CN102181582A - Method for detecting classical swine fever resistance character of swine and special kit thereof - Google Patents
Method for detecting classical swine fever resistance character of swine and special kit thereof Download PDFInfo
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Abstract
The invention discloses a method for detecting a classical swine fever resistance character of a swine and a special kit thereof. A specific primer pair which is provided by the invention for assisting detection of the classical swine fever resistance character of the swine is shown as (a) or (b): (a) the primer pair consisting of a DNA (Deoxyribonucleic Acid) fragment shown as a sequence 5 of a sequence table and a DNA fragment shown as the sequence 6 of the sequence table; and (b) the primer pair consisting of the DNA fragment shown as the sequence 3 of the sequence table and the DNA fragment shown as the sequence 4 of the sequence table. In the method, the primers are designed at a swine NEAT1 homologous sequence conservation region to amplify genome DNA fragments of different types of swine; the obtained fragment comprises a C/G mutation site; and the classical swine fever virus antibody level of the swine is detected by using polymorphism of a BfaI enzyme cutting site caused by the mutation site. Experiments prove that the mutation site is obviously related to the classical swine fever virus antibody level, so that the method for evaluating the classical swine fever virus antibody level by using the mutation site is accurate and feasible.
Description
Technical Field
The invention relates to a method for detecting swine fever resistance of a pig and a special kit thereof.
Background
Pork is the most important animal protein food source for residents in China, and the pig industry plays a significant role in the animal husbandry production chain. For decades, modern breeding techniques have significantly improved the productivity of pigs, but mainly aimed at the production traits (growth traits and reproductive traits). Compared with the production traits, the research on the health and disease resistance of the pigs is weak. The loss or frequency of alleles caused by the breeding target of high lean-type pigs is reduced, so that the physique and resistance of modern commercial breeds are reduced, and with the popularization of intensive breeding modes, swine diseases, particularly certain recurrent infectious diseases, bring great loss to pig production. Classical Swine Fever (CSF) is a highly contagious and fatal epidemic disease caused by swine fever virus, and brings great economic loss to the swine industry all over the world. The swine fever always threatens the safety of the pig industry due to the factors of vaccine quality, non-strict immune program, the group spread of wild pigs carrying viruses and the like.
At present, the research on pig disease resistance breeding mainly searches for major genes or QTL for controlling disease resistance. Through candidate genes, comparative genome and genome scanning and other methods, a plurality of functional genes and QTLs which affect the pig diarrhea resistance and edema disease resistance, foot-and-mouth disease resistance, swine fever resistance and influenza virus resistance, including K88 and/or F18 receptors, FUT, interferon and receptors thereof, MHC and Mxl genes, have been identified. Wherein: (1) k88 or F18 receptor-deficient strains block the attachment of cell surface antigens of strains of E.coli K88 or F18 to small intestinal mucosal epithelial cell surface receptors and thus confer resistance to diarrhea or edema disease caused by the corresponding E.coli (Bertschinger, H.and Pohlenz, J. (1983) Bacterial physiology and toxicity of the intestine in pore Escherichia coli enterotoxemia (edema disease). Veterinary Pathology.20, 99.).
