CN110669847A - Detection method and kit for animal sexual control sperm purity and application - Google Patents
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Abstract
The invention relates to the field of biological detection, and particularly provides a detection method and a kit for the purity of animal sexual control semen and application. The invention provides a method for detecting the purity of animal sexual control semen, which respectively carries out real-time PCR detection on X sperm and Y sperm in a standard substance containing X sperm and Y sperm with different known concentration ratios, prepares a standard curve by taking Ct values respectively obtained by the X sperm and the Y sperm as variable factors and the concentration ratios of the X sperm and the Y sperm, and then prepares an average Ct value of the X sperm and the Y sperm in a sample to be detected(i)And comparing with a standard curve to obtain the concentration ratio of the X sperm to the Y sperm in the sample to be detected, thereby obtaining the purity of the sample to be detected. The detection method solves the problem that the detection method in the prior art has different amplification efficiencies of different target genes and different primer sequencesThe problem that the system error exists between the measured result and the actual sample purity is solved, so that the detection result is more accurate.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a detection method, a kit and application of animal sex control semen purity.
Background
Animal sex directly influences the economic benefits of livestock production. Sex control is a biological technique for female animals to produce offspring of a desired sex by human intervention in the normal reproductive process of the animal. X, Y sperm separation is the most fundamental technical means for sex control, and for sperm separation, the establishment of the method and the optimization of the method can not be separated from the evaluation of the semen purity.
In the prior art, the identification method of sperm separation purity mainly comprises the following steps: identification is carried out through sex ratio of offspring, the method has long period, is not beneficial to optimization of a separation method, and has high cost; the embryo cytology identification and evaluation method has the advantages that the technical difficulty of microdissection and karyotype analysis of embryo cells is high, and the metaphase chromosome split phase of the embryo cells is not easy to obtain; the embryo immunology identification and evaluation method has the defects of large species difference and poor accuracy due to the specificity and immunogenicity of the antigen; the method needs to wash out the embryo in vitro for detection, and has longer time consumption and larger risk; flow cytometry re-analysis of the evaluation method, which is less accurate when X, Y sperm-sperm DNA content differences are small. In addition, the fluorescence in situ hybridization evaluation method and the PCR evaluation method are also widely applied to sperm separation purity determination, however, the methods do not consider the influence of different target gene sequences and amplification efficiencies of different primer pairs on the determination result, and both have systematic errors.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for detecting the purity of animal sex-controlled semen, so as to solve the problem of systematic errors of the detection method in the prior art.
The second purpose of the invention is to provide a kit for detecting the purity of animal sexual control semen, and provide a product which has good accuracy, high sensitivity, short detection time and high flux.
The third purpose of the invention is to provide the application of the detection method or the kit.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a method for detecting the purity of animal sexual control semen includes such steps as respectively using X sperm primer pair and Y sperm primer pair to perform real-time PCR on specimen to be detected to obtain Ct(xi)And Ct(yi)Then Ct is obtained(xi)And Ct(yi)Average value Ct of(i)Ct of the sample to be detected(i)Comparing with a standard curve to obtain the purity of the sample to be detected;
the standard curve comprises a corresponding relation between standard product purity and a standard product Ct, wherein the standard product Ct is the Ct in the first standard product(x)And Ct in the second standard(y)Average value of (d);
performing real-time PCR on the first standard substance by using X sperm primer pair to obtain Ct(x)；
Performing real-time PCR on the second standard substance by using the Y sperm primer pair to obtain Ct(y)；
The standards include X sperm to Y sperm at different known concentration ratios, the concentration of X sperm in the first standard being the same as the concentration of Y sperm in the second standard.
Further, the animal sexual control semen comprises bovine sexual control semen, the X sperm primer pair is preferably a PLP gene primer pair, and the Y sperm primer pair is preferably an SRY gene primer pair.
Further, the sequences of the PLP gene primer pairs are as follows:
PLP-F：5’-TGCTGCTCTCGTTTCACC-3’(SEQ ID NO.1)；
PLP-R：5’-CCCCACATTTGTCTTACC-3’(SEQ ID NO.2)；
preferably, the sequence of the SRY gene primer pair is as follows:
SRY-F：5’-GCCACAGAAATCGCTTCC-3’(SEQ ID NO.3)；
SRY-R：5’-CCGTGTAGCCAATGTTACCTT-3’(SEQ ID NO.4)。
furthermore, the X sperm and the Y sperm in the standard sample are independently recombinant plasmids containing X sperm unique genes and Y sperm unique genes;
preferably, the X sperm-unique gene comprises a PLP gene;
preferably, the Y sperm unique gene comprises an SRY gene.
A kit for detecting the purity of animal sexual control semen comprises an X sperm primer pair, a Y sperm primer pair and a standard substance.
Further, the animal sexual control semen comprises bovine sexual control semen, the X sperm primer pair is preferably a PLP gene primer pair, and the Y sperm primer pair is preferably an SRY gene primer pair.
