CN112195252A - Multiple PCR primers for pseudobagrus ussuriensis gender detection and detection method - Google Patents
Multiple PCR primers for pseudobagrus ussuriensis gender detection and detection method Download PDFInfo
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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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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention belongs to the technical field of identification of neutral species in aquaculture, and discloses a multiplex PCR primer and a detection method for pseudobagrus ussuriensis gender detection, which provide 3 male specific sites of pseudobagrus ussuriensis, wherein the nucleotide sequence of the male specific sites is SEQ ID NO: 1-3; the invention also provides a multiple PCR primer designed from the sex specific locus, and the nucleotide sequence of the multiple PCR primer is SEQ ID NO: 4-9; the invention integrates 3 pseudobagrus ussuriensis male specific markers, develops the multiple PCR primers for sex detection and the detection method thereof, effectively eliminates the interference of zero allele, can efficiently and accurately realize the sex detection of the pseudobagrus ussuriensis, and can be used for detecting and analyzing the sex ratio and the sex structure of each pseudobagrus ussuriensis group; the invention also discloses a kit for the pseudobagrus ussuriensis gender detection; the method is high in detection efficiency and accurate in identification result, and has important significance for promoting the process of pseudobagrus ussuriensis all-male breeding.
Description
Technical Field
The invention belongs to the technical field of identification of the sex in aquaculture, and particularly relates to a multiple PCR primer for pseudobagrus ussuriensis sex detection and a detection method thereof in pseudobagrus ussuriensis sex identification.
Background
Multiplex PCR (multiplex PCR) is a rapid and efficient PCR technology developed on the basis of ordinary PCR. The technology is that more than two pairs of primers are added into a PCR system, and more than two target fragments can be amplified simultaneously. Compared with the common PCR, the technology can achieve the effect of multiple PCR by one-time PCR, can 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 identification, disease diagnosis, sex detection and the like of organisms.
Pseudobagrus ussuriensis (Pseudobagrus ussuriensis) is a species of Pseudobagrus species distributed in the water system from Heilongjiang to Zhujiang in China. The fish is delicious in taste and rich in nutrition, is deeply loved by consumers, has low difficulty in breeding technology, few breeding diseases and continuously expanded breeding range, and is bred in more than ten provinces nationwide at present. Pseudobagrus ussuriensis has obvious growth advantages of males, and can reach more than 3 times of females at the second age, so that the Pseudobagrus ussuriensis has great full-male breeding potential.
The rapid and accurate sex identification of target individuals is particularly important in all-male breeding. Although the female and male individuals can be identified by morphology, this is limited to the identification of physiological sex. In breeding work, sex reversal treatment of a target population by using female or male hormones is often required, and the genetic sex of sex-reversed individuals cannot be identified through morphological characteristics. In addition, the morphological characteristics of male and female individuals are not very obvious until one year, and the sex cannot be identified by morphology. Therefore, the development of sex-specific molecular markers is an effective way to perform rapid and accurate sex identification of pseudobagrus ussuriensis. We have developed 1 Male-Specific SCAR Markers (Sequence-ordered Amplified Regions) (Pan ZJ, Li XY, ZHou FJ, Qiang XG, Gui JF. identification of Sex-Specific Markers alternatives, Large heterogeneous Determination in pseudo-diagnosis in Marine Biotechnology,2015,17(4):441- & 451.), and 1 Male-Specific microsatellite marker (grant: CN106811540B) based on AFLP technology (Amplified Fragment Length Polymorphism). However, in practical applications, we have found that both of these markers have certain drawbacks: because the SCAR marker identifies the sex by judging whether an individual has an amplification band at a corresponding site, if the individual has point mutation in a flanking sequence of a primer, no amplification band exists in the individual, namely a Null Allele (Null Allele) appears, and errors can occur in the sex judgment of the individual by utilizing the marker; the microsatellite marker identifies the sex by judging whether one or two amplified bands of a certain individual exist, and if the certain individual has zero allele at the position of the microsatellite marker primer, the microsatellite marker has no amplified band in the certain individual, so that the sex of the individual cannot be identified.
Disclosure of Invention
The invention aims to provide a multiple PCR primer for pseudobagrus ussuriensis gender detection and a detection method, namely, a group of pseudobagrus ussuriensis gender specific multiple PCR primers are provided, and the primers are used as a beneficial tool for accurately and efficiently detecting the pseudobagrus ussuriensis gender and can effectively solve the problems in the background technology.
