CN104561279B - Method of improving quality of chicken semen, primer used for method, kit and using method of kit - Google Patents

Abstract

A primer for genes closely related to chicken semen quality, including a mixture of upstream and downstream primers for PCR amplification of vasoactive intestinal peptide receptor 1 (VIPR1) gene, and upstream and downstream primers for PCR amplification of vasoactive intestinal peptide receptor 2 (VIPR2) gene Mixture, upstream and downstream primer mix for PCR amplification of the dopamine receptor 2 (DAR2) gene. The method to improve the quality of chicken semen: During breeding and screening, extract the chicken genomic DNA of the sample to be tested, and amplify the gene sequence with specific primers to obtain target fragments of 192, 250 and 270 bp. The target fragments are analyzed by SSCP and gel electrophoresis. The SSCP map of DNA was obtained by silver staining, and the individuals with TT/GG/OQ three-gene composite genotype were selected according to the band patterns in the three maps. The kit includes upstream and downstream primer mixtures for PCR amplification of VIPR1, VIP2 and DAR2 genes, and reagents required for PCR. Kit usage method: PCR amplification of the fragment where the SNP site is located; PCR‑SSCP detection; SSCP analysis. The method of the invention has remarkable effect, and the detection method is simple and quick, and is not affected by the external environment.

Description

Method for improving quality of chicken semen, primers, kit and method of use thereof

technical field

The invention relates to a high-quality chicken semen quality closely related gene diagnosis kit and a use method thereof, which belong to the technical field of molecular biology and are applied to the early screening of roosters.

Background technique

Human beings have experienced thousands of years of unconscious and conscious selection of poultry production traits, but the process from phenotypic selection to genetic basis research and genotype selection is only a matter of recent decades. Excellent breeding roosters are one of the keys to improving economic benefits. On the one hand, they can reduce production costs, especially the breeding costs of breeding chickens. Adopting artificial insemination can increase the breeding ratio of chickens by about 3 times, and reduce 3/4 of the breeding cocks, which can greatly reduce the cost. On the other hand, it can greatly increase the utilization rate of excellent breeding cocks, improve production efficiency and product quality . The current selection of roosters still adopts conventional selection. Although conventional breeding methods have made some progress in the genetic improvement of poultry, the selection of quantitative traits requires a certain period of individual growth and development. The breeding cycle is long and the cost is high. The semen quality traits of high-quality chickens are quantitative traits, and the results of individual selection based on the phenotype values of individuals or relatives are not very reliable. Therefore, genetic markers are needed for assisted selection. With the completion of human genome sequencing, the screening and detection of SNP has officially become the focus of extensive attention of researchers. This technology has the characteristics of high specificity, rapid detection, strong versatility, wide application range, and low cost. more important role.

After searching the existing domestic and foreign literature and patents, there has been no report on the correlation between VIPR1, VIPR2 and DAR2 gene polymorphism sites and chicken semen quality traits.

Contents of the invention

The object of the present invention is to provide a method for improving the quality of chicken semen, primers, a kit and a method of use thereof. The method of the present invention has remarkable effects, and the detection method is simple and quick, and is not affected by the external environment.

The invention provides a primer for genes closely related to chicken semen quality, including a mixture of upstream and downstream primers for the PCR amplification of the vasoactive intestinal peptide receptor 1 (VIPR1) gene, and a PCR amplification of the vasoactive intestinal peptide receptor 2 (VIPR2) gene. The upstream and downstream primer mixtures, the upstream and downstream primer mixtures of dopamine receptor 2 (DAR2) gene PCR amplification;

The upstream primer sequence in the upstream and downstream primer mixture of the vasoactive intestinal peptide receptor 1 (VIPR1) gene PCR amplification: 5'-gctcccgcagatatatggaa-3'; the downstream primer sequence: 5'-tgggaggagacaaaacaaca-3';

The upstream primer sequence of the upstream and downstream primer mixture of the vasoactive intestinal peptide receptor 2 (VIPR2) gene PCR amplification: 5'-gtgttcacttttgggcacct-3'; the downstream primer sequence: R:5'-cagccaaaaacattggtgtg-3';

The upstream and downstream primer mixture for PCR amplification of the dopamine receptor 2 (DAR2) gene, the upstream primer sequence: 5'-gttggggagtggaggttcag-3'; the downstream primer sequence: 5'-attgggctggagatggcaaa-3'.

