CN113373244A - Method for rapidly detecting genetic sex of channel catfish based on SNP site-specific primer extension reaction - Google Patents
Method for rapidly detecting genetic sex of channel catfish based on SNP site-specific primer extension reaction Download PDFInfo
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Abstract
The invention discloses a method for rapidly detecting the genetic sex of channel catfish based on SNP site specific primer extension reaction, which comprises the steps of designing a peripheral primer, a specific extension primer (ASE) and a DNA probe (ASE-DP) for amplifying SNP sites linked with the channel catfish sex, carrying out extension reaction and nucleic acid hybridization by using the DNA, the primer and the DNA probe of the channel catfish to be detected, and judging the genetic sex of a sample to be detected according to the color development result of a rapid nucleic acid detection test strip. The accuracy of the invention reaches 100%, and the invention has good application prospect.
Description
Technical Field
The invention belongs to the technical field of molecular markers of fish genetic breeding, and particularly relates to a method for rapidly detecting genetic sex based on a spot ietalurus punetaus sex-linked SNP site-specific primer extension reaction.
Background
Ictalurus punctatus (Ictalurus puncatus) belongs to the order of Parasilurus, belongs to the family of Ictalurus, is native to North America, is the most mature freshwater fish culture breed with the highest yield in the American culture technology, and has become a worldwide aquaculture breed due to the advantages of strong environmental adaptability, good muscle quality, easy processing and the like.
The channel catfish is a diploid organism, has the chromosome number of 58(2 n-58), consists of 28 pairs of autosomes and 1 pair of sex chromosomes (X and Y chromosomes), and belongs to an XY/XX sex determination system. The growth of male and female individuals of the channel catfish has obvious growth bimorph phenomenon, namely the growth speed of the male channel catfish after sexual maturity is obviously higher than that of the female channel catfish individuals, and the edible proportion of the adult female is far lower than that of the male channel catfish along with the development of the ovary of the female individuals. Therefore, the research on the technology for identifying the genetic sex of the channel catfish is actively developed, the rapid, accurate and efficient method for identifying the genetic sex is developed, and the method has important application values for controlling the population ratio of the channel catfish breeding, culturing all-male seedlings and the like.
Disclosure of Invention
The purpose of the invention is as follows: in order to overcome the defects in the prior art, the invention provides a method for rapidly detecting genetic sex based on a site-specific primer extension reaction of SNP (single nucleotide polymorphism) linked with the sex of channel catfish.
The technical scheme is as follows: the invention aims to provide a method for rapidly detecting genetic sex by applying a primer extension reaction of sex-linked SNP locus specificity of channel catfish. Mainly realized by the color reaction on test paper after the SNP site-specific primer extension reaction.
The technical scheme that this patent adopted: a method for rapidly detecting the genetic sex of channel catfish comprises the following steps:
(1) designing and amplifying a peripheral Primer (WF/R) of sex-linked SNP locus of the channel catfish, a specific extension Primer (ASE) and a DNA probe (ASE-DP) by using Primer BLAST online software in NCBI, wherein the sequence of the sex-linked SNP locus interval is shown as SEQ ID NO.1, the sequences of the peripheral Primer, the ASE Primer and the probe ASE-DP are shown as SEQ ID NO.2-SEQ ID NO.5, and the last base at the 3' end of the ASE Primer is male specific base of the channel catfish;
(2) marking FAM fluorescent group at 5 'end of ASE primer, marking Biotin at 3' end of probe ASE-DP, and detecting the correlation between the secondary structures of ASE primer and DNA probe by using Oligo software to avoid generating primer dimer;
(3) carrying out PCR amplification by using the DNA of the fish sample to be detected and the peripheral primer in the step (1) to obtain a first round of PCR amplification product;
(4) performing extension reaction and nucleic acid hybridization by using the PCR amplification product in the step (3), the ASE primer in the step (1) and a probe ASE-DP to obtain a hybridized nucleic acid product of a second round PCR amplification product and a DNA probe;
(5) and detecting the hybrid nucleic acid product of the second round PCR amplification product and the DNA probe by using a rapid nucleic acid detection test strip, and judging the genetic sex of the fish sample to be detected according to the test strip color development result.
