TWI308595B - Probe set, kit and method for sex determination of bovine or ovine cells - Google Patents

Description

1308595 IX. Description of the invention: [Technical field to which the invention pertains] The present invention relates to a gender identification technique. In particular, the present invention relates to a technique for detecting the sex of cattle or sheep cells using a glutinous rice needle. [Prior Art] In animal husbandry, gender control of livestock is an important key. For example, dairy farmers need cows to produce milk, while breeding companies that supply frozen semen expect to produce semen to supply semen, such as controlling sex during embryonic period. Can improve industrial competitiveness. At present, in the reproductive technology of dairy cows, the application of gender identification and embryo displacement technology can enable cows with excellent performance to obtain more offspring in a short period of time, increase the milk production of cattle and accelerate genetic improvement. In the gender identification of animal embryos before bed, the karyotype analysis of sex chromosomes and the determination of Ηγγ antigen present on the surface of male embryos have been developed (White et al., Biol·Repr〇d 37:867 873, 1987). Determination of the amount of X-chromosome enzymes and heterozygous reactions using Y_chromosome-specific probes (Bondioli' J. Anim. Sci 7〇 (suppl): 19 29, 1992), etc. These methods are faced in practical applications. The detection operation steps are cumbersome, the accuracy and sensitivity are insufficient, and special factors, instruments and equipment are required, which are not easy to apply. In recent years, because of the rapid progress of molecular biotechnology, plus

105007.doc 1308595

Biotechnology 8: 1279-1281, 1990; Bredbacka,

Theriogenology 55: 23-34, 2001). After the traditional polymerase chain reaction is completed, electrophoresis analysis must be performed to determine the results, and an instant polymerase chain reaction is developed to apply for foreign gene detection and paternity testing in gene transfer animals (Ballester et al., Biotechniques 37:61). -630, 2004) or gender identification (Alonso and Martin, Methods Mol. Biol. 297.3 1- 44 '2004), qualitative or quantitative sensitivity analysis can be completed in a short time, greatly improving the sensitivity of traditional polymerase linkage deficiency And reduce the time and cost of colloidal electrophoresis.

Virta et al. (Theriogenology 57: 2229-2236, 2002) revealed a method for identifying the sex of bovine embryos using an instant polymerase chain reaction, first with P1-5EZ (SEQ ID NO: 3) and p3_3MZ (SEQ ID NO: 4) Amplify the ZFX/ZFY gene fragment in bovine embryos, and then use the instant polymerase chain reaction of TaqMan8 system to identify the sex of the embryo; the primer used is ZFX/ZFY forward primer (SEQ ID NO: 5) and zfx/zfy reverse primer. (SEQ ID NO: 6) can further amplify the specific fragment of the male gene (ZFX) and the female gene in the pre-amplified fragment of P1_5EZ and p3_3MZ, and design a probe suitable for the TaqMan8 system for ζ; ργ specific fragment ( Sequence identification number 9) and probe for ZFX specific fragment (sequence identification number 1 G) to detect the sex of the embryo. However, the slave needle for the ZFγ-specific fragment (SEQ ID NO: 9) is a probe with 33 bases, and the probe for the ZFX-specific fragment (the sequence identification number is a probe with a Chuangu test). In terms of the heterozygous reaction in the instant polymerase chain reaction, sub-difficult to operate 'again, the cost of the probe with more bases is higher than that of 105007.doc 1308595, and it is easier to make mistakes in the synthesis process. The accuracy of the detection is reduced. Furthermore, when the instant polymerase chain reaction is performed, the design of the probe is obvious—the sensitivity and accuracy of the gender identification. The bonding temperature of the pf used by Virta et al. is too close to the primer. The accuracy of cattle and sheep blasts == accuracy is not ideal. For the above reasons, the industry still needs a method that is accurate, fast and economical to detect the sex of cattle or sheep embryos.

