CN108384845A - Duck sex identification RT-PCR primer, kit and identification method - Google Patents
Duck sex identification RT-PCR primer, kit and identification method Download PDFInfo
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Abstract
The present invention relates to a kind of duck sex identification RT PCR primers, the nucleotide sequence such as sequence table SEQ ID NO of primer:1 and SEQ ID NO:Shown in 2, in addition duck property method for distinguishing is identified the present invention also provides a kind of kit comprising duck sex identification RT PCR primers and using the kit.The present invention can carry out the sex identification of duck individual on rna level, easy to operate, differentiate quickly, be of great significance particularly with the sex identification of rare cDNA samples.
Description
Technical field
The present invention relates to biological detection identification technology fields, and in particular to a kind of duck sex identification PCR, identification method and
Kit.
Background technology
Sex identification plays an important role in poultry farming, by identifying the male and female of 1 age in days duckling, can eliminate non-purpose
Gender reduces feeding cost, increases economic efficiency.With the difference of people's living habit and region, to different sexes meat duck
Demand has apparent lack of uniformity, and male and female are also different for the reaction of environment, nutritional condition etc., realizes that male and female divide group feeding
It supports, convenient for scientific management, improves production performance and carcass quality, while sex identification has more important meaning in basic research field
Justice.
Currently, the identification method of duck gender is generally divided into shape differential method, syrinx differential method, touches anus differential method and molecule
Horizontal identification method.Wherein shape differential method is distinguished according to the appearance traits feature of male and female duck, often there is erroneous judgement；Syrinx differential method needs
The syrinx of duck develop it is good could distinguish, go out shell to the adolescence be difficult distinguish；Although it is higher to touch anus differential method accuracy rate, deposit
It in the risk of some vertical transmission germ cross-infections, and touches anus and technology is required relatively high, need production experience abundant
It just can guarantee accuracy rate.Molecular methods are prejudgementing character using the clip size difference of special gene on sex chromosome W and Z
Not, for poultry W chromosomes there is only with female individuals, there are many conserved sequences on W chromosomes, can be right using these sequences
Fowl Sex is identified.Wpkc i genes on duck W chromosomes have well-conserved, can be used for duck gender mirror
It is fixed.But Molecular Identification is focused on DNA level at present, it is special for some single or mixing sample the cDNA obtained
It is not precious sample, gender is unknown, but gender is particularly important for subsequent research, its gender can not be identified by cDNA.
Sometimes some samples have carried out sex identification in DNA level, but during extracting RNA and reverse transcription is cDNA,
It is possible that individual sample numbers, which are obscured, leads to the uncertain problem of gender, sex identification can not be carried out in cDNA levels.
Invention content
Technical problem to be solved by the invention is to provide a kind of duck sex identification RT-PCR primer, identification method and examinations
Agent box can carry out duck sex identification in rna level.
The technical solution that the present invention solves above-mentioned technical problem is as follows：A kind of duck sex identification RT-PCR primer, including
Sense primer DWL and downstream primer DWR, sequence is respectively such as SEQ ID NO:1 and SEQ ID NO:Shown in 2.
