CN108441550A - Primer combination, detection kit and its application of pigeon sex identification - Google Patents
Primer combination, detection kit and its application of pigeon sex identification Download PDFInfo
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- CN108441550A CN108441550A CN201810491784.0A CN201810491784A CN108441550A CN 108441550 A CN108441550 A CN 108441550A CN 201810491784 A CN201810491784 A CN 201810491784A CN 108441550 A CN108441550 A CN 108441550A
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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Abstract
The present invention provides the combination of the primer of pigeon sex identification, detection kit and its applications, belong to biomolecular science detection field.The primer combination of pigeon sex identification provided by the invention accurately identifies the gender of pigeon, it is combined by using the primer of identification, the gender of young age pigeon can fast and accurately be identified, facilitate raising and breeding production, therefore the combination of the primer of identification is applied to pigeon sex identification and prepared in the kit for identifying pigeon gender, identification result can be improved, there is higher actual application value and promotional value.
Description
Technical field
The present invention relates to molecular Biological Detection fields, in particular to primer combination, the detection of pigeon sex identification
Kit and its application.
Background technology
With the continuous improvement of people's living standards, pigeon meat is increasingly becoming the delicacies together in ordinary citizen house as the popular vehicles with pigeon eggs,
More and more businessmans also put into the production of dove product, and the development of pigeon for meat and egg dove has obtained wider concern.Pigeon
It is a kind of altrices, sex identification cannot be carried out as general poultry, can only can just differentiates male and female to or so 5 monthly ages.
If squab identifies gender before 28 ages in days, extra male dove can be used as squab listing.
Pigeon is a kind of species that typical ZW types chromosomal sex determines, just with XY types on the contrary, the property of female individuals
Genome becomes ZW, and the sex chromosome group of male becomes ZZ, and therefore, the sex chromosome for the sperm that male generates is only
There is one kind --- Z chromosome, female individuals can generate two kinds of ovums containing Z or W, thus the gender of offspring depends on sperm
Which type of (Z) combined with egg cell (contain Z, or contain W).This mode is prevalent in lepidopterous insects, amphibian animal, creeps
Among class and birds.
Currently, there are no the effective ways and means for carrying out sex identification for young age pigeon, it is badly in need of developing one
The noninvasive detection means of kind.
Invention content
The first object of the present invention is that the primer combination for providing pigeon sex identification can be accurately using the detection reagent
The gender for identifying pigeon, can apply it to pigeon raising or feeding field.
The second object of the present invention is that the primer for providing above-mentioned pigeon sex identification combines in pigeon sex identification
Application.
The third object of the present invention is that the primer combination for providing above-mentioned pigeon sex identification is preparing pigeon gender mirror
Application in fixed detection kit.
The fourth object of the present invention is to provide the detection kit of pigeon sex identification, a variety of identifications use of kit concentration
Reagent can conveniently serve detection by kit, improve testing result stability.
The fifth object of the present invention is to provide the detection kit of above-mentioned pigeon sex identification in identification pigeon gender
In application.
In order to realize the above-mentioned purpose of the present invention, using following technical scheme：
The primer of pigeon sex identification combines, and primer combination includes detection primer pair, and the base sequence of detection primer pair is such as
Shown in SEQ ID NO.1-2.
The primer of above-mentioned pigeon sex identification combines the application in pigeon sex identification.
Application of the primer combination of above-mentioned pigeon sex identification in the detection kit for preparing pigeon sex identification.
The detection kit of pigeon sex identification, detection kit include the primer combination of above-mentioned pigeon sex identification.
Application of the detection kit of above-mentioned pigeon sex identification in identifying pigeon gender.
Beneficial effects of the present invention are：The primer combination of pigeon sex identification provided by the invention accurately identifies pigeon
Gender, by using identification primer combination, can fast and accurately identify the gender of young age pigeon, facilitate raising and cultivation life
Production, therefore the combination of the primer of identification is applied to pigeon sex identification and is prepared in the kit for identifying pigeon gender, all
Identification result can be improved, there is higher actual application value and promotional value.
