CN113373244B - Method for rapidly detecting genetic sex of channel catfish based on SNP locus specific primer extension reaction - Google Patents
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Abstract
The invention discloses a method for rapidly detecting the genetic sex of channel catfish based on SNP locus specific primer extension reaction, which comprises the steps of designing and amplifying peripheral primers, specific extension primers (ASE) and DNA probes (ASE-DP) of channel catfish sex-linked SNP loci, carrying out extension reaction and nucleic acid hybridization by utilizing the DNA, primers and DNA probes 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 for fish genetic breeding, and particularly relates to a method for rapidly detecting genetic sex based on a channel catfish sex-linked SNP locus specific primer extension reaction.
Background
Channel catfish (Ictalurus punctatus), belonging to the order of catfish, belonging to the family of catfish, which is native to North America, is the most mature freshwater fish culture variety with highest yield in the United states culture technique, and has the advantages of stronger environment adaptability, better muscle quality, easy processing and the like, thus becoming a worldwide aquaculture variety.
Channel catfish is a diploid organism with a chromosome number of 58 (2n=58), consisting of 28 pairs of autosomes and 1 pair of sex chromosomes (X and Y chromosomes), belonging to the XY/XX sex determination system. The growth of male and female individuals of the channel catfish has obvious growth dichotomy phenomenon, namely the growth speed of the male channel catfish after sexual maturity is obviously faster than that of the female channel catfish, and the edible proportion of adult females is far lower than that of males along with the development of ovaries of the female individuals. Therefore, research on the genetic sex identification technology of the channel catfish is actively developed, a rapid, accurate and efficient genetic sex identification method is developed, and the method has important application value for sex ratio control of channel catfish breeding population, cultivation of all male offspring seeds and the like.
Disclosure of Invention
The invention aims to: in order to overcome the defects in the prior art, the invention provides a method for rapidly detecting the genetic sex based on the channel catfish sex-linked SNP locus specific primer extension reaction.
The technical scheme is as follows: the invention aims to provide a method for rapidly detecting genetic sex by applying a channel catfish sex-linked SNP locus specific primer extension reaction. The method is realized mainly by the color development reaction on test paper after the SNP locus specific primer extension reaction.
The technical scheme that this patent adopted: a method for rapidly detecting the genetic sex of channel catfish, comprising the following steps:
(1) Designing and amplifying peripheral primers (WF/R) and specific extension primers (ASE) and DNA probes (ASE-DP) of sex-linked SNP loci of channel catfish by using Primer BLAST on-line software in NCBI, wherein the sequence of the sex-linked SNP loci is shown as SEQ ID NO.1, the sequences of the peripheral primers, the ASE primers and the probes ASE-DP are shown as SEQ ID NO.2-SEQ ID NO.5, and the last base at the 3' -end of the ASE primers is channel catfish male specific base;
(2) The 5 'end of the ASE primer is marked with FAM fluorescent groups, the 3' end of the probe ASE-DP is marked with Biotin, and Oligo software is used for checking the correlation between the ASE primer and the secondary structure of the DNA probe, so that primer dimer is avoided;
(3) Performing 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 extension reaction and nucleic acid hybridization by utilizing the PCR amplification product in the step (3), the ASE primer in the step (1) and the probe ASE-DP to obtain a hybridized nucleic acid product of the PCR amplification product and the DNA probe in the second round;
(5) And detecting the hybridized nucleic acid product of the second 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 color development result of the test strip.
Preferably, in step (3), the PCR amplification reaction is performed in a reaction system of 40. Mu.L, including 2X Phanta Max Master Mix. Mu.L, 1. Mu.L of the template DNA, 1. Mu.L of each of the peripheral primers (WF and WR), H 2 O17 μl; the PCR amplification procedure is as follows:
pre-denaturation at 95 ℃ for 5min; denaturation at 94℃for 30s, annealing at 57℃to 53℃for 30s (each cycle minus 1 ℃) and elongation at 72℃for 60s, each annealing temperature being 2 cycles; denaturation at 94℃for 30s, annealing at 55℃for 30s, elongation at 72℃for 60s,15 cycles; extending at 72℃for 10min.