The present invention also provides a method for improving the quality of chicken semen by using compound genotypes. The chicken genome DNA of the sample to be tested is extracted. The primers were used to amplify the fragment where the SNP site is located, and the target fragments of 192, 250 and 270 bp were amplified. The target fragments were first detected by PCR-SSCP, and then stained with silver staining after gel electrophoresis to obtain the SSCP maps of the DNA respectively. , select individuals with TT/GG/OQ trigene compound genotype according to band pattern selection in the 3 maps, that is, individuals with T, G, T and A/G alleles at 73352, 36127, 201 and 208, respectively .

Preferably, 20 μL of the reaction system for PCR amplifying the fragment where the SNP site is located: add 2.0 μL of upstream and downstream primer mixture, 3 μL of PCR reaction reagent, 1.0 μL of 50 ng/μL DNA template into the PCR reaction tube, add ultrapure water to 20 μL , mix well and centrifuge briefly.

Preferably, the reaction conditions of the PCR amplification are first denaturation at 94°C for 5 minutes; then 35 cycles, each cycle including 94°C for 30s, 60°C for 15s, and 72°C for 30s; then extending at 72°C for 10 minutes, and finally storing at 4°C .

Preferably, the PCR-SSCP detection includes: adding 2 μL of PCR amplification products to 7 μL of loading buffer, denaturing at 98° C. for 10 minutes, and then ice-bathing for 5 minutes; the polyacrylamide gel concentration of 3 pairs of primers is 10%, and the gel cross The coupling ratio was 29:1, the voltage was 150V, and electrophoresis was performed overnight.

In addition, the present invention also provides a chicken semen quality three-gene combined effect diagnostic kit, which includes a box body and a liner in the box body, and the vasoactive intestinal peptide receptor 1 VIPR1 gene is provided in the cavity of the liner The upstream and downstream primer mixture P for PCR amplification, the upstream and downstream primer mixture P for the PCR amplification of the vasoactive intestinal peptide receptor 2 VIPR2 gene, the upstream and downstream primer mixture P for the PCR amplification of the dopamine receptor 2 DAR2 gene, and PCR reaction reagents , ultrapure water, loading buffer; the PCR reaction reagent is a mixture of 10×buffer, dNTP and Taq enzyme.

The present invention further provides a method for using the chicken semen quality three-gene combined effect diagnostic kit, comprising the following steps:

(1) PCR amplification of the fragment where the SNP site is located: use a 20 μL reaction system: add 2.0 μL of upstream and downstream primer mixture, 3 μL of PCR reaction reagent, 1.0 μL of 50 ng/μL DNA template, and add ultrapure water to 20 μL in a PCR reaction tube. Centrifuge briefly after thorough mixing;

(2) Put the PCR reaction tube into the PCR instrument for reaction amplification: first denature at 94°C for 5 minutes; then go through 35 cycles, each cycle including 94°C for 30s, 60°C for 15s, and 72°C for 30s; then extend at 72°C for 10 minutes, Finally, store at 4°C.

(3) PCR-SSCP detection: 2 μL of PCR amplification product was added to 7 μL of loading buffer, denatured at 98° C. for 10 min, and then kept on ice for 5 min. The polyacrylamide gel concentration of the 3 pairs of primers is 10%, the gel cross-linking degree is 29:1, the voltage is 150V, and electrophoresis is carried out overnight;

(4) The target fragment was analyzed by SSCP, and after gel electrophoresis, it was developed by silver staining to obtain the SSCP map of the DNA. According to the band patterns in the three maps, the genotypes of VIPR1, VIPR2 and DAR2 were selected as TT/GG/OQ Individuals with a trigene compound genotype, ie individuals with T, G, T and A/G alleles at 73352, 36127, 201 and 208, respectively.