Etc. (
P.，Meijerink，E.，Fries，R.，Neuenschwander，S.，Vorl nder，N.，Stranzinger，G.and Bertschinger，H.(1997)Amolecular test for the detection of E.coli F18 receptors：a breakthrough in the struggleagainst edema disease and post-weather Diarrhae in swine, Schweizer Archiv fur Tieerheilkund, 139, 479.) the dunaliella salina glycosyltransferase genes FUT1 and FUT2, which are located in the 6q11 segment, were also found to be 1 candidate gene for controlling F18 adhesion by linkage analysis. Us ARS uses this marker to announce the development of resistant pigs in about grams in 7 months 1999. (2) Porcine interferon is proved to have broad-spectrum defense and inhibition effects on various major infectious diseases such as reproductive failure and respiratory syndrome virus, foot and mouth disease virus, swine fever virus and the like (Buddaert, W., Van Reeth, K.and Pensaert, M. (1998) In vivo and In vitro Interferons (IFN) students with the bacterial reproduction and respiratory syndrome viruses (PRRSV), Coronavir and arylviruses.440, 461.), so that the interferon and receptor genes thereof are important candidate genes for variety breeding. (3) MHC as a molecular marker for breeding against disease has been proved to be related to the responses of various antigens, bacteriophagy of bacteria, infection of trichina and toxoplasma, and characters such as melanoma and tumorigenesis, and is an important selection site of nonspecific disease resistance. (4) Studies have shown that Transgenic pigs Transgenic for the mouse MX1 gene have increased resistance to influenza virus (Muller, M., Brenig, B., Winnacker, E.L. and Brem, G. (1992) Transgenic pig coding cDNA encoding the vaccine Mx1 protein tissue restriction infection. Gene.121, 263-270.). Nakajima et al (Nakajima, E., Morozu, T., Tsukamoto, K., Watanabe, T., Plasstow, G.and Mitsuhashi, T. (2007) A naturally occingvariate of gene Mx1 associated with the use of secreted to influenza virus in vitro. biochemical genetics.45, 11-24.) the in vitro study found that a natural variation in the porcine MX1 gene may increase susceptibility to influenza. (5) SLC11A1 (also known as NRAMP1) is a disease-resistant gene that has been studied more recently. SLC11A1 was first reported to be associated with the development of tuberculosis and sarcoidosis (Bellamy, R., Ruwende, C., Corrah, T., McAdam, K.P.W.J., Whittle, H.C.and Hill, A.V.S. (1998) variaaions in the NRAMP1 gene and susceptability to tuboculosis in West Africans.New Englandd Journal of medicine.338, 640-. The gene plays a Role in the regulation of Toll-like receptor 7 ligand-induced signal transduction (Moisan, J., Thuraiingam, T., Henault, J., Santis, J.D. and Radzioch, D. (2006) Role of SLC11A1(for example NRAMP1) in modulation of signal transduction induced by Toll-like receptor 7 ligands&Medical microbiology.47, 138- & 147)), and thus may be involved in a broad range of non-specific immune responses. It has been found that the SLC11A1 gene is an important molecular marker related to immune traits, productivity and the like of swine (Wu, H., Cheng, D.and Wang, L. (2008) Association of polymorphisms of Nram 1 gene with immune function and production performance of large white pig. journal of Genetics and genomics.35, 91-95.). Further development of candidate gene research on disease resistance traits will be important content of future pig disease resistance breeding research.
Genome-wide analysis found that many non-coding RNAs were transcribed in mammalian genomes, and there is increasing evidence that these non-coding RNAs are not simply "noise" of transcription, but can participate in the regulation of transcription as precursors of structural or small RNAs (Ponting, c.p., over, p.l.and Reik, W. (2009) Evolution and functions of coding RNAs, cell.136, 629 641.). NEAT1 is a potential non-coding RNA transcript found by Peyman et al (Peyman, J.A. (1999) replication of major histocompatibility complex genes by a human tropiphoblast nucleic acid of biology of reproduction.60, 23.) to be expressed predominantly in trophoblast cells. The human NEAT1 gene can inhibit the class II transactivator promoter, thereby inhibiting the expression of the major histocompatibility complex, and significantly inhibit the B cell allogenic response (Geirsson, A., Bothwell, A.L.M.and Hammond, G.L. (2004) Inhibition of allogenic reaction by allogenic by a human trophosphatno-coding RNA deletion class II transactivator III and macrohistocompatibility class II ex
Disclosure of Invention
The invention aims to provide a specific primer pair for assisting in detecting the swine fever resistance of a pig.
The specific primer pair for assisting in detecting the swine fever resistance of the pigs, provided by the invention, is different from the following primer pairs in a) or b):
a) a primer pair consisting of a DNA fragment shown in a sequence 5 of the sequence table and a DNA fragment shown in a sequence 6 of the sequence table;
b) a primer pair consisting of a DNA fragment shown in a sequence 3 of the sequence table and a DNA fragment shown in a sequence 4 of the sequence table.
The application of the specific primer pair in the auxiliary detection of the swine fever resistance of the pigs also belongs to the protection scope of the invention.
The application of the specific primer pair in the preparation of the kit for the auxiliary detection of the swine fever resistance of the pigs also belongs to the protection scope of the invention.
The invention also aims to provide a kit for auxiliary detection of the swine fever resistance of the pigs.
The kit for the auxiliary detection of the swine fever resistance of the pigs, provided by the invention, comprises the following specific primer pair shown in a) or b):
a) a primer pair consisting of a DNA fragment shown in a sequence 5 of the sequence table and a DNA fragment shown in a sequence 6 of the sequence table;
b) a primer pair consisting of a DNA fragment shown in a sequence 3 of the sequence table and a DNA fragment shown in a sequence 4 of the sequence table.