SUMMARY OF THE INVENTION One object of the present invention is to provide a scorpion needle set for identifying the sex of a bovine or ovine cell, which comprises a ZFY probe and a ZFX probe. It is still another object of the present invention to provide a kit for identifying bovine or amniocyte cell characteristics comprising the probe set of the present invention. A further object of the present invention is to provide a method for identifying the sex of a bovine or ovine cell, which uses the probe set of the present invention. DETAILED DESCRIPTION OF THE INVENTION The present invention first provides a probe set for identifying the sex of a bovine or ovine cell, comprising: a ZFY probe having a sequence as indicated by a sequence identification number; and a ZFX needle having a sequence Identify the sequence shown in number 2. ZFX and ZFY are sex-specific genes, in which ZFx is located on the X chromosome and ZFY is located on the γ chromosome. In female animals, there are only X-stained bodies, so they are (four) homozygous; in male animals, they have both; and the dyes are so ZFY heterozygous. In the gender identification, the presence of the 105007.doc 1308595 ZFX gene was identified as a female animal; if the presence of the ZFX and ZFY genes was detected simultaneously, it was identified as a male animal. In the genome of cattle, the ZFX gene and the ZFY gene have three different base sequences, wherein the specific fragment of ZFX is shown in the sequence identification number ;; the specific fragment of zfy is shown in sequence identification number 12. The probe set according to the present invention, wherein the ZFY probe has 18 bases and is completely hybridized with a specific fragment of ZFY; the ZFX probe also has 18 base numbers, which can be completely hybridized with a specific fragment of ZFX . The probe set according to the present invention is compared with the probe for the ZFY-specific fragment (SEQ ID NO: 9) and the probe identification for the ZFX-specific fragment (sequence identification number i〇) contained in the prior literature (see Figure 1). The detection base is greatly reduced, and it is easy to operate on the sex of the identified cells. Furthermore, the probes with fewer bases are less costly to synthesize, and the errors in the synthesis process are less likely to occur, and the detection accuracy can be greatly improved. degree. On the other hand, the probe according to the present invention has a bonding temperature which is close to 10 C ' of the primer, thereby effectively improving the accuracy of the sex identification and enhancing the sensitivity of the identification. The cell according to the present invention may be a cell derived from cattle or sheep. Preferably, the cell line is selected from the group consisting of embryonic cells, somatic cells, embryonic leaf cells, and fertilized eggs; more preferably, the cell line is an embryonic cell. . On the other hand, the probe set according to the present invention can be simultaneously applied to the sex identification of bovine cells and sheep cells, and its applicability is extensive. Because of the high degree of similarity between the genomic sequences of cattle and sheep (Moore et al., 1991), the use of the sex-specific sequence of the Y-chromosome or somatic chromosome of cattle can also be used in the sex identification of sheep. For example, γ-chromosome-specific sequences such as bovine ZFY/ZFX and Sry gene 105007.doc 1308595 have been shown to be useful for sex identification of sheep embryos (Gutierrez-Adan et al., 1997; Ng et al., 1996). The probe set according to the present invention can be used in a heterozygous reaction to detect the Ζ]ρχ and ZFY genes. Preferably, the probe set is applied to an immediate polymerase chain reaction. The "polymerase chain reaction" as used herein includes four steps: (1) denaturation of a template to form two single strands; (2) adhesion of the two primers to the two strands of step (1) (anneaHng) (3) extending the primers with a DNA polymerase; and (4) obtaining two pairs of DNA. The above steps are repeated, and amplification is obtained for a specific DNA fragment. The "Instant Polymerase Chain Reaction" is the use of fluorescence to detect the "polymerase chain reaction" and can be quantified and can be used to infer the amount of template involved in the reaction. The current real-time polymerase chain reaction is developed in a closed reaction tube. In addition to the primers and assays required for the polymerase chain reaction, a fluorescent signal is added to the reaction tube. The function of simultaneously amplifying pure nuclear nucleic acid and simultaneously detecting signals can be achieved. Therefore, the immediate polymerase chain reaction has the advantages of direct amplification and no post-treatment, which not only saves 4 labor, but also avoids the pollution caused by post-treatment. At the same time, because of the cycle-by-cycle DNA fluorescence signal, and at the same time depicting the integer pattern of the gene copy, it has better accuracy and sensitivity. On the other hand, the 'instant polymerase chain reaction can complete a large number of samples in a few minutes', for example, processing % or 384 samples at the same time, which not only saves time, but also automatically controls the operation of the umbrella - thousands of Reduce errors caused by human operations. In a specific embodiment of the invention, TaqMan9 instant polymerization is used 105007.doc 1308595

