CN110564863B - Gosling sex identification method - Google Patents
Gosling sex identification method Download PDFInfo
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- CN110564863B CN110564863B CN201910849375.8A CN201910849375A CN110564863B CN 110564863 B CN110564863 B CN 110564863B CN 201910849375 A CN201910849375 A CN 201910849375A CN 110564863 B CN110564863 B CN 110564863B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
Abstract
The invention relates to the field of biological detection, and particularly discloses a reagent kitThe identification method of the gender of the breeding gosling comprises the following steps: s1, collecting blood sample of gosling to be identified by ddH 2 Diluting by 500-1000 times to obtain goose blood sample diluent; s2, taking the dilution of the blood sample of the gosling as a template, carrying out PCR amplification reaction by using primers with sequences shown in SEQ NO.1 and SEQ NO.2, wherein 20 mu L of PCR amplification reaction system comprises 0.5-4.0 mM of Mg 2+ 2.0 mu L; s3, detecting the PCR amplification product through agarose gel electrophoresis, wherein two bands of 455bp and 300bp appear in the amplification product band, the female goose is the female goose, and only one band of 455bp appears in the amplification product band, and the male goose is the male goose. The invention omits the traditional step of DNA extraction, and can directly carry out PCR on the blood sample only by adopting the common Taq polymerase, thereby simplifying the identification process, shortening the identification time and reducing the identification cost.
Description
Technical Field
The invention relates to the field of biological detection, in particular to biological molecular detection, and more particularly relates to a gosling sex identification method.
Background
Due to differences in growth rate, growth performance and the like among individuals of different sexes, it is often necessary to breed them in groups according to their sexes in the modern poultry industry, and it is therefore necessary to perform sex determination on poultry at an early stage. At present, the traditional goose sex identification mainly comprises appearance identification and anus turning identification. In the gosling stage, the difference of the appearance of the male goose and the appearance of the female goose is small, the difficulty in sex identification of the goose according to the appearance is large, and the identification accuracy is low. The method for identifying the anus turning over has high accuracy, the accuracy can reach 98-99% generally, but the method is completed within 24 hours after the gosling emerges, and the shrinkage of the procreation process of the gosling is difficult to observe after 24 hours. Meanwhile, the method has high technical requirements on operators, otherwise, the gosling is greatly damaged, and the misjudgment rate is increased. Therefore, the method has important significance for identifying the sex by adopting goose feathers, blood and the like and a molecular biological means.
The avian sex-determining system is ZW type, male chromosome is ZZ type, and female chromosome is ZW type. Therefore, the PCR identification method for avian sex is based on searching specific genes or sequences on the W chromosome. Sex-linked genes that have been found to be useful for sex determination include chromosomal helix protein gene (CHD 1), adenosine triphosphate 5A1 (ATP 5 A1), pseudogene sequence (EE 0.6 sequence), and the like. The CHD1 gene has 2 homologous copies (CHD-W and CHD-Z) in non-ratites, linked to the W chromosome and Z chromosome, respectively. The exon sequences of these two homologous copies are similar, but the sizes of the introns are very different. Therefore, after PCR amplification using the differential design specific primers and electrophoresis detection of the PCR product, 2 bands are generated in female (ZW) and only 1 band is generated in male (ZZ). The method judges the male and female through double positive results, eliminates the possibility of false negative, and has more reliable and accurate results.
At present, the sex identification of poultry and birds by applying a PCR technology has been reported more, generally, genomic DNA is extracted firstly, and then PCR identification is carried out, for example, CN103468825B and CN107354219A respectively disclose that PCR primers are used for sex identification of geese and penguin, the DNA extraction process is carried out, and the DNA extraction is more complicated, so the whole identification process consumes more time. In the PCR reaction system of the technical scheme, omni Klenaq is selected as Taq polymerase, the enzyme is Taq or Klenaq mutant which is screened from a library and has high tolerance, the target gene can be directly amplified in one step by taking materials such as whole blood, serum and the like as a template, the complicated nucleic acid DNA extraction process of geese is avoided, but the enzyme is expensive, and the cost for identifying the sex by using the enzyme is too high. Therefore, there is a need for a method of determining the sex of poultry that is less time consuming and less costly for poultry growers, especially for poultry growers.