Preferably, in the step (4), the extension reaction is performed in a reaction system of 20. Mu.L, including 2X Phanta Max Master Mix. Mu.L, 1. Mu.L of template DNA, 1. Mu.L of ASE primer (ASE-F) and 1. Mu.L of DNA probe (ASE-DP), H 2 O7 μl; the PCR amplification procedure is as follows: 95 ℃ for 5min;94 ℃,30sec;66 ℃,30sec,20 cycles; 94 ℃,30sec;50 ℃,30sec,25 cycles.
Preferably, in step (5), the hybridized 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, wherein 10 μl of the hybridized nucleic acid product is dripped onto a sample pad of the rapid nucleic acid detection test strip, and then the sample pad is put into 20 μl of 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 invention, the invention provides a primer and a kit for rapidly detecting the genetic sex of channel catfish, which are characterized in that: the primers comprise a peripheral primer, a specific ASE primer and a probe ASE-DP, and the peripheral primer, the ASE primer and the ASE-DP are shown as SEQ ID NO.2-SEQ ID NO. 5.
The beneficial effects of the invention are as follows:
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 reaches 100%;
2. the whole process does not use the traditional agarose gel electrophoresis method, omits complicated steps such as gel preparation, gel running and the like, does not use nucleic acid dye, and is safe and environment-friendly in experimental process.
Drawings
FIG. 1 shows the results of detection of female individuals of Ictalurus punctatus;
FIG. 2 shows the results of male individual detection of Ictalurus punctatus;
FIG. 3 shows the color development of channel catfish nucleic acid strips, wherein: test strip No. 1: nucleic acid test strip color development condition of channel catfish with female genetic sex, test strip No. 2: the color development condition of the nucleic acid test paper of the channel catfish with female genetic sex; (1) the detection line (2) is a quality inspection line, and the detection line (3) is a quality inspection line.
Detailed Description
The present invention will be further described below.
Example 1
In practice, 96 channel catfish were randomly harvested from the mouth of the culture pond, their tail fins were taken in 1.5mL EP tubes and kept with absolute ethanol. 10 samples of the tail fin tissue of each male and female of 96 individuals of channel catfish with determined genetic sex were randomly selected for the experiment of example 1.
1. Channel catfish DNA extraction
1.1 digestion and cleavage of Ictalurus punctatus tail fins
20mg of preserved skein tissue was cut and placed in a 1.5mL EP tube. DNA extraction was performed using FastPure Cell/Tissue DNA Isolation Mini Kit (Nanjinovain Biotech Co., ltd.). 230. Mu.L of Buffer GA and 20. Mu.L of PK protein working solution are added, mixed by vortex for 15sec and placed in a 55 ℃ water bath for overnight digestion. After complete enzymolysis of the tissue, 250. Mu.L Buffer GB is added into the digestive juice, and the mixture is stirred and mixed for 20sec, and water bath is carried out for 10min at 70 ℃.
1.2 column purification of the digestate
180 mu L of absolute ethyl alcohol is added into the digestion liquid, and the mixture is stirred and mixed for 15-20sec. The gDNA Columns were placed in Collection Tubes 2mL, and the mixture obtained in the previous step was transferred to the Columns and centrifuged at 12,000rpm (13,400 Xg) for 1min. The filtrate was discarded and the column was placed in a collection tube.
500 mu L Washing Buffer A was added to the column. Centrifuge at 12,000rpm for 1min.
The filtrate was discarded, the column was placed in a collection tube, 650. Mu. L Washing Buffer B was added to the column, and the column was centrifuged at 12,000rpm for 1min.
The above steps are repeated.
The filtrate was discarded and the column was placed in a collection tube. The tube was centrifuged at 12,000rpm for 2min.
The column was placed in a new 1.5ml centrifuge tube. 50. Mu.L of an Elution Buffer preheated to 70℃was added to the center of the membrane of the adsorption column and left at room temperature for 3min. Centrifuge at 12,000rpm (13,400 Xg) for 1min.
The adsorption column was discarded and the DNA was stored at-20℃for a long period of time.
2. SNP locus screening and primer design for channel catfish genetic sex identification
Sex-linked SNP locus g.zbtb38ycds 366G > A was selected as a SNP locus for developing rapid identification of the genetic sex of channel catfish, which is a locus on zbtb38-Y and whose sequence is TTCTTGAGAARCTTCTTGAGA. Designing and amplifying peripheral primers (WF/R) and specific extension primers (ASE) and DNA probes (ASE-DP) of the SNP locus interval by using Primer BLAST in NCBI on-line software, wherein the last base at the 3' end of the ASE primers is a male specific base of channel catfish, the 5' end of the ASE primers is marked with FAM fluorescent groups, the 3' end of the probes is marked with Biotin, and the correlation between the ASE primers and the secondary structure of the DNA probes is checked by using Oligo software, so that Primer dimers are avoided. The primer sequences are shown in Table 1.
