CN112195252B - Multiplex PCR primer and detection method for detecting pseudobagrus ussuriensis gender - Google Patents
Multiplex PCR primer and detection method for detecting pseudobagrus ussuriensis gender Download PDFInfo
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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
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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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- 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
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention belongs to the technical field of neutral identification in aquaculture, and discloses a multiplex PCR primer for detecting the sex of pseudobagrus ussuriensis and a detection method, and provides 3 male specific loci of pseudobagrus ussuriensis, the nucleotide sequence of which is SEQ ID NO:1-3; the invention also provides a multiplex PCR primer designed from the sex-specific site, and the nucleotide sequence of the multiplex PCR primer is SEQ ID NO:4-9; according to the invention, 3 male specific markers of the pseudobagrus ussuriensis are integrated, a multiplex PCR primer for sex detection and a detection method thereof are developed, so that interference of zero alleles is effectively eliminated, sex detection of the pseudobagrus ussuriensis can be efficiently and accurately realized, and the method can be used for detection and analysis of sex proportion and sex structure of various pseudobagrus ussuriensis populations; the invention also discloses a kit for detecting the gender of the pseudobagrus ussuriensis; the invention has high detection efficiency and accurate identification result, and has important significance for promoting the whole male breeding process of the pseudobagrus ussuriensis.
Description
Technical Field
The invention belongs to the technical field of sex identification in aquaculture, and particularly relates to a multiplex PCR primer for detecting the sex of pseudobagrus ussuriensis and a detection method thereof in sex identification of pseudobagrus ussuriensis.
Background
Multiple PCR (multiplex PCR) is a rapid and efficient PCR technique developed on the basis of common PCR. The technology is that more than two pairs of primers are added into a PCR system, so that more than two target fragments can be amplified simultaneously. Compared with the common PCR, the technology can achieve the effect of multiple times of PCR through one time of PCR, effectively reduce the detection cost and improve the detection efficiency. The method is widely applied to a plurality of research fields of population genetic diversity analysis, species identification, paternity test, disease diagnosis, sex detection and the like of organisms.
Pseudobagrus ussuriensis (Pseudobagrus ussuriensis) is a fish belonging to the family of the Pseudobagrus ussuriensis which is distributed in the river system from Heilongjiang to Zhujiang in China. The method has delicious taste and rich nutrition, is deeply favored by consumers, has little difficulty in cultivation technology and few cultivation diseases, and continuously expands cultivation range, so that cultivation is carried out in more than ten provinces nationwide. The Pseudobagrus ussuriensis has obvious male growth advantage, and can reach more than 3 times of female at two ages, so that the Pseudobagrus ussuriensis has great full male breeding potential.
Rapid and accurate sexing of target individuals is particularly important in all-male breeding. Although female and male individuals can be identified by morphology, this is limited to the identification of physiological sexes. In breeding, it is often necessary to perform sex reversal treatment on a target population with an estrogen or an androgen, and the genetic sex of the sex-reversed individual cannot be identified by morphological characteristics. In addition, morphological characteristics of female and male individuals are not obvious before one age, and sex cannot be identified by morphology. Therefore, developing sex-specific molecular markers is an effective way to rapidly and accurately identify the sex of pseudobagrus ussuriensis. We have developed 1 male-specific SCAR marker （Sequence Characterized Amplified Regions）(Pan ZJ, Li XY, Zhou FJ, Qiang XG, Gui JF. Identification of Sex-Specific Markers Reveals Male Heterogametic Sex Determination in Pseudobagrus ussuriensis. Marine Biotechnology, 2015,17(4): 441-451.）, and 1 male-specific microsatellite marker (grant bulletin number: CN 106811540B) based on AFLP technology (AMPLIFIED FRAGMENT LENGTH polymorphs). However, in practical applications, we find that both markers have certain drawbacks: because SCAR markers identify sex by judging whether an individual has an amplified band at a corresponding site, if a specific individual has point mutation at a flanking sequence where a primer is located, no amplified band exists in the individual, namely zero Allele (Null Allele) appears, and the sex judgment of the individual by using the markers is possibly wrong; the microsatellite marker is used for sex identification by whether a certain individual has one or two amplified bands, and if a certain individual has zero alleles at the position of a microsatellite marker primer, the microsatellite marker has no amplified band in the individual, so that sex identification of the individual cannot be carried out.