Wherein SEQ ID NO:1 sequence is ATGGCCGACGGGATTGTT
SEQ ID NO:2 sequence is CTATTCGGCGGCGATAGT
A kind of duck sex identification kit, including the duck sex identification PCR primer, further includes for expanding β-
Internal control primer, Taq DNA polymerase, buffer buffer solutions and the dNTP of actin reference genes, the internal control primer includes upstream
Primer actL and downstream primer actR, sequence are respectively SEQ ID NO:3 and SEQ ID NO:4, wherein
SEQ ID NO:3 sequence is GCCATCTTTCTTGGGTAT
SEQ ID NO:4 sequence is CTTGATTTTCATCGTGCT
A kind of duck sex appraisal method, this method are：The total serum IgE for extracting sample to be tested, with the duck sex identification RT-
PCR primer, internal control primer, Taq DNA polymerase, buffer buffer solutions and dNTP carry out RT-PCR, then use Ago-Gel
Electrophoresis detection pcr amplification product is determined as female when occurring the specific amplification band of 363bp and 255bp in amplified production
Duck is determined as male duck when not occurring the specific amplification band of 363bp and 255bp in amplified production.The sample to be tested
Including but not limited to duck chest muscle, heart, liver, spleen, kidney, stomach, small intestine, testis, ovary, blood tissues.The RT-PCR
In the process, the response procedures of cDNA amplifications are：98 DEG C of pre-degenerations 2 minutes, 98 DEG C are denaturalized 10 seconds, and 59 DEG C of renaturation 30 seconds, 72 DEG C are prolonged
It stretches 1 minute, totally 35 recycle, and last 72 DEG C extend 5 minutes, and the nucleotides sequence of the specific amplification band of the 363bp is classified as
SEQ ID NO:5。
Wherein SEQ ID NO：5 sequence is
ATGGCCGACGGGATTGTTAGGGCGCAGGCCGCCTGGCCCGGTGGCGGCGCTGCCGCTGCTTTCGGAGAG
GTCGCCTGCAAGGGCAAGGAGGTCCCCGCCAACGTTCTCCGTGAGGATGAGCGGTCGTGGACGAGGAGTGCCTTGCG
TTCCGTGATAGTTCGCCTCAGGCTCCAGTGTTTTTTCCTAGTCCTTCCTGAGAAGGTAGTTGTCCGGTTATGTGAAG
CAGAAGATTCTGGCGGACCCCTTCTTGGGCGTTTCGTGGTTGTTGGCAAGAAGTGTGCTGCTAACCTGGACCTGACC
AATGGATTCCGGATGGCTGTGAATGCCCACCCTCGGGCCCTTCAGACTATCGCCGCCGAATAG
The beneficial effects of the invention are as follows：Duck sex identification PCR primer, kit and the identification side provided through the invention
Method can carry out duck sex identification on rna level, especially for some precious the sample cDNA, Ke Yijian obtained
Its fixed gender carries out follow-up study.
Description of the drawings
Fig. 1 is the agar expanded with primer pair female duck Wpkci genes using 1 duck sex identification of the embodiment of the present invention
Sugared gel electrophoresis figure, wherein Figure 1A are the product expanded using cDNA, and Figure 1B is the product expanded using DNA；
Fig. 2 is the amplification figure that the embodiment of the present invention 2 carries out reference gene β-actin using cDNA；
Fig. 3 is that the embodiment of the present invention 2 identifies duck property using duck sex identification kit of the present invention by duck different tissues
Other agarose gel electrophoresis result figure, wherein 1-4 swimming lanes are respectively female chest muscle, heart, liver, renal tissue cDNA genders
Identify that PCR product, 5-10 are male chest muscle, heart, liver, kidney, muscular stomach, testis tissue cDNA sex identification PCR products；
Fig. 4 is to identify duck property by the blood tissues of different ducks using 3 duck sex identification kit of the embodiment of the present invention
Other agarose gel electrophoresis result figure, wherein 1-5 swimming lanes are respectively 5 known male blood sample cDNA sex identifications
PCR product, 6-10 swimming lanes are respectively 5 known female individuals blood sample cDNA sex identification PCR products；
Fig. 5 is that duck Different Individual PCR is compared with RT-PCR qualification results, and wherein Fig. 5 A are the rna level RT- of sample 1-12
PCR testing results, Fig. 5 B are the DNA level PCR testing results of sample 1-12.
Specific implementation mode
Principles and features of the present invention are described below in conjunction with drawings and the specific embodiments, example is served only for solving
The present invention is released, is not intended to limit the scope of the present invention.
1 duck sex identification of embodiment is designed with PCR primer
According to the Partial cDNA Sequence (accession number of duck Wpkci genes in GeneBank：AB033883.1), by with duck base
Because of a group comparison, analysis prediction, the RT-PCR primer of design duck Wpkci, the wherein sequence of sense primer DWL and downstream primer DWR
Respectively SEQ ID NO:1 and SEQ ID NO:2.
CDNA, DNA of the Adult female cherry valley duck musculature preserved respectively using laboratory is templates, with primer DWL
Wpkci genes are expanded with DWD.PCR reactions are carried out according to TAKARA Taq (being purchased from TAKARA companies) specification, response procedures
For：98 DEG C of 2min, 98 DEG C of 10s, 59 DEG C of 30s, 72 DEG C of 1min, totally 35 recycle, 72 DEG C of extension 5min.