Further, the sequences of the PLP gene primer pairs are as follows:
PLP-F：5’-TGCTGCTCTCGTTTCACC-3’(SEQ ID NO.1)；
PLP-R：5’-CCCCACATTTGTCTTACC-3’(SEQ ID NO.2)；
preferably, the sequence of the SRY gene primer pair is as follows:
SRY-F：5’-GCCACAGAAATCGCTTCC-3’(SEQ ID NO.3)；
SRY-R：5’-CCGTGTAGCCAATGTTACCTT-3’(SEQ ID NO.4)。
further, the standard substance is X sperm and Y sperm which are independently packaged or packaged in combination;
preferably, the standard is a combination package of X sperm and Y sperm at different known concentration ratios.
Furthermore, the X sperm and the Y sperm in the standard sample are independently recombinant plasmids containing X sperm unique genes and Y sperm unique genes;
preferably, the X sperm-unique gene comprises a PLP gene;
preferably, the Y sperm unique gene comprises an SRY gene.
The detection method or the kit is applied to the evaluation of the quality of the cattle sex control semen product.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a method for detecting the purity of animal sexual control semen, which respectively carries out real-time PCR detection on X sperm and Y sperm in a standard substance containing X sperm and Y sperm with different known concentration ratios, prepares a standard curve by taking Ct values respectively obtained by the X sperm and the Y sperm as variable factors and the concentration ratio of the X sperm to the Y sperm, and then prepares an average Ct value of the X sperm and the Y sperm in a sample to be detected(i)And comparing with a standard curve to obtain the concentration ratio of the X sperm to the Y sperm in the sample to be detected, thereby obtaining the purity of the sample to be detected. The detection method simultaneously takes the detection data of the X sperm and the Y sperm as variable factors to prepare a standard curve, solves the problem that the detection method in the prior art has system errors between the detection result and the actual sample purity due to different amplification efficiencies of different target genes and different primer sequences, enables the detection result to be more accurate and closer to the actual purity of the sample, and simultaneously has good sensitivity, short detection time and low cost.
The kit provided by the invention comprises an X sperm primer pair, a Y sperm primer pair and a standard substance. The use method of the kit refers to the detection method provided by the invention, so that the kit has the advantages of good accuracy, high sensitivity, short detection time and high throughput.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph showing the result of DNA extraction from bovine blood in example 1 of the present invention;
FIG. 2 is a graph showing the result of DNA extraction from bovine semen in example 1 of the present invention;
FIG. 3 is a diagram showing the result of amplification of the PLP gene in example 2 of the present invention, in which 1 to 5 are all the PLP genes, and 6 is a negative control;
FIG. 4 is a graph showing the results of amplification of SRY genes in example 2 of the present invention, in which 1 to 5 are SRY genes, 6 are the results of amplification of a DNA sample from a cow, and 7 are negative controls;
FIG. 5 is a standard curve diagram in example 4 of the present invention.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.
Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.
A method for detecting the purity of animal sexual control semen includes such steps as respectively using X sperm primer pair and Y sperm primer pair to perform real-time PCR on specimen to be detected to obtain Ct(xi)And Ct(yi)Then Ct is obtained(xi)And Ct(yi)Average value Ct of(i)Ct of the sample to be detected(i)Comparing with a standard curve to obtain the purity of the sample to be detected;
the standard curve comprises the corresponding relation between the standard product purity and the standard product Ct, wherein the standard product Ct is the Ct in the first standard product(x)And Ct in the second standard(y)Using the X sperm primer pair to perform real-time PCR on the first standard substance to obtain Ct(x)(ii) a Performing real-time PCR on the second standard substance by using the Y sperm primer pair to obtain Ct(y)；
The standards include X sperm to Y sperm at different known concentration ratios, the concentration of X sperm in the first standard being the same as the concentration of Y sperm in the second standard.
The detection method respectively carries out real-time PCR detection on X sperms and Y sperms in standard products containing the X sperms and the Y sperms with different known concentration ratios, and the X sperms and the Y sperms are detectedCt values respectively obtained by the sperms and the Y sperms are used as variable factors to prepare standard curves according to the concentration ratio of the X sperms to the Y sperms, and then the average Ct value of the X sperms and the Y sperms in a sample to be detected is used as the Ct value(i)And comparing with a standard curve to obtain the concentration ratio of the X sperm to the Y sperm in the sample to be detected, thereby obtaining the purity of the sample to be detected. The detection method simultaneously takes the detection data of the X sperm and the Y sperm as variable factors to prepare a standard curve, solves the problem that the detection method in the prior art has system errors between the detection result and the actual sample purity due to different amplification efficiencies of different target genes and different primer sequences, enables the detection result to be more accurate and closer to the actual purity of the sample, and simultaneously has good sensitivity, short detection time and low cost. The concentration ratio of X sperm to Y sperm in the calibration curve is preferably obtained as a common logarithmic value (Lg) by conversion. The concentration ratio of the X sperms to the Y sperms can visually reflect the purity of the sample to be detected, for example, the smaller the concentration ratio of the X sperms to the Y sperms is, the more the Y sperms are, the higher the purity of the Y sperms is; on the contrary, if the sample to be detected is X-type semen control, the obtained concentration ratio of the X sperms to the Y sperms is larger, the more X sperms are, the higher the purity of the X-type semen control is
The X sperm primer pair is specially designed for an X sperm genetic sequence, and can specifically amplify a certain segment in the X sperm genetic sequence to reflect the content of X sperm; similarly, the Y sperm primer pair is specially designed for the genetic sequence of the Y sperm, and the primer pair can specifically amplify a certain segment in the genetic sequence of the Y sperm and is used for reflecting the content of the Y sperm. For example, when the animal sex control semen is bovine sex control semen, the X sperm primer pair is preferably a primer pair that detects the PLP gene, and the Y sperm primer pair is preferably a primer pair that detects the SRY gene.