Enzyme chain reaction system. Preferably, the 5 and 3 ends of the ZFY probe respectively have a first reporter and a first quencher; the 5 and 3 ends of the ZFX probe respectively have a second reporter and a The second quencher' and the first reporter are different from the second reporter, and the reporter and the quencher are preferably two moonlight groups to detect a polymerase chain reaction product, for example, to report protein In the fluorescent portion, the commonly used luminescent group is FAWIC; the quenching agent is usually MGBNFq or TAMRA. If the probe is not hybridized with the template, its quencher fluorescent base (usually a long wavelength color, such as red) is transferred by fluorescence resonance energy transfer (F 丨 escence Res〇nance L milk Transfer ' FRET) reduces the fluorescence of the reporter's fluorescent base (usually - short, long color, such as green) at the 5th end. If the probe is denatured and adhered to the template: On a single-strand stencil, the polymerase can add the nucleating acid according to the sequence of the template, and remove the target probe from the 5th end, thus making the 3' end quencher The fluorescent base is separated from the reporter at the 5' end, so that the reporter can diverge its fluorescent energy, such as the polymerase chain reaction, the more times there are, the more reports = glory divergence, so by measuring the reporter Fluorescence, the product of the chain reaction of the polymerase can be determined. According to the present invention, the cell genomic or 1 magnified column is used as a template, and the instant polymerase reaction is carried out using a ZFX probe and a ZFY probe. The conditions of the instant polymerization-lock reaction according to the present invention are determined by those of ordinary skill in the art to be more expensive, and the 94C reaction is 15 seconds and 6 〇t reaction] minutes. Mt. Month also provides a kit for identifying the sex of bovine or ovine cells. Its 105007.doc 1308595 contains the reagents required for the above probes. It is preferred to use one of the P1-5EZ primers of the present invention. In addition, it also contains an instant polymerase chain reaction to cooperate with the immediate polymerase chain reaction. In a specific embodiment, the kit further comprises: having a sequence as shown in sequence identification number 3; a P3-3MZ primer, a Ganesh _ having a sequence as shown in sequence identification number 4. According to the present invention, the cell genome amplification sequence of the instant polymerase chain reaction template is based on the P1.5EZ primer and the p3_3Mz primer, and the polymerase chain reaction is carried out by using the cell genome as a template, and the dish is P3 3MZ. The two primers can pre-amplify the ZFX/ZFY gene fragment in the bovine embryo, which can improve the quantification and sensitivity of the instant polymerase chain reaction. The conditions of the ts enzyme chain reaction are determined by those skilled in the art. It is preferably determined by a 95t reaction for 5 minutes, followed by a 95C reaction for 30 seconds, a reaction of 30 seconds at 56 ° C for 30 seconds, and 72 cycles. The reaction was carried out at ° C for 1 minute; and further at 72 ° C for 7 minutes. In another embodiment of the present invention, the kit further comprises: a ZFX/ZFY forward primer having a sequence as shown in sequence identification number 5; and a ZFX/ZFY reverse primer having sequence identification number 6 The sequence shown in the ZFX/ZFY forward and reverse primers can further amplify the specific fragment of the male gene (ZFX) and the female gene (ZFY) in the pre-amplified fragment of P1_5EZ and p3_3MZ and combine with the instant polymerase chain reaction. The ZFX and ZFY genes were identified for the probe set according to the invention. 105007.doc 12· 1308595 Preferably, in addition to detecting the ZFX and ZFY genes, a kit according to the present invention can assist in identifying the sex of a cell by detecting other sex-specific genes. Therefore, the kit according to the present invention preferably further comprises a primer for amplifying a sex-specific gene.

