Primer, kit and method for early pregnancy diagnosis of sows and application

Abstract

The invention discloses a primer for diagnosis of early pregnancy of sows. The nucleotide sequence of the primer is shown as follows: SRY-O-F: 5 '-CAGCAAAATATTCTCGCCTTGG-3', and the nucleotide sequence of the primer is shown as follows. And SRY-O-R: 5 '-CATCCTCTCTCTACGC-3', and SRY-O-R: 5 '- SRY-I-F: 5 '-ATATTCTCGCCTTGGGG-3', SRY-I-F: 5 '- And SRY-I-R is 5 '-GCTTTCGGCTTCTGTA-3', and the formula is shown in the description. Two pairs of primers with high sensitivity and strong specificity are designed through comparative analysis of pig SRY gene sequences, fetal free DNA in peripheral blood of sows is used as a template, and the specificity and sensitivity of PCR are enhanced through amplification of the two pairs of primers. Through two rounds of PCR amplification, whether the sow is pregnant or not can be detected on the 18th day of pregnancy, and the accuracy of the detection result is extremely high and reaches 100%. Compared with the prior art that the ZFY gene is used for detecting the sows on the twentieth breeding day, the method has the advantages that the detection time can be advanced to the eighteenth breeding day, and the detection accuracy is greatly improved (100% vs 10%). According to the method, powerful technical support is provided for early and accurate identification of the non-pregnant sows, reduction of non-production days and feeding cost and smooth execution of a production plan.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology, in particular to a primer, kit and method for early pregnancy diagnosis of sows and application. BACKGROUND

[0002] Pigs belong to polytocous animals, and the ratio of male and female fetuses per pregnancy is close to 1:1 without human intervention, and studies have shown that fetal DNA can enter the maternal blood through the placenta. Therefore, if the Y chromosome specific gene of the male fetus can be detected in the peripheral blood of the sow, it can be determined that the sow is pregnant. Based on the above reasons, the Y chromosome specific gene can be detected by PCR in the peripheral blood of the sow to determine whether the sow is pregnant. In the patent CN115961058A, a method for determining whether a sow is pregnant by detecting the expression of ZFY gene in the peripheral blood of the sow is disclosed, but the accuracy of the patent is higher for sows mated for 20 days, and the detection accuracy is not high for sows at an earlier stage. Therefore, it is urgent to find a new primer, kit and method for early pregnancy diagnosis of sows with higher detection accuracy and earlier detection time. SUMMARY

[0003] The purpose of the present application is to provide a primer, kit and method for early pregnancy diagnosis of sows and application, so as to diagnose whether the sow is pregnant at an early mating stage and improve the detection accuracy.

[0004] According to the first aspect of the present application, a primer for early pregnancy diagnosis of sows is provided, and the nucleotide sequence of the primer is as follows: SRY-O-F: 5'-CAGCAGCAAAATATTCTCGCCTTGG-3'; SRY-O-R: 5'-CATCCTCTCCTCTACGC-3'; SRY-I-F: 5'-ATATTCTCGCCTTGGGG-3'; SRY-I-R: 5'-GCTTTTCGGCTTCTGTA-3'. Thus, through the alignment analysis of the pig SRY gene sequence, two pairs of primers with high sensitivity and strong specificity are designed, and the primers can be used to detect whether the sow is pregnant at the 18th day of mating, and the accuracy is as high as 100%. The primer can accurately identify barren sows earlier, reduce the non-production days of sows and feeding costs, and provide strong technical support for the smooth implementation of production plans.

[0005] According to a second aspect of the present application, a kit containing the primers is provided, the nucleotide sequences of the primers are as follows: SRY-O-F: 5'-CAGCAGCAAAATATTCTCGCCTTGG-3'; SRY-O-R: 5'-CATCCTCTCCTCTACGC-3'; SRY-I-F: 5'-ATATTCTCGCCTTGGGG-3'; SRY-I-R: 5'-GCTTTTCGGCTTCTGTA-3'. Thus, the kit can be used to detect whether a sow is pregnant on the 18th day of mating, and the accuracy is as high as 100%. The kit can more accurately identify barren sows, reduce the non-production days of sows and feeding costs, and provide strong technical support for the smooth execution of production plans.

