CN116287173B - Application of sex tags, primers and kit of high-body Seriola exosome microRNAs - Google Patents
Application of sex tags, primers and kit of high-body Seriola exosome microRNAs Download PDFInfo
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Abstract
The invention discloses a sex tag of high-body Seriola exosome microRNAs, a primer for amplifying the sex tag of high-body Seriola exosome microRNAs and application of a kit comprising the primer in a sex identification product of high-body Seriola, wherein the sex tag of the microRNAs is dre-miR-223, and the base sequence of the sex tag is shown in SEQ ID NO: 1. The serum exosome microRNAs are used for judging the high-body female and male Seriola, the identification result is reliable, and the fish body is not injured; the invention designs the amplification primer aiming at the label by utilizing the identification label, and develops a detection kit, thereby being convenient, quick, simple and efficient.
Description
Technical Field
The invention belongs to the technical field of aquaculture, and particularly relates to application of sex labels, primers and kits of high-body quince-quince exosome microRNAs, in particular to application of sex labels, primers and kits of high-body quince-quince exosome microRNAs in high-body quince sex identification products.
Background
High body quince (Seriola dumerili) is a worldwide deep-open sea economic fish with high growth speed, good meat quality and high nutritive value. The high body of quiniola is a female-male variant fish, which has no phenotypic sex, and thus it is difficult to directly identify the sex of fish from the surface of fish body. In addition, under the condition of artificial cultivation, the high-body Seriola exhibits serious reproductive dysfunction, and the problem of difficult sex identification greatly hinders the industrial cultivation of the high-body Seriola.
The sex identification of fish includes physiological anatomy observation, gonad section, gonad cell observation, female specific molecular marker and other methods. The exosome internal content microRNAs are highly conserved in evolution, stable in property, easy to quantitatively detect, have the condition of biological tags, and have important significance for fish sex identification.
Disclosure of Invention
The invention aims to provide a sex-indicating sex tag of high-body Seriola quinquefolia exosome microRNAs, and a primer and a kit for amplifying the sex-indicating sex tag of the high-body Seriola quince exosome microRNAs, so as to solve the problem that high-body Seriola quince female fish and high-body Seriola male fish are difficult to directly identify from the surface of the fish body.
The invention also aims to provide the application of the sex tag, primer or kit of the high body Seriola exosome microRNAs in preparing high body Seriola sex identification products.
The first object of the present invention can be achieved by the following technical means: sex tags of high-body Seriola exosomes microRNAs are dre-miR-223, and the base sequence of the sex tags is shown in SEQ ID NO: 1.
Aiming at the problem of identifying female and male high-body Seriola quinquefoil, the invention provides exosome microRNAs derived from serum of high-body Seriola quinquefoil as biomarkers for identifying female and male fish.
When the high-body Seriola serum exosome microRNA sex tag is applied to high-body Seriola sex identification, the microRNA sex tag is dre-miR-223, and the sequence of the microRNA sex tag is TGTCAGTTTGTCAAATACCCC.
The miRNAs sex tags have the characteristic of obvious differential expression in female fish and male fish, and are specific molecular recognition markers.
The sex tags of the high-body Seriola exosome microRNAs in the invention are preferably obtained by screening by the following method:
1) After serum exosomes are separated and identified, sequencing analysis is carried out on serum exosomes small RNA of 3 female fish and 3 male fish, raw reads are filtered to obtain clean reads, and the clean reads are sequentially compared with an Rfam database, a cDNA sequence, a species repeated sequence library and a miRBase database for annotation; counting known microRNAs and predicting new microRNAs; and carrying out differential expression analysis on microRNAs, carrying out Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis on target genes of differential expression miRNAs, and finally carrying out correlation matching with female fish and male fish samples, screening one or more microRNAs with indication effect on the two samples, and taking the microRNAs as candidate microRNAs biomarkers.
2) Candidate tagged microRNAs were filtered according to the following five criteria: (1) There was a significant difference in expression in the two groups of samples of the female group (3 females) and of the male group (3 males), i.e. P-value less than 0.05; (2) Comparing with a known database to confirm the existence of microRNAs; (3) at least one of the samples has a TPM value greater than 0; (4) A foldChange value greater than 2, the foldChange value = TPM sample_Male ./TPM Sample female The method comprises the steps of carrying out a first treatment on the surface of the (5) From the four criteria selected from the microRNAs randomly choose 11 microRNAs.