Preferably, the sex-specific gene is selected from the group consisting of AMX/Y BOV97M, BRY.1, BtYl, FBNY, 0Y11_1, S4 and Sry. AMX/Y as Res vet sci. 1999, 67(1): 111-2; BC1.2 as indicated in the Gene Database Acceptance Number 56119; B〇v97M as Res Vet Sci. 1999 '67( 1): 111 -2 and Theriogenology. 200 1, 1; 55(9): 1 843-53 Both documents; BRY1 such as Zyg〇te. 2〇〇3, 11(1): 17-22 and Res VetSci 1999, 67( 1): 1u_2 described in the two literatures; BtYl such as the gene database accepts the number A1 848 1; fbny* gene database accepts the number AJ002548; ΟΥίΜ such as the gene database accepted compilation U303307; S4 such as the gene database The acceptance number is ^"; and Sry is as described in J Anim Sci. 1995, 73(5): 1408_15, each of which is incorporated herein by reference. The appropriate primers are designed to carry out the polymerase chain reaction and to identify the sex of the cells by the amplification of the genes. Preferably, the polymerase chain reaction is linked to the pre-polymerase chain reaction according to the invention. - the same as in the present - in a preferred embodiment - the sex-specific gene line is Υ Υ It is a specific gene of Y-chromosome. Therefore, if the Sry gene is detected, the cell line can be determined to be male. More preferably, the primer for amplifying a sex-specific gene comprises: ^ y-direction primer, which has a sequence Identification of the sequence shown in number 7; 105007.doc 13 1308595 and Sry extension primers, which have the sequence shown in sequence identification number 8. : Sry forward primer and Sry reverse primer are primer pairs, and the cell genome The polymerase chain reaction is carried out for the template. For example, the (four)^ forward and reverse primers can be identified as male by the polymerase chain reaction, and the cells are identified as male; if not amplified, the female is identified as a female. In the case, when the obtained bovine and goat cells to be tested = the number of more than 10 cells, the accuracy of sex identification can reach 1%; if only one cell is obtained, the accuracy rate can be 70% when performing gender identification. Therefore, it can be seen that the present invention can obtain a satisfactory accuracy by using a very small number of samples. The present invention further provides a method for identifying the sex of a bovine or ovine cell, which uses the aforementioned probe set. The following examples are intended to describe the present invention in detail, but are not intended to limit the invention to the disclosure of the examples. [Embodiment] Treatment of embryonic cells: After obtaining a sex-identified bovine ameloblast or a general cell sample 'Cleaved three times with calcium-magnesium-free phosphate buffer (PBS), and immediately placed 10 μl of cell lysate [(MgCh-free polymerase chain reaction buffer, proteinase Κ) 0·15 5 μg/μl of 0·25 5 liter microcentrifuge tube for cell lysis reaction, the reaction conditions were set at 56 ° C for 30 minutes, 94 ° C for 15 minutes, and finally the sample was maintained at 4 ° C. After the cell sample is treated with the cell lysate, it can be used for polymerization. Pre-amplification amplification of the 4 1308595 synthase chain reaction. Pre-amplification and Sry polymerase chain reaction: template 〇, 5, 1, 或, 15 or 2 制) and P1-5EZ primer (SEQ ID NO: 3) and p3_3MZ primer (SEQ ID NO: 4) Or simultaneously use the Sry forward primer (SEQ ID NO: 7) and Sry$ to the primer (SEQ ID NO: 8) for polymerase chain reaction. The reaction solution used was 1 μl of 1〇〇pM primer, 0.5 μl of DNA polymerase (5 U/μl), 1 μl of polymerase chain reaction buffer 3 μl, 2.5 mM dNTP 1 μl and 25 mM MgCl2 3 μl and water in a total volume of 30 μl. The reaction conditions were 95. (: reaction for 5 minutes; followed by 95 cycles of 95. (: reaction for 30 seconds, reaction at 56 ° C for 30 seconds, and reaction at 72 ° C for 1 minute; reaction at 72 ° C for 7 minutes. The product obtained was passed through agar. The results of gel electrophoresis analysis are shown in Fig. 2 and Fig. 3. Fig. 2 is a result of the synthesis using ZFX/ZFY primer alone, and it can be seen that when the number of cells is 1, 5, 10 or 20, the gene can be specifically combined. In order to combine the results of ZFX/ZDY primer and Sry primer synthesis, it can be seen that the sample of male embryo as template can synthesize SFX gene in addition to ZFX/ZFY gene. Instant polymerase chain reaction: The above is introduced by P1-5EZ Different cell numbers (1, 10 or 20) or 25 ng/μL of ZFX/ZFY gene fragment pre-amplified with P3-3MZ primer are template 'ZFX/ZFY forward primer (SEQ ID NO: 5) and ZFX/ZFY reverse primer (SEQ ID NO: 6) is the primer, ZFY probe (SEQ ID NO: 1) and 105007.doc -15· 1308595 ZFX probe (SEQ ID NO: 2) for immediate polymerase chain reaction of the probe; wherein ZFY probe The 5' end has a FAM fluorescent reporter, and the 5' end of the ZFX probe has a VIC fluorescent reporter, ZFX and ZFY. The probe has a MGBNFQ quencher at the 31 end. The reaction solution used is 1 time TaqMan® Universal PCR Master Mix, 100 nM probe amount and 70〇nM2 primer. The conditions used are 1 〇 cycle. 5 〇 ° c reaction for 2 minutes and 95 reaction for five minutes, followed by 35 to 40 cycles of 94 it reacts for 15 seconds and 6 〇 t: reaction clock. The instrument used is ABI® pRISM 7900HT sequence detection system The real-time polymerase chain reaction instrument, the SeqUence Detection System (SDS) analysis software will generate v] [c-light sample is determined as female, and the FAM-fluorescent sample is judged as male. Instant polymerase chain After the reaction, the instrument automatically scans the fluorescence at a wavelength of 550 to _ nm, and then automatically sets the scale to determine the sex of the sample. The results are shown in Figure 4 and Figure 5. Figure 4 shows the immediate poly-enzyme chain reaction of male and female embryos. The amplification curve | ° 曰田踝, Figure 5 is the instant polymerase junction map. It can be seen from Figure 5 that the male anti-red female embryo samples are concentrated in the upper part of the sputum 'in the lower left'. Judging "concentration and sensitivity. It is true that the accuracy of the sex identification can be achieved on the Niu Ping cell to be sampled, and the I μ Jianye cell can reach 100% in thousands of sheep. If only the cell is used, the sex identification can be as early as a fine + Up to 70 〇 / 〇. The above examples are merely illustrative of the principles and effects of the present invention this month, and are not intended to limit the invention of J05007.doc 1308595. Modifications and variations made by the above-described techniques are not inconsistent with the spirit of the present invention. The right of the local month should be as listed in the scope of the patent of Shen 26 as described later. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a comparison diagram of a probe of the prior art. Fig. 2 is a graph showing the results of electrophoresis analysis of agar agaric gelatin with a P1-5EZ primer and a P3_3MZ primer pre-amplified by the zfx/zfy gene, wherein the Μ is a 100 bp molecular marker; Nc is a negative control group. Ding ° ′ Figure 3 is a P1_5EZ primer, P3_3MZ primer, ^ forward primer more w reverse primer pre-amplification of the ZFX / ZFUSry gene agar gelatin electrophoresis analysis results, Μ is 1 〇〇 bp molecular marker; ^ is a negative control group. Figure 4 is an amplification curve of the instant polymerase chain reaction, the left-to-right amplification curve is 25 ng / microliter of DNA, the stencil reaction concentration prepared by 20, 1 〇 or 1 cell number; (A) is female (B) is male. Figure 5 is a plot of the real-time polymerase chain reaction of 1, 5, 1 or 20 individual cell numbers; NTC is the negative control group; Female is the female sample and μ is the male sample. I05007.doc 1308595 Sequence Listing <11〇> Executive Yuan Agricultural Committee Livestock Laboratory<120> Probe set, kit and method for identifying the sex of cattle or sheep cells<130>None<160>< 1 70> Patentln version 3.2 <210> 1 <211> 18 <212> DNA <213> Artificial sequence