Disclosure of Invention
In order to eliminate the defects of the prior art, the invention optimizes a reaction system for gradient of Taq polymerase, blood sample, magnesium ion concentration and the like, thereby not only eliminating the step of genome DNA extraction and greatly saving time and labor cost, but also greatly reducing the identification cost by adopting the common Taq polymerase to carry out blood sample PCR.
A gosling sex identification method comprises the following steps:
s1, collecting blood sample of gosling to be identified by ddH 2 Diluting by 500-1000 times with O to obtain goose blood sample diluent;
s2, taking the dilution of the blood sample of the gosling as a template, carrying out PCR amplification reaction by using primers with sequences shown in SEQ NO.1 and SEQ NO.2, wherein 20 mu L of PCR amplification reaction system comprises 0.5-4.0 mM of Mg 2+ 2.0μL；
S3, detecting the PCR amplification product through agarose gel electrophoresis, wherein two bands of 455bp and 300bp appear in the amplification product band, the female goose is the female goose, and only one band of 455bp appears in the amplification product band, and the male goose is the male goose.
The invention can avoid genome DNA extraction and can use common Taq polymerase to carry out PCR of the blood sample, and the key points are the design of primers, the dilution multiple of the blood sample and Mg in a PCR reaction system 2+ Optimization of concentration and dosage. The primer pair of the sequences shown in SEQ NO.1 and SEQ NO.2 is designed according to the difference of different copy introns of the goose CHD1 gene on Z chromosome and W chromosome, and the specific positions are shown in figure 1. The invention uses different Mg 2+ The PCR reactions were carried out at concentrations (0.5 mM, 1.0mM, 2.0mM and 4.0 mM) in combination with different dilution factors (50, 100, 200, 500 and 1000 fold) of the blood samples and Mg was found 2+ The concentration is 0.5-4.0 mM, the blood sample is diluted by 500-1000 times, and the primer with a specific sequence is combined, so that the extraction of genome DNA is avoided, and the accurate molecular identification of the blood sample can be carried out by using common Taq polymerase.
The invention finds that the dilution multiple of goose blood sample can obviously influence the accuracy and sensitivity of detection, and preferably ddH is used in S1 2 Diluting by 500 times with O to obtain goose blood sample diluent.
The invention discovers Mg in a PCR amplification reaction system 2+ The concentration of (A) in the goose blood sample can influence the accuracy and sensitivity of the detection together with the dilution factor of the goose blood sample, and preferably, 2.0mM of Mg is included in a 20 mu L PCR amplification reaction system 2+ 2.0μL。
Preferably, the blood sample is anticoagulated with EDTA prior to dilution.
Preferably, the 20 μ L PCR amplification reaction system is: 10 XPCR Buffer 2.0. Mu.L, 2.0mM Mg 2+ 2.0. Mu.L, 200. Mu.M dNTP mix 1.0. Mu.L, 1.0U Taq DNA polymerase 0.2. Mu.L, 4pmol primer 0.4. Mu.L shown in SEQ NO.1, 4pmol primer 0.4. Mu.L shown in SEQ NO.2, goose blood sample diluent 10. Mu.L, ddH 2 O 4μL；
Preferably, the PCR reaction conditions are: pre-denaturation at 94 deg.C for 5min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 30s, and 33 cycles; extending for 5min at 72 ℃; storing at 12 deg.C.
Compared with the prior art, the invention has the following beneficial effects:
the method can rapidly complete the molecular identification of the gender of the gosling within 2h, and the accuracy rate is 100%. The invention only needs a common PCR instrument and agarose gel electrophoresis equipment, directly adopts the blood sample as the PCR template, avoids the traditional process of extracting DNA and greatly improves the efficiency.