Disclosure of Invention
The invention aims to provide a multiplex PCR primer and a detection method for detecting the sex of pseudobagrus ussuriensis, namely a group of specific multiplex PCR primers for detecting the sex of the pseudobagrus ussuriensis, which are used as a favorable tool for accurately and efficiently detecting the sex of the pseudobagrus ussuriensis, and can effectively solve the problems in the background technology.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a multiplex PCR primer for detecting the sex of pseudobagrus ussuriensis features that its nucleotide sequence is SEQ ID No. 1-3.
A set of primers capable of specifically amplifying at least one sequence of SEQ ID NO. 1-3.
The primer sequences of the primer groups are shown in SEQ ID NO. 4-9.
A kit for detecting the sex of pseudobagrus ussuriensis, which comprises the primer set.
Further, the kit also comprises reagents commonly used in PCR technology, such as Taq DNA polymerase, 10×PCR Buffer, dNTPs, redistilled water and MgCl 2 according to practical situations. The solvents for each of the above reagents were sterile ultrapure water.
A sex detection method of multiple PCR primer group or kit in pseudobagrus ussuriensis.
A method for detecting the sex of pseudobagrus ussuriensis comprises the following steps:
S1, DNA extraction: extracting genome DNA of a target individual;
s2, multiplex PCR amplification: performing PCR amplification by using the multiple PCR primer group and the target individual genome DNA obtained in the step S1 as a template to obtain a target individual multiple PCR amplification product;
S3, electrophoresis detection of amplification products: detecting multiple PCR amplified products obtained in the S2 by agarose gel electrophoresis and GoldView staining method;
S4, sex identification: sex determination is carried out according to the presence or absence of multiplex PCR amplification products of each individual, and if one individual has no amplification band at 246 bp, 597 bp and 823 bp, the individual is female; if an individual has an amplified band in at least one of three places, the individual is male.
S1, extracting genomic DNA of tail fin tissues of a target individual by adopting a general phenol-chloroform DNA extraction method; but is not limited thereto.
As a preferred embodiment, the reaction system for PCR amplification: the total volume is 13-25 [ mu ] L, the template DNA (about 0.8-1.5 [ mu ] L) comprises 60-80 ng, 1U Taq DNA polymerase (0.1-0.2 [ mu ] L), 1.3-2.5 [ mu ] L of 10 xPCR Buffer,0.4-1 [ mu ] L dNTPs (2.5 mM), 0.3-0.5 [ mu ] L of each of SEQ4 and SEQ5 primers, 0.2-0.4 [ mu ] L of each of SEQ6 and SEQ7 primers, 0.4-0.6 [ mu ] L of each of SEQ8 and SEQ9 primers (the concentration of each primer is 2.5 [ mu ] M), and the sterilized ultrapure water is 9-18 [ mu ] L; the PCR amplification procedure was: pre-denaturation at 94 ℃ for 5-7 min; denaturation at 94℃for 35-45 seconds, annealing at 65℃to 50℃for 35-45 seconds, and extension at 72℃for 40 seconds-1 minute, each cycle of annealing temperature reduction by 1℃and running for 15-17 cycles; denaturation at 94℃for 35-45 seconds, annealing at 50℃for 35-45 seconds, elongation at 72℃for 40-50 seconds, run for 18-22 cycles; finally, the PCR product was stored at 4℃for a final extension of 8-15 minutes at 72 ℃.
As a preferred technical scheme, the detailed process of S3 is: multiplex PCR amplified products were separated by electrophoresis in 1% -2% agarose gel with GoldView stain added, at a constant voltage of 150-200V for 15-30 minutes.