Furthermore, the present invention is illustrated by the following specific examples of the specific procedures of the detection method, and the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention:
(a) detecting the purity of the cattle sex-controlled semen, and respectively designing a detection primer pair aiming at a PLP gene (X sperm) and an SRY gene (Y sperm);
(b) standards included X sperm to Y sperm at different known concentration ratios:
preparing a recombinant plasmid containing a PLP gene and a recombinant plasmid containing an SRY gene into standard substances with different concentration ratios according to the ratio of 1: 17, 1: 12, 1: 9, 1: 6, 6: 1, 9: 1, 12: 1 and 17: 1;
(c) performing real-time PCR on the first standard substance by using X sperm primer pair to obtain Ct(x)：
Performing real-time PCR detection on the above standard substances sequentially by using PLP gene primer pair to obtain 8 Ct values respectively of A1 (1: 17), A2 (1: 12), A3 (1: 9), A4 (1: 6), A5 (6: 1), A6 (9: 1), A7 (12: 1) and A8 (17: 1);
(d) performing real-time PCR on the second standard substance by using the Y sperm primer pair to obtain Ct(y)：
Respectively carrying out real-time PCR detection on the standard substances by using SRY gene primer pairs in sequence to obtain 8 Ct values which are respectively B1 (1: 17), B2 (1: 12), B3 (1: 9), B4 (1: 6), B5 (6: 1), B6 (9: 1), B7 (12: 1) and B8 (17: 1);
(e) the Ct of the standard is the Ct of the first standard(x)And Ct in the second standard(y)The X sperm concentration in the first standard is the same as the Y sperm concentration in the second standard:
calculating the average values of C1 for a1 and B8, C2 for a2 and B7, C3 for A3 and B6, C4 for a4 and B5, C5 for a5 and B4, C6 for a6 and B3, C7 for a7 and B2, and C8 for A8 and B3;
(f) preparing a standard curve, including the corresponding relation between the standard product purity and the standard product Ct:
values of 1: 17, 1: 12, 1: 9, 1: 6, 6: 1, 9: 1, 12: 1 and 17: 1 were designated as P1-P8, respectively, in Lg(Pi, i ═ 1, 2, 3, 4, 5, 6, 7, or 8)The standard curve is obtained by using the abscissa C1-C8 as the ordinate;
(g) and (3) detecting the purity of the sample to be detected:
performing real-time PCR detection on the sample to be detected by utilizing PLP gene primer pair to obtain Ct(xi)(ii) a Using SRY gene primer to carry out real-time PCR detection on a sample to be detected to obtain Ct(yi)To obtain Ct(xi)And Ct(yi)Average value Ct of(i)Ct of the sample to be detected(i)And comparing with the standard curve to obtain the purity of the sample to be detected.
In a preferred embodiment, when the animal sexual control sperm is bovine sexual control sperm, the X sperm primer pair is preferably a PLP gene primer pair and the Y sperm primer pair is preferably an SRY gene primer pair. The PLP gene is a marker gene specific to the genetic information of the X sperm of the cattle; the SRY gene is a specific marker gene of the genetic information of the cattle Y sperm.
In a preferred embodiment, the sequences of the PLP gene primer pairs are as follows:
PLP-F：5’-TGCTGCTCTCGTTTCACC-3’(SEQ ID NO.1)；
PLP-R：5’-CCCCACATTTGTCTTACC-3’(SEQ ID NO.2)；
in a preferred embodiment, the sequences of the primer pairs for the SRY genes are as follows:
SRY-F：5’-GCCACAGAAATCGCTTCC-3’(SEQ ID NO.3)；
SRY-R：5’-CCGTGTAGCCAATGTTACCTT-3’(SEQ ID NO.4)。
in a preferred embodiment, the X sperm and the Y sperm in the standard are each independently a recombinant plasmid comprising an X sperm unique gene and a recombinant plasmid comprising a Y sperm unique gene.
In preferred embodiments, the X sperm unique gene comprises a PLP gene;
in preferred embodiments, the Y sperm unique gene comprises an SRY gene.
A kit for detecting the purity of animal sexual control semen comprises an X sperm primer pair, a Y sperm primer pair and a standard substance. The use method of the kit refers to the detection method provided by the invention, so that the kit has the advantages of good accuracy, high sensitivity, short detection time and high throughput.
In a preferred embodiment, the animal sexual control semen comprises bovine sexual control semen, the X sperm primer pair is preferably a PLP gene primer pair, and the Y sperm primer pair is preferably an SRY gene primer pair.
In a preferred embodiment, the sequences of the PLP gene primer pairs are as follows:
PLP-F：5’-TGCTGCTCTCGTTTCACC-3’(SEQ ID NO.1)；
PLP-R：5’-CCCCACATTTGTCTTACC-3’(SEQ ID NO.2)；
in a preferred embodiment, the sequences of the primer pairs for the SRY genes are as follows:
SRY-F：5’-GCCACAGAAATCGCTTCC-3’(SEQ ID NO.3)；
SRY-R：5’-CCGTGTAGCCAATGTTACCTT-3’(SEQ ID NO.4)。
in preferred embodiments, the standards are X sperm and Y sperm, each packaged individually or in combination.