Description of the drawings
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 is the electrophoresis result figure for the primer 1 that experimental example of the present invention provides；
Fig. 2 is the electrophoresis result figure for the primer 2 that experimental example of the present invention provides；
Fig. 3 is the electrophoresis result figure for the primer 3 that experimental example of the present invention provides；
Fig. 4 is the electrophoresis result figure for the primer 4 that experimental example of the present invention provides；
Fig. 5 is the electrophoresis result figure for the primer 5 that experimental example of the present invention provides；
Fig. 6 is the electrophoresis result figure for the primer 6 that experimental example of the present invention provides；
Fig. 7 is the electrophoresis result figure for the primer S that experimental example of the present invention provides.
Specific implementation mode
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Birds during evolution, the most segment of W chromosome diminutions, but sequence preservative, and Z chromosome gene
Mutability is higher, exactly because CHD-Z and CHD-W have differences during evolution, just there is the base by PCR sector point gender
Plinth.The research report of pigeon CHD complete genome sequences is had no at present, so have design degeneracy base during design of primers,
It tests and carries out trial test referring still to classical P2/P8 and 2550F/2718RCHD specific primers early period, the results showed that obtained
The accuracy of product is not high, it is difficult to identify, or an only band, be consistent with report before, with polyacrylamide gel electricity
Swimming can not distinguish its banding pattern, therefore cannot be used as the gender molecular genetic marker of pigeon.Therefore, the present invention is with reference to chicken
The sequence of CHD1-Z and CHD1-W genes, in conjunction with concrete practice, design obtains the primer combination of the present invention.
The combination of the primer of the pigeon sex identification of the embodiment of the present invention, detection kit and its application are carried out below specific
Explanation.
The primer of pigeon sex identification combines, and primer combination includes detection primer pair, and the base sequence of detection primer pair is such as
Shown in SEQ ID NO.1-2.
The primer of above-mentioned pigeon sex identification combines the application in pigeon sex identification.
Further, in preferred embodiments of the present invention, including PCR reactions are carried out, the annealing temperature of PCR reactions is
50-58℃。
Application of the primer combination of above-mentioned pigeon sex identification in the detection kit for preparing pigeon sex identification.
The detection kit of pigeon sex identification, detection kit include the primer combination of above-mentioned pigeon sex identification.
Further, in preferred embodiments of the present invention, further include PCR reaction buffers, Taq archaeal dna polymerases,
DNTPs, 2 × Super Real PreMix, fluorescent reagent and Mg2+At least one of；And DNA extracts reagents.
In kit, detection primer, and related mating chemical reagent, convenient detection and mirror to pigeon gender are integrated
It is fixed；Meanwhile corresponding detection reagent is provided by kit, stability, the reliability and accuracy of detection can be improved.
Further, in preferred embodiments of the present invention, fluorescent reagent includes SYBR Green I or SYBR
GreenII。
SYBR Green I are a kind of dyes with green excitation wavelength being incorporated into all dsDNA minor grooves region
Material.Under free state, SYBR Green I send out faint fluorescence, but once combined with double-stranded DNA, fluorescence increases greatly
By force.Therefore, the fluorescence signal intensity of SYBR Green I and the quantity of double-stranded DNA are related, can be detected according to fluorescence signal
Double-stranded DNA quantity existing for PCR system.The maximum absorption wavelength of SYBR Green I is about 497nm, and launch wavelength is about
520nm。
SYBR Green II dyestuffs are a kind of nucleic acid staining reagents of high sensitivity, can be to DNA either single-stranded RNA
It is dyed.
Further, in preferred embodiments of the present invention, DNA extracts reagents include PBS solution, DNA eluents and egg
At least one of white enzyme K.
Application of the detection kit of above-mentioned pigeon sex identification in identifying pigeon gender.
Further, in preferred embodiments of the present invention, include the following steps：
To identify that the primer combination of pigeon gender carries out PCR reactions, the annealing temperature of PCR reactions is 50-58 DEG C.
The feature and performance of the present invention are described in further detail with reference to embodiments.
Embodiment 1
The present embodiment provides the combination of the primer of pigeon sex identification, primer combination includes detection primer pair, detection primer pair
Base sequence as shown in SEQ ID NO.1-2.
Regular-PCR or quantitative fluorescent PCR can be passed through by above-mentioned detection primer to being applied in pigeon sex identification
The identification to pigeon gender may be implemented.
When carrying out PCR, the annealing temperature selected is 50-58 DEG C.
It can also be by above-mentioned detection primer to being applied in the detection kit of pigeon sex identification.