Preferably, in step (3), the PCR amplification reaction has a reaction system of 40. mu.L, including 2 XPPhanta Max Master Mix 20. mu.L, template DNA 1. mu.L, and peripheral primers (WF and WR) each 1. mu.L, H2O17 mu L; the PCR amplification procedure is as follows:
pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 57-53 ℃ (1 ℃ per cycle) for 30s, extension at 72 ℃ for 60s, and annealing temperature for 2 cycles; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 60s, for 15 cycles; extension at 72 ℃ for 10 min.
Preferably, in step (4), the extension reaction has a reaction system of 20. mu.L, including 2. mu.L of 2 XPPhanta Max Master Mix 10. mu.L, 1. mu.L of template DNA, 1. mu.L each of ASE primer (ASE-F) and DNA probe (ASE-DP), and H2O7 mu L; the PCR amplification procedure is as follows: 95 ℃ for 5 min; 94 ℃, 30 sec; 30sec at 66 ℃ for 20 cycles; 94 deg.C30 sec; 50 ℃, 30sec, 25 cycles.
Preferably, in the step (5), the hybrid nucleic acid product of the second round of PCR amplification product and the DNA probe is detected by using a rapid nucleic acid detection test strip, in which 10 μ L of the hybrid nucleic acid product is dropped onto a sample pad of the rapid nucleic acid detection test strip, and then the sample pad is placed into 20 μ L of a buffer solution compatible with the rapid nucleic acid detection test strip, and the result is interpreted within 10-15 min.
As another aspect of the present invention, the present invention provides a primer and a kit for rapidly detecting the genetic sex of ictalurus punctatus, wherein the primer and the kit are characterized in that: the primers comprise a peripheral primer, a specific ASE primer and a probe ASE-DP, and the sequences of the peripheral primer, the ASE primer and the ASE-DP are shown as SEQ ID NO.2-SEQ ID NO. 5.
The invention has the following beneficial effects:
1. the rapid identification of the genetic sex of the channel catfish is realized by using simple PCR reaction and nucleic acid detection test paper, the result is stable, and the accuracy rate reaches 100%;
2. the traditional agarose gel electrophoresis method is not applied in the whole process, so that the complicated steps of gel preparation, gel running and the like are omitted, nucleic acid dye is not used, and the experimental process is safe and environment-friendly.
Drawings
FIG. 1 shows the detection results of female individuals of Ictalurus punctatus;
FIG. 2 shows the results of the male individual detection of Ictalurus punctatus;
fig. 3 is a color development condition of the nucleic acid test strip of channel catfish, wherein: test strip No. 1: the nucleic acid test strip of the channel catfish with female genetic sex shows the color, and the No.2 test strip: the nucleic acid test paper of the channel catfish with female genetic sex shows the color condition; the first is a quality inspection line, the second is a detection line and the third is a quality inspection line.
Detailed Description
The present invention will be further described below.
Example 1
In the implementation, 96 channel catfishes are randomly harvested from the mouth of the culture pond, tail fins of the channel catfishes are taken out and put into a 1.5mL EP tube, and the channel catfishes are stored by absolute ethyl alcohol. In 96 channel catfish individuals with judged genetic sex, 10 samples of tail fin tissues with male genetic sex and female genetic sex are randomly selected for carrying out the experiment of example 1.
1. DNA extraction of channel catfish
1.1 digestive cleavage of the tail fin of Ictalurus punctatus
20mg of the preserved tail fin tissue was minced and placed in a 1.5mL EP tube. DNA extraction was performed using the FastPure Cell/Tissue DNA Isolation Mini Kit (Nanjing Novovisan Biotech Co., Ltd.). Add 230. mu.L Buffer GA and 20. mu.L PK protein working solution, vortex and mix for 15sec, and place in a 55 ℃ water bath for overnight digestion. Adding 250 μ L Buffer GB into the digestive juice after complete enzymolysis of the tissue, mixing uniformly by vortex for 20sec, and carrying out water bath at 70 ℃ for 10 min.