In a preferred embodiment, the standard is a combination package of X sperm and Y sperm at different known concentration ratios.
In a preferred embodiment, the X sperm and the Y sperm in the standard are each independently a recombinant plasmid comprising an X sperm unique gene and a recombinant plasmid comprising a Y sperm unique gene; wherein the X sperm unique gene preferably comprises a PLP gene; preferably, the Y sperm unique gene comprises an SRY gene.
The detection method or the kit is applied to the evaluation of the quality of animal sex control semen products.
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
The blood sample of the Chinese Heibai cow is provided by Xushui county, Cao river town Xushui Dairy cooperative. Selecting 6 heads of 10-month-old Holstein bulls and cows respectively, collecting 7mL of blood at the tail root of a disposable blood collecting and anticoagulating tube, subpackaging the blood into 1.5mL of sterile EP tubes in a laboratory, and freezing and storing the tubes in a refrigerator at the temperature of-20 ℃.
The sex control semen of Chinese Holstein cow is purchased from Nemontage Corsai Biotechnology GmbH, and the semen is preserved at liquid nitrogen-196 deg.C. Commercial semen is from 3 bulls, the bull numbers are 15516051, 15516055 and 15517034 respectively, each bull provides 4 branches of X, Y sex sorted semen and unsorted semen respectively, the X sorted semen of the 15516051 bull is 93 percent in trademark purity, the Y sorted semen is 92 percent in trademark purity, and the unsorted semen is 50 percent in trademark purity; the trademark purity of X-sorted semen of the No. 15516055 bull is 90%, the trademark purity of Y-sorted semen is 90%, and the trademark purity of unsorted semen is 50%; the trademark purity of the X-sorted semen of the No. 15517034 bull is 92%, the trademark purity of the Y-sorted semen is 90%, and the trademark purity of the unsorted semen is 50%.
Escherichia coli (Escherichia coli) Trans 5. alpha. is competent for ampicillin resistance. pMD19-T (simple) vector has a full length of 2692 bp.
Example 1 DNA extraction and quality testing
(a) Blood DNA extraction and quality testing
Taking 300 mu L of a bovine blood sample added with an anticoagulant, adding 10 mu L of RNase A into the sample to remove RNA in the sample, adding 20 mu L of proteinase K and 500 mu L of BB3 into the sample, fully mixing the sample by swirling for 30s, sealing the port of the mixed sample, putting the port of the mixed sample into a water bath kettle for overnight digestion at 55 ℃, carrying out short-time centrifugation on the completely digested sample, adding the whole solution into a centrifugal column, centrifuging for 60s at 12000 Xg, and pouring off the liquid passing through the column. 500. mu.L of CB3 solution (to which absolute ethanol has been added) was added, and the mixture was centrifuged at 12000 Xg for 30 seconds to discard the liquid. Then 500. mu.L of WB3 solution (with absolute ethanol added) was added, centrifuged at 12000 Xg for 30s and the liquid was discarded (this step was repeated 2 times). The residue of WB3 was removed by centrifugation at 12000 Xg for 120 s. Placing the centrifugal column into a clean 1.5mL centrifuge tube, adding 100 μ L preheated EB (preheating in a 60 ℃ water bath), standing at room temperature for 120s, centrifuging at 10000 Xg for 120s, eluting DNA (passing through the column for many times to obtain high concentration DNA), and storing the collected DNA at-20 ℃ for later use. Measuring the concentration of DNA, weighing 0.2g of agarose, putting the agarose into a triangular flask, adding 20mL of 0.5 xTBE, setting the concentration of gel to be 1%, heating to ensure that agarose particles disappear, and cooling to 50-60 ℃ after the agarose particles are completely dissolved; slowly poured into the slab to avoid air bubbles, and cooled at room temperature for about 30min until the gel sets. And slowly placing the rubber block with the rubber plate in an electrophoresis tank, covering the rubber surface with TBE, and carrying out electrophoresis from the negative electrode to the positive electrode. And uniformly mixing the sample and the dye, adding the DNA component quality standard into a sample application hole beside the sample, and performing electrophoresis under the voltage condition of 100V until the strip runs out. The gel block was placed in a gel imaging system, and compared with a molecular weight standard to observe the fragment size and to observe the presence or absence of other electrophoretic bands except the position of the band of interest. The results are shown in FIG. 1, where the electrophoretic image clearly shows a single band with little degradation. The concentration and purity are detected by a Nanodrop 2000 ultramicro spectrophotometer, the average DNA concentration is between 210 and 320 ng/mu L, the value of A260/280 is between 1.8 and 1.83, and the value of A260/230 is between 2.0 and 2.25, which indicates that the DNA extraction is successful and can be used for subsequent experiments.