3) Two sets of validation samples were extracted: designing reverse transcription primers and qPCR primers of 11 microRNAs and internal reference genes oni-let-7a by a conventional stem-loop method, quantitatively detecting the differential expression condition of the microRNAs filtered in the step 2) by using a microRNA quantitative analysis kit, and screening out the miRNAs with the most label indication function as final labeled microRNAs; according to the principle of statistical analysis, P values smaller than 0.05 are significant differences, and finally dre-miR-223 is selected, and the sequence is TGTCAGTTTGTCAAATACCCC. The expression level of the tag in a male fish sample is higher, while the expression level in a female fish sample is lower. And judging female fish and male fish according to the microRNA quantitative analysis result.
The invention also provides a primer for amplifying sex tags of the high-body Seriola exosome microRNAs, wherein the primer is a sex tag dre-miR-223 quantitative detection primer, the dre-miR-223 quantitative detection primer comprises a forward primer dre-miR-223-F and a reverse primer dre-miR-223-R, and the base sequence of the forward primer dre-miR-223-F is shown as SEQ ID NO:2, the base sequence of the reverse primer dre-miR-223-R is shown as SEQ ID NO: 3.
Specific:
the base sequence of the primer for quantitatively detecting dre-miR-223 is as follows:
dre-miR-223-F：CGCGTGTCAGTTTGTCAAA(SEQ ID NO：2)；
dre-miR-223-R：AGTGCAGGGTCCGAGGTATT(SEQ ID NO：3)。
the invention also provides a kit for sex identification of high body Seriola, which comprises the dre-miR-223 quantitative detection primer, dre-miR-223 reverse transcription primer dre-miR-223-RT, internal reference gene oni-let-7a quantitative detection primer, oni-let-7a reverse transcription primer oni-let-7a-RT, reverse transcriptase polymerase chain reaction (RT-PCR) and real-time fluorescence quantitative PCR (qPCR) conventional reagents.
Preferably, the base sequence of the dre-miR-223 reverse transcription primer dre-miR-223-RT is shown in SEQ ID NO:4 is shown in the figure; the quantitative detection primers of the reference gene oni-let-7a comprise a forward primer oni-let-7a-F and a reverse primer oni-let-7a-R, wherein the base sequence of the oni-let-7a forward primer oni-let-7a-F is shown as SEQ ID NO:5, the base sequence of the oni-let-7a reverse primer oni-let-7a-R is shown in SEQ ID NO:6 is shown in the figure; the base sequence of the oni-let-7a reverse transcription primer oni-let-7a-RT is shown in SEQ ID NO: shown at 7.
Specific:
the dre-miR-223 reverse transcription primer is dre-miR-223-RT, and the base is as follows:
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGGGGTA(SEQ ID NO：4)。
the quantitative detection primers of the reference gene oni-let-7a are as follows:
oni-let-7a-F：GCGCGTGAGGTAGTAGGTTGT(SEQ ID NO：5)；
oni-let-7a-R：AGTGCAGGGTCCGAGGTATT(SEQ ID NO：6)；
the oni-let-7a reverse transcription primer is oni-let-7a-RT, and the base sequence is as follows:
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACTAT(SEQ ID NO：7)。
the second object of the present invention can be achieved by the following technical means: the application of the sex tag of the high-body Seriola exosome microRNAs in preparing the high-body Seriola sex identification product.
The invention further discloses application of the primer or the kit in preparation of high-body Seriola sex identification products.
In the high body seriolae sex identification, the following steps can be adopted: collecting a serum sample of the quiniola quinquera, extracting the RNA of the quiniola quinquera exosome, carrying out RT-PCR through a specific miRNA reverse transcription primer or the kit to obtain a cDNA template, carrying out qPCR through the specific miRNA forward primer and reverse primer or the kit to obtain the expression condition of microRNA (preferably, carrying out miRNA quantitative reaction through a SYBR Green I chimeric fluorescence method), and identifying the sex of the quiniola quinquera according to the expression quantity of a sex tag dre-miR-223 in female and male fish, wherein the expression quantity in the male fish sample is higher, and the expression quantity in the female fish sample is lower.