1.2 column purification of the digestive juice
Add 180. mu.L of absolute ethanol to the digest and mix by vortexing for 15-20 sec. The gDNA Columns were placed in a Collection tube of 2mL Collection Tubes, and the mixture from the previous step was transferred to the Columns and centrifuged at 12,000rpm (13,400 Xg) for 1 min. The filtrate was discarded and the adsorption column was placed in the collection tube.
Add 500. mu.L Washing Buffer A to the adsorption column. Centrifuge at 12,000rpm for 1 min.
The filtrate was discarded, the column was placed in a collection tube, 650. mu.L of Washing Buffer B was added to the column, and centrifuged at 12,000rpm for 1 min.
And repeating the previous step.
The filtrate was discarded and the adsorption column was placed in the collection tube. The mixture was centrifuged at 12,000rpm for 2min in an empty tube.
The column was placed in a new 1.5ml centrifuge tube. Add 50. mu.L of Elution Buffer preheated to 70 ℃ to the center of the membrane of the adsorption column and leave it at room temperature for 3 min. Centrifuge at 12,000rpm (13,400 Xg) for 1 min.
The adsorption column was discarded and the DNA was stored at-20 ℃ for a long period.
2. SNP locus screening and primer design for identifying genetic sex of channel catfish
Selecting a sex-linked SNP locus g.zbt38ycds 366G > A as an SNP locus for developing and rapidly identifying the genetic sex of the channel catfish, wherein the locus is a locus on zbtb38-Y, and the sequence of the locus is TTCTTGAGAARCTTCTTGAGA. Peripheral primers (WF/R), specific extension primers (ASE) and DNA probes (ASE-DP) of the SNP locus interval are designed and amplified by using Primer BLAST online software in NCBI, the last base at the 3 ' end of the ASE Primer is a male specific base of channel catfish, the 5 ' end of the ASE Primer is marked with FAM fluorescent group, the 3 ' end of the probe ASE-DP is marked with Biotin, and Oligo software is used for detecting the mutual relationship between the secondary structures of the ASE Primer and the DNA probes to avoid generating Primer dimers. The primer sequences are shown in Table 1.
TABLE 1 sex-linked SNP site-specific extension reaction primer table for channel catfish
Note: WF, peripheral forward primer; WR, peripheral reverse primer; ASE, specific forward primer; ASE-DP, DNA probe.
3. Amplification of sex-linked SNP (single nucleotide polymorphism) sites of channel catfish
3.1 elongation reaction of primer at periphery of sex-linked SNP locus of channel catfish
The reaction system was 40. mu.L, in which 2X Phanta Max Master Mix (20. mu.L, Nyvowed Biotech Co., Ltd., Nanjing), template DNA 1. mu.L, and peripheral primers (WF and WR) each were 1. mu.L, H2O 17μL。
PCR amplification procedure (first round PCR amplification reaction): pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 57-53 ℃ (1 ℃ per cycle) for 30s, extension at 72 ℃ for 60s, and annealing temperature for 2 cycles; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 60s, for 15 cycles; extension at 72 ℃ for 10 min.
3.2 sex-linked SNP site-specific primer extension reaction of Ictalurus punctatus
The product of the first PCR amplification reaction is diluted by 10 times and then used as a template of the second PCR amplification reaction. The reaction system was 20. mu.L, in which 2X Phanta Max Master Mix (Nanjing Novodax Biotech Co., Ltd.) was 10. mu.L, template DNA was 1. mu.L, ASE primer (ASE-F) and DNA probe (ASE-DP) were each 1. mu.L, H2O 7μL。
PCR amplification procedure (second round PCR amplification reaction): 95 ℃ for 5 min; 94 ℃, 30 sec; 30sec at 66 ℃ for 20 cycles; 94 ℃, 30 sec; 50 ℃, 30sec, 25 cycles.