(b) Semen DNA extraction and quality detection
The bovine semen DNA extraction was carried out using the DNA extraction Kit TIANAmp Genomic DNA Kit (spin columns) DP304 from Tiangen Biotechnology (Beijing) Ltd, and the experimental procedures were as follows: sucking 150 mu L of semen into a 1.5mL centrifuge tube, and adding 50 mu L of buffer GA to make the final volume 200 mu L; adding 20 μ L PK and 20 μ L1M DTT, shaking for 15s to mix well, incubating at 56 deg.C for 6h, and shaking every 30min for 2 times. After the mixture is centrifuged for 10s for a short time, 220 mu L of buffer solution GB is added, the mixture is shaken for 15s and mixed evenly, and the mixture is incubated for 10min at 70 ℃; centrifuging for 10s for a while, adding 200 μ L ethanol, mixing by inversion for 10s, and incubating at room temperature for 3 min; after a brief 10s centrifugation, the lysate was carefully transferred to a core tube, followed by centrifugation at 12000r/min for 1min and the filtrate discarded. 480 mu L of buffer GD is added into a column tube, the mixture is centrifuged for 70s at 11000r/min, and the filtrate is discarded. 580 μ L of buffer PW was added to the column tube, and the mixture was centrifuged at 11000r/min for 70s, and the filtrate was discarded. 580. mu.L of buffer PW was added again, the mixture was centrifuged at 11000r/min for 70s, and the filtrate was discarded. 14000 r/min full speed centrifugation for 3min, the column tube was placed into a 1.5mL centrifuge tube, 30. mu.L of buffer TE was added to the center of the membrane of the column tube, and incubation was performed at room temperature for 5 min. Centrifuging at 14000 r/min for 2min, extracting DNA, and storing at-20 deg.C. Measuring the concentration of DNA, weighing 0.2g of agarose, putting the agarose into a triangular flask, adding 20mL of 0.5 xTBE, setting the concentration of gel to be 1%, heating to ensure that agarose particles disappear, and cooling to 50-60 ℃ after complete dissolution; slowly poured into the slab to avoid air bubbles, and cooled at room temperature for about 30min until the gel sets. And slowly placing the rubber block with the rubber plate in an electrophoresis tank, covering the rubber surface with TBE, and carrying out electrophoresis from the negative electrode to the positive electrode. And uniformly mixing the sample and the dye, adding the DNA component quality standard into a sample application hole beside the sample, and performing electrophoresis under the voltage condition of 100V until the strip runs out. The gel block was placed in a gel imaging system, and compared with a molecular weight standard to observe the fragment size and to observe the presence or absence of other electrophoretic bands except the position of the band of interest. As shown in FIG. 2, only a clear band is observed in the electrophoresis image, and the semen DNA concentration is detected by a Nanodrop 2000 ultramicro spectrophotometer, wherein the semen DNA concentration is between 180 and 220 ng/. mu.L on average, A260/280 is between 1.78 and 1.85, and A260/230 is between 2.0 and 2.05. The method proves that the bovine semen DNA has better integrity and no obvious degradation, and can be used for subsequent tests.
Example 2 preparation of standards
(a) Primer design
The sequence of the downloaded PLP gene of cattle (GenBank accession number is shown in the following table) is obtained from NCBI database, the Primer Premier 5.0 is used for designing specific primers, the sequence of the primers used in the test is shown in the following table, and the primers are synthesized by Beijing Liuhe Huada Gene science and technology Co.
The sequence of the downloaded SRY gene of the cattle SRY gene (GenBank accession number is shown in the following table) is obtained from the NCBI database, the Primer Premier 5.0 is used for designing a specific Primer, the sequence of the Primer used in the test is shown in the following table, and the Primer is synthesized by Beijing Liuhe Huada Gene science and technology Co., Ltd.
(b) PCR amplification
Centrifuging 12000r of a centrifugal tube filled with the primers for 3min, slightly opening the cover, adding a proper amount of sterile ultrapure water according to the instructions, mixing uniformly by vortex, and performing PCR amplification system:
PCR conditions for PLP gene:
SRY gene PCR reaction conditions:
(c) electrophoretic detection of PCR products
The PCR product was detected by 1.5% agarose gel electrophoresis.
Weighing 0.3g agarose and placing in a triangular flask, weighing 20mL 0.5 × TBE solution, preparing into 1.5% gel, heating to dissolve agarose powder, and cooling to 50-60 deg.C after the powder is completely dissolved.
a) Slowly pouring into a rubber plate to avoid bubbles, and cooling at room temperature for about 30min until the rubber is solidified.
b) The block with the slab was slowly placed in an electrophoresis tank with a TBE that had to be able to cover the gel surface.
c) The sample to be tested and the dye are mixed and carefully added to the wells, and the DNA quality standard is added to the wells next to the sample to be tested as a reference.
d) Electrophoresis was performed using a 120V voltage condition until the band ran off.
e) And after the electrophoresis is finished, placing the gel block in a gel imaging system, comparing the gel block with the molecular weight standard to observe the size of the fragment, and observing whether other electrophoresis bands exist except the position of a target band.
The cow DNA sample is used for carrying out PCR amplification on the cow X chromosome specific primer PLP, the PCR product is detected by 1.5 percent gel electrophoresis, the result is shown in figure 3, the size of the amplification target band is 214bp and is the same as the size of the expected fragment. The ox Y chromosome specific primer SRY is subjected to PCR amplification by using ox and cow DNA samples, PCR products are detected by 1.5% gel electrophoresis, and the result is shown in figure 4, wherein the size of an amplification target band is 229bp and is the same as the size of an expected fragment.