Compared with the prior art, the present invention has the following beneficial effects:

First, a pair of primers were designed for VIPR1, VIPR2 and DAR2 genes respectively. The amplified products of VIPR1 primers were analyzed by SSCP to generate three genotypes (CC, CT and TT). A C→T mutation occurred at bp; the amplified product of VIPR2 primer was analyzed by SSCP to produce three genotypes (AA, AG and GG), GG type and AA type had A→G mutation at 36127 bp; DAR2 primer The amplified product was analyzed by SSCP to produce 5 genotypes (OO, OP, OQ, PP and PQ). Compared with the OO type, the C→T mutation was detected at 201 in the QQ type, and 208 in the PP type compared with the OO type. A → G mutation. The correlation analysis of the different compound genotypes of the three genes and the high-quality broiler comb and semen quality showed that the combination of the three gene compound genotypes TT/GG/OQ genotypes had the best semen quality, and the semen volume was significantly higher than that of TT/AA/PP, CC/AA/OO, CC/AA/OP and CT/AA/OO individuals (P<0.05); sperm density was significantly higher than that of CT/AA/PP, CC/GG/OP, CC/AA/OO, CC/ GG/OO and CT/AA/OO type individuals (P<0.05); sperm motility was significantly better than CT/AA/OO and CC/AA/OP type individuals (P<0.05);

Second, the present invention provides a high-accuracy, rapid , Inexpensive genotyping kits. The kit of the invention comprises the upstream and downstream primer mixtures for PCR amplification of VIPR1, VIP2 and DAR2 genes, and reagents required for PCR. During breeding and screening, the chicken genome DNA of the sample to be tested was extracted, and the gene sequence was amplified by specific primers to obtain the target fragments of 192, 250 and 270 bp. The target fragments were analyzed by SSCP and developed by silver staining after gel electrophoresis. The SSCP map of DNA, according to the band pattern selection in the three maps, select individuals with TT/GG/OQ three-gene compound genotype. The method of the invention has remarkable effect, and the detection method is simple and quick, and is not affected by the external environment.

Description of drawings

Figure 1 is the SSCP map of the VIPR1 gene; in the figure 1, 2: CC type; 3, 4: CT type; 5, 6: TT type.

Figure 2 is a sequence diagram of the VIPR1 gene.

Figure 3 is the SSCP map of the VIPR2 gene; in the figure 2, 3, 5: AA type; 4, 6: GG type; 1: AG type.

Figure 4 is a sequence diagram of the VIPR-2 gene;

Figure 5 is the SSCP map of the DAR2 gene; in the figure 1: QQ type; 2: PP type; 3: PQ type; 4, 5: OQ type; 6, 8: OP type; 7: OO type.

Figure 6 is a sequence diagram of the DAR2 gene.

detailed description

A primer for genes closely related to chicken semen quality, including a mixture of upstream and downstream primers for PCR amplification of vasoactive intestinal peptide receptor 1 VIPR1 gene, a mixture of upstream and downstream primers for PCR amplification of vasoactive intestinal peptide receptor 2 VIPR2 gene, and dopamine receptor 2 DAR2 gene PCR amplification upstream and downstream primer mix.

In the upstream and downstream primer mixture for PCR amplification of vasoactive intestinal peptide receptor 1 VIPR1 gene, the upstream primer sequence: 5'-gctcccgcagatatatggaa-3'; the downstream primer sequence: 5'-tgggaggagacaaaacaaca-3'.

The upstream primer sequence of the upstream and downstream primer mixture for PCR amplification of vasoactive intestinal peptide receptor 2 VIPR2 gene: 5'-gtgttcacttttgggcacct-3'; the downstream primer sequence: R: 5'-cagccaaaaacattggtgtg-3'.

A mixture of upstream and downstream primers for PCR amplification of the dopamine receptor 2 DAR2 gene. The upstream primer sequence: 5'-gttggggagtggaggttcag-3'; the downstream primer sequence: 5'-attgggctggagatggcaaa-3'.