Still another object of the present invention is to provide a method for the auxiliary detection of swine fever resistance traits in pigs.
The method for auxiliary detection of the swine fever resistance of the pigs is shown in the following 1) or 2):
1) detecting whether the genotype of the pig to be detected is CC genotype, GG genotype or CG genotype; the CC genotype is a homozygote of a 112 th nucleotide from the 5' end in a DNA fragment shown in a sequence 2 in a sequence table in a genome DNA; the GG genotype is a homozygote of a 112 th nucleotide from the 5' end in a DNA fragment shown in a sequence 2 in a sequence table in a genome DNA, and is G; the CG genotype is a heterozygote of a 112 th nucleotide from the 5' end in a DNA fragment shown in a sequence 2 in a sequence table in a genome DNA, wherein the 112 th nucleotide is C and G; compared with the pig with the GG genotype, the pig with the CC genotype and the CG genotype has stronger swine fever resistance;
2) detecting whether the genotype of the pig to be detected is CC genotype, GG genotype or CG genotype; the CC genotype is a homozygote of nucleotide 451 th from the 5' end in a DNA fragment shown in a sequence 1 in a sequence table in a genome DNA, which is C; the GG genotype is a homozygote of nucleotide 451 th from the 5' end in a DNA fragment shown in a sequence 1 in a sequence table in a genome DNA, which is G; the CG genotype is a heterozygote of nucleotide 451 th from the 5' end in a DNA fragment shown in a sequence 1 in a sequence table in a genome DNA, which is C and G; compared with the pig with the GG genotype, the pig with the CC genotype and the CG genotype has stronger swine fever resistance.
The method for detecting whether the genotype of the pig to be detected is CC genotype, GG genotype or CG genotype comprises the following steps:
1) taking the genome DNA of the pig to be detected as a template, and carrying out PCR amplification by using a specific primer pair to obtain a PCR amplification product; the specific primer pair is shown as a) or b) as follows:
a) a primer pair consisting of a DNA fragment shown in a sequence 5 of the sequence table and a DNA fragment shown in a sequence 6 of the sequence table;
b) a primer pair consisting of a DNA fragment shown in a sequence 3 of the sequence table and a DNA fragment shown in a sequence 4 of the sequence table;
2) detecting the PCR amplification product, and determining whether the genotype of the pig to be detected is CC genotype, GG genotype or CG genotype according to the detection result;
the method for detecting the PCR amplification product is shown as A) or B) as follows:
A) sequencing the PCR amplification product, and determining whether the genotype of the pig to be detected is a CC genotype, a GG genotype or a CG genotype according to a sequencing result;
B) carrying out enzyme digestion on the PCR amplification product by using BfaI restriction enzyme, and detecting the enzyme digestion product; if the enzyme digestion product is two fragments of 112bp and 108bp, determining the genotype of the pig to be detected as the CC genotype; if the enzyme digestion product is a fragment of 220bp, determining that the genotype of the pig to be detected is GG genotype; and if the enzyme digestion product is three fragments of 112bp, 108bp and 220bp, determining that the genotype of the pig to be detected is CG genotype.
Detecting the enzyme digestion product in the step 2) by agarose gel electrophoresis; if the enzyme digestion product shows two bands of 100bp-200bp and 100bp-200bp on the gel, determining that the genotype of the pig to be detected is a CC genotype; if the enzyme digestion product is a strip which is displayed on the gel as 200bp-300bp, determining that the genotype of the pig to be detected is GG genotype; and if the enzyme digestion product is three bands which are displayed on gel as 100bp-200bp, 100bp-200bp and 200bp-300bp, determining that the genotype of the pig to be detected is CG genotype.
The swine fever is caused by swine fever virus;
compared with the pig with the GG genotype, the pig with the CC genotype and the CG genotype has stronger swine fever resistance: the antibody titer of the classical swine fever virus in the blood of the pig with the CC genotype and the CG genotype is higher than that of the pig with the GG genotype.
The application of the specific primer pair, the test kit and/or the method in breeding pigs also belongs to the protection scope of the invention.
Still another object of the present invention is to provide a breeding method of pigs.
The breeding method of the pig provided by the invention is to select the pig with the CC genotype or CG genotype detected by the method for breeding.