(d) PCR product purification or gel recovery
When the PCR product is electrophoretically detected to have other non-target electrophoretic bands, gel cutting recovery is carried out, and when only the target band exists, PCR product purification is carried out.
And (3) cutting and recycling:
a) scrubbing and disinfecting the blade by using an alcohol cotton ball, slightly burning the blade on an alcohol lamp, cutting off agarose gel containing a target band under the irradiation of an ultraviolet lamp after the blade is cooled, putting the agarose gel into a prepared centrifugal tube, weighing again, and calculating the weight of a target fragment gel block.
b) Adding B2 in an amount of 3 times the weight of the gelatin block, placing in a water bath at 50 deg.C, and mixing uniformly every 2min until the gelatin block is dissolved.
c) Sucking the solution into an adsorption column, setting the rotation speed of a centrifugal machine to be 8000 g, quickly centrifuging for 30s, and pouring out waste liquid.
d) Add 500. mu.L of washing solution, centrifuge set to 9000 g for 30s and discard the waste (repeat this step twice).
e) The adsorption column was rapidly centrifuged at 9000 g for 1 min.
f) Placing the adsorption column into a sterile centrifuge tube, adding 20 μ L (preheated at 65 deg.C) of the eluate, standing at room temperature for 1min, rapidly centrifuging at 9000 g for 1min, and freezing at-20 deg.C.
And (3) PCR purification step:
a) and (3) adding 250 mu L of binding buffer solution into 50 mu L of PCR product, gently mixing uniformly, standing in a centrifugal column for 1min, setting the rotation speed of a centrifugal machine to 10000 g, quickly centrifuging for 60s, and discarding the effluent.
b) 650. mu.L of washing buffer was added, the rotation speed was set to 10000 g and the effluent was discarded by flash centrifugation for 60 s.
c) The centrifuge is set to rotate at 10000 g for 2min to remove residual liquid completely.
d) The column was placed into a sterile dry 1.5mL centrifuge tube, 30. mu.L of elution buffer (pre-heated to 65 ℃) was added, and frozen at-20 ℃ for use.
(e) Connecting the recovered product with T carrier
Agarose gel cutting and purificationPCR products were ligated with pMD19-T (simple) vector in a 10. mu.L system: mu.L of DNA recovery product, 5. mu.L of ligation solution, 1. mu.L of pMD19-T (simple) vector, 3. mu.L of sterile ddH2O mixing, 16 ℃ connection, the connection time is determined according to the fragment size, and 0.5h to overnight.
(f) Transformation and propagation of ligation products
a) Trans5 α competent cells were thawed on ice, aliquoted into 50 μ L per tube, 10 μ L of ligation product was added to the tube, gently mixed with a pipette, and allowed to stand on an ice bath for 0.5 h.
b) Heat shock was applied for 45s in a 42 ℃ water bath and then rapidly iced for 2min without shaking.
c) 890. mu.L of liquid medium LB (without ampicillin) was added thereto, the temperature was set at 37 ℃ and the rotation speed was 210r, and the mixture was cultured for 1 hour.
d) Operating on a super clean bench, taking 250 mu L of liquid culture medium LB (without ampicillin) for coating, uniformly coating on a culture plate (preheating at 37 ℃ in advance) until the bacteria liquid absorbs and the water disappears, and carrying out inverted culture at 37 ℃ for about 13-15 h.
e) Selecting a single round colony, placing the colony in a sterile centrifuge tube, adding 900 mu L of a culture medium LB (containing ampicillin), setting the temperature at 37 ℃, culturing for 2-4h at the rotating speed of 210r, carrying out PCR by taking a bacterial solution as a template, and selecting the bacterial solution meeting the conditions for sequencing.
f) 200. mu.L of a bacterial suspension with a sequencing result of the objective band was added to 5mL of a liquid medium LB (containing ampicillin), and cultured at a set temperature of 37 ℃ and a rotation speed of 210r for about 12-16 h.
The size of the target fragment of the PLP gene is 214bp, the target fragment is connected into a carrier, and the PCR result of the bacterial liquid meets the requirement and is sent to a sequencing company for sequencing. After sequencing, sequencing results are compared by using BioEdit, and can be obtained by comparison, so that the requirements of subsequent tests are met.
The size of the target fragment of the SRY gene is 229bp, the target fragment is connected to a carrier, and the PCR result of the bacterial liquid meets the requirement and is sent to a sequencing company for sequencing. After sequencing, sequencing results are compared by using BioEdit, and can be obtained by comparison, so that the requirements of subsequent tests are met.