Embodiment 2
The present embodiment provides the detection kit of pigeon sex identification, which includes drawing for pigeon sex identification
Object combines, and primer combination includes detection primer pair, and the base sequence of detection primer pair is as shown in SEQ ID NO.1-2.
Certainly, kit can also include PCR reaction buffers, Taq archaeal dna polymerases, dNTPs, 2 × Super Real
At least one of PreMix, fluorescent reagent and Mg2+.
Embodiment 3
The present embodiment provides the detection kit of pigeon sex identification, which includes drawing for pigeon sex identification
Object combines, and primer combination includes detection primer pair, and the base sequence of detection primer pair is as shown in SEQ ID NO.1-2.
Certainly, kit may include PCR reaction buffers, Taq archaeal dna polymerases, dNTPs, 2 × Super Real
PreMix, fluorescent reagent and Mg2+At least one of.
Wherein fluorescent reagent is can to carry out fluorescent quantitative PCR experiment；Fluorescent reagent include SYBR Green I or
SYBR GreenII。
Kit can also include pigeon extracting genome DNA reagent.
DNA extracts reagents include at least one of PBS solution, DNA eluents and Proteinase K.
Experimental example 1
The detection kit for the pigeon sex identification that this experimental example provides embodiment 3, to the detection accuracy of kit
It is verified.
Sampling and sample treatment prepare
The EP pipes of 250 good 1.5mL of high pressure steam sterilization are numbered in advance, 1mL is perfused through high pressure steam sterilization in the inside
0.75% physiological saline.3 ice bags, disposable glove are got out, alcohol swab sterilizes blade, tweezers, the tools such as scissors.
206 are randomly choosed from the squab batch of 28 age in days of yueyang, hunan whole people Ge Ye Co., Ltds, wear number not
Same foot ring, every is pulled out nascent feather 3-5 roots, removes epimere feather, and every feather leaves lower end band hair follicle about 2cm or so and is put into
In EP pipes, and record foot ring number and corresponding EP pipes number.Pigeon is butchered after the completion of sampling, carries out physiology dissection, according to
Sexual gland judges male and female and is recorded in corresponding numbered positions, the recording materials as laboratory sex identification.
Sample box and ice bag are put into bubble chamber together, prevent DNA degradation rotten, laboratory is taken back and carries out subsequent experimental behaviour
It performs an analysis, -20 DEG C save backup.
Pigeon extracting genome DNA, steps are as follows：
1.1 take squab feather, and after being rinsed with distilled water, absolute ethyl alcohol embathes, drying at room temperature, are use up pen feather with knife blade
It may shred, be placed in 1.5mL sterile centrifugation tubes；
0.5mL lysis buffers and 20 μ L of Proteinase K (10mg/mL) are added in 1.2 pipes；
1.3 digest in 55 DEG C of air bath shaken overnights to the transparency liquid for wadding precipitation of floaing containing white；
1.4 are added isometric (0.5mL) saturated phenol, and 5min is stood after fully shaking up, and 12000rpm centrifuges 8min, repeats 1-
It 2 times, is subject at boundary and sees white egg white matter, take supernatant for use；
1.5 add isometric chloroform/isoamyl alcohol (24:1) 0.5mL is stripped, and 5min, 12000rpm are stood after fully shaking up
8min is centrifuged, supernatant is taken, if also protein, is repeated, general extracting is twice；
1.6 add supernatant the absolute ethyl alcohol that the ice of two volumes is pre-chilled, about lmL to overturn mixing, 8000rpm centrifugations
2min, it is DNA that white precipitate, which occurs, in tube bottom；
1.7 75% alcohol 1mL of addition are washed, and 12000rpm centrifuges 5min, and volatilization is inverted after gently outwelling ethyl alcohol, natural
Drying is placed in 4 DEG C of refrigerators；
1.8 are added 200 μ L of TE buffer solutions or distilled water after ethyl alcohol is evaporated completely, and are placed in 55 DEG C of water-bath 3-4h dissolving DNAs ,-
20 DEG C of preservations；
1.9 measure absorbance of the DNA sample in 260nm and 280nm, calculate its concentration and purity；
1.10 take partial mother liquid, are diluted to 100ng/ μ L working solutions with TE or distilled water, save backup.
