05-08 | University Of Guelph | Methods of determining risk and severity of disease in pigs |
| CN101392297A (en) * | 2008-11-12 | 2009-03-25 | 中国农业科学院北京畜牧兽医研究所 | Resistance breeding method of porcine respiratory syndrome disease and special kit thereof |
| CN102181582A (en) * | 2011-04-21 | 2011-09-14 | 中国农业科学院北京畜牧兽医研究所 | Method for detecting classical swine fever resistance character of swine and special kit thereof |
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2017
- 2017-07-24 CN CN201710607206.4A patent/CN107267635B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008052335A1 (en) * | 2006-10-31 | 2008-05-08 | University Of Guelph | Methods of determining risk and severity of disease in pigs |
| CN101392297A (en) * | 2008-11-12 | 2009-03-25 | 中国农业科学院北京畜牧兽医研究所 | Resistance breeding method of porcine respiratory syndrome disease and special kit thereof |
| CN102181582A (en) * | 2011-04-21 | 2011-09-14 | 中国农业科学院北京畜牧兽医研究所 | Method for detecting classical swine fever resistance character of swine and special kit thereof |
Non-Patent Citations (4)
| Title |
|---|
| Antiviral activity of recombinant porcine surfactant protein A against porcine reproductive and respiratory syndrome virus in vitro;Lan Li等;《ARCHIVES OF VIROLOGY》;20160421;第161卷(第7期);第1883-1890页 * |
| rs322522578;rs322522578;《Ensemble》;20161231;第1页 * |
| Single nucleotide polymorphisms in collagenous lectins and other innate immune genes in pigs with common infectious diseases;N.D. Keirsteada等;《Veterinary Immunology and Immunopathology》;20110715;第142卷;第1-13页 * |
| 猪繁殖与呼吸综合征的抗病育种研究进展;王晓杜等;《浙江农业学报》;20141231;第26卷(第5期);第1394-1398页 * |
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