In order to achieve the purpose, the invention adopts the technical scheme that:
a multiple PCR primer for detecting the sex of Pseudobagrus ussuriensis and its detection method features that its nucleotide sequence is SEQ ID NO. 1-3.
A group of primers capable of specifically amplifying at least one sequence shown in SEQ ID NO. 1-3.
The sequence of the primer group primer is shown as SEQ ID NO. 4-9.
A kit for the sex detection 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 XPCR Buffer, dNTPs, redistilled water and MgCl2. The solvents of the above reagents are all sterile ultrapure water.
A method for sex detection of a multiplex PCR primer set or kit in Pseudobagrus ussuriensis.
A pseudobagrus ussuriensis gender detection method comprises the following steps:
s1, DNA extraction: extracting the genome DNA of a target individual;
s2, multiple PCR amplification: performing PCR amplification by using the multiple PCR primer group and the target individual genome DNA obtained in S1 as a template to obtain a target individual multiple PCR amplification product;
s3, electrophoresis detection of the amplification product: detecting the multiple PCR amplification products obtained by S2 by agarose gel electrophoresis and a GoldView staining method;
s4, sex identification: performing sex identification according to the existence of the multiple PCR amplification product of each individual, wherein if a certain individual has no amplification band at 246bp, 597bp and 823bp, the individual is female; if an individual has an amplified band in at least one of the three locations, the individual is male.
Extracting the genome DNA of the tail fin tissue of the target individual by adopting a general phenol-chloroform DNA extraction method in S1; but is not limited thereto.
As a preferred technical scheme, the reaction system of the PCR amplification comprises the following steps: a total volume of 13-25. mu.L, containing 60-80ng of template DNA (about 0.8-1.5. mu.L), 1U of Taq DNA polymerase (0.1-0.2. mu.L), 1.3-2.5. mu.L of 10 XPCR Buffer, 0.4-1. mu.L of dNTP (2.5mM), 0.3-0.5. mu.L of each of primers SEQ4 and SEQ5, 0.2-0.4. mu.L of each of primers SEQ6 and SEQ7, 0.4-0.6. mu.L of each of primers SEQ8 and SEQ9 (each primer concentration is 2.5. mu.M), and 9-18. mu.L of sterilized ultrapure water; the procedure of PCR amplification is as follows: pre-denaturation at 94 ℃ for 5-7 minutes; denaturation at 94 ℃ for 35-45 seconds, annealing at 65 ℃ → 50 ℃ for 35-45 seconds, and extension at 72 ℃ for 40 seconds-1 minute, wherein the annealing temperature is reduced by 1 ℃ in each cycle, and the operation is carried out for 15-17 cycles; denaturation at 94 ℃ for 35-45 seconds, annealing at 50 ℃ for 35-45 seconds, extension at 72 ℃ for 40-50 seconds, and running for 18-22 cycles; finally, final extension at 72 ℃ for 8-15 min, and the PCR product was stored at 4 ℃.
As a preferred technical solution, the detailed process of S3 is: the multiplex PCR amplification products were separated by electrophoresis in a 1% -2% agarose gel to which a GoldView stain had been added for 15-30 minutes at a constant voltage of 150-200V.
Compared with the prior art, the invention has the following beneficial effects:
firstly, the effect of multiple PCR detection can be achieved by one-time PCR, the detection efficiency is high, and the identification result is accurate;
secondly, the problems of misjudgment, incapability of identifying and the like in the prior art caused by zero allele in part of individuals to be detected are effectively eliminated, and the detection result is more reliable and stable;
the invention overcomes the defects of the prior art and provides a multiple PCR technology and a detection method for the pseudobagrus ussuriensis gender detection, and the multiple PCR technology and the detection method comprise the steps of multiple PCR primer amplification, amplification product electrophoresis detection, banding pattern analysis, gender identification and the like. According to the invention, by integrating the three sex-specific markers, a multiple PCR system for pseudobagrus ussuriensis sex detection is constructed, the efficiency and accuracy of pseudobagrus ussuriensis sex detection can be further improved, a favorable tool is provided for real-time monitoring of the sex structures of various groups of pseudobagrus ussuriensis, and the method has important significance for promoting the all-male breeding process of pseudobagrus ussuriensis;
therefore, the invention develops a group of multi-PCR primers for the pseudobagrus ussuriensis gender detection, the primer group can not only eliminate the gender identification error caused by the zero allele, but also eliminate the situation that the gender cannot be identified caused by the zero allele, and the pseudobagrus ussuriensis gender detection can be realized more accurately and efficiently.