The method for improving the quality of chicken semen by using compound genotypes: extract the chicken genome DNA of the sample to be tested, and the chicken genome DNA gene sequence of the sample to be tested is subjected to PCR amplification of the SNP position with primers for genes closely related to the quality of chicken semen described in claim 1 Click on the fragments, and amplify the target fragments of 192, 250 and 270 bp. After the target fragments are detected by PCR-SSCP, after gel electrophoresis, they are developed by silver staining, and the SSCP maps of DNA are obtained respectively. According to the bands in the three maps Type selection selects individuals with TT/GG/OQ trigene compound genotype, that is, individuals with T, G, T and A/G alleles at 73352, 36127, 201 and 208, respectively.

20 μL reaction system for PCR amplifying the fragment where the SNP site is located: add 2.0 μL of upstream and downstream primer mixtures to the PCR reaction tube (each upstream and downstream primer mixture corresponds to a PCR reaction tube), 3 μL of PCR reaction reagent, 50 ng/μL DNA template 1.0 μL, add ultrapure water to 20 μL, mix thoroughly and centrifuge briefly.

The reaction conditions for PCR amplification were denaturation at 94°C for 5 minutes; then 35 cycles, each cycle including 94°C for 30s, 60°C for 15s, and 72°C for 30s; then extension at 72°C for 10 minutes, and finally storage at 4°C.

PCR-SSCP detection includes: add 2 μL of PCR amplification product to 7 μL of loading buffer, denature at 98°C for 10 minutes, and then ice-bath for 5 minutes; the polyacrylamide gel concentration of 3 pairs of primers is 10%, and the gel cross-linking degree is 29: 1. The voltage is 150V, electrophoresis overnight.

A chicken semen quality three-gene combined effect diagnostic kit, including box body 1, liner 2, VIPR1 primer mixture P3, VIPR2 primer mixture P4, VAR2 primer mixture P5, PCR reaction reagent 6: 10×buffer, dNTP and Mixture of Taq enzymes, ultrapure water7, loading buffer8.

The upstream and downstream primer mixture of VIPR1 gene PCR amplification, the upstream primer sequence 5'-gctcccgcagatatatggaa-3'; the downstream primer sequence: 5'-tgggaggagacaaaacaaca-3'.

A mixture of upstream and downstream primers for PCR amplification of VIPR2 gene, upstream primer sequence: 5'-gtgttcacttttgggcacct-3'; downstream primer sequence: R: 5'-cagccaaaaacattggtgtg-3'.

DAR2 gene PCR amplification upstream and downstream primer mixture, upstream primer sequence: 5'-gttggggagtggaggttcag-3'; downstream primer sequence: 5'-attgggctggagatggcaaa-3'.

Chicken semen quality closely related gene diagnosis kit, its operation method is as follows:

1. PCR amplification of the fragment where the SNP site is located: use 20 μL reaction system: add 2.0 μL of upstream and downstream primer mixture, 3 μL of PCR reaction reagent, 1.0 μL of 50 ng/μL DNA template in the PCR reaction tube, add ultrapure water to 20 μL, fully Mix and centrifuge briefly. Each upstream and downstream primer mix corresponds to one PCR reaction tube.

Put the PCR reaction tube into the PCR instrument for reaction amplification: first denature at 94°C for 5 minutes; then go through 35 cycles, each cycle includes 94°C for 30s, 60°C for 15s, 72°C for 30s; then extend at 72°C for 10 minutes, and finally 4°C save.

PCR-SSCP detection: Add 2 μL of PCR amplification product to 7 μL of loading buffer, denature at 98°C for 10 minutes, and then ice-bath for 5 minutes. The polyacrylamide gel concentration of the 3 pairs of primers was 10%, the gel cross-linking degree was 29:1, the voltage was 150V, and electrophoresis was carried out overnight.

The target fragment was analyzed by SSCP, and after gel electrophoresis, it was developed by silver staining to obtain the SSCP map of the DNA. According to the band patterns in the three maps, the genotypes of the VIPR1, VIPR2 and DAR2 genes were selected as TT/GG/OQ three-gene composite Individuals with genotypes, ie individuals with T, G, T and A/G alleles at 73352, 36127, 201 and 208, respectively.