Preferably, when RT-PCR is used, a two-step method is used. The first step of removing genomic DNA, the reaction system is a 10. Mu.L system,comprises 4 mu L ddH 2 O, 2. Mu.L gDNA Wiper Mix and 4. Mu.L RNA, the reaction procedure was: 42 ℃ for 2min; in the second step, cDNA was synthesized in a 20. Mu.L reaction system comprising 2. Mu.LddH 2 O, 10. Mu.L of the mixture in the first step, 4. Mu.L of reverse transcription primer, 2. Mu.L of 10 XRT Mix and 2. Mu.L of LHiScript II Enzyme Mix, the reaction procedure is: 25 ℃ for 5min,50 ℃ for 15min and 85 ℃ for 5min.
Wherein, the reverse transcription primer is dre-miR-223 reverse transcription primer dre-miR-223-RT or oni-let-7a reverse transcription primer oni-let-7a-RT.
Preferably, when qPCR (preferably SYBR Green I chimeric fluorescence method) is used for quantitative reaction of miRNA, the reaction system is 10. Mu.L system comprising 0.5. Mu.L 10 XmiRNA-F primer, 0.5. Mu.L 10 XmiRNA-R primer, 5. Mu.L 2X miRNA Universal SYBR qPCR Master Mix, 3. Mu.L ddH 2 O, 1. Mu.L cDNA template.
Wherein the miRNA-F primer is dre-miR-223-F or oni-let-7a-F, and the miRNA-R primer is dre-miR-223-R or oni-let-7a-R.
Preferably, when qPCR (preferably SYBR Green i chimeric fluorescence method is used for miRNA quantitative reaction), the reaction procedure is: 95 ℃ for 5min; cycling for 40 times at 94 ℃ for 30s,58 ℃ for 20s and 72 ℃ for 20 s; 15s at 95 ℃, 1min at 65 ℃ and 97 ℃; 30s at 37 ℃.
Compared with the prior art, the invention has the following advantages:
(1) The serum exosome microRNAs are used for judging the high-body female and male Seriola, the identification result is reliable, and the fish body is not injured;
(2) The invention designs the amplification primer aiming at the label by utilizing the identification label, and develops a detection kit, thereby being convenient, quick, simple and efficient.
Drawings
FIG. 1 shows 18 microRNAs and log-based thereof selected by microRNA sequencing in example 1 of the present invention 2 The relative expression quantity calculated by the FoldChange value;
fig. 2 shows qRT-PCR expression levels of dre-miR-223 in example 3 of the present invention, n=9, after verification by 18 verification samples;
fig. 3 is a qRT-PCR expression level mean value of dre-miR-223 in example 3 in two groups of samples after verification by 18 verification samples, n=9, P <0.05;
FIG. 4 is a ROC graph of the evaluation of the effect of dre-miR-223 on high-body Seriola sexing in example 3.
Detailed Description
The invention will be further elaborated in connection with the drawings and the specific embodiments described below, which are intended to illustrate the invention only and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
Example 1
Screening and determining sex tags of high-body Seriola exosome microRNAs as biomarkers
1. Collection of serum and gonadal samples
Sex differentiated blood of one-year-old high body of Seriola is collected to obtain serum, and 0.5mL of serum is obtained from each fish for subsequent exosome separation and identification experiments. Dissecting the fish body, observing and collecting gonads to identify the sex of the fish body.
2. Extraction of exosomes
(1) Serum samples were taken from 0.5mL transfer 1.5mLEP tubes, added to 1mL total exosome separation reagent (from cell culture medium) (4478359,Thermofisher Scientific), mixed thoroughly, and left overnight at 4 ℃;
(2) Centrifuging at 4deg.C and 10000rpm/min for 1 hr, discarding supernatant, and inverting the centrifuge tube on filter paper for 2min;
(3) The pellet was resuspended in 50. Mu.L PBS and stored at-20 ℃.
3. Identification of exosomes:
nanoparticle Tracking Analysis (NTA) using a malvern Nanosight NS 300; western Blot (WB) analysis using antibodies CD63, CD9 and HSP 70; transmission Electron Microscopy (TEM) analysis was performed using Tecnai G2 Spirit, and after analysis and identification of exosomes, the remaining samples were used for RNA extraction and sequencing analysis.