[0006] According to a third aspect of the present application, the primers or the kit are used for non-therapeutic purposes in early pregnancy diagnosis of sows. Thus, through the application, it can be detected whether a sow is pregnant on the 18th day of mating, and the accuracy is as high as 100%.

[0007] According to a fourth aspect of the present application, a non-therapeutic method for early pregnancy diagnosis of sows is provided, the method comprising the following steps: S1: collecting peripheral blood samples of sows mated for 18 days, and extracting free DNA in the peripheral blood samples of sows; S2: synthesizing the following primers: SRY-O-F: 5'-CAGCAGCAAAATATTCTCGCCTTGG-3'; SRY-O-R: 5'-CATCCTCTCCTCTACGC-3'; SRY-I-F: 5'-ATATTCTCGCCTTGGGG-3'; SRY-I-R: 5'-GCTTTTCGGCTTCTGTA-3'; S3: using the DNA extracted in step S1 as a template, and performing first round PCR amplification with outer primers SRY-O-F and SRY-O-R; S4: using the first round PCR amplification product as a template, and performing second round amplification with inner primers SRY-I-F and SRY-I-R; S5: performing electrophoresis detection on the second round product, and if a 249bp electrophoresis band appears, it is judged that the sow is pregnant, and if there is no 249bp electrophoresis band, it is judged that the sow is not pregnant. The method is simple to operate and non-invasive to sows. The detection method has high sensitivity, good specificity, and high accuracy. Through the method, it can be detected whether a sow is successfully pregnant on the 18th day of mating, and the detection accuracy is as high as 100%. The method can more accurately identify barren sows, reduce the non-production days of sows and feeding costs, and provide strong technical support for the smooth execution of production plans.

[0008] In some embodiments, the reaction system of the first round of PCR amplification in step S3 is 2 × Green Taq Mix 10 μL, ddH2O 7 μL, 10 μmol / L of primers SRY-O-F and SRY-O-R each 0.5 μL, and template DNA 2 μL.

[0009] In some embodiments, the reaction procedure of the first round of PCR amplification in step S3 comprises: 95°C pre-denaturation for 5 min; 95°C denaturation for 15 s, 60°C annealing for 15 s, 72°C extension for 15 s, repeating for 35 cycles; 72°C extension for 10 min; and 4°C storage for 2 h.

[0010] In some embodiments, the reaction system of the second round of PCR amplification in step S4 is 2 × Green Taq Mix 10 μL, ddH2O 6 μL, 10 μmol / L of primers SRY-I-F and SRY-I-R each 1 μL, and template DNA 2 μL.

[0011] In some embodiments, the reaction procedure of the second round of PCR amplification in step S4 is 95°C pre-denaturation for 5 min; 95°C denaturation for 15 s, 60°C annealing for 15 s, 72°C extension for 15 s, repeating for 35 cycles; 72°C extension for 10 min; and 4°C storage for 2 h.

[0012] In some embodiments, in step S5, if both 417 bp and 249 bp electrophoresis bands or only 249 bp electrophoresis band appear in the electrophoresis detection, it is judged that the sow is pregnant, and if there is no 249 bp electrophoresis band, it is judged that the sow is not pregnant.

[0013] According to the fifth aspect of the present application, the above method is provided for non-therapeutic purposes in the early pregnancy diagnosis of sows. Thus, the application of the method can detect whether the sow is successfully pregnant on the 18th day of mating, and the detection accuracy is as high as 100%. The method can more accurately identify barren sows, reduce the non-production days of sows and feeding costs, and provide strong technical support for the smooth implementation of production plans.