With the genomic DNA of the pigeon sample of extraction, randomly chooses 24 parts of genomic DNA samples and carry out PCR experimental examinations.
PCR reaction total system be：20μL；
Wherein：PCR Mix 10 μ L, ddH2O, 8 μ L；Each 0.5 μ L of upstream and downstream primer；1 μ L of DNA profiling (100 μ g/ μ L).
PCR response procedures are：
95 DEG C of pre-degeneration 5min；95 DEG C of denaturation 30s, 50-58 DEG C of annealing 30s, 72 DEG C of extension 1min；35 cycles；72℃
Extend 10min, 4 DEG C of preservations.
Primer screening
The phase designs 7 pairs of primer sequences to the present invention with reference to chicken CHD genes before the test, is respectively designated as primer 1-6 and draws
Object S.The base sequence of seven pairs of primers is as shown in table 1.
Table 1 tests primer information
Carry out PCR amplification experiment for the primer in above-mentioned table 1, grope suitable annealing temperature, and to amplified production into
The agarose gel electrophoresis and gel imaging of row 1% are observed.
The result is shown in Figure 1 analyzes result to obtain the conclusion of following primer to Fig. 6：Primer 1, primer 2, primer 3 exist
Expanding effect is bad, and apparent DNA bands are not observed in gel imaging system.Primer 4 is expanded without polymorphism, DNA bands
It is random；Primer 5, the amplification of primer 6 show as all DNA bands without specificity；Primer S amplification polymorphisms and special
Property it is good, Pass Test purpose requirement.Therefore selection primer S carries out next step experiment.
Primer S is the identification primer combination that the present invention protects.
It carries out a concentration of 1% Ago-Gel, SYBR Green coloring agents is added, glue and be plugged comb, wait for that glue is cold
But comb is gently taken out after, the intact of loading wells is kept, glue is placed in electrophoresis tank, is drawn each PCR to be checked with pipettor and is produced
5 μ L of object are gently inserted into loading wells, carry out point sample.Buffer concentration is 1 × TBE, is 120V, electric current 110mA electrophoresis with voltage
35min.Electrophoresis terminate by gel be placed in gel imaging system observation the results are shown in Figure 7, have 2 bands be ZW types, only 1 band
It is ZZ types, comparison is original to butcher gender record, and as a result matching is consistent.
Then PCR amplification is carried out to 206 samples, is carried out by Gel electrophoresis results, and with practical sexual gland anatomical results
Compare, obtains the accuracy rate by primer amplification and Molecular Identification.Specific experiment the results are shown in Table 2.
The comparison of the result and anatomical results of 2 Molecular Identification of table
From the results shown in Table 2, the result identified by primer and the result that sexual gland is dissected are almost the same；It compares and slaughters
Kill original gender record, the sex identification accuracy of the primer is 97%, it is not anticipated that 100%.It is likely due to sample
Or the reasons such as experiment lead to the deviation of inspection result；But since deviation is minimum, the influence to actual production is also smaller, error
In range, caused by being perhaps not due to primer inaccuracy itself, therefore later stage Optimal Experimental condition can also be passed through；Or
Optimize the preparation of sample, further Optimal Experimental result so that testing result is more accurate and reliable.Offer of the present invention is also provided
Pigeon sex identification primer combination, have higher accuracy, have the effect of actual use；It can make in actual production
With.
Experiment reference frame is provided to study the higher site of accuracy rate from now on, test result is that dove farm carries out breast
The Sex judging of dove, divides group, eliminates hero dove and double female match-place technologies provide the reliable appraisal basis of science.Test result simultaneously
Meet the theory of avian sex determination mechanism CHD-W, but have no the gene order of pigeon CHD gene clusters now, so result
It also fails to reach notional result 100%.
In conclusion the primer combination of pigeon sex identification provided in an embodiment of the present invention has very high identification accurate
Property, by primer detection, energy Rapid identification goes out the gender of squab, provides reliable data for large-scale cultivation etc. and technology is protected
Barrier and support；Identification primer is used to prepare detection kit, it is simple and convenient, the stability of detection can be effectively improved, is had
Higher practicability and higher application value.
Embodiments described above is a part of the embodiment of the present invention, instead of all the embodiments.The reality of the present invention
The detailed description for applying example is not intended to limit the range of claimed invention, but is merely representative of the selected implementation of the present invention
Example.Based on the embodiments of the present invention, those of ordinary skill in the art are obtained without creative efforts
Every other embodiment, shall fall within the protection scope of the present invention.