4. MicroRNA sequencing analysis of exosomes
Extracting RNA of 3 female fish and 3 male fish exosomes by using a Trizol method, constructing a small RNA library after quality inspection, sequencing based on an illumine platform, filtering raw reads after sequencing to obtain 23.98-24.69M clean reads, and comparing and annotating the clean reads with an Rfam database, a cDNA sequence, a species repeated sequence library and a miRBase database in sequence; counting 476 known microRNAs and predicting new microRNAs; performing differential expression analysis on known microRNAs, performing GO enrichment analysis and KEGG enrichment analysis on target genes of differential expression miRNA, performing correlation matching with female and male fish samples, and screening one or more microRNAs with indication effect on the female and male fish samples as candidate tag microRNAs.
5. Candidate tagged microRNAs were filtered according to the following five criteria:
(1) There was a significant difference in expression in the two groups of samples of the female group (3 females) and of the male group (3 males), i.e. P-value less than 0.05;
(2) Comparing with a known database to confirm the existence of microRNAs;
(3) At least one of the two samples has a TPM value greater than 0;
(4) A foldChange value greater than 2, the foldChange value = TPM sample_Male ./TPM Sample female ；
(5) From the 18 microRNAs screened out from the four standards, 11 microRNAs are randomly selected.
The expression levels of the 18 microRNAs were counted as shown in FIG. 1, and the results are shown in FIG. 1.
6. Two random sets of validation samples were extracted: extracting serum-derived exosome total RNA of 3 male fish and 3 female fish identified by gonadal dissection; designing reverse transcription primers and qPCR primers of 11 microRNAs and internal reference gene oni-let-7a by a conventional stem-loop method; RT-PCR is carried out by using a microRNA reverse transcription kit (miRNA 1st Strand cDNA Synthesis Kit (by stem-op), and the company of vazyme) to obtain cDNA templates of 11 candidate microRNAs and reference gene oni-let-7 a; quantitative analysis kit (miRNA Universal SYBRgPCR Master Mix, vazyme#MQ1) was used with microRNA01 Quantitatively detecting the differential expression condition of the 11 candidate microRNAs filtered in the step (5); select 2 -ΔΔCt And (3) carrying out calculation analysis by using a method, and screening out the microRNAs with the most label indication function as final labeled microRNAs.
According to the principle of statistical analysis, P values smaller than 0.05 are significant differences, and finally dre-miR-223 is selected, and the sequence is TGTCAGTTTGTCAAATACCCC. The expression level of the tag in the male fish sample is higher, the expression level in the female fish sample is lower, the expression level is consistent with the miRNA sequencing result, and the expression level of the tag between the male fish and the male fish has obvious difference (P < 0.05).
Example 2
1) Oni-let-7a was used as an internal reference gene.
Verification of stability of the reference gene oni-let-7 a: total RNA of 8 tissues including gonad, hypothalamus, pituitary, heart, liver, intestinal tract, muscle, serum exosome and the like of 3 male fishes and 3 female fishes is extracted, wherein the concentration of RNA of the serum exosome is 10 ng/mu L, and the concentration of RNA of 7 tissues is 400 ng/mu L. RT-PCR is carried out by utilizing a microRNA reverse transcription kit to obtain a cDNA template of oni-let-7a, and the expression condition (C) of oni-let-7a in 8 different tissues is quantitatively detected by utilizing a microRNA quantitative analysis kit T Values). oni let-7a is expressed stably in 8 tissues such as gonad, hypothalamus, pituitary, heart, liver, intestinal tract, muscle and serum exosome (C T Small value changes) can be used as reference genes as shown in table 1.
TABLE 1 internal reference gene oni-let-7a C in 8 different tissues T Values.
2) Primers for amplifying sex tags of the seriolae serum exosomes microRNAs were designed as follows:
reverse transcription primers and qPCR primers of the dr-miR-223 and reference gene oni-let-7a were designed by stem-loop method using miRNA Design (Version 1.01) and Primer3 (Version 0.4.0) based on RNA sequences of dr-miR-223 and reference gene oni-let-7 a.
The method is characterized in that a specific primer is designed based on a serum exosome microRNA marker dre-miR-223, the primer is a sex tag dre-miR-223 quantitative detection primer, the dre-miR-223 quantitative detection primer comprises a forward primer dre-miR-223-F and a reverse primer dre-miR-223-R, and the base sequence of the forward primer dre-miR-223-F is shown as SEQ ID NO:2, the base sequence of the reverse primer dre-miR-223-R is shown as SEQ ID NO: 3.