Example 1

1. Three-gene SNP detection of VIPR-1, VIPR-2 and DAR2

The genotypes of three genes of 218 high-quality broiler chickens—Shaobo chickens were detected. Blood samples were collected from the Jiangsu Institute of Poultry Science, 1.5 mL of blood samples were collected from the wing vein, anticoagulated with sodium heparin, and stored at -20°C. Genomic DNA was extracted by phenol-chloroform extraction, dissolved in TE, and the concentration of genomic DNA was measured for later use.

According to the operation steps of this kit, the SSCP map of VIPR1 primers is shown in Figure 1, showing 3 genotypes (the band pattern indicates the formation of 3 genotypes, the two homozygous types are represented by CC and TT respectively, and the heterozygous type Denoted by CT. Sequencing showed that the TT type had a C→T mutation at 73352 bp compared with the CC type (Figure 2).

The SSCP map of VIPR2 primers is shown in Figure 3, showing three genotypes (the band pattern indicates the formation of three genotypes, the two homozygous types are represented by AA and GG, and the heterozygous type is represented by AG, and sequencing shows that the GG type A G→A mutation occurred at 36127bp (Fig. 4).

The SSCP map of DAR2 primers is shown in Figure 5, showing 6 genotypes (the band pattern indicates that 6 genotypes have been formed, and the three homozygous types are represented by OO, PP and QQ respectively, and the corresponding heterozygous types are represented by OP, OQ and PQ means that the alleles at 201 and 208bp of the OO type are C and A respectively, the PP type is C and A at the corresponding positions, and the QQ type is T and G at the corresponding positions, respectively, which are defined as OO , OP, OQ, PP, PQ, and QQ genotypes. The PCR product fragments of OO, PP, and QQ genotypes were sequenced. The results found that compared with OO, QQ had a C→T mutation at 201 places, and A at 208 places. → G mutation (Fig. 6).

2. Analysis of the effect of composite genotypes of VIPR1, VIPR2 and DAR2 genes on comb and semen quality of high-quality broilers

Cooperate with the following model to analyze the effect of VIPR-1, VIPR-2 and DAR2 compound genotypes on high-quality broiler comb and semen quality: Y=μ+Genotype effect of VIPR1 gene+Genotype effect of VIPR2 gene+Genotype effect of DAR2 gene + residual effects. The comparisons of comb and semen quality between compound genotype TT/GG/OQ and different compound genotypes of high-quality broilers are shown in Table 1.

Table 1 Comparison of semen quality between compound genotype TT/GG/OQ and different compound genotypes of high-quality broilers

composite genotype Density (100 million/ml) Semen volume (μl) vitality Deformation rate% TT/GG/OP 35.98±1.02 ^a 621±22 ^a 8.33±0.13 ^a 4.01±0.33 CC/GG/OQ 35.44±1.31 ^a 582± ^25ab 8.31±0.18 ^a 4.21±0.35 TT/AA/PP 35.12±1.23 ^a 551± ^21b 8.12± ^0.15ab 4.12±0.28 CT/AA/PP 34.62± ^1.52b 579± ^24ab 8.30±0.16 ^a 4.20±0.31 CT/GG/OP 35.02± ^1.21ab 584± ^23ab 8.24±0.23 ^a 4.13±0.36 CC/GG/OP 34.48± ^1.22b 587± ^25ab 8.16± ^0.12ab 4.10±0.18 CC/AA/OO 34.07± ^1.23b 536± ^42b 7.50± ^0.15b 4.20±0.36 CT/AA/OO 34.54± ^1.28b 554± ^25b 7.86± ^0.19b 4.44±0.68 CT/AA/OP 35.04± ^1.36ab 583± ^34ab 8.11± ^0.18ab 4.18±0.47 CC/AG/OO 35.54±1.29 ^a 587± ^28ab 8.11± ^0.31ab 4.18±0.39 TT/GG/OO 35.68± ^1.47ab 608± ^16ab 8.11± ^0.07ab 4.02±0.62 CC/GG/OO 34.59± ^1.39b 575± ^31ab 8.63±0.11 ^a 4.20±0.58 TT/AA/OO 34.81± ^1.42ab 581± ^28ab 8.37±0.23 ^a 4.21±0.39 TT/AG/OP 35.59±1.18 ^a 606± ^37a 8.11± ^0.26ab 4.18±0.47 CC/AA/OP 34.24± ^1.37b 568± ^36b 7.99± ^0.29b 4.24±0.52 TT/AA/OP 35.68±1.31 ^a 603±25 ^a 8.31±0.18 ^a 4.22±0.35 CT/GG/OO 35.12± ^1.23ab 572± ^21ab 8.12± ^0.15ab 4.02±0.28