Drawings
FIG. 1 shows agarose electrophoresis patterns of male and female 10-tailed Pseudobagrus ussuriensis using the multiplex PCR primers disclosed herein, and lane M shows DM2000 DNA Marker.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
The following examples are not given to specific experimental conditions and methods, generally following conventional conditions such as: J. SummBruk et al, science publishers, 2002, molecular cloning guidelines (third edition), or following the conditions recommended by the manufacturer.
Example 1 composition and formulation of a multiplex PCR System for the sex detection of Pseudobagrus ussuriensis
(1) The PCR system comprises:
dNTPs (10mM) are products of the company Limited for the engineering bioengineering (Shanghai);
taq DNA polymerase (5U/. mu.L), 10 XPCR Buffer for Beijing kang century products;
the primers were synthesized by the company of Biotechnology (Shanghai).
(2)10 × PCR Buffer composition:
Tris-HCl(pH8.3)100mM、KCl 500mM、MgCl2 15mM。
(3) preparing a PCR reaction system:
a total volume of 25. mu.L, containing 1.5. mu.L of 60-80ng template DNA, 1U Taq DNA polymerase, 2.5. mu.L 10 XPCR Buffer, 1. mu.L dNTP (2.5mM), 0.3. mu.L each of primers SEQ4 and SEQ5, 0.2. mu.L each of primers SEQ6 and SEQ7, 0.4. mu.L each of primers SEQ8 and SEQ9 (each primer concentration is 2.5. mu.M), and 18. mu.L of sterilized ultrapure water.
(4) Preparing agarose gel:
agarose 0.8g, TAE 40mL, GoldView stain (Beijing kang, a century product) 3. mu.L.
Example 2 assessment of the effects of sex detection in pseudobagrus ussuriensis breeding parents of known sex
S1, randomly selecting 100 tails of each female and male parent of the pseudobagrus ussuriensis judged according to morphological characteristics in a breeding farm, shearing a small amount of tail fin tissues, respectively extracting genome DNA by adopting a phenol-chloroform method, and diluting the DNA concentration to 60-80 ng/mu L by using sterilized ultrapure water.
S2, a PCR reaction mixture was prepared in the same manner as in (3) in example 1, using the genomic DNA extracted in S1 as a template.
S3, carrying out amplification on the PCR instrument, and pre-denaturing at 94 ℃ for 5 minutes; denaturation at 94 ℃ for 45 seconds, annealing at 65 ℃ → 50 ℃ for 40 seconds, and extension at 72 ℃ for 1 minute, wherein the annealing temperature is reduced by 1 ℃ in each cycle, and 15 cycles are performed; denaturation at 94 ℃ for 40 seconds, annealing at 50 ℃ for 40 seconds, extension at 72 ℃ for 50 seconds, and running for 22 cycles; final extension at 72 ℃ for 10 min and storage of the PCR product at 4 ℃.
S4.PCR products were separated by electrophoresis at 150V for 30 minutes in 2% agarose Gel prepared according to the system of (4) in example 1, and each Gel was scanned and stored by photographing using a Gel DocTM EZ (Bio-RAD, USA) Gel imager.
The sex identification method comprises the following steps: performing sex identification according to the existence of the electrophoresis band of the multiple PCR amplification product of each individual, wherein if one individual has no amplification band at 246bp, 597bp and 823bp, the individual is female; an individual is male if it has an amplified band at least one of the three band positions, as shown in FIG. 1. The results of the evaluation are shown in Table 1.
Table 1 evaluation results of the effects of multiplex PCR for the detection of pseudobagrus ussuriensis gender according to the present invention
From the results, the female and male individuals identified by the multiplex PCR primer are identical with the morphologically identified sex, and the extremely high accuracy of sex detection of the multiplex PCR primer is reflected.
Comparative example comparison of the sex determination Effect of multiplex PCR of the present invention and Male specific microsatellite (CN106811540B)
S1, randomly selecting 216 fishes of pseudobagrus ussuriensis with unknown gender, clipping a small amount of tail fin tissues, respectively extracting genomic DNA by adopting a phenol-chloroform method, and diluting the DNA concentration to 60-80 ng/mu L by using sterilized ultrapure water.