The method of the invention is to design primers in the conservation region of the homologous sequence of the pig NEAT1 to amplify the DNA fragments of the genome of different pig breeds, the obtained fragments contain a C/G mutation, and the polymorphism of the BfaI enzyme cutting site caused by the mutation site is used for detecting the immune character of the pig, namely the antibody level of the classical swine fever virus. Experiments prove that the mutation site is obviously related to the antibody level of the classical swine fever virus, and the method for evaluating the antibody level of the classical swine fever virus by using the mutation site is an accurate and feasible method. The method can be used for evaluating the titer of the antibody of the classical swine fever virus, thereby providing a new method for detecting the genetic character of the swine pest virus for the molecular breeding of the swine, and playing an important role in the breeding of the swine.
Drawings
FIG. 1 shows the results of electrophoresis of samples of three genotypes.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 method for detecting swine fever resistance traits of pigs by RFLP polymorphism of pig NEAT1 gene
First, identification of pig NEAT1 gene RFLP polymorphic site
Designing primers according to sequences of conserved regions of the pig NEAT1 gene, wherein the sequences of the primers are as follows:
neat1 a: 5'-GGAGGTGGACAAAATTGACC-3' (sequence 3 in the sequence table),
neat1 b: 5'-GACACGGGACATTGGAGAAC-3' (SEQ ID NO: 4 in the sequence Listing).
Respectively taking 3 genome DNAs of a Tongcheng pig, a big white pig, a Changbai pig, a Wuzhishan pig, a Laiwu pig and a Bama miniature pig as templates and using Neat1a and Neat1b as primers to carry out PCR amplification, wherein the total volume of PCR reaction is 20 mu L, the template DNA is 50ng, and the MgCl contains 1 xbuffer and 1.5mmol/L2dNTP final concentration of 200. mu. mol/L, primer final concentration of 0.2. mu. mol/L, 1.0U Taq DNA polymerase (TaKaRa). The PCR amplification procedure was: pre-denaturation at 95 ℃ for 5min, followed by 95 ℃ for 30s, 60 ℃ for 30s, 72 ℃ for 30s, 35 cycles, and final extension at 72 ℃ for 5 min.
The length of the primer amplification fragment is 559bp, namely the sequence 1 in the sequence table. The amplified product is purified, recovered, cloned and sequenced, and the SeqMan module in DNAstar7.0 software is used for aligning the sequence and analyzing possible polymorphic sites. The analysis and sequencing result shows that the 451 th site of the sequence 1 in the sequence table from the 5' end has C or G mutation, and when the 451 th site nucleotide is C, 1 BfaI enzyme cutting site (C ↓ TAG) exists, which can cause the polymorphism of the BfaI enzyme cutting fragment in the sequence 1 in the sequence table.
Second, establishment of pig NEAT1 gene RFLP polymorphism detection method
1. Primer design
Primers are designed on both sides of the polymorphic site, and the primer sequences are as follows:
BfaIa: 5'-GTACCCGCTGAAAGCTACGC-3' (SEQ ID NO: 5 in the sequence listing),
BfaIb: 5'-GGCCTAGACACGGGACATTG-3' (SEQ ID NO: 6 in the sequence Listing).
2. Experimental Material
DNA samples for gene polymorphism detection were from 7 swine groups (Table 1). Extracting pig blood genome DNA by phenol chloroform method, and storing at-20 deg.C for use.
TABLE 1 population and sample number for RFLP polymorphism detection
(3) PCR amplification conditions
The PCR reaction was performed in a total volume of 20. mu.L, in which about 50ng of pig genomic DNA was contained in 1 XBuffer, 1.5mmol/LMgCl2dNTP final concentration of 200U mol/L, primer final concentration of 0.2U mol/L, 1U Taq DNA polymerase. The PCR amplification procedure was: pre-denaturation at 95 ℃ for 5min, followed by 35 cycles of 95 ℃ for 30s, 60 ℃ for 30s, and 72 ℃ for 30s, and final extension at 72 ℃ for 5 min. The PCR reaction products were detected by electrophoresis on 2% strength agarose gel and photographed, and the sequence recovered. The sequencing result shows that the nucleotide sequence of the primer amplification fragment is shown as a sequence 2 in a sequence table, and the length is 226 bp. The sequence analysis result shows that the 112 th site of the sequence 2 in the sequence table has C or G mutation from the 5' end. When the nucleotide at this site is homozygote of C, 1 BfaI cleavage site (C ↓ TAG) exists. In addition, another BfaI enzyme recognition site exists from 220 th site to 223 th site of the sequence 2 in the sequence table from the 5 'end, and a small fragment of 6bp can be cut by the 3' end of the sequence 2 in the sequence table of the BfaI enzyme, and can be ignored in RFLP detection.