<400> 1 cacacttgaa agacattt 18 <210> 2 <211> 18 <212> DNA <213> Artificial sequence <400> 2 tcacacttga atggcatc 18 <210> 3

<211> 24 <212> DNA <213> Artificial sequence <400> 3 24 ataatcacat ggagagccac aagc <210> 4 <211> 24 <212> DNA <213> Artificial sequence <400> 4 gagcctcttt ggtatctgag aaagt 24 105007.doc 1308595 <210> 5 <211> 23 <212> DNA <213> Artificial sequence <400> 5 aaagcacatg cgaatccata ctg <210> 6 <211> 29 <212> DNA <213> Artificial sequence <400> 6 ttggtatctg agaaagtcag aagacaaat <210> 7 <211> 22 <212> DNA <213> Artificial sequence <400> 7 tgttcagagt attgaacgac ga <210> 8 <211> 22 <212> DNA <213> Artificial sequence <400> 8 aataagcaca agaaagtcca gg <210> 9 <211> 33 <212> DNA <213> Artificial sequence 105007.doc 1308595 <400> 9 aaaactaagc atagtaaaga aatgtctttc aag <210><211><212><213> 10 31 DNA artificial sequence <400> 10 agcatagtaa agagatgcca ttcaagtgtg a <210><211><212><213> 11 34 DNA Cattle <400> 11 aaaactaagc atagtaaaga gatgccatgt gtga <210><211><212><213> 2 4 1 3 <400> 12 aaaactaagc atagtaaaga aatgtcttgt gtga 105007.doc

Claims (1)