TABLE 1 primer list for sex-linked SNP locus specific extension reaction of Ictalurus punctatus
Note that: WF, peripheral forward primer; WR, peripheral reverse primer; ASE, specific forward primer; ASE-DP, DNA probe.
3. Sex-linked SNP locus amplification of channel catfish
3.1 sex-linked SNP locus peripheral primer extension reaction of Ictalurus punctatus
The reaction system was 40. Mu.L, of which 2X Phanta Max Master Mix (Nanjinovain Biotech Co., ltd.) was 20. Mu.L, 1. Mu.L of template DNA, 1. Mu.L of peripheral primers (WF and WR) each, H 2 O 17μL。
PCR amplification procedure (first round PCR amplification reaction): pre-denaturation at 95 ℃ for 5min; denaturation at 94℃for 30s, annealing at 57℃to 53℃for 30s (each cycle minus 1 ℃) and elongation at 72℃for 60s, each annealing temperature being 2 cycles; denaturation at 94℃for 30s, annealing at 55℃for 30s, elongation at 72℃for 60s,15 cycles; extending at 72℃for 10min.
3.2 sex-linked SNP locus specific primer extension reaction of Ictalurus punctatus
The products of the first round of PCR amplification reaction are diluted by 10 times and then serve as templates for the second round of PCR amplification reaction. The reaction system was 20. Mu.L, in which 2X Phanta Max Master Mix (Nanjinovain Biotech Co., ltd.) was 10. Mu.L, template DNA was 1. Mu.L, and ASE primer (ASE-F) and DNA probe (ASE-DP) were each 1. Mu.L, H 2 O 7μL。
PCR amplification procedure (second round PCR amplification reaction): 95 ℃ for 5min;94 ℃,30sec;66 ℃,30sec,20 cycles; 94 ℃,30sec;50 ℃,30sec,25 cycles.
Preserving the hybridization nucleic acid product of the final second round PCR amplified product and the DNA probe at-20deg.C
4. Identification of channel catfish genetic sex
4.1 detection of amplified products
20 mu L of buffer solution (Sangzhou Yongda biotechnology Co., ltd.) used for matching the rapid nucleic acid detection test paper is dripped into 100 mu L of micro-pore plates, and 10 mu L of the hybridized nucleic acid product of the second round PCR amplification product and the DNA probe is dripped onto a sample pad of the rapid nucleic acid detection test paper. The nucleic acid detection test paper into which the hybridized nucleic acid product has been dripped is inserted into a 100 mu L microplate containing a buffer solution, so that the lowest end of the sample pad is ensured to be contacted with the buffer solution.
4.2 result determination
And judging the result within 10-15min after the completion of the process. The red band was observed in the interpretation zone on the rapid nucleic acid test strip. In the interpretation of 20 sample results, red stripes appear in the quality control region of the interpretation region of the rapid nucleic acid detection test paper, and red stripes appear in the detection region of the DNA samples of 10 channel catfish male individuals; in the DNA samples of the female individuals of the 10 channel catfish, red bands do not appear in the detection areas.