(4) BfaI-RFLP detection
The PCR product was digested with BfaI in 20. mu.L, 10 XBuffer 2. mu.L, the PCR product 8. mu.L, BfaI restriction enzyme 1. mu.L (from NEB) using ddH2O make up to 20. mu.L. The sample is mixed evenly and centrifuged, and the mixture is bathed at 37 ℃ for 2.5 h. The cleavage products were subjected to agarose gel electrophoresis at 2% concentration to detect the cleavage results, and the gel imaging system photographed and recorded the genotypes, the results being shown in FIG. 1 and Table 2.
When the 112 th nucleotide from the 5' end of the sequence 2 in the sequence table is a homozygote of C, the gene is controlled by C allele (the BfaI enzyme cutting product has two fragments of 112bp and 108 bp); when the 112 th nucleotide from the 5 'end of the sequence 2 in the sequence table is a homozygote of G, the gene is controlled by a G allele (the BfaI enzyme digestion product has only 220 fragments), when the 112 th nucleotide from the 5' end of the sequence 2 in the sequence table is a heterozygote of C and G, the gene is controlled by the C allele and the G allele (the BfaI enzyme digestion product has three fragments of 112bp, 108bp and 220 bp), and the two alleles can form three genotypes of CC, GG and CG.
The BfaI digestion results of the three genotype samples are shown in FIG. 1 (M: DNA molecular weight standard (100bp-1500bpladder)), and the samples show 3 band types in total). The BfaI enzyme digestion result of the sample is that two fragments of 112bp and 108bp are obtained, and the two fragments are named as CC genotypes; the BfaI enzyme cutting result of the sample is to obtain a 220bp fragment, and the fragment is named as genotype GG; the BfaI enzyme cutting result of the sample is that three fragments of 112bp, 108bp and 220bp are obtained, and the three fragments are named as genotype CG.
TABLE 2 results of BfaI-RFLP detection in different pig populations
The results of genotype and gene frequency according to table 2 show that of the several breeds tested, the C allele predominates in all but the two alleles of the long and white pigs (C vs.g. ═ 0.412 vs.0.588).
Third, the correlation analysis of different genotypes of the pig NEAT1 gene and the swine fever resistant character
The experimental herd used for association analysis was a resource family consisting of 376 pure breed Changbai pigs (purchased from Guangdong Wen group), with 17 boars and 36 sows as parents and 323 as offspring. All offspring were from the same pig farm but were distributed to 2 production lines for feeding.
And (3) genotype detection: and (3) detecting according to the method in the second experiment, sequencing and enzyme cutting the PCR product respectively, wherein the genotype result obtained by result sequencing is consistent with the genotype result obtained by enzyme cutting. As shown in table 3.
Detecting the swine fever resistance character: the measured indexes include titer of swine fever antibody (CSFV-AB) in blood, and blood routine indexes such as erythrocyte, leukocyte and platelet.
The titer of CSFV-AB is calculated after detecting the blocking rate of the swine fever antibody by a swine fever antibody enzyme-linked immunosorbent assay Kit (CSFV-AB ELISA Kit, purchased from Indexx); hog cholera antibody blocking rate (PI) ═ ODN-ODT)/(ODN-ODP) Wherein ODN、ODTAnd ODPThe OD values measured by the negative control, the test sample and the positive control are respectively (the negative control and the positive control are provided by the kit); the CSFV antibody titer (Y) is calculated by the following method: log2Y＝-7.56+56.41PI-83.85PI2+44.66PI3。
The measurement of the conventional indices of blood (red blood cells, white blood cells and platelets) was performed by a hematology analyzer (purchased from sysmex, model kx-21N). The indexes are influenced by sex, feeding environment and number of fetuses and belong to a fixed effect; meanwhile, the effects of families and structures in groups, such as male livestock or female livestock effects in male livestock, belong to random effects. Therefore, according to the population structure and the character characteristics of the collected sample, a mixed model is selected to analyze the genotype effect of the candidate gene and the relation between the genotype effect and the character, and the modeling is as follows:
Y＝Xβ+Zb+ε
wherein,
y is a character observed value vector;
x is a fixed effect incidence matrix;
beta is a parameter vector of fixed effect, including genotype effect, gender effect, fetal time effect and environmental effect (genotyps, six, Litter, and environmental) of candidate genes;
a correlation matrix of Z random effects;
b parameter vectors of random effects, including Sire effect and dam (Sire, dam (Sire) effect within Sire;
epsilon is a random error effect vector, and epsilon is independent of each other.