Note: Different lowercase letters on the shoulders of peer data indicate significant differences (P<0.05).

It can be seen from the table that the semen quality of individuals with the three-gene compound genotype TT/GG/OQ is the best, and the semen volume is 15.9% higher than that of individuals with CC/AA/OO genotype (P<0.05), and that of individuals with genotype CT/AA/OO 12.1% more (P<0.05); the sperm density of TT/GG/OQ individuals was 5.6% more than CC/AA/OO individuals (P<0.05), and 5.1% more than CC/AA/OP individuals (P<0.05); Sperm motility was significantly different between TT/GG/OP individuals and CC/AA/OO, CT/AA/OO, CC/AA/OP individuals (P<0.05); although there was no significant difference in sperm deformity rate between genotype individuals, However, the deformity rate of TT/GG/OQ individuals was lower than that of other genotypes. Screening the compound genotype TT/GG/OQ in the population can improve the quality of high-quality chicken semen, so the screening of the TT/GG/OQ three-gene compound genotype according to the SSCP map of the VIPR1, VIPR2 and DAR2 genes can be used as an early method for high-quality broiler combs and semen quality Methods.

<110> Jiangsu Institute of Poultry Science

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Claims (7)

1. Primers for genes closely related to chicken semen quality, including upstream and downstream primer mixtures for PCR amplification of vasoactive intestinal peptide receptor 1 VIPR1 gene, upstream and downstream primer mixtures for PCR amplification of vasoactive intestinal peptide receptor 2 VIPR2 gene, and dopamine receptor 2 DAR2 gene PCR amplification upstream and downstream primer mix; In the upstream and downstream primer mixture of the vasoactive intestinal peptide receptor 1 VIPR1 gene PCR amplification, the upstream primer sequence: 5'-gctcccgcagatatatggaa-3'; the downstream primer sequence: 5'-tgggaggagacaaaacaaca-3'; The upstream primer sequence of the upstream and downstream primer mixture of the vasoactive intestinal peptide receptor 2 VIPR2 gene PCR amplification: 5'-gtgttcacttttgggcacct-3'; downstream primer sequence : R:5'-cagccaaaaacattggtgtg-3'; The upstream and downstream primer mixture of the PCR amplification of the dopamine receptor 2DAR2 gene, the upstream primer sequence: 5'-gttggggagtggaggttcag-3'; the downstream primer sequence : 5'-attgggctggagatggcaaa-3'. 2. Utilize the method for compound genotype to improve chicken semen quality, it is characterized in that, extract the chicken genome DNA of sample to be tested, the chicken genome DNA gene sequence of sample to be tested passes through chicken semen quality described in claim 1 closely related gene with primer The fragments where the SNP sites are located were amplified by PCR, and the target fragments of 192, 250 and 270 bp were amplified. After the target fragments were detected by PCR-SSCP, they were developed by silver staining after gel electrophoresis, and the SSCP maps of the DNA were obtained respectively. Individuals with TT/GG/OQ trigene compound genotype were selected for the band patterns in the three maps, that is, individuals with T, G, T and A/G alleles at 73352, 36127, 201 and 208, respectively. 3. the method utilizing composite genotype to improve chicken semen quality according to claim 2, is characterized in that, the reaction system 20 of described PCR amplification SNP site fragment μL: Add the upstream and downstream primer mixture for PCR amplification of vasoactive intestinal peptide receptor 1 VIPR1 gene or the upstream and downstream primer mixture for PCR amplification of vasoactive intestinal peptide receptor 2 VIPR2 gene or the PCR amplification of dopamine receptor 2 DAR2 gene in the PCR reaction tube Added upstream and downstream primer mix 2.0 µL, PCR reaction reagent 3 µL, 50 ng/µL DNA template 1.0 µL, add ultrapure water to 20 µL, mix well and centrifuge briefly. 