(g) Plasmid small grape
a) The rotation speed of the centrifuge is set to 8000 g, 5mL of bacterial liquid is rapidly centrifuged for 2min, and waste liquid is poured out.
b) Add 250. mu. L P1 buffer (containing RNaseA), blow and shake to suspend the cells.
c) Add 250 u L P2 buffer solution, shake 10 times, mix well, stand for 3 min.
d) Add 350 u L P3 buffer solution, shake 10 times up and down, mix well with the rotational speed set to 12000 g, centrifuge rapidly for 10 min.
e) Transferring the supernatant into an adsorption column, setting the rotating speed at 8000 g, quickly centrifuging for 30s, and pouring out the waste liquid.
f) Add 500. mu.L DW1 buffer, set the speed 9000 g, centrifuge rapidly for 30s, pour out the waste.
g) Add 500. mu.L of wash and centrifuge at 9000 g for 30s and drain off (this step was repeated twice).
h) And (3) placing the collecting pipe and the adsorption column into a centrifugal machine, setting the rotating speed to 9000 g, and quickly centrifuging for 1 min.
i) The adsorption column was transferred to a 1.5mL sterile centrifuge tube, 50. mu.L of the eluent (preheated at 60 ℃) was added to the adsorption column, allowed to stand for 2min, set at 9000 g, and centrifuged rapidly for 1min to obtain a plasmid DNA solution, which was stored at-20 ℃ for cryopreservation.
(h) Standard quality particle copy number calculation and dilution
The concentration and quality of the extracted standard plasmid are detected by a NaNoDrop 2000 spectrophotometer, the values of A260/280 and A260/230 are obtained, the purity is between 1.82 and 2.0, and a standard curve can be constructed. The plasmid copy number calculation formula is as follows: plasmid copy number (copies. mu.L)-1)＝6.02×1023(copies·mol-1) X plasmid concentration (ng. mu.L)-1) Plasmid molecular weight (g. mol)-1)。
Example 3RT-qPCR reaction procedure and Condition determination
Centrifuging the primer after synthesizing the primer, dissolving the primer, slowly opening a tube cover (the primer is powder which can not be seen by naked eyes, centrifuging for 3min at the speed of 10,000 Xg before opening), adding corresponding sterilized water according to the requirement of the primer, covering, fully shaking and uniformly mixing by using an oscillator, and if the primer is frequently used at 4 ℃, keeping the final concentration of the primer at 10 mu M, and not storing at-20 ℃ for a long time.
Setting corresponding temperature gradient according to the recommended temperature of the TM value on the primer synthesis report sheet, and searching the conditions for PCR of the primers to find the optimal annealing temperature.
PCR assay kit reference: TB GreenTMPremix Ex TaqTMII(Tli RNaseH Plus)
And (3) PCR reaction system:
| TB Green Premix Ex Taq II(Tli RNaseH Plus(2×) | 5μL |
| ROX Reference Dye(50×) | 0.2μL |
| DNA Sample | 1μL |
| upstream primer (Forward primer) (10. mu. mol/L) | 0.4μL |
| Downstream primer (Reverse primer) (10. mu. mol/L) | 0.4μL |
| ddH2O | 3μL |
| Total Volume (Total Volume) | 10μL |
The test conditions are as follows:
example 4 construction of Standard Curve and establishment of detection method
PLP and SRY plasmids with different proportions are subjected to real-time fluorescent quantitative PCR amplification according to the ratio of 1: 17, 1: 12, 1: 9, 1: 6, 6: 1, 9: 1, 12: 1 and 17: 1:
performing real-time PCR detection on the above standard substances sequentially by using PLP gene primer pair to obtain 8 Ct values respectively of A1 (1: 17), A2 (1: 12), A3 (1: 9), A4 (1: 6), A5 (6: 1), A6 (9: 1), A7 (12: 1) and A8 (17: 1);
respectively carrying out real-time PCR detection on the standard substances by using SRY gene primer pairs in sequence to obtain 8 Ct values which are respectively B1 (1: 17), B2 (1: 12), B3 (1: 9), B4 (1: 6), B5 (6: 1), B6 (9: 1), B7 (12: 1) and B8 (17: 1);
calculating the average values of C1 for a1 and B8, C2 for a2 and B7, C3 for A3 and B6, C4 for a4 and B5, C5 for a5 and B4, C6 for a6 and B3, C7 for a7 and B2, and C8 for A8 and B3;
values of 1: 17, 1: 12, 1: 9, 1: 6, 6: 1, 9: 1, 12: 1 and 17: 1 were designated as P1-P8, respectively, in Lg(Pi, i-1, 2, 3, 4, 5, 6, 7, or 8)The abscissa and the ordinate represent C1-C8, and a standard curve is obtained, the result is shown in FIG. 5, and the standard curve equation is that y is-0.9569 x +25.92, and R is2This standard curve can be used to calculate sexed semen purity, 0.9948.
Performing real-time PCR detection on the sample to be detected by utilizing PLP gene primer pair to obtain Ct(xi)(ii) a Using SRY gene primer to carry out real-time PCR detection on a sample to be detected to obtain Ct(yi)To obtain Ct(xi)And Ct(yi)Average value Ct of(i)Ct of the sample to be detected(i)And (5) substituting a standard curve to obtain the ratio of the PLP gene to the SRY gene, namely obtaining the ratio of X to Y, and obtaining the purity of the sample to be detected.
Example 5 commercial methods for semen control
And (3) carrying out purity detection on the purchased commercial sex-controlled semen by using the established semen purity evaluation method (the reaction conditions and the procedures are the same as above), and according to the relation between the Ct value and the copy number of the specific DNA template of each sex. The sum of the X and Y content in each sample, calculated using established methods, is equal to 100% and is used to estimate the percentage of X sperm content (X%) and Y sperm content (Y%) in commercial seminal control fluid.