Compared with the prior art, the invention has the following beneficial effects:
1. the effect of multiple PCR detection can be achieved by one-time PCR, the detection efficiency is high, and the identification result is accurate;
2. the problems of misjudgment, incapability of identification and the like in the prior art caused by zero alleles in part of individuals to be detected are effectively solved, and the detection result is more reliable and stable;
3. the invention overcomes the defects of the prior art and provides a multiplex PCR technology and a detection method for detecting the sex of pseudobagrus ussuriensis, which comprise the steps of multiplex PCR primer amplification, amplification product electrophoresis detection, band analysis, sex identification and the like. The invention constructs a multiplex PCR system for detecting the sex of the pseudobagrus ussuriensis by integrating three specific markers, can further improve the efficiency and accuracy of the sex detection of the pseudobagrus ussuriensis, provides a favorable tool for real-time monitoring of the sex structures of various groups of the pseudobagrus ussuriensis, and has important significance for promoting the whole male breeding process of the pseudobagrus ussuriensis;
therefore, the invention develops a group of multiplex PCR primers for detecting the sex of the Uperzia serrata, and the primer group not only can exclude the sex identification error caused by zero alleles, but also can exclude the situation that the sex cannot be identified caused by the zero alleles, and can realize the sex detection of the Uperzia serrata more accurately and efficiently.
Drawings
FIG. 1 shows agarose gel electrophoresis patterns in each of 10 Zusanli pairs of female and male PCR primers disclosed by the invention, and lane M is DM2000 DNA MARKER.
Detailed Description
The invention is further described in connection with the following detailed description, in order to make the technical means, the creation characteristics, the achievement of the purpose and the effect of the invention easy to understand.
The specific experimental conditions and methods are not noted in the examples below, and are generally in accordance with conventional conditions such as: J. sambrook et al, scientific press, 2002, guidance on molecular cloning experiments (third edition), or take care of conditions recommended by the manufacturer.
EXAMPLE 1 composition and formulation of multiple PCR System for detecting the sex of Pseudobagrus ussuriensis
(1) The PCR system comprises:
dNTPs (10 mM) are a product of the division of biological engineering (Shanghai);
Taq DNA polymerase (5U/[ mu ] L), 10 XPCR Buffer is Beijing kang is a century company product;
primers were synthesized by the division of biological engineering (Shanghai) Co.
(2) 10 XPCR Buffer composition:
Tris-HCl (pH8.3) 100 mM、KCl 500 mM、MgCl2 15 mM。
(3) Preparing a PCR reaction system:
The total volume is 25 [ mu ] L, template DNA containing 60-80 ng [ mu ] L, 1U Taq DNA polymerase, 2.5 [ mu ] L10×PCR Buffer,1 [ mu ] L dNTP (2.5 mM), each of SEQ4 and SEQ5 primers is 0.3 [ mu ] L, each of SEQ6 and SEQ7 primers is 0.2 [ mu ] L, each of SEQ8 and SEQ9 primers is 0.4 [ mu ] L (each primer concentration is 2.5 [ mu ] M), and sterilized ultrapure water is 18 [ mu ] L.
(4) Agarose gel preparation:
agarose 0.8 g, 0.5×TAE 40 mL, goldView stain (Beijing kang is century company product) 3 μl.
Example 2 evaluation of sex detection Effect in propagation parents of Pseudobagrus ussuriensis of known sex
S1, randomly selecting 100 tails of each of Usnea philippinensis female and male parents judged according to morphological characteristics in a breeding field, shearing a small amount of tail fin tissues, respectively extracting genome DNA by a phenol-chloroform method, and diluting the DNA concentration to 60-80 ng/mu L by sterilized ultrapure water.
S2, preparing a PCR reaction mixture by using the genomic DNA extracted in the S1 as a template according to the system of the (3) in the embodiment 1.
S3, performing amplification on a PCR instrument, and performing pre-denaturation at 94 ℃ for 5 minutes; denaturation at 94 ℃ for 45 seconds, annealing at 65 ℃ to 50 ℃ for 40 seconds, and extension at 72 ℃ for 1 minute, annealing temperature reduction of 1 ℃ per cycle, and 15 cycles are run; denaturation at 94 ℃ for 40 seconds, annealing at 50 ℃ for 40 seconds, extension at 72 ℃ for 50 seconds, run 22 cycles; final extension at 72 ℃ for 10min and PCR products were stored at 4 ℃.