S2. multiplex PCR assay was carried out according to the methods shown in examples 1 and 2 of the present invention, and the detection of male-specific microsatellite markers was carried out according to the detection method described in CN 106811540B. The detection results are shown in table 2:
TABLE 2 comparison of the multiplex PCR of the present invention and the Male specific microsatellite marker for Pseudobagrus ussuriensis sex detection
The comparison result shows that the multiple PCR can accurately detect the sex of each individual, the detection success rate reaches 100%, and the male specific microsatellite marker cannot detect the sex of 2 individuals in 216 individuals, and the detection success rate is 99.1%.
Therefore, the multiple PCR primers of the present invention can effectively eliminate the cases of wrong or impossible sex identification due to the null allele. The reason is that the multiplex PCR primer integrates three male specific markers, can amplify three male specific bands of 246bp, 597bp and 823bp, can carry out sex identification by at least one amplified band, and has extremely low probability of zero allele appearing at the positions of the three pairs of primers, so that the interference of the zero allele on the sex identification can be effectively avoided, and the accuracy and the reliability of the sex identification are improved.
Claims (9)
1. A multiple PCR primer and a detection method for the sex detection of Pseudobagrus ussuriensis are characterized in that: the nucleotide sequence of the multiplex PCR primer is SEQ ID NO 1-3.
2. A multiplex PCR primer set capable of amplifying at least one sequence of SEQ ID NO. 1-3.
3. The primer set according to claim 2, 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'。
4. Pseudobagrus ussuriensis sex identification multiplex PCR primer kit is characterized in that: the kit comprises the primer set according to claim 2 or 3.
5. The kit of claim 4, wherein: the kit also comprises reagents commonly used in PCR technology.
6. Use of the multiplex PCR primer of claim 1, the primer set of claim 2 or 3, the kit of claim 4 or 5 for pseudobagrus ussuriensis gender identification.
7. A multiple PCR primer and a detection method for the sex detection of Pseudobagrus ussuriensis are characterized in that: 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 of claim 2 or 3 and the target individual genomic DNA obtained in S1 as a template to obtain a target individual multiplex PCR amplification product;
s3, electrophoresis detection of an amplification product: detecting the multiple PCR amplification products obtained by S2 by agarose gel electrophoresis and a GoldView staining method;
s4, sex identification: performing sex identification according to the existence of the multiple PCR amplification product of each individual, wherein if one individual has no amplification band at 246bp, 597bp and 823bp, the individual is female; if an individual has an amplified band in at least one of the three locations, the individual is male.
8. The multiplex PCR primer and detection method for Pseudobagrus ussuriensis gender detection as claimed in claim 7, wherein: the reaction system of the PCR amplification comprises: a total volume of 13-25 μ L, comprising 60-80ng of template DNA (about 0.8-1.5 μ L), 1U Taq DNA polymerase (0.1-0.2 μ L), 1.3-2.5 μ L10 XPCR Buffer, 0.4-1 μ L dNTP (2.5mM), 0.3-0.5 μ L for SEQ4 and SEQ5 primers, 0.2-0.4 μ L for SEQ6 and SEQ7 primers, 0.4-0.6 μ L for SEQ8 and SEQ9 primers (each primer concentration is 2.5 μ M), and 9-18 μ L of sterilized ultrapure water; the procedure of PCR amplification is as follows: pre-denaturation at 94 ℃ for 5-7 min; denaturation at 94 ℃ for 35-45 seconds, annealing at 65 → 50 ℃ for 35-45 seconds, and extension at 72 ℃ for 40 seconds-1 minute, wherein the annealing temperature is reduced by 1 ℃ in each cycle, and the operation is carried out for 15-17 cycles; denaturation at 94 ℃ for 35-45 seconds, annealing at 50 ℃ for 35-45 seconds, extension at 72 ℃ for 40-50 seconds, running for 18-22 cycles; final extension at 72 ℃ for 8-15 min, and the PCR product was stored at 4 ℃.
9. The multiplex PCR primer and detection method for Pseudobagrus ussuriensis gender detection as claimed in claim 7, wherein: the detailed process of S3 is: the multiplex PCR amplification products were separated by electrophoresis in a 1% -2% agarose gel to which a GoldView stain had been added for 15-30 minutes at a constant voltage of 150-200V.
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| CN114058713A (en) * | 2021-11-30 | 2022-02-18 | 淮阴师范学院 | Acquisition method of Y chromosome sequence of Pseudobagrus ussuriensis |
| CN114058713B (en) * | 2021-11-30 | 2023-09-01 | 淮阴师范学院 | Method for obtaining Y chromosome sequence of Pseudobagrus ussuriensis |
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