The accuracy of the method was verified by real-time fluorescent quantitative PCR detection of commercially available X, Y sex-controlled semen and unseparated semen, the results of which are shown in the following table. The average purity of the detected X sorted semen is 90.73 percent, the trademark marked average purity of the commercially available X sorted semen is 91.67 percent, and the difference between the two is not significant (P is more than 0.05); detecting that the average purity of the commercial Y sorted semen is 89.04 percent, the commercial Y sorted semen has the average purity marked by a trademark of 90.67 percent, and the difference between the commercial Y sorted semen and the commercial Y sorted semen is not obvious (P is more than 0.05); meanwhile, the average purity of the commercial unsorted semen is 49.84% through detection, the commercial labeling theoretical purity of the commercial unsorted semen is 50%, and the difference between the two is not significant through comparison (P is greater than 0.05). The result of the real-time fluorescence quantitative PCR for semen detection is compared with the commercial quality control semen brand labeling result, the difference between the two results is not obvious, and the higher accuracy of the real-time fluorescence quantitative PCR for semen detection is proved.
Example 6 mixed semen method validation
DNA extracted from X, Y commercially available sorted semen is mixed according to a corresponding ratio, the mixing ratio of X, Y is 1:20, 1:10, 1:5, 1:1, 5:1, 10:1 and 20:1, real-time fluorescence quantitative PCR detection (the reaction conditions and procedures are the same as above) is carried out on the mixed semen by using the established semen purity evaluation method, and the proportion of X, Y sperms in the mixed semen is calculated.
While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
SEQUENCE LISTING
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Claims (10)
1. The method for detecting the purity of animal sexual control semen is characterized in that real-time PCR is carried out on a sample to be detected by respectively utilizing an X sperm primer pair and a Y sperm primer pair to obtain Ct(xi)And Ct(yi)Then Ct is obtained(xi)And Ct(yi)Average value Ct of(i)Ct of the sample to be detected(i)Comparing with a standard curve to obtain the purity of the sample to be detected;
the standard curve comprises a corresponding relation between standard product purity and a standard product Ct, wherein the standard product Ct is the Ct in the first standard product(x)And Ct in the second standard(y)Average value of (d);
performing real-time PCR on the first standard substance by using X sperm primer pair to obtain Ct(x)；
Performing real-time PCR on the second standard substance by using the Y sperm primer pair to obtain Ct(y)；
The standards include X sperm to Y sperm at different known concentration ratios, the concentration of X sperm in the first standard being the same as the concentration of Y sperm in the second standard.
2. The detection method according to claim 1, wherein the animal sex-controlled semen comprises bovine sex-controlled semen, the X sperm primer pair is preferably a PLP gene primer pair, and the Y sperm primer pair is preferably an SRY gene primer pair.
3. The detection method according to claim 2, wherein the sequences of the PLP gene primer pairs are as follows:
PLP-F：5’-TGCTGCTCTCGTTTCACC-3’(SEQ ID NO.1)；
PLP-R：5’-CCCCACATTTGTCTTACC-3’(SEQ ID NO.2)；
preferably, the sequence of the SRY gene primer pair is as follows:
SRY-F：5’-GCCACAGAAATCGCTTCC-3’(SEQ ID NO.3)；
SRY-R：5’-CCGTGTAGCCAATGTTACCTT-3’(SEQ ID NO.4)。
4. the detection method according to any one of claims 1 to 3, wherein each of X sperm and Y sperm in the standard is independently a recombinant plasmid containing an X sperm-specific gene and a recombinant plasmid containing a Y sperm-specific gene;
preferably, the X sperm-unique gene comprises a PLP gene;
preferably, the Y sperm unique gene comprises an SRY gene.
5. A kit for detecting the purity of animal sexual control semen is characterized by comprising an X sperm primer pair, a Y sperm primer pair and a standard substance.
6. The kit according to claim 5, wherein the animal sexual control semen comprises bovine sexual control semen, the X sperm primer pair is preferably a PLP gene primer pair, and the Y sperm primer pair is preferably an SRY gene primer pair.
7. The kit of claim 6, wherein the sequence of the PLP gene primer pair is as follows:
PLP-F：5’-TGCTGCTCTCGTTTCACC-3’(SEQ ID NO.1)；
PLP-R：5’-CCCCACATTTGTCTTACC-3’(SEQ ID NO.2)；
preferably, the sequence of the SRY gene primer pair is as follows:
SRY-F：5’-GCCACAGAAATCGCTTCC-3’(SEQ ID NO.3)；
SRY-R：5’-CCGTGTAGCCAATGTTACCTT-3’(SEQ ID NO.4)。
8. the kit according to claim 5, wherein the standards are X sperm and Y sperm, each of which are individually or in combination packaged;
preferably, the standard is a combination package of X sperm and Y sperm at different known concentration ratios.
9. The kit according to any one of claims 5 to 8, wherein each of X sperm and Y sperm in the standard is independently a recombinant plasmid containing a unique gene for X sperm and a recombinant plasmid containing a unique gene for Y sperm;
preferably, the X sperm-unique gene comprises a PLP gene;
preferably, the Y sperm unique gene comprises an SRY gene.
10. Use of the detection method according to any one of claims 1 to 4 or the kit according to any one of claims 5 to 9 for assessing the quality of an animal's sexual control semen product.
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