S4. PCR products were separated by electrophoresis in a 2% agarose gel prepared according to the system of (4) in example 1 at a constant voltage of 150V for 30 minutes and each gel was scanned and stored by photographing using a Gel DocTM EZ (Bio-RAD) gel imager.
Sex identification method: sex identification is carried out according to the presence or absence of electrophoresis bands of multiplex PCR amplification products of each individual, and if one individual does not have amplification bands at 246 bp, 597 bp and 823 bp, the individual is female; if an individual has an amplified band in at least one of the three band positions, the individual is male, as shown in FIG. 1. The results of the identification are shown in Table 1.
TABLE 1 evaluation results of the effect of multiple PCR detection of the sex of pseudobagrus ussuriensis according to the present invention
From the results, the female and male individuals identified by the multiplex PCR primer are matched with the morphologically identified sex, and the accuracy of the multiplex PCR primer identification detection is extremely high.
Comparative example multiple PCR of the present invention was compared with Male specific microsatellite (CN 106811540B) sex identification effect
S1, randomly selecting 216-tail Usnea pseudolaris with unknown gender, shearing a small amount of tail fin tissues, extracting genome DNA by a phenol-chloroform method, and diluting the DNA concentration to 60-80 ng/MuL by sterilized ultrapure water.
S2, multiplex PCR detection is carried out according to the method shown in the embodiment 1 and the embodiment 2, and male-specific microsatellite marker detection is carried out according to the detection method described in CN 106811540B. The pair of detection results are shown in table 2:
TABLE 2 comparison of the sex detection effect of multiple PCR and Male specific microsatellite markers of the invention on Pseudobagrus ussuriensis
The comparison result shows that the multiple PCR can accurately detect the sex of each individual, the detection success rate reaches 100 percent, and the male specific microsatellite marker can not detect the sex of 2 individuals in 216 individuals, and the detection success rate is 99.1 percent.
Therefore, the multiplex PCR primer of the invention can effectively eliminate the situations of wrong sex identification or incapacity of sex identification caused by zero alleles, and the like. The reason is that the multiplex PCR primer is a composite mark integrated with three male specific marks, can amplify three male specific bands of 246 bp, 597 bp and 823 bp, can carry out sex identification by amplifying the bands at least at one position, has extremely low probability of zero allele at the position of the three pairs of primers, and can effectively avoid interference of the zero allele on sex identification, thereby improving the accuracy and reliability of sex identification.
SEQ1
<110> Huaiyin academy of teachers and students
<120> Multiplex PCR primer for detecting the sex of pseudobagrus ussuriensis and detection method
<130> 2020
<170> PatentIn version 3.3
<211> 823
<212> DNA
<213> SEQ1
cagtaacggg tatcgtccaa tataacacaa cgattgatgc tggcggaggc aacttgtatc 60
gccagtggct gatagactct gcccaccaag tcgtcactaa cactacgtgt gtggtgtgtc 120
acgacaggtc caagcagata cctcatataa tgcctgttag gggcgtaagg gaatgtgata 180