Storing the hybrid nucleic acid product of the finally obtained second round PCR amplification product and the DNA probe at-20 DEG C
4. Channel catfish genetic sex identification
4.1 detection of amplification products
20 mul of buffer solution (Yosida Biotechnology, Hangzhou) compatible with the rapid nucleic acid detection test paper is dripped into a 100 mul of microplate, and 10 mul of hybrid nucleic acid products of the second round PCR amplification products and the DNA probes are dripped onto a sample pad of the rapid nucleic acid detection test paper. The nucleic acid test paper to which the hybridized nucleic acid product is added is inserted into a 100 muL micro-porous plate containing a buffer solution, and the lowest end of the sample pad is ensured to be contacted with the buffer solution.
4.2 determination of results
The result is interpreted within 10-15min after the above process is finished. The red band was observed in the interpretation zone on the rapid nucleic acid detection strip. In the interpretation of 20 sample results, red strips appear in the quality control zone of the interpretation zone of the rapid nucleic acid detection test paper, and red strips appear in the detection zone of 10 channel catfish male individuals in DNA samples; in the DNA samples of 10 female Ictalurus punctatus individuals, no red strip appears in the detection area.
5. Verification of genetic sex identification method
And randomly selecting 96 samples from a sample library of the channel catfish genetic breeding center to carry out sex identification so as to verify the accuracy of the genetic sex identification method. And (3) detecting the hybrid nucleic acid product of the second round PCR amplification product and the DNA probe by using a rapid nucleic acid detection test strip, and comparing the genetic sex identification result of the selected sample with sex information recorded in a database, wherein the result shows that the accuracy rate of the genetic sex identification method is 100%.
The invention relates to a method for rapidly detecting the genetic sex of channel catfish based on SNP site-specific primer extension reaction, belonging to the technical field of fish molecular markers. By extracting genome DNA of a channel tissue of the channel catfish, then enriching a sequence where a sex-linked SNP locus is located through peripheral PCR amplification, and then carrying out SNP locus specific amplification and probe hybridization, the obtained hybrid nucleic acid product is used for carrying out SNP locus detection by using a nucleic acid test strip, thereby realizing the rapid identification of the genetic sex of the channel catfish. The result shows that the method can effectively identify the genetic sex of the channel catfish and the accuracy rate reaches 100%.
Sequence listing
<110> research institute for fresh water and aquatic products in Jiangsu province
<120> method for rapidly detecting genetic sex of channel catfish based on SNP site-specific primer extension reaction
<141> 2021-07-13
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ttcttgagaa rcttcttgag a 21
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<213> Artificial Sequence
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gaaatgtctt ggctgcgact 20
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<212> DNA
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ctcttgctcc tcaagccgtg 20
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<212> DNA
<213> Artificial Sequence
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tcccctttct tgagaaa 17
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<212> DNA
<213> Artificial Sequence
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gcctttcaat ctcaag 16
Claims (6)
1. A method for rapidly detecting the genetic sex of channel catfish is characterized by comprising the following steps: comprises the following steps of (a) carrying out,
(1) designing a peripheral primer, a specific extension primer and a DNA probe for amplifying sex-linked SNP sites of the channel catfish, wherein the interval sequence of the SNP sites is shown as SEQ ID NO.1, the sequences of the peripheral primer, the ASE primer and the DNA probe are shown as SEQ ID NO.2-SEQ ID NO.5, and the last base at the 3' end of the ASE primer is the male specific base of the channel catfish;
(2) the 5 'end of the ASE primer is marked with FAM fluorophore, the 3' end of the probe ASE-DP is marked with Biotin, and Oligo software is applied to check the mutual relation between the secondary structures of the ASE primer and the probe ASE-DP so as to avoid generating primer dimer.