[0014] The application has the following beneficial effects: through sequence alignment analysis of the pig SRY gene, two pairs of primers with high sensitivity and specificity are designed, and the specificity and sensitivity of PCR are improved by using the fetal free DNA in the peripheral blood of sows as a template and amplifying the two pairs of primers. Through two rounds of PCR amplification, it can be detected whether the sow is pregnant on the 18th day of mating, and the accuracy of the detection result is extremely high, reaching 100%. Compared with the prior art of using ZFY gene to detect sows on the 20th day of mating, the application not only advances the detection time to the 18th day of mating, but also greatly improves the accuracy of detection (100% vs. 10%). The application provides strong technical support for earlier and more accurate identification of barren sows, reduction of non-production days and feeding costs, and smooth implementation of production plans. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Figure 1 is a first round of PCR amplification detection result chart of the first to third number of mating sows to be tested and non-mating sows and boars, wherein "1-3" represents the first to third number of mating sows to be tested, "female" represents non-mating sows, "male" represents boars, and "M" represents Marker; Figure 2 Figure 2 is a first round of PCR amplification detection result chart of the fourth to tenth number of mating sows to be tested and non-mating sows and boars, wherein "4-10" represents the fourth to tenth number of mating sows to be tested, "female" represents non-mating sows, "male" represents boars, and "M" represents Marker; Figure 3 Figure 3 is a first round of PCR amplification detection result chart of the eleventh to twentieth number of mating sows to be tested and non-mating sows and boars, wherein "11-20" represents the eleventh to twentieth number of mating sows to be tested, "female" represents non-mating sows, "male" represents boars, and "M" represents Marker; Figure 4 Figure 4 is a second round of PCR amplification detection result chart of the first to third number of mating sows to be tested and non-mating sows and boars, wherein "1-3" represents the first to third number of mating sows to be tested, "female" represents non-mating sows, "male" represents boars, "M" represents Marker, and "GAPDH" represents the detection result of the internal reference gene GAPDH; Figure 5 Figure 5 is a second round of PCR amplification detection result chart of the fourth to tenth number of mating sows to be tested and non-mating sows and boars, wherein "4-10" represents the fourth to tenth number of mating sows to be tested, "female" represents non-mating sows, "male" represents boars, "M" represents Marker, and "GAPDH" represents the detection result of the internal reference gene GAPDH; Figure 6The first and second round PCR amplification detection result chart of the 1-10 number of test mating sows and unmated sows and boars using ZFY gene for detection: wherein, "1-10" represents the 1-10 number of test mating sows, "sow" represents the unmated sow, "male" represents the boar, "M" represents Marker, and "GAPDH" represents the detection result of the internal reference gene GAPDH. Figure 7 The first and second round PCR amplification detection result chart of the 1-10 number of test mating sows and unmated sows and boars using ZFY gene for detection: wherein, "1-10" represents the 1-10 number of test mating sows, "sow" represents the unmated sow, "male" represents the boar, "M" represents Marker, and "GAPDH" represents the detection result of the internal reference gene GAPDH. DETAILED DESCRIPTION

[0016] The present application will be described in detail below with specific examples. The following examples will help those skilled in the art to further understand the present application, but do not limit the present application in any form. It should be noted that for those skilled in the art, without departing from the concept of the present application, a number of changes and improvements can be made, which are within the scope of protection of the present application. Chemical substances, proteins, enzymes or kits used in the present application, if not otherwise specified, can be obtained commercially.

[0017] Example 1, nested PCR specific primer design.

[0018] Pigs belong to polytocous animals, and the ratio of male and female fetuses per litter is close to 1:1 without human intervention, and studies have shown that fetal DNA can enter the maternal blood through the placenta. Therefore, if the male fetus Y chromosome specific gene can be detected in the peripheral blood of sows, it can be determined that the sow has been pregnant. SRY gene is a key gene for male sex determination located in Yp11.3 of Y chromosome short arm, which activates the testis formation pathway in early embryonic development. Therefore, the expression of SRY gene in the peripheral blood of sows can be detected to determine whether the sow is pregnant.

[0019] According to the pig SRY gene sequence (NM_214452.3) published in the NCBI database, two pairs of specific detection primers were designed for the gene using Primer 6.0 software, and the nucleotide sequences are shown in Table 1 (SEQ ID No: 1-SEQ ID No: 4): Table 1 Nested PCR detection primer sequence list

[0020] Example 2, early pregnancy diagnosis method of sows based on nested PCR detection of SRY gene.

[0021] 2.1 Collecting peripheral blood samples of sows, extracting free DNA in peripheral blood samples of sows.

[0022] Select 20 sows at 18 days after mating (the mating day is recorded as day 0), 1 sow for each of non-mated sows (negative control) and boars (positive control), and detect whether each of the sows is pregnant after mating by B-ultrasound and record the results. Collect the blood of the sows from the anterior vena cava into an anticoagulant blood collection tube, centrifuge at 4℃ 3000 rpm / min for 10 min, carefully remove the upper plasma into a centrifuge tube, take 1.5 mL to extract free DNA of the sample by a serum / plasma free DNA extraction kit, and determine the concentration for standby.