ggcccccgta caatgacacc catctatgcc cagcactttg tctattagga tcagcggcca 240
gaacacatgg gccaaaatgg atgggtaaca taagtagatc ctgcacgtat caacccgtaa 300
tctctccaat cccaacagat atgacagtta ggacgtgccc taaccaagag tatcccctgt 360
gcatacgttc aaagggagct gtgtgggtgg ggttccttcc taaggtggcc tgtaaagata 420
cgttagatgc ccatcatcct atagtaaatg tgctcccgcc atgtaacaca tcagaatatg 480
ggtgtgagac ggctatccca ggcttaatga ccgtccccag tcttagattt ggcaccctgc 540
cactagcaga tattttttgg tactgtggag acggcacagc cctccaaatg ctcccccgaa 600
tgtggcatgg cttgtgtgcc ccagttgtat tggaaggagg ccttacagta ctggcgttaa 660
atgactccat cttagaccga cttctcactc agactcccac tcgcgccata cgcactaggc 720
gtgatgtctc aagctacctg gagcacacca actcctctgt gctccctgcc tggtcagcag 780
atgcacatag actggaaagg agagcccgta gtgtgaatag aaa 823
SEQ 2
<110> Huaiyin academy of teachers and students
<120> Multiplex PCR primer for detecting the sex of pseudobagrus ussuriensis and detection method
<130> 2020
<170> PatentIn version 3.3
<211> 597
<212> DNA
<213> SEQ2
gtgagttgcc ctgaggtctt ctgtgcttag caaacacagt ccagttgtgg tagtaaatgc 60
gtcgactgat cgaggacaaa gatgttgttt ctattcagtt tgaaataaac cttccagatg 120
tttcctaaac ttttgcttag ctgtatttca gtttgttgaa ctgttactat tctgccgttc 180
tatatcgaat agatattacc ataccttatt gtttattaat gtaaacaaat taaacccttg 240
tagaaaagat tcatgacctt caaccaatgt ttaattgatt attccatgaa taaaattcta 300
agctaaagtt gatattgtac ttaacatcac cagtgtgttt gtattcataa taaaatggga 360
gaactctcat ttacattaat atttattatt aatgatcact agcaactagg agatcatgtg 420
cagtgtataa gctagaatca tgactgtaga aggaaattaa agataccctt cgggtttctt 480
tagcacacag gtaaaccccc ttttgctttt ctaacagtct tcactcattt gtaaaggctt 540
ttcacttgaa tttgaatcaa ccacaagagt atttctaaag tcaagcaccg atgtcag 597
SEQ 3
<110> Huaiyin academy of teachers and students
<120> Multiplex PCR primer for detecting the sex of pseudobagrus ussuriensis and detection method
<130> 2020
<170> PatentIn version 3.3
<211> 246
<212> DNA
<213> SEQ3
gaagtcaatg ttccctgttg ttttaaagca gtcctgcctt ttgcttatct aatcttttat 60
agattataca tcttatatgt ctattcagtc tattatagag catacaactt tatacatata 120
gagcatacat atacttatgg ttatctctag acttgtgatt atcaaaggag tataaaatgg 180
tacacataag gtgaatttct ttattgaggt cataaagata tatatgcaag agttcgtggt 240
tgagaa 246
SEQ1
<110> Huaiyin academy of teachers and students
<120> Multiplex PCR primer for detecting the sex of pseudobagrus ussuriensis and detection method
<130> 2020
<170> PatentIn version 3.3
<211> 823
<212> RNA
<213> SEQ1
cagtaacggg tatcgtccaa tataacacaa cgattgatgc tggcggaggc aacttgtatc 60
gccagtggct gatagactct gcccaccaag tcgtcactaa cactacgtgt gtggtgtgtc 120
acgacaggtc caagcagata cctcatataa tgcctgttag gggcgtaagg gaatgtgata 180
ggcccccgta caatgacacc catctatgcc cagcactttg tctattagga tcagcggcca 240
gaacacatgg gccaaaatgg atgggtaaca taagtagatc ctgcacgtat caacccgtaa 300
tctctccaat cccaacagat atgacagtta ggacgtgccc taaccaagag tatcccctgt 360
gcatacgttc aaagggagct gtgtgggtgg ggttccttcc taaggtggcc tgtaaagata 420
cgttagatgc ccatcatcct atagtaaatg tgctcccgcc atgtaacaca tcagaatatg 480
ggtgtgagac ggctatccca ggcttaatga ccgtccccag tcttagattt ggcaccctgc 540
cactagcaga tattttttgg tactgtggag acggcacagc cctccaaatg ctcccccgaa 600
tgtggcatgg cttgtgtgcc ccagttgtat tggaaggagg ccttacagta ctggcgttaa 660
atgactccat cttagaccga cttctcactc agactcccac tcgcgccata cgcactaggc 720
gtgatgtctc aagctacctg gagcacacca actcctctgt gctccctgcc tggtcagcag 780
atgcacatag actggaaagg agagcccgta gtgtgaatag aaa 823
SEQ 2
<110> Huaiyin academy of teachers and students