Drawings
FIG. 1 is a diagram showing the location of a target sequence and primers amplified in the CHD1 gene of goose according to the present invention; the goose CHD1 gene is located in 1990082-2046480 of a genome and has a total length of 56699 bp; the black arrow indicates the position of the primer, F indicates the upstream primer, R indicates the downstream primer, and the PCR product is 2022022022023-2022477.
FIG. 2 the present invention with different Mg 2+ Gel electrophoresis profiles of PCR products at concentrations (0.5 mM, 1.0mM, 2.0mM, and 4.0 mM) and at different blood sample dilutions (50, 100, 200, 500, and 1000-fold); m is Marker DL2,000; A-E are 50, 100, 200, 500 and 1000 fold dilutions of the blood sample, respectively; 1 to 4 are each Mg 2+ Concentrations 0.5mM, 1.0mM, 2.0mM and 4.0mM.
FIG. 3 is a gel electrophoresis diagram of PCR sex determination of a gosling blood sample in the present invention; m is Marker DL2,000,1-6 are detection samples, NC1 is negative control without adding template, and NC2 is negative control without adding primer.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. The reagents, methods and apparatus employed in the present invention are conventional in the art, except as otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1
A method for identifying the gender of a gosling comprises the following steps:
1. collecting and diluting a blood sample of the gosling: the gosling to be identified is subjected to sub-fin venous micro blood sampling (about 0.1 mL), 2 mu L of blood sample is directly taken and ddH is used 2 Diluting the O by 50-1000 times, and directly using the diluted O as a template for PCR reaction.
If the number of goslings to be identified is large, blood samples can be stored firstly and then identified, at the moment, anticoagulation treatment is needed after the blood samples are collected, and the specific implementation mode is as follows: a subplantar venous microcentre (about 0.1 mL) was taken, and the blood was poured into a 1.5mL centrifuge tube containing 15. Mu.L of 2% sterile EDTA anticoagulant (ethidene EDTA), shaken gently, numbered, and stored at-20 ℃ for further use. 2 μ L of blood sample was taken and ddH was applied 2 Diluting by 50-1000 times with O, and directly using as a template for PCR reaction.
2. Taking the dilution of the blood sample of the gosling as a template, and carrying out PCR amplification reaction by using a PCR primer pair
Primers containing different copies of intron fragments were designed based on the genomic DNA sequence of the goose CHD1 gene (see FIG. 1).
The upstream primer nucleotide sequence SEQ NO.1 of the PCR primer pair is: 5 'AATGAGTGCACTGCAGAAA-3'; the nucleotide sequence of the downstream primer SEQ NO.2 is: 5 'TAATGAGGTAGCAATGGGTT-containing 3'.
The invention uses different Mg 2+ The PCR reaction was carried out at a concentration (0.5 mM, 1.0mM, 2.0mM, or 4.0 mM) in combination with a dilution (50, 100, 200, 500, or 1000-fold) of different blood samples, and the composition of the 20. Mu.L PCR reaction system is shown in Table 1.
TABLE 1 goose CHD1 Gene blood sample PCR reaction System
The PCR reaction program is: pre-denaturation at 94 deg.C for 5min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 30s, and 33 cycles; extending for 5min at 72 ℃; storing at 12 deg.C.
3. Detecting the PCR amplification product by agarose gel electrophoresis. The blood sample was found by comparison to have a dilution factor of 500,mg 2+ The resulting band was detected only when the concentration was 2.0mM (FIG. 2).
Example 2
A method for identifying the gender of a gosling comprises the following steps:
1. collecting and diluting a blood sample of the gosling: the gosling to be identified is subjected to sub-fin venous micro blood sampling (about 0.1 mL), 2 mu L of blood sample is directly taken and ddH is used 2 Diluted 500 times with O and used directly as a template for PCR reaction.
If the number of goslings to be identified is large, blood samples can be stored firstly and then identified, at the moment, anticoagulation treatment is needed after the blood samples are collected, and the specific implementation mode is as follows: a subplantar venous microcentre (about 0.1 mL) was taken, and the blood was poured into a 1.5mL centrifuge tube containing 15. Mu.L of 2% sterile EDTA anticoagulant (ethidene EDTA), shaken gently, numbered, and stored at-20 ℃ for further use. Taking 2. Mu.L of blood sample, using ddH 2 Diluted 500 times with O and used directly as a template for PCR reaction.