[0023] 2.2 The outer primers in the nested PCR specific primers in Table 1 are used for first round amplification.

[0024] For the SRY gene, the outer primers SRY-O-F and SRY-O-R in Table 1 are used for first round amplification. The PCR reaction system includes: 2 × Green Taq Mix (purchased from Novozyme) 10 μL, ddH2O 7 μL, 10 μmol / L of the upstream primer SRY-O-F and the downstream primer SRY-O-R each 0.5 μL, template DNA 2 μL; the PCR reaction program includes: 95℃ pre-denaturation for 5 min, (95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 15 s) × 35 cycles, 72℃ extension for 10 min, 4℃ storage for 2 h. The PCR product of the first round amplification is detected by 1.5% agarose gel electrophoresis, and the electrophoresis result is shown in Table 1: Figures 1-3 Figure 1 The detection results of the 1-3 sows to be tested (indicated as “1-3” in the figure), the non-mated sows (indicated as “sow” in the figure) and the boars (indicated as “boar” in the figure) are shown in Table 1, and it can be seen that the boar can clearly see the 417 bp PCR product band, and the 1-3 sows to be tested can see the 417 bp PCR product band, and the non-mated sows cannot detect the 417 bp PCR product band; Figure 2 The detection results of the 4-10 sows to be tested (indicated as “4-10” in the figure), the non-mated sows (indicated as “sow” in the figure) and the boars (indicated as “boar” in the figure) are shown in Table 1, and it can be seen that the boar can clearly see the 417 bp PCR product band, and the 4-9 sows to be tested can see the 417 bp PCR product band, and the 10 sows to be tested cannot see the 417 bp PCR product band, and the non-mated sows cannot detect the 417 bp target band; Figure 3 ​The results of the detection of the 11-20 number of sows to be mated (indicated as "11-20" in the figure) and non-mated sows (indicated as "female" in the figure) and boars (indicated as "male" in the figure) can be seen that the boar can be clearly seen the 417bp size of the PCR product band, and the 11-20 number of sows to be mated can be seen the 417bp size of the PCR product band, and the non-mated sows have not detected the 417bp size of the target band.

[0025] For the first round of detection results, whether there is a target band (417bp size) or not, the second round of amplification is carried out. Since the ratio of fetal cells to maternal cells in maternal blood is 1:20,000-50,000, the content of fetal free DNA in the peripheral blood of pregnant sows is very low, which does not reach the minimum template amount required for PCR amplification, resulting in that the first round of amplification usually cannot show a band or a barely visible band. Although there is no target band in the first amplification, the copy number of the SRY gene target fragment (417bp) in the product is greatly increased, which provides a high concentration of templates for the second amplification and improves the specificity of the reaction.

[0026] 2.3 The second round of amplification is carried out using the inner primer in the nested PCR specific primer in Table 1.

[0027] 2μL of the first round of amplification PCR product is taken as a template, and the inner primer SRY-I-F, SRY-I-R in Table 1 is used for the second round of amplification. The PCR reaction system includes: 2 × Green Taq Mix (purchased from Novozyme) 10μL, ddH2O 6μL, 10μmol / L of upstream primer SRY-I-F and downstream primer SRY-I-R each 1μL, template DNA 2μL; the PCR reaction program includes: 95℃ pre-denaturation for 5min, (95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 15s) × 35 cycles, 72℃ extension for 10min, 4℃ storage for 2h.

[0028] 2.4 The PCR product of the second round of amplification is detected to determine whether the sow is pregnant.