SEQUENCE LISTING
<110>Agricultural University Of Hunan
<120>Primer combination, detection kit and its application of pigeon sex identification
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 1
cgagaacgtg gcaacagagt 20
<210> 2
<211> 23
<212> DNA
<213>It is artificial synthesized
<400> 2
cccttttatt gatccatcaa gtc 23
Claims (10)
1. the primer of pigeon sex identification combines, which is characterized in that the primer combination includes detection primer pair, and the detection is drawn
The base sequence of object pair is as shown in SEQ ID NO.1-2.
2. the primer of pigeon sex identification as described in claim 1 combines the application in pigeon sex identification.
3. application according to claim 2, which is characterized in that including carrying out PCR reactions, the annealing temperature of the PCR reactions
Degree is 50-58 DEG C.
4. the primer combination of pigeon sex identification as described in claim 1 is in the detection kit for preparing pigeon sex identification
Application.
5. the detection kit of pigeon sex identification, which is characterized in that the detection kit includes dove described in claim 1
The primer of sub- sex identification combines.
6. the detection kit of pigeon sex identification according to claim 5, which is characterized in that further include that PCR reactions are slow
Fliud flushing, Taq archaeal dna polymerases, dNTPs, 2 × Super Real PreMix, fluorescent reagent and Mg2+At least one of；And
DNA extracts reagents.
7. the detection kit of pigeon sex identification according to claim 6, which is characterized in that the fluorescent reagent includes
SYBR GreenI or SYBR GreenII.
8. the detection kit of pigeon sex identification according to claim 7, which is characterized in that the DNA extracts reagents
Including at least one of PBS solution, DNA eluents and Proteinase K.
9. such as detection kit the answering in identifying pigeon gender of claim 5-8 any one of them pigeon sex identifications
With.
10. application according to claim 9, which is characterized in that include the following steps：
To identify that the primer combination of pigeon gender carries out PCR reactions, the annealing temperature of the PCR reactions is 50-58 DEG C.
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| CN201810491784.0A CN108441550A (en) | 2018-05-21 | 2018-05-21 | Primer combination, detection kit and its application of pigeon sex identification |
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| CN201810491784.0A Pending CN108441550A (en) | 2018-05-21 | 2018-05-21 | Primer combination, detection kit and its application of pigeon sex identification |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109913556A (en) * | 2019-01-08 | 2019-06-21 | 北京市农林科学院 | A kind of primer, kit and its method for Rapid identification dove gender |
| CN117535392A (en) * | 2023-11-27 | 2024-02-09 | 广州动物园 | RPA primer and kit for identifying sex of swan and application |
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| CN105925690A (en) * | 2016-05-16 | 2016-09-07 | 华南农业大学 | Primer, kit and method for identifying dove sex |
| CN106434952A (en) * | 2016-11-02 | 2017-02-22 | 湖南农业大学 | Pigeon sex molecular identification method and primer pair used by method |
| CN106701910A (en) * | 2015-11-18 | 2017-05-24 | 深圳华大农业与循环经济科技有限公司 | Primer pair, kit and method for identifying gender of dove |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101333563A (en) * | 2008-07-23 | 2008-12-31 | 扬州大学 | Sex appraisal process for pigeon for meat |
| CN101935710A (en) * | 2010-10-14 | 2011-01-05 | 江苏省农业科学院 | Pigeon sex discriminating method |
| CN103397091A (en) * | 2013-07-30 | 2013-11-20 | 华中农业大学 | Polymerase chain reaction (PCR) method for identifying sex of young pigeons |
| CN203846028U (en) * | 2014-05-07 | 2014-09-24 | 江苏省家禽科学研究所 | Kit for pigeon sex determination |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109913556A (en) * | 2019-01-08 | 2019-06-21 | 北京市农林科学院 | A kind of primer, kit and its method for Rapid identification dove gender |
| CN117535392A (en) * | 2023-11-27 | 2024-02-09 | 广州动物园 | RPA primer and kit for identifying sex of swan and application |
| CN117535392B (en) * | 2023-11-27 | 2024-05-07 | 广州动物园 | RPA primer and kit for identifying sex of swan and application |
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