2, (3) carrying out PCR amplification by using the DNA of the fish sample to be detected and the peripheral primer in the step (1) to obtain a first round of PCR amplification product;
(4) carrying out second round PCR extension reaction and nucleic acid hybridization by using the first round amplification product in the step (3), the ASE primer in the step (1) and ASE-DP to obtain a second round PCR amplification product and an ASE-DP hybridized nucleic acid product;
(5) and detecting the hybrid nucleic acid product by using a rapid nucleic acid detection test strip, and judging the genetic sex of the fish sample to be detected according to the test strip color development result.
3. The method for rapidly detecting the genetic sex of ictalurus punctatus according to claim 1, wherein the method comprises the following steps: in the step (3), the reaction system of the first round of PCR reaction is 40 muL, and comprises 2 XPhanta Max Master Mix 20 muL, template DNA 1 muL and peripheral primers (WF and WR) which are 1 muL and H respectively2O17 muL; the PCR amplification program is: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 57-53 ℃ (1 ℃ minus each cycle) for 30s, extension at 72 ℃ for 60s, and annealing temperature for 2 cycles; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 60s, for 15 cycles; extension at 72 ℃ for 10 min.
4. The method for rapidly detecting the genetic sex of ictalurus punctatus according to claim 1, wherein the method comprises the following steps: in the step (4), the extension reaction and the nucleic acid hybridization have a reaction system of 20 muL, including 2 XPhanta Max Master Mix 10 muL, 1 muL of template DNA, 1 muL of ASE primer (ASE) and DNA probe (ASE-DP) each, and H muL2O7 muL; the PCR amplification procedure is as follows: 95 ℃ for 5 min; 94 ℃, 30 sec; 30sec at 66 ℃ for 20 cycles; 94 ℃, 30 sec; 50 ℃, 30sec, 25 cycles.
5. The method for rapidly detecting the genetic sex of ictalurus punctatus according to claim 1, wherein the method comprises the following steps: in the step (5), 10 mu L of the hybrid nucleic acid product is dripped onto a sample pad of the rapid nucleic acid detection test strip, then the hybrid nucleic acid product is put into a buffer solution used by compatibility of 20 mu L of the rapid nucleic acid detection test strip, and the result is interpreted within 10-15 min.
6. A primer and a kit for rapidly detecting the genetic sex of channel catfish are characterized in that: the primers comprise peripheral primers, specific ASE primers and DNA probes, and the sequences of the peripheral primers, the specific ASE primers and the DNA probes are shown in SEQ ID NO.2-SEQ ID NO. 5.
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| US17/845,893 US20220411882A1 (en) | 2021-06-21 | 2022-06-21 | Snp molecular marker for weight gain trait selection and genetic sex identification of ictalurus punctatus as well as screening method and application of snp molecular marker |
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| CN109161603A (en) * | 2018-10-31 | 2019-01-08 | 江苏省淡水水产研究所 | The microsatellite marker primer and genetic sex identification method of channel catfish gunther sex-linked |
| CN111304338A (en) * | 2020-03-13 | 2020-06-19 | 江苏省淡水水产研究所 | SNP molecular marker linked with sex of channel catfish and genetic sex identification method |
| CN112342214A (en) * | 2020-11-11 | 2021-02-09 | 江苏省淡水水产研究所 | sgRNA sequence for targeted knockout of channel catfish zbtb38 gene and screening method thereof |
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| Title |
|---|
| NAIRONG PAN等: "Identification of male-specific SNP markers and development of rapid PCR-based genetic sex identification method in channel catfish (Ictalurus punctatus)", 《AQUACULTURE》 * |
| SHIYONG ZHANG等: "Construction of a High-Density Linkage Map and QTL Fine Mapping for Growth- and Sex-Related Traits in Channel Catfish (Ictalurus punctatus)", 《FRONTIERS IN GENETICS》 * |
| 单洪波等: "基于PCR和位点特异性引物延伸反应的SNP检测方法的建立", 《检验医学》 * |
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