[0029] The second round of amplification PCR product is detected by 1.5% agarose gel electrophoresis, and the results of the nested PCR amplification of pig SRY gene are shown in Figures 4-6 Figure 4 ​The detection results of the first to third test mating sows (indicated as "1-3" in the figure), non-mating sows (indicated as "female" in the figure) and boars (indicated as "male" in the figure) are shown in the figure. It can be seen that the first round (417bp) and second round (249bp) PCR product bands of the boar can be clearly seen. The first round product (417bp) and second round product (249bp) bands of the first to third test mating sows are clearly visible, while the first round and second round product bands of the non-mating sows are not visible. The GAPDH band of the internal reference gene is clearly visible. Figure 5 The detection results of the fourth to tenth test mating sows (indicated as "4-10" in the figure), non-mating sows (indicated as "female" in the figure) and boars (indicated as "male" in the figure) are shown in the figure. It can be seen that the first round (417bp) and second round (249bp) PCR product bands of the boar can be clearly seen. The first round product (417bp) and second round product (249bp) bands of the fourth to ninth test mating sows are clearly visible, while the first round and second round product bands of the tenth test mating sow and non-mating sow are not visible. The GAPDH band of the internal reference gene is clearly visible. Figure 6 The detection results of the eleventh to twentieth test mating sows (indicated as "11-20" in the figure), non-mating sows (indicated as "female" in the figure) and boars (indicated as "male" in the figure) are shown in the figure. It can be seen that the first round (417bp) and second round (249bp) PCR product bands of the boar can be clearly seen. The first round product (417bp) and second round product (249bp) bands of the eleventh to twentieth test mating sows are clearly visible, while the first round and second round product bands of the non-mating sow are not visible. The GAPDH band of the internal reference gene is clearly visible.

[0030] According to the above results, the internal reference GAPDH of the 1st to 20th to-be-tested mating sows, boars and unmated sows has a band, indicating that the DNA extraction is successful; the unmated sows have no SRY band in the first and second rounds of nested amplification, and the electrophoresis has no impurity band, indicating that the detection primer and detection method have good specificity; the 1st to 9th and 11th to 20th to-be-tested mating sows have SRY bands in the first and second rounds of nested amplification, of which the detection band after the first round of amplification is barely visible, and the detection band after the second round of amplification is clear and bright, indicating that the 19 sows (1st to 9th and 11th to 20th) are all pregnant; the 10th to-be-tested mating sow has no SRY band in the first and second rounds of amplification, indicating that the sow is not pregnant; the boar as a positive control well verifies the position of the SRY band in the first and second rounds of PCR electrophoresis. The bands of the 1st to 9th and 11th to 20th to-be-tested mating sows are consistent with those of the boar, indicating that the detection result is accurate. The 1st to 9th and 11th to 20th to-be-tested mating sows were detected by B-ultrasound at 28 days after mating, and the results showed that they were all pregnant, while the 10th to-be-tested mating sow was detected by B-ultrasound at 28 days after mating, and the result showed that it was not pregnant. The nested PCR detection result in the present application is completely consistent with the B-ultrasound examination result, indicating that the accuracy rate of the sow early diagnosis method in the present application is 100%.

[0031] Example 3, comparison of sow early pregnancy diagnosis method of ZFY and SRY genes.

[0032] According to the detection primer sequence of ZFY gene disclosed in CN115961058A patent: ZFY-O-F: 5'-CACAAGACTTACCTTTCAGCG-3'(SEQ ID No: 5); ZFY-O-R: 5'-CCTGACCAAGGTGCCTATTC-3'(SEQ ID No: 6); ZFY-I-F: 5'-GGGCTCCCAAATCCTCA-3'(SEQ ID No: 7); ZFY-I-R: 5'-GTGGGCGAGAGGAAAAGA-3'(SEQ ID No: 8).

[0033] According to the above primer synthesis sequence, and according to the method disclosed in CN115961058A patent, the results are as follows: Figure 7As shown in the middle: 10 sows (randomly selected from 1~20 sows in Example 2) were selected, and the plasma free DNA of the sows was detected by PCR 18 days after mating, 1 boar genomic DNA, 1 non-mated sow free DNA, the results showed that GAPDH was normally amplified, only No. 7 sow had a faint band in the first round of product, and the band was more mixed, and only No. 7 sow band was obvious after the second round of amplification, which could determine that No. 7 sow was pregnant, indicating that the final detection accuracy of ZFY primer for pregnancy was 1 / 10 (10%). And the 10 sows selected in this example were randomly selected from 1~20 sows in Example 2 (all the 10 sows selected for testing were confirmed to be pregnant sows), and the pregnancy accuracy was 100% when the SRY gene primer was used for detection in Example 2, indicating that the SRY gene detection accuracy was higher than that of ZFY gene (100% vs 10%). Moreover, the results also verified that the ZFY gene mentioned in CN115961058A patent is suitable for detection at 20 days after mating, and the detection effect is not good at 18 days.