PCR product is detected with 1.2% agarose gel electrophoresis, as a result as shown in Figure 1A, 1B, from Ago-Gel respectively
The purpose band of recovery purifying 363bp, 3000bp-5000bp, and sample presentation cloning and sequencing, it is SEQ ID NO to respectively obtain sequence：
5 and SEQ ID NO：6 segment.Agarose gel electrophoresis from female sample the results show that obtain the purpose band of 363bp, together
When also obtain the non-specific band of 255bp, to the temperature in amplification system, Mg2+, the conditions such as cycle-index carried out it is excellent one by one
Change, the non-specific band of 255bp exists always, is due to there are other genes with Wpkci DNA homologs on W chromosomes, drawing
Caused by object generates mispairing with it, but this non-specific band has no effect on sex identification result.
SEQ ID NO:6 sequence is
ATGGCCGACGGGATTGTTAGGGCGCAGGCCGCCTGGCCCGGTGGCGGCGCTGCCGCTGCTTTCGGAGAG
GTCGCCTGCAAGGGCAAGGAGGTCCCCGCCAACGTTCTCCGTGAGGATGAGCGGTCGTGGACGAGGAGGTAGCCGCG
GCGTTGTTGTGCGAGTCTTTCCTTCCTTTCCCTCGGCCGCTCGCAGGCCCTCTGAAACGAGGGCCCGTCGCGGGTCC
CGGGAGTGCGGCAGGGCGGGTGGTGGCCTTGCTGCGGGAGGGAGGGAGGGAGGGACGGTCGGTCGGTCGGTCGGTCG
GGGTCTCTCCGGCTCGTGGCGCTCTCTACTGCAGCGCCTCTAAAACGCCGCGCTTCTTCGGTGCCTGCCCCCGGGGT
GCCACGGCGCGTACTGGGTCGCCGGCGCAGTCGGTCTCCTTGGGGGTTCGGACTGTATGCATATAGGCAGTGAGCAT
CGCAGTAGTGACCGTAATCAGTCGTTTCTGCGCTGGTGATAGCGTTGCAGAGACGCCCCGGCTCGGCTGTGCTAGGC
TTTGGTTATGGCACCACCCCCACCGCGCACACGAGCGCACGCTCTCTCCAAAGTGGCTTGTGAAAGGGAAGAATTGC
AGCTTTTCCTAGCTTTCTTAAACGCCTAGAAACCAGTCAATCCCGCAGGCTGCGAGAGCTTCCCAGCTCTCATTTCC
GTGCTGGAGGTGGGCCGTGATAAGAGCATGCTGGCAAGGTTCACTGATGCTGGTGCGCCGCAACGTGCTAGGAGCTA
AGTGAGCAGCTGCGGAGAGTTATGAAAAAGTTGCAGATGTTATCTGCAAGGTGCTAACTTTAAGTTAGTGTCCAGCC
TAACCGTTGTTGCTGTTCTTAAATTTGAAATGCTAAAGATAGGACAGTTAGGGATTTCCTCTGCTTCTGACGGCAGG
GTTTATCTGCAGCGTGGAAAACAGGCCCTTAGTGAAAGGGGCTGCGTTGCCCGCTCTGCGTGCTGACTCGTAGAATG
GTTAGGCTTGGAAGGCACCCTTAAAGATCATCTAGCCCAACCCGCCTGCCGCGGTCAGGGACGTGTTTCACTAGATC
GGGTTGCTCAAAGCGTTGACGCGTTGCTTGGAACAGCGCTTTCTTAATAGGTCTCGGTAATGTTGCAGCTGCCGCCC
GTTTGGAGTACTGGAAGCGTTCAACTGCTCTCCGGAAACCGTGCTTGCGGTAGCGCCCTGCCTTTACCAAAGGGCGC
GTTTCCTGTCTCCTTCGTGTTGAAGGCTGCGCGAGAAGTCTGTGTGTGTGTGTGCGCGTGCGCGCATCTGTTTTGAC
TCGGCCGTACTGGCTGCAGCAACGTATGTAACCTGAACGCTGTGCTTGATTGGAGAACCAGGCCGTTAACTTGAGTC
TGATGAGTGACTTGAGAGCAGACAACCTACTCTGACCGCTCTTAGTGCGACACGTTTCCTGGAGGACGAATTAGTTG
CAGTTTCTCGCTTGGTCTTAACTTGAGGCGGGGGGAGGGGGCGGGGGGCTTAGAACCACTTCTGGAGCGTAGTTGGC
TCTAGCGAATGGTCTTGAATTTGCTTAGCTCACTGTACCGAAGGCCTGTTTCACGGTTGAGCGATGTTATAGGGAGG
AGCGTGAGGAAGGCTTATAATTCTCGATTGTCGTGCAGTGAAAGGCGATGGTGAGTAAGAAGTACAAAGCATCTGTG
GAATCGAGTCTTATCGGCGGTGTTTCAGTCGCATCTAAAACACATAAAAATAGCGGAAGTCGCTAACCTGAAGTACT
CGGCTGTCTCGCTGCCCCCGTCACCTTGCGCGCTGGATCCTGCTTCTGGTGCCGTATGCAGTTTCTGTCTGCTGGTG
ATTTGGGGAGTTCCCTCGCAATTAGTTGCGAGTTGATGGTTTCCTACAGGTCGGTGGTATTCGTGTTTCGAAGGTGA