Specific:
the base sequence of the primer for quantitatively detecting dre-miR-223 is as follows:
dre-miR-223-F：CGCGTGTCAGTTTGTCAAA(SEQ ID NO：2)；
dre-miR-223-R：AGTGCAGGGTCCGAGGTATT(SEQ ID NO：3)。
the embodiment also provides a kit for high body Seriola sex determination, which comprises a dre-miR-223 quantitative detection primer, a dre-miR-223 reverse transcription primer dre-miR-223-RT, an internal reference gene oni-let-7a quantitative detection primer, a oni-let-7a reverse transcription primer oni-let-7a-RT and RT-PCR and qPCR conventional reagents.
The base sequence of the dre-miR-223 reverse transcription primer dre-miR-223-RT is shown in SEQ ID NO: 4.
The quantitative detection primers of the reference gene oni-let-7a comprise a forward primer oni-let-7a-F and a reverse primer oni-let-7a-R, wherein the base sequence of the forward primer oni-let-7a-F is shown as SEQ ID NO:5, the base sequence of the reverse primer oni-let-7a-R is shown in SEQ ID NO: shown at 6.
The base sequence of the internal reference gene oni-let-7a reverse transcription primer oni-let-7a-RT is shown in SEQ ID NO: shown at 7.
Specific:
the base sequence of the primer for quantitatively detecting dre-miR-223 is as follows:
dre-miR-223-F：CGCGTGTCAGTTTGTCAAA(SEQ ID NO：2)；
dre-miR-223-R：AGTGCAGGGTCCGAGGTATT(SEQ ID NO：3)。
the dre-miR-223 reverse transcription primer is dre-miR-223-RT, and the base is as follows:
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGGGG TA(SEQ ID NO：4)。
the primers of the reference gene oni-let-7a are:
oni-let-7a-F：GCGCGTGAGGTAGTAGGTTGT(SEQ ID NO：5)；
oni-let-7a-R：AGTGCAGGGTCCGAGGTATT(SEQ ID NO：6)；
the reference gene oni-let-7a reverse transcription primer is oni-let-7a-RT, and the base sequence is as follows:
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACT AT(SEQ ID NO：7)。
the sequence of the reference gene oni-let-7a was TGAGGTAGTAGGTTGTATAGTT (SEQ ID NO: 8).
Therefore, the serum exosome microRNA marker dre-miR-223-based identification kit comprises a dre-miR-223 quantitative detection primer and a reverse transcription primer thereof, a quantitative detection primer of an internal reference gene oni-let-7a and a reverse transcription primer thereof, and other conventional reagents of RT-PCR and qPCR: such as RNase-free ddH 2 O、gDNA Wiper Mix、RT Mix、HiScriptⅡEnzyme Mix、dNTP、miRNA Universal SYBR qPCR Master Mix、RNase inhibitor、Ace Taq DNA polymerase、Mg 2+ SYBR Green I, specific ROX Reference, etc.
Example 3
The high body quince sex detection kit of example 2 was used to detect high body quince sex, comprising the steps of:
(1) Collecting a serum sample of the high-body Seriola, and extracting the external RNA;
(2) Carrying out RT-PCR on the exosome RNA by using reverse transcription primers and reagents in the high-body Seriola gender detection kit of the example 2 to obtain a cDNA template;
(3) qPCR is carried out on cDNA obtained through reverse transcription to obtain the expression condition of miRNA;
(4) Judging the sex of the high body quince according to the expression condition of miRNA.
For step (2):
wherein, RT-PCR is carried out by adopting a two-step method so as to obtain a cDNA template;
further, the two-step method comprises the following steps:
the first step of removing genomic DNA, the reaction system was 10. Mu.mL System comprising 4. Mu.L ddH 2 O, 2. Mu.L gDNA Wiper Mix and 4. Mu.L RNA, the reaction procedure was: 42 ℃ for 2min;
in the second step, cDNA was synthesized in a 20. Mu.L reaction system comprising 2. Mu.L ddH 2 O, 10. Mu.L of the mixture in the first step, 4. Mu.L of reverse transcription primer, 2. Mu.L of 10 XRT Mix and 2. Mu.L of LHiScript II Enzyme Mix, the reaction procedure is: 25 ℃ for 5min,50 ℃ for 15min and 85 ℃ for 5min.