5. Verification of genetic sex identification method
96 samples are randomly selected from a sample library of a channel catfish genetic breeding center for sex identification so as to verify the accuracy of the genetic sex identification method. The hybridization nucleic acid products of the PCR amplification products and the DNA probes of the second round are detected by using a rapid nucleic acid detection test strip, and the genetic sex identification result of the selected sample is compared with sex information recorded in a database, so that the result shows that the accuracy 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 locus specific primer extension reaction, and belongs to the technical field of fish molecular markers. Genomic DNA of channel catfish tail fin tissues is extracted, sequences in which sex-linked SNP loci are located are enriched through peripheral PCR amplification, SNP locus specific amplification and probe hybridization are carried out, and the obtained hybridized nucleic acid products are subjected to SNP locus detection by using a nucleic acid test strip, so that the rapid identification of the genetic sex of channel catfish is realized. 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> fresh water aquatic institute of Jiangsu province
<120> method for rapidly detecting genetic sex of Ictalurus punctatus based on SNP locus specific primer extension reaction
<141> 2021-07-13
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gaaatgtctt ggctgcgact 20
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ctcttgctcc tcaagccgtg 20
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Claims (5)
1. The method for rapidly detecting the genetic sex of the channel catfish is characterized by comprising the following steps of:
(1) Designing peripheral primers WF and WR for amplifying sex-linked SNP loci of channel catfish, a specific extension primer ASE and a DNA probe ASE-DP, wherein the interval sequence of the SNP loci is shown as SEQ ID NO.1, the peripheral primer WF and WR sequences are shown as SEQ ID NO.2-SEQ ID NO.3, the specific extension primer ASE and the DNA probe ASE-DP sequences are shown as SEQ ID NO.4-SEQ ID NO.5, wherein 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, and the 3' end of the DNA probe ASE-DP is marked with Biotin;
(2) The correlation between ASE primer and probe ASE-DP secondary structure is checked by Oligo software, so as to avoid the generation of primer dimer;
(3) Performing a first round of 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 a second PCR extension reaction and nucleic acid hybridization by using the first round of amplification product obtained in the step (3) and the ASE primer and the probe ASE-DP of the step (1) to obtain a second round of PCR amplification product and ASE-DP hybridized nucleic acid product;
(5) And detecting the hybridized 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 color development result of the test strip.
2. The method for rapidly detecting the genetic sex of channel catfish according to claim 1, wherein the method comprises the following steps: in the step (3), the first round of PCR amplification is performed, wherein a reaction system is 40 [ mu ] L and comprises 2× Phanta Max Master Mix 20 [ mu ] L, 1 [ mu ] L template DNA, and 1 [ mu ] L peripheral primers WF and WR, and H 2 O17 [ mu ] L; the PCR amplification procedure is as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 94 ℃ for 30s, annealing at 57-53 ℃ for 30s, and extension at 72 ℃ for 60s, wherein each annealing temperature is 2 cycles; denaturation at 94℃for 30s, annealing at 55℃for 30s,72℃Extending for 60s,15 cycles; extending at 72℃for 10min.
3. The method for rapidly detecting the genetic sex of channel catfish according to claim 1, wherein the method comprises the following steps: in the step (4), the second PCR extension reaction and nucleic acid hybridization are performed in a reaction system of 20 [ mu ] L, wherein the reaction system comprises 2X Phanta Max Master Mix [ mu ] L, 1 [ mu ] L of template DNA, 1 [ mu ] L of ASE primer and 1 [ mu ] L of DNA probe ASE-DP, and H 2 O7 [ mu ] L; the PCR amplification procedure is as follows: 95 ℃ for 5min;94 ℃ for 30s,66 ℃ for 30s,20 cycles; 94℃for 30s,50℃for 30s,25 cycles.
4. The method for rapidly detecting the genetic sex of channel catfish according to claim 1, wherein the method comprises the following steps: in the step (5), the rapid nucleic acid detection test strip is used for detecting the hybridized nucleic acid product, wherein 10 mu L of the hybridized nucleic acid product is dripped on a sample pad of the rapid nucleic acid detection test strip, and then the sample pad is put into a buffer solution compatible with 20 mu L of the rapid nucleic acid detection test strip, and the result is interpreted within 10-15 min.
5. The primer for rapidly detecting the genetic sex of the channel catfish is characterized in that: the primers comprise peripheral primers WF and WR, a specific extension primer ASE and a DNA probe ASE-DP, wherein the sequences of the peripheral primers WF and WR are shown as SEQ ID NO.2-SEQ ID NO.3, the sequences of the specific extension primer ASE and the DNA probe ASE-DP are shown as SEQ ID NO.4-SEQ ID NO.5, wherein the 5 'end of the ASE primer is marked with a FAM fluorescent group, and the 3' end of the DNA probe ASE-DP is marked with Biotin.
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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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| 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 |
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| Identification of male-specific SNP markers and development of rapid PCR-based genetic sex identification method in channel catfish (Ictalurus punctatus);Nairong Pan等;《Aquaculture》;第547卷;https://doi.org/10.1016/j.aquaculture.2021.737535 * |
| 基于PCR和位点特异性引物延伸反应的SNP检测方法的建立;单洪波等;《检验医学》;第33卷(第6期);第530-535页 * |
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