3. The gosling blood sample diluent is used as a template, and PCR amplification reaction is carried out by using a PCR primer pair, wherein the PCR primers are the same as those in example 1.
The composition of the 20. Mu.L PCR reaction system is shown in Table 2.
TABLE 2 goose CHD1 Gene blood sample PCR reaction System
The PCR reaction program is: pre-denaturation at 94 deg.C for 5min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 30s, and 33 cycles; extending for 5min at 72 ℃; storing at 12 deg.C.
3. Detecting PCR amplification product by agarose gel electrophoresis, and judging sex according to the band of the amplification product
Take 5. Mu.L of PCR product (which can be directly spotted), detect it by 1.5% agarose gel electrophoresis, and use DNA marker 2000 as reference. The sex was judged from the electrophoresis results (FIG. 3), and it was a female goose when the product showed two bands of 455bp and 300bp, and a male goose when the product showed one band of 455 bp. The total number of 6 male and female individuals is identified, the identification result is consistent with the result of anatomical identification or the identification result of anus turning by professionals according to the patent method of the invention, and the identification accuracy rate is 100 percent.
Claims (2)
1. The gosling sex identification method is characterized by comprising the following steps:
s1, collecting blood sample of gosling to be identified by ddH 2 Diluting by 500 times with O to obtain goose blood sample diluent; s2, taking the dilution of the gosling blood sample as a template, carrying out PCR amplification reaction by using primers with sequences shown in SEQ NO.1 and SEQ NO.2, wherein a 20 mu L PCR amplification reaction system comprises 2.0mM Mg 2+ 2.0 mu L; s3, detecting a PCR amplification product through agarose gel electrophoresis, wherein two bands of 455bp and 300bp appear in the amplification product band, the female goose is the female goose, and only one band of 455bp appears in the amplification product band, and the male goose is the male goose;
the 20 mu L PCR amplification reaction system is as follows: 10 XPCR Buffer 2.0. Mu.L, 2.0mM Mg 2+ 2.0. Mu.L, 200. Mu.M dNTP mix 1.0. Mu.L, 1.0U TaqDNA polymerase 0.2. Mu.L, 4pmol primer 0.4. Mu.L shown in SEQ NO.1, 4pmol primer 0.4. Mu.L shown in SEQ NO.2, goose blood sample diluent 10. Mu.L, ddH 2 O 4μL；
The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 30s, and 33 cycles; extending for 5min at 72 ℃; storing at 12 ℃;
the nucleotide sequence of the upstream primer SEQ NO.1 is: 5 'AATGAGTGCACTGCAGAAA-3'; the nucleotide sequence of the downstream primer SEQ NO.2 is: 5 'TAATGAGGTAGCAATGGGTT-containing 3'.
2. The method for sexing a gosling according to claim 1, wherein said blood sample is anticoagulated with EDTA before dilution.
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| TWI372784B (en) * | 2007-12-31 | 2012-09-21 | Academia Sinica | Oligonucleotides for avian sex identification |
| CN103468825B (en) * | 2013-10-14 | 2015-03-04 | 扬州大学 | Primer, kit and method used for goose sex identification |
| CN204079950U (en) * | 2014-06-16 | 2015-01-07 | 江苏省家禽科学研究所 | Fowl Sex Rapid identification test kit |
| CN204111736U (en) * | 2014-09-12 | 2015-01-21 | 江苏省家禽科学研究所 | A kind of test kit for goose sex identification |
| CN105925690B (en) * | 2016-05-16 | 2019-07-19 | 华南农业大学 | It is a kind of for identifying the primer, kit and its discrimination method of pigeon gender |
| CN108893547A (en) * | 2018-07-24 | 2018-11-27 | 温氏食品集团股份有限公司 | It is a kind of for the primer pair of the quick Sex judging of squab, kit and quick Sex judging method |
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