The correlation analysis of the genotype and the character of the experimental swinery is completed by using a SAS V8 GLM program, and the difference of the character among different genotypes is expressed by least square variance. The simple mean and standard error analysis results of the intergenotype traits after eliminating the differences between the breeds, slaughter batches and sex are summarized in table 3.
TABLE 3 correlation analysis of pig NEAT1 genotype with population blood and immune indices
Note: different shoulder markers (a, b) in the same row represent significant differences (P < 0.05) and asterisks represent significant levels of gene effect (P < 0.05 or P < 0.01).
The results show that the genotype effect reaches a significant level (p is less than 0.05) on the measured traits; the antibody titer of swine fever at 0 day and 17 days of age of CC and CG genotype individuals is obviously higher than that of GG genotype individuals, and the CC genotype individuals also show higher erythrocyte parameters and lower platelet parameters (p is less than 0.05) than other genotype individuals. According to the judgment of the correlation analysis result, the C allele is the dominant allele of the swine fever virus resistance, and the CC genotype is the favorable genotype of the swine fever virus resistance. Compared with the pig with the GG genotype, the pig with the CC genotype and the CG genotype has stronger swine fever resistance.
Claims (10)
1. The specific primer pair for auxiliary detection of the swine fever resistance traits of the pigs is shown in a) or b) as follows:
a) a primer pair consisting of a DNA fragment shown in a sequence 5 of the sequence table and a DNA fragment shown in a sequence 6 of the sequence table;
b) a primer pair consisting of a DNA fragment shown in a sequence 3 of the sequence table and a DNA fragment shown in a sequence 4 of the sequence table.
2. The use of the specific primer pair of claim 2 for the auxiliary detection of swine fever resistance in swine.
3. Use of the specific primer pair of claim 1 in the preparation of a kit for the assisted detection of swine fever resistance in swine.
4. A kit for auxiliary detection of swine fever resistance traits of pigs comprises a specific primer pair shown in the following a) or b):
a) a primer pair consisting of a DNA fragment shown in a sequence 5 of the sequence table and a DNA fragment shown in a sequence 6 of the sequence table;
b) a primer pair consisting of a DNA fragment shown in a sequence 3 of the sequence table and a DNA fragment shown in a sequence 4 of the sequence table.
5. The method for auxiliary detection of the swine fever resistance traits of the pigs is shown in the following 1) or 2):
1) detecting whether the genotype of the pig to be detected is CC genotype, GG genotype or CG genotype; the CC genotype is a homozygote of a 112 th nucleotide from the 5' end in a DNA fragment shown in a sequence 2 in a sequence table in a genome DNA; the GG genotype is a homozygote of a 112 th nucleotide from the 5' end in a DNA fragment shown in a sequence 2 in a sequence table in a genome DNA, and is G; the CG genotype is a heterozygote of a 112 th nucleotide from the 5' end in a DNA fragment shown in a sequence 2 in a sequence table in a genome DNA, wherein the 112 th nucleotide is C and G; compared with the pig with the GG genotype, the pig with the CC genotype and the CG genotype has stronger swine fever resistance;
2) detecting whether the genotype of the pig to be detected is CC genotype, GG genotype or CG genotype; the CC genotype is a homozygote of nucleotide 451 th from the 5' end in a DNA fragment shown in a sequence 1 in a sequence table in a genome DNA, which is C; the GG genotype is a homozygote of nucleotide 451 th from the 5' end in a DNA fragment shown in a sequence 1 in a sequence table in a genome DNA, which is G; the CG genotype is a heterozygote of nucleotide 451 th from the 5' end in a DNA fragment shown in a sequence 1 in a sequence table in a genome DNA, which is C and G; compared with the pig with the GG genotype, the pig with the CC genotype and the CG genotype has stronger swine fever resistance.