[0034] Although the DNA of the fetus enters the maternal blood through the placenta, the fetal free DNA contained in the maternal peripheral blood is less, and the sensitivity of ordinary PCR detection is very low, which is easy to cause false negative. Nested PCR is a variant of polymerase chain reaction, which uses two pairs of primers for PCR amplification, which can greatly improve the sensitivity and specificity of PCR. Therefore, in view of the technical bottleneck that the prior art lacks a pregnancy diagnosis technology within 21 days after mating, and cannot shorten the non-production days of sows and increase the annual production of fetal times through early pregnancy diagnosis, based on the fact that fetal free DNA exists in the peripheral blood of pregnant sows and the male and female fetus ratio of each litter is close to 1:1, a universal nested PCR specific primer is proposed for detecting whether the SRY gene specific to male fetus exists in the peripheral blood of pregnant sows, so as to realize early pregnancy diagnosis.

[0035] Through the sequence alignment analysis of pig SRY gene, two pairs of primers with high sensitivity and specificity (sequences in Table 1) are designed, and the free DNA in the peripheral blood of sows is used as a template, and the two pairs of primers increase the specificity and sensitivity of PCR. Through two rounds of amplification, the detection time can be advanced to 18 days after mating, and the detection result accuracy is very high (100%), which can more accurately identify empty sows, reduce non-production days and feeding costs, and provide strong technical support for the smooth implementation of production plan. The detection primer and detection method are non-invasive and efficient, and are convenient for large-scale use, which can greatly improve the detection efficiency of early pregnancy of sows.

Claims

1. A primer for early pregnancy diagnosis in sows, wherein, The nucleotide sequence of the primer is shown below: SRY-OF: 5'-CAGCAGCAAAATATTCTCGCCTTGG-3'; SRY-OR: 5'-CATCCTCTCCTCTACGC-3'; SRY-IF: 5'-ATATTCTCGCCTTGGGG-3'; SRY-IR: 5'-GCTTTTTCGGCTTCTGTA-3'.

2. A kit containing the primers described in claim 1.

3. The use of the primers or kits described in claim 1 or 2 for non-therapeutic purposes in the diagnosis of early pregnancy in sows.

4. A non-treatment method for diagnosing early pregnancy in sows, wherein, The method includes the following steps: S1: Collect peripheral blood samples from sows 18 days after mating and extract cell-free DNA from the peripheral blood samples; S2: Synthesize the primers described in claim 1; S3: Using the DNA extracted in step S1 as a template, the first round of PCR amplification was performed using the outer primers SRY-OF and SRY-OR; S4: Using the first round of PCR amplification products as templates, perform the second round of amplification using the inner primers SRY-IF and SRY-IR; S5: Perform electrophoretic detection on the second batch of products. If a 249bp electrophoretic band appears, the sow is considered pregnant. If no 249bp electrophoretic band appears, the sow is considered not pregnant.

5. The method according to claim 4, wherein, The reaction system for the first round of PCR amplification in step S3 consists of 10 μL of 2×Green Taq Mix, 7 μL of ddH2O, 0.5 μL each of 10 μmol / L primers SRY-OF and SRY-OR, ​​and 2 μL of template DNA.

6. The method according to claim 5, wherein, The reaction procedure for the first round of PCR amplification in step S3 includes: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 15 s, annealing at 60℃ for 15 s, extension at 72℃ for 15 s, repeated for 35 cycles; extension at 72℃ for 10 min; storage at 4℃ for 2 h.

7. The method according to claim 4, wherein, The reaction system for the second round of PCR amplification in step S4 consists of 10 μL of 2×Green Taq Mix, 6 μL of ddH2O, 1 μL each of 10 μmol / L primers SRY-IF and SRY-IR, and 2 μL of template DNA.

8. The method according to claim 7, wherein, The reaction program for the second round of PCR amplification in step S4 is as follows: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 15 s, annealing at 60℃ for 15 s, extension at 72℃ for 15 s, repeated for 35 cycles; extension at 72℃ for 10 min; storage at 4℃ for 2 h.

9. The method according to claim 4, wherein, In step S5, if both 417bp and 249bp electrophoretic bands appear in the electrophoresis test, or only a 249bp electrophoretic band appears, the sow can be determined to be pregnant. If there is no 249bp electrophoretic band, the sow is determined not to be pregnant.

10. The application of the method according to any one of claims 4-9 for non-therapeutic purposes in the diagnosis of early pregnancy in sows.

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