TGCCTTGGCCTCGGTCTGGAGGTGTCTGACGTCGGGCCGGCCGTAACGGGCTAATTTGGAACGTTCCTTCCCCTGAG
TAGGTGGCGTTCTCGAGGACGGAGAGTTAATACGGCCTCAGCCGGATTACTCCAGGTTCTTTGAAGATGCCTTCCTG
CTTCTGGCCGAGGCAATATTTGGACGTAACCGAAGGACGGTTAGCCATTGTTTGCCGCTTGTGTGTCTTGTGCGTAG
AAGGTAGAGAACTTTGCCTATTCAAAGCTCGCTTAAGAAGAATGGCTGTTTTGTCGTGCGCGTTTGGGGCGCCTGAT
AATCTGCTTCGGTAGGGCGAGTTCAGTGGGGCATAGTGATTTGCTTAAGCTGTTGCCTTTCTCTGAAGCCGACTACG
TTTTAACTGTTTACTCTTCCTCCAGTGCCTTGCGTTCCGTGATAGTTCGCCTCAGGCTCCAGTGTTTTTTCCTAGTC
CTTCCTGAGAAGGTAGTTGTCCGGTTATGTGAAGCAGAAGATTCTGGCGGACCCGTGAGTACTGTGGGAGCTTTCTG
TTCGCTAGGCTGGCTGTACCTGTAACTGGTTTGTAACTTCCTAGACTCTTTCCCGTCCCCTCTTTTCCTCTGGATGT
TGCAGAGGGGCGCACAGTAGGCTATTTGGGGTTGTCCGATGCTGTAGTCTTTCGCCTCTAGCTTGTAATGGAGCTGA
GCTTTTTGACTGAGAGCAGTGATATAAAGCGATGCTCGTCTCCAGCTACGCGCTTGCAGTGCAAAAAGCAGCGTTGT
TCCGACGTGCGGTCTGATGCCCGTTTTCACGCTTTCCGGACAGCGGGGGTGGTGCCTTGACTATGCAGAAGAGGGCT
TGGGGTTTGGTCAGGTCAGAGGCGAGGAAAGGGAAAGCTGGTTGAGCGGCGCTTGGGTGTTTGTTTCGCTGTTAGCT
ATGCAAGTCAAGTTCCAAGGCTCTGTGAGCTCACGCAAGAGAAAAGGCACCGTGAAGCTTTTATTTTCCGCCAGAGC
GGCTTACGTTGCCCGAAGCTATGACGGGGGCTTTAAGCCCCGAGACAAATTCAGCTTCTGTGACTGTTTTGTAGTCT
GTTACTGTCTAGAGGGTAATTCAAAGAGCAGCGGTTGAATTGTGAAGGATTAGCTAACCGTGTTCTGTGGGTGGTTC
TCTGCTCGCGTTCATTCTGTGCGTCGTGGCGCAGCACGTTAGGTGACGTTATTTCCTTTTTCCAGCTTCTTGGGCGT
TTCGTGGTTGTTGGCAAGAAGTGTGCTGCTAACCTGGACCTGACCAATGGATTCCGGATGGCTGTGAATGCCCACCC
TCGGGCCCTTCAGACTATCGCCGCCGAATAG
By SEQ ID NO：5 sequences and duck Wpkci Gene Partial cDNA Sequence (accession number：AB033883.1 it) compares, phase
It is 99% like property；By SEQ ID NO：5 sequences and SEQ ID NO：6 sequence alignments, it is 100% to have 3 sections of sequence similarities；It will
SEQ ID NO：5 sequences, SEQ ID NO：6 sequences are compared with Duck genome respectively, with biology such as DNASTAR, Augustus
Software analysis prediction, knows SEQ ID NO：5 sequences are the overall length CDS sequences of duck Wpkci genes, the length of 363bp,
SEQ ID NO：6 sequences are the full length DNA sequence of duck Wpkci genes, the length of 3257bp.SEQ ID NO：5 sequences
1-137 is exons 1 (137bp), 138-243 is exon 2 (106bp), 244-363 is exon 3 (120bp)；SEQ ID
NO：The 1-137 of 6 sequences is exons 1 (137bp), 138-2327 is introne 1 (2190bp), 2328-2433 is exon 2
(106bp), 2434-3137 intrones 2 (704bp), 3138-3257 are exon 3 (120bp)
2 duck different tissues sex identification of embodiment
1, Adult female, male overflowing sun sheldrake (animal and veterinary research institute of Academy of Agricultural Sciences of Wuhan City experiment duck are acquired respectively
) chest muscle, heart, liver, spleen, kidney, muscular stomach, small intestine, testis, ovary tissue, 5 individual (property of each tissue sampling
It is unknown), each total tissue RNA is extracted respectively using Trizol reagents (being purchased from Thermo Fisher companies), with the RNA of extraction
For the first chain of templated synthesis cDNA.