<120> Multiplex PCR primer for detecting the sex of pseudobagrus ussuriensis and detection method
<130> 2020
<170> PatentIn version 3.3
<211> 597
<212> RNA
<213> SEQ2
gtgagttgcc ctgaggtctt ctgtgcttag caaacacagt ccagttgtgg tagtaaatgc 60
gtcgactgat cgaggacaaa gatgttgttt ctattcagtt tgaaataaac cttccagatg 120
tttcctaaac ttttgcttag ctgtatttca gtttgttgaa ctgttactat tctgccgttc 180
tatatcgaat agatattacc ataccttatt gtttattaat gtaaacaaat taaacccttg 240
tagaaaagat tcatgacctt caaccaatgt ttaattgatt attccatgaa taaaattcta 300
agctaaagtt gatattgtac ttaacatcac cagtgtgttt gtattcataa taaaatggga 360
gaactctcat ttacattaat atttattatt aatgatcact agcaactagg agatcatgtg 420
cagtgtataa gctagaatca tgactgtaga aggaaattaa agataccctt cgggtttctt 480
tagcacacag gtaaaccccc ttttgctttt ctaacagtct tcactcattt gtaaaggctt 540
ttcacttgaa tttgaatcaa ccacaagagt atttctaaag tcaagcaccg atgtcag 597
SEQ 3
<110> Huaiyin academy of teachers and students
<120> Multiplex PCR primer for detecting the sex of pseudobagrus ussuriensis and detection method
<130> 2020
<170> PatentIn version 3.3
<211> 246
<212> RNA
<213> SEQ3
gaagtcaatg ttccctgttg ttttaaagca gtcctgcctt ttgcttatct aatcttttat 60
agattataca tcttatatgt ctattcagtc tattatagag catacaactt tatacatata 120
gagcatacat atacttatgg ttatctctag acttgtgatt atcaaaggag tataaaatgg 180
tacacataag gtgaatttct ttattgaggt cataaagata tatatgcaag agttcgtggt 240
tgagaa 246
SEQ4
<110> Huaiyin academy of teachers and students
<120> Multiplex PCR primer for detecting the sex of pseudobagrus ussuriensis and detection method
<130> 2020
<170> PatentIn version 3.3
<211> 20
<212> DNA
<213> SEQ4
CAGTAACGGG TATCGTCCAA 20
SEQ5
<110> Huaiyin academy of teachers and students
<120> Multiplex PCR primer for detecting the sex of pseudobagrus ussuriensis and detection method
<130> 2020
<170> PatentIn version 3.3
<211> 21
<212> DNA
<213> SEQ5
TTTCTATTCA CACTACGGGC T 21
SEQ6
<110> Huaiyin academy of teachers and students
<120> Multiplex PCR primer for detecting the sex of pseudobagrus ussuriensis and detection method
<130> 2020
<170> PatentIn version 3.3
<211> 20
<212> DNA
<213> SEQ6
GTGAGTTGCC CTGAGGTCTT 20
SEQ7
<110> Huaiyin academy of teachers and students
<120> Multiplex PCR primer for detecting the sex of pseudobagrus ussuriensis and detection method
<130> 2020
<170> PatentIn version 3.3
<211> 20
<212> DNA
<213> SEQ7
CTGACATCGG TGCTTGACTT 20
SEQ8
<110> Huaiyin academy of teachers and students
<120> Multiplex PCR primer for detecting the sex of pseudobagrus ussuriensis and detection method
<130> 2020
<170> PatentIn version 3.3
<211> 19
<212> DNA
<213> SEQ8
GAAGTCAATG TTCCCTGTT 19
SEQ9
<110> Huaiyin academy of teachers and students
<120> Multiplex PCR primer for detecting the sex of pseudobagrus ussuriensis and detection method
<130> 2020
<170> PatentIn version 3.3
<211> 19
<212> DNA
<213> SEQ9
TTCTCAACCA CGAACTCTT 19
Claims (7)
1. A multiplex PCR primer set capable of amplifying at least one sequence of SEQ ID NO. 1-3.
2. The primer set according to claim 1, wherein: the sequence of the primer group is
SEQ4: 5'-CAGTAACGGGTATCGTCCAA -3'
SEQ5: 5'-TTTCTATTCACACTACGGGCT -3'
SEQ6: 5'-GTGAGTTGCCCTGAGGTCTT -3'
SEQ7: 5'-CTGACATCGGTGCTTGACTT -3'
SEQ8: 5'-GAAGTCAATGTTCCCTGTT -3'
SEQ9: 5'-TTCTCAACCACGAACTCTT -3'。
3. The multiple PCR primer kit for identifying the gender of the pseudobagrus ussuriensis is characterized by comprising the following components: the kit comprises the primer set of claim 1 or 2.