'刟 〇 〇 3144 Patent Application Patent Application No. 97 IZ B ___ Announcement 1. A probe set for identifying the sex of a bovine or sheep cell, comprising: a ZFY probe having a sequence identification number The sequence shown in Figure 1; and the ZFX probe 'which has the sequence shown in Sequence Identification Number 2. 2. The probe set according to claim 1, wherein the 5' and 3' ends of the ZFY probe respectively have a first reporter and a first quencher; and 5, 3, and 3 of the ZFX probe respectively There is a second reporter and a second quencher, and the first reporter is different from the second reporter. 3. The probe set according to the item 1 wherein the cell line is selected from the group consisting of embryonic cells, somatic cells, embryonic leaf cells, and fertilized eggs. 4. The probe set according to claim 3, wherein the cell line is an embryonic cell. A kit for identifying the sex of a bovine or ovine cell, comprising a probe set according to any one of claims 1 to 4. 6. A kit according to claim 5, further comprising a reagent required for an instant polymerase chain reaction. 7. The kit according to claim 5, further comprising: a P1-5EZ primer having a sequence as shown in sequence identification number 3; and a P3-3MZ primer having a sequence as shown in sequence identification number 4. 8. The kit of claim 5, further comprising: a ZFX/ZFY forward primer having a sequence as indicated by sequence identification number $; and a ZFX/ZFY reverse primer having sequence identification number 6 sequence. 9. According to the state of clarification item 5 έθ j ^ —r- t #,. and it further includes 105007 (replacement) 971212.doc 1308595 ' BC1.2 ' S4 and Sry cited ' to enlarge the sex-specific gene The sex-specific gene is selected from the group consisting of amX/y B〇V97M, BRY1, BtYl, FBNY, OYii.i. 10. According to the kit of claim 9, the genus of the genus is Sry. 11. The kit according to the present invention, wherein the primer for amplifying the sex-specific gene comprises: a Sry forward primer having a sequence as shown in sequence identification number 7; and a Sry reverse primer having sequence recognition The sequence shown in number 8. 12. A method for identifying the sex of a bovine or ovine cell, comprising: performing a heterozygous reaction or a polymerase chain reaction with a probe sample according to any one of claims 1 to 4 to detect ZFX and ZFY The existence of genes. 13. According to the method of claim 12, it utilizes an instant polymerase chain reaction to identify sex. 14. According to the method of claim 13, which comprises using a ZFX/ZFY forward primer and a ZFX/ZFY reverse primer as a primer pair, using a cell genome or an amplified sequence thereof as a template, and using a ZFX probe and a ZFY probe. An instant polymerase chain reaction in which the ZFX/ZFY forward primer has the sequence as shown in SEQ ID NO: 5 'ZFX/ZFY reverse primer has a sequence as shown by sequence recognition to identify the sex of the bovine or sheep embryo. 15. The method according to claim 14, wherein the conditions of the immediate polymerase chain reaction are 10 cycles of 50 ° C for 2 minutes and 95 ° C for 1 minute; followed by a 35 to 40 cycle of 94 ° C for 15 seconds. And react at 60 ° C for 1 minute. 16. The method according to claim 14 wherein the cell genomic amplification sequence as a template uses a P1-5EZ primer and a P3-3MZ primer as a primer pair, and the cell 105007 (replace) 971212.doc 1308595 gene body is used as a template for polymerization. The enzyme chain reaction is obtained, wherein the P1_5EZ primer has a sequence as shown in SEQ ID NO: 3, and the p3_3MZ primer has a sequence as shown in SEQ ID NO: 4. 17) The method according to claim 16, wherein the conditions of the polymerase chain reaction are 95 ° C for 5 minutes; followed by 30 cycles of 95 t of reaction for 3 sec, "it reacts for 30 seconds and reacts at 72 ° C for 1 minute; At 72. (: 7 minutes of reaction. 18. According to the method of claim 12, which further comprises amplifying a sex-specific gene by a polymerase chain reaction, wherein the sex-specific gene is selected from the group consisting of Amx/y, BC1.2, BOV97M, BRY_1 A group consisting of BtYl, FBNY, OY11.1, S4 and Sry 19. The method according to claim 18, wherein the sex-specific gene system is Sry. 20. According to the method of claim 19, wherein the sry forward is introduced The Sry reverse primer is a primer pair and polymerase chain reaction is performed with a cell genome as a template. The Sry forward primer has a sequence as shown in sequence identification number 7 'Sry reverse primer, which has a sequence identification number of 8 The sequence shown is 0 105007 (replace) 971212.doc