2, using the first chains of the cDNA of synthesis as template, PCR expansions are carried out with the primer actL and actR of reference gene β-actin
Increase while adjusting the concentration of cDNA, PCR reactions are carried out according to TAKARA Taq (being purchased from TAKARA companies) specification, response procedures
For：98 DEG C of 2min, 98 DEG C of 10s, 59 DEG C of 10s, 72 DEG C of 10s, totally 35 recycle, 72 DEG C of extension 5min.1.5% fine jade of PCR product
Sepharose electrophoresis detection, the results are shown in Figure 2, according to the corresponding cDNA concentration of PCR product band brightness adjustment, makes all cDNA
Concentration is almost the same.
3, using the cDNA of adjustment as template, with duck sex identification primer DWL and DWR and reference gene primer actL and
ActR expands Wpkci genes and β-actin reference genes (196bp) simultaneously.PCR reaction systems (15 μ l of total volume)：10×
1.5 μ L of Buffer, 0.3 μ L of dNTP (2.5mM), sense primer DWL (10 μM) 0.5 μ L, sense primer actL (10 μM) 0.3 μ L,
Downstream primer DWR (10 μM) 0.5 μ L, downstream primer actR (10 μM) 0.3 μ L, 0.2 μ L of Taq enzyme (2.5U), 1 cDNA μ L, sterilizing
10.4 μ L of deionized water.PCR response procedures are：98 DEG C of 2min, 98 DEG C of 10s, 59 DEG C of 10s, 72 DEG C of 10s, totally 35 recycle, 72 DEG C
Extend 5min.PCR product is detected with 1.5% agarose gel electrophoresis.
The results are shown in Figure 3, chest muscle, heart, liver, spleen, kidney, muscular stomach, small intestine, the testis tissue of all males
CDNA samples detect a band (196bp), chest muscle, heart, liver, spleen, kidney, muscular stomach, small intestine, the ovum of all females
Nest tissue cDNA sample detects three bands (363bp, 255bp, 196bp), accuracy rate 100%.The result shows that with duck difference
Tissue primer, method and the kit of the present invention can be used to carry out the duck sex identification based on RT-PCR.
The sex identification of the different duck blood samples of embodiment 3
Adult female, male overflowing sun sheldrake, Liancheng white duck, cherry valley duck, Jiang-Han Area meat duck A strains, Jiang-Han Area meat are acquired respectively
Duck B strains (duck is tested by animal and veterinary research institute of Academy of Agricultural Sciences of Wuhan City) blood sample, each kind/strain female, hero
Property each 5 individuals (known to gender) of acquisition, extract each blood sample total serum IgE respectively using Trizol reagents, following step with
Embodiment 2 is identical.
The results are shown in Figure 4, and the blood cDNA samples of all males detect a band (196bp), all females
Blood cDNA samples detect three bands (363bp, 255bp, 196bp), accuracy rate 100%.
4 duck Different Individual PCR of embodiment is compared with RT-PCR qualification results
Acquire overflowing sun sheldrake (duck is tested by animal and veterinary research institute of Academy of Agricultural Sciences of Wuhan City) blood of 1 week old male and female mixed breeding
Liquid sample acquires 50 individuals (gender is unknown) altogether, and DNA extraction kit (being purchased from Tiangeng company), Trizol is used to try respectively
The DNA and RNA of each blood sample of agent (being purchased from Thermo Fisher companies) extraction.