4. the method utilizing compound genotype to improve chicken semen quality according to claim 2, is characterized in that, the reaction condition of described PCR amplification is first 94 ℃ denaturation 5min; Through 35 cycles again, each cycle comprises 94 30s at ℃, 15s at 60℃, 30s at 72℃; then extend at 72℃ for 10 minutes, and finally store at 4℃. 5. The method for improving chicken semen quality by compound genotype according to claim 2, characterized in that, the PCR-SSCP detection comprises: 2 μL PCR amplification product is added to 7 μL The loading buffer was denatured at 98°C for 10 min, and then kept in ice bath for 5 min; the polyacrylamide gel concentration of the 3 pairs of primers was 10%, the gel cross-linking degree was 29:1, and the voltage was 150V, and electrophoresis was performed overnight. 6. A chicken semen quality three-gene combined effect diagnostic kit, comprising a box body (1) and a liner (2) in the box body, characterized in that the cavity of the liner (2) is provided with the claim 1 The upstream and downstream primer mixture P (3) for the PCR amplification of the vasoactive intestinal peptide receptor 1 VIPR1 gene, the upstream and downstream primer mixture P (4) for the PCR amplification of the vasoactive intestinal peptide receptor 2 VIPR2 gene, the The above-mentioned dopamine receptor 2 DAR2 gene PCR amplification upstream and downstream primer mixture P (5), PCR reaction reagent (6), ultrapure water (7), loading buffer (8); the PCR reaction reagent (6) It is a mixture of 10×buffer, dNTP and Taq enzyme. 7. The method for using the chicken semen quality three-gene combined effect diagnostic kit is characterized in that it comprises the following steps: (1) PCR amplification of the fragment where the SNP site is located: use 20 μL reaction system: add the upstream and downstream primer mixture for PCR amplification of vasoactive intestinal peptide receptor 1 VIPR1 gene or PCR of vasoactive intestinal peptide receptor 2 VIPR2 gene in the PCR reaction tube Amplified upstream and downstream primer mixture or dopamine receptor 2 DAR2 gene PCR amplification upstream and downstream primer mixture 2.0 µL, PCR reaction reagent 3 µL, 50 ng/µL DNA template 1.0 µL, add ultrapure water to 20 µL, mix well brief centrifugation; the PCR reaction reagent (6) is a mixture of 10×buffer, dNTP and Taq enzyme; (2) Put the PCR reaction tube into the PCR instrument for reaction amplification: denature at 94°C for 5 minutes; then go through 35 cycles, each cycle including 94°C for 30s, 60°C for 15s, and 72°C for 30s; then extend at 72°C for 10 minutes , and finally stored at 4°C; (3) PCR-SSCP detection: Add 2 μL PCR amplification product to 7 μL Loading buffer, denatured at 98°C for 10 min, then ice-bathed for 5 min; the polyacrylamide gel concentration of 3 pairs of primers was 10%, the gel cross-linking degree was 29:1, the voltage was 150V, and electrophoresis was carried out overnight; (4) The target fragment was analyzed by SSCP, and after gel electrophoresis, it was developed by silver staining to obtain the SSCP map of the DNA. According to the band patterns in the three maps, the genotypes of VIPR1, VIPR2 and DAR2 were selected as TT/GG/OQ Individuals with a trigene compound genotype, ie individuals with T, G, T and A/G alleles at 73352, 36127, 201 and 208, respectively.