6. The method of claim 5, wherein: the method for detecting whether the genotype of the pig to be detected is CC genotype, GG genotype or CG genotype comprises the following steps:
1) taking the genome DNA of the pig to be detected as a template, and carrying out PCR amplification by using a specific primer pair to obtain a PCR amplification product; the specific primer pair is shown as a) or b) as follows:
a) a primer pair consisting of a DNA fragment shown in a sequence 5 of the sequence table and a DNA fragment shown in a sequence 6 of the sequence table;
b) a primer pair consisting of a DNA fragment shown in a sequence 3 of the sequence table and a DNA fragment shown in a sequence 4 of the sequence table;
2) detecting the PCR amplification product, and determining whether the genotype of the pig to be detected is CC genotype, GG genotype or CG genotype according to the detection result;
the method for detecting the PCR amplification product is shown as A) or B) as follows:
A) sequencing the PCR amplification product, and determining whether the genotype of the pig to be detected is a CC genotype, a GG genotype or a CG genotype according to a sequencing result;
B) carrying out enzyme digestion on the PCR amplification product by using BfaI restriction enzyme, and detecting the enzyme digestion product; if the enzyme digestion product is two fragments of 112bp and 108bp, determining the genotype of the pig to be detected as the CC genotype; if the enzyme digestion product is a fragment of 220bp, determining that the genotype of the pig to be detected is GG genotype; and if the enzyme digestion product is three fragments of 112bp, 108bp and 220bp, determining that the genotype of the pig to be detected is CG genotype.
7. The method of claim 6, wherein:
detecting the enzyme digestion product in the step 2) by agarose gel electrophoresis; if the enzyme digestion product shows two bands of 100bp-200bp and 100bp-200bp on the gel, determining that the genotype of the pig to be detected is a CC genotype; if the enzyme digestion product is a strip which is displayed on the gel as 200bp-300bp, determining that the genotype of the pig to be detected is GG genotype; and if the enzyme digestion product is three bands which are displayed on gel as 100bp-200bp, 100bp-200bp and 200bp-300bp, determining that the genotype of the pig to be detected is CG genotype.
8. The method according to any one of claims 5-7, wherein:
the swine fever is caused by swine fever virus;
compared with the pig with the GG genotype, the pig with the CC genotype and the CG genotype has stronger swine fever resistance: the antibody titer of the classical swine fever virus in the blood of the pig with the CC genotype and the CG genotype is higher than that of the pig with the GG genotype.
9. Use of a specific primer pair according to claim 1, a test kit according to claim 4 and/or a method according to any one of claims 5 to 8 for breeding pigs.
10. A breeding method of pigs, which is to select pigs of which CC genotype or CG genotype is detected by the method of any one of claims 5 to 8 for breeding.
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| CN102399910A (en) * | 2011-12-13 | 2012-04-04 | 华南农业大学 | Primers and method for identifying swine fever virus vaccine strains and wild strains |
| CN107267635A (en) * | 2017-07-24 | 2017-10-20 | 北京农业职业学院 | For detecting kit of the pig to be measured to PRRS virus resistance |
| CN111662990A (en) * | 2020-07-10 | 2020-09-15 | 内蒙古农业大学职业技术学院 | Method and primer pair for quantitatively detecting different levels of swine fever antibody |
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| CN101762705A (en) * | 2010-01-21 | 2010-06-30 | 中国农业科学院哈尔滨兽医研究所 | Colloidal gold immunochromatographic test strip for detecting wild-type classical swine fever virus |
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| CN101762705A (en) * | 2010-01-21 | 2010-06-30 | 中国农业科学院哈尔滨兽医研究所 | Colloidal gold immunochromatographic test strip for detecting wild-type classical swine fever virus |
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| CN102399910A (en) * | 2011-12-13 | 2012-04-04 | 华南农业大学 | Primers and method for identifying swine fever virus vaccine strains and wild strains |
| CN102399910B (en) * | 2011-12-13 | 2013-06-05 | 华南农业大学 | Primers and method for identifying swine fever virus vaccine strains and wild strains |
| CN107267635A (en) * | 2017-07-24 | 2017-10-20 | 北京农业职业学院 | For detecting kit of the pig to be measured to PRRS virus resistance |
| CN107267635B (en) * | 2017-07-24 | 2020-08-21 | 北京农业职业学院 | Kit for detecting resistance of pig to be detected to porcine reproductive and respiratory syndrome virus |
| CN111662990A (en) * | 2020-07-10 | 2020-09-15 | 内蒙古农业大学职业技术学院 | Method and primer pair for quantitatively detecting different levels of swine fever antibody |
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