The method identified using RNA as template is with embodiment 2 and embodiment 3, as a result as shown in Figure 5A；
According to chicken CHD1-Z (accession number in GenBank：) and CHD1-W (accession number AC186875.2：AC177807.2) base
Because of sequence, design primer (bibliography：Liu Hongxiang etc., 2014, new general of a pair of the main poultry gender of Rapid identification draws
Object), the sequence of sense primer CHDF and downstream primer CHDR are respectively SEQ ID NO:7 and SEQ ID NO:8,
Wherein SEQ ID NO:7 sequence is TGCAGAAGCAATATTACAAGT3
SEQ ID NO:8 sequence is AATTCATTATCATCTGGTGG
The DNA of extraction is template, with primer CHDF and CHDR amplification duck CHD1-Z (467bp) and CHD1-W genes
(326bp) identifies gender from DNA level.PCR reactions are carried out according to TAKARA Taq (being purchased from TAKARA companies) specification, instead
The program is answered to be：98 DEG C of 2min, 98 DEG C of 10s, 49 DEG C of 20s, 72 DEG C of 30s, totally 35 recycle, 72 DEG C of extension 5min.PCR product is used
1.5% agarose gel electrophoresis detects, as a result as shown in Figure 5 B, according to the band number Sex estimation of PCR product band：Detection
It is male to go out a band (467bp), detects that two bands (467bp and 326bp) are female.
Fig. 5 A and Fig. 5 B the result shows that, with the present invention the duck sex identification primer based on RT-PCR, identification method and
Kit is consistent with the result of sex identification is carried out with DNA.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent replacement, improvement and so on should all be included in the protection scope of the present invention.
Claims (8)
1. a kind of duck sex identification RT-PCR primer, which is characterized in that including
Sense primer DWL, sequence are SEQ ID NO:1；
Downstream primer DWR, sequence are SEQ ID NO:2.
2. a kind of duck sex identification kit, which is characterized in that include duck sex identification RT-PCR as described in claim 1
Primer.
3. a kind of duck sex identification kit according to claim 2, which is characterized in that further include for expanding β-
The internal control primer of actin reference genes, the internal control primer include sense primer actL and downstream primer actR, sequence difference
For SEQ ID NO:3 and SEQ ID NO:4.
4. a kind of duck sex identification kit according to claim 3, which is characterized in that further include TaqDNA polymerizations
Enzyme, buffer buffer solutions and dNTP.
5. a kind of duck sex appraisal method, which is characterized in that, should using the duck sex identification kit described in claim 4
Method is：The total serum IgE for extracting sample to be tested is polymerize with the duck sex identification RT-PCR primer, internal control primer, TaqDNA
Enzyme, buffer buffer solutions and dNTP carry out RT-PCR, then agarose gel electrophoresis are used to detect pcr amplification product, work as amplification
Occur being determined as female duck when the specific amplification band of 363bp and 255bp in product, when not occurring 363bp in amplified production
It is determined as male duck when with the specific amplification band of 255bp.
6. a kind of duck sex appraisal method according to claim 5, which is characterized in that the sample to be tested is duck to be measured
One or more of chest muscle, heart, liver, spleen, kidney, stomach, small intestine, testis, ovary, blood tissues.
7. a kind of duck sex appraisal method according to claim 5, which is characterized in that during the RT-PCR, cDNA
The response procedures of amplification are：98 DEG C of pre-degenerations 2 minutes, 98 DEG C are denaturalized 10 seconds, 59 DEG C of renaturation 30 seconds, and 72 DEG C extend 1 minute, and totally 35
A cycle, last 72 DEG C extend 5 minutes.
8. a kind of duck sex appraisal method according to claim 5, which is characterized in that the specific amplification of the 363bp
The nucleotides sequence of band is classified as SEQ ID NO:5.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111387134A (en) * | 2020-03-06 | 2020-07-10 | 武汉市农业科学院 | Method for improving reproductive performance of small and medium-sized meat ducks cultured in cages |
| 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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| CN111387134A (en) * | 2020-03-06 | 2020-07-10 | 武汉市农业科学院 | Method for improving reproductive performance of small and medium-sized meat ducks cultured in cages |
| 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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