4. A kit according to claim 3, wherein: the kit also comprises reagents commonly used in PCR technology.
5. The method for detecting the sex of the pseudobagrus ussuriensis by using the multiplex PCR primer is characterized by comprising the following steps of: the method comprises the following steps: s1, DNA extraction: extracting the genome DNA of a target individual;
s2, multiplex PCR amplification: performing PCR amplification by using the primer set according to claim 2 and taking the target individual genome DNA obtained in the step S1 as a template to obtain a target individual multiplex PCR amplification product;
s3, electrophoresis detection of amplification products: detecting multiple PCR amplified products obtained in the S2 by agarose gel electrophoresis and GoldView staining method;
S4, sex identification: sex determination is carried out according to the presence or absence of multiplex PCR amplification products of each individual, and if one individual has no amplification band at 246 bp, 597 bp and 823 bp, the individual is female; if an individual has an amplified band in at least one of three places, the individual is male.
6. The method for detecting the sex of the pseudobagrus ussuriensis by using the multiplex PCR primer according to claim 5, which is characterized by comprising the following steps: the reaction system of PCR amplification: the total volume is 13-25 [ mu ] L, the template DNA comprises 60-80 ng [ mu ] L, 1U Taq DNA polymerase 0.1-0.2 [ mu ] L,1.3-2.5 [ mu ] L,10 XPCR Buffer,0.4-1 [ mu ] L dNTP 2.5mM, SEQ4, 0.3-0.5 [ mu ] L each of SEQ5 primers, 0.2-0.4 [ mu ] L each of SEQ6 and SEQ7 primers, 0.4-0.6 [ mu ] L each of SEQ8 and SEQ9 primers, the concentration of each primer is 2.5 [ mu ] M, and sterilized ultrapure water; the PCR amplification procedure was: pre-denaturation at 94 ℃ for 5-7 min; denaturation at 94℃for 35-45 seconds, annealing at 65℃to 50℃for 35-45 seconds, and extension at 72℃for 40 seconds-1 minute, each cycle annealing temperature being reduced by 1℃and running for 15-16 cycles; denaturation at 94℃for 35-45 seconds, annealing at 50℃for 35-45 seconds, elongation at 72℃for 40-50 seconds, run for 18-22 cycles; finally, the PCR product was stored at 4℃for a final extension of 8-15 minutes at 72 ℃.
7. The method for detecting the sex of the pseudobagrus ussuriensis by using the multiplex PCR primer according to claim 5, which is characterized by comprising the following steps: the detailed process of the S3 is as follows: multiplex PCR amplified products were separated by electrophoresis in 1% -2% agarose gel with GoldView stain added, at a constant voltage of 150-200V for 15-30 minutes.
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| CN106011300A (en) * | 2016-08-04 | 2016-10-12 | 淮阴师范学院 | PCR primer pair used for identifying genetic sex of Pseudobagrus ussuriensis, and rapid identification method thereof |
| CN106811540A (en) * | 2017-03-22 | 2017-06-09 | 淮阴师范学院 | It is a kind of to identify female ussuriensis, male individual microsatellite marker and specific primer and application |
| CN107996468A (en) * | 2017-12-20 | 2018-05-08 | 淮阴师范学院 | A kind of ussuriensis Genetic male reverses physiology female fish to educate screen method |
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| CN106011300A (en) * | 2016-08-04 | 2016-10-12 | 淮阴师范学院 | PCR primer pair used for identifying genetic sex of Pseudobagrus ussuriensis, and rapid identification method thereof |
| CN106811540A (en) * | 2017-03-22 | 2017-06-09 | 淮阴师范学院 | It is a kind of to identify female ussuriensis, male individual microsatellite marker and specific primer and application |
| CN107996468A (en) * | 2017-12-20 | 2018-05-08 | 淮阴师范学院 | A kind of ussuriensis Genetic male reverses physiology female fish to educate screen method |
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