Wherein the reverse transcription primer is dre-miR-223 reverse transcription primer dre-miR-223-RT or reference gene oni-let-7a reverse transcription primer oni-let-7a-RT.
Specifically, RT-PCR of miRNA was performed by Veriti TM The 96-well rapid thermal cycler was performed using a two-step method.
For step (3):
when qPCR (SYBR Green I chimeric fluorescence method) is used for carrying out miRNA quantitative reaction, the reaction system is a 10 mu L system, and comprises 0.5 mu L of 10 xmiRNA-F primer, 0.5 mu L of 10 xmiRNA-R primer, 5 mu L of 2 x miRNA Universal SYBR qPCR Master Mix and 3 mu L of ddH 2 O, 1. Mu.L cDNA template. Wherein the miRNA-F primer is dre-miR-223-F or oni-let-7a-F, and the miRNA-R primer is dre-miR-223-R or oni-let-7a-R.
qPCR detection of mirnas was performed using Roche Applied Science LightCycler 480, the procedure of the reaction was: 95 ℃ for 5min; cycling for 40 times at 94 ℃ for 30s,58 ℃ for 20s and 72 ℃ for 20 s; 15s at 95 ℃, 1min at 65 ℃ and 97 ℃; 30s at 37 ℃.
The method specifically comprises the following steps:
collecting serum samples of 9 high-body Seriola quinquefoil and 9 female Seriola quinquefoil after gonad dissection identification, dissecting fish bodies, observing and collecting gonads to identify the sex of the fish bodies, and extracting serum exosome RNA;
RT-PCR is carried out through specific miRNA reverse transcription primers or kits to obtain cDNA templates of dre-miR-223 and internal reference gene oni-let-7 a;
qPCR is carried out by adopting specific miRNA forward and reverse primers or a kit to obtain the expression condition of dre-miR-223 (miRNA quantitative reaction is carried out by a SYBR Green I chimeric fluorescence method);
and judging the sex of the high-body Seriola according to the expression level of the label dre-miR-223 in the two groups of samples.
Samples were tested in 3 replicates. The specific primer is used for detecting the expression condition of the label microRNA of 9 male fishes and 9 female fishes, namely dre-miR-223, the results are shown in table 2 and figure 2, the expression quantity of the label dre-miR-223 in a male fish sample is higher, the expression quantity of the label in a female fish sample is lower, and the expression of the label in two groups of samples has obvious difference (P < 0.05), as shown in figure 3.
Table 2dre-miR-223 expression in serum exosomes, n=9.
Diagnostic value of the tag dre-miR-223 in the identification of the sex of high body quince:
ROC curve analysis (subject working characteristic curve, receiver Operator CharacteristicCurve, abbreviated as ROC curve) analysis is carried out according to the expression level of dre-miR-223 in 18 high body Seriola serum exosome samples in table 2, so that the diagnostic value of dre-miR-223 in high body Seriola sex identification is achieved. ROC analysis and AUC (area size under ROC curve, area under the curve, AUC for short) and boulder index (correct index) calculations were done by MedCalc statistical software (Version 20.217). The results are shown in FIG. 4, in which the vertical axis represents sensitivity (%), and the horizontal axis represents 100-specificity (%), i.e., false positive rate. The area under the curve AUC is 0.926 (male/female), which indicates that the diagnosis of the tag dre-miR-223 in sex identification of high body quince is high. When the about dengue index is maximum, the critical value of the expression quantity of dre-miR-223 is 81.94969454. According to the standard, the high-body Seriola serum exosome dre-miR-223 is used for identifying high-body Seriola female fish and high-body Seriola male fish, the expression quantity identification result of 16 samples accords with the gonad identification result, and the accuracy of dre-miR-223 in the verified samples is 88.89%.
For those skilled in the art, the conception and technical gist of the present invention are modified, and corresponding modifications should fall within the claims of the present invention. The invention is not limited to the specific embodiments described above, which are only intended to be able to describe in detail the course of use of the invention, and also the production methods and technical details with equivalent functions are part of the present disclosure. Indeed, those skilled in the art will be able to find different adjustment schemes according to the needs of each, and these adjustments are within the scope of the claims appended hereto.
Claims (4)
1. The application of the reagent for detecting the sex tag of the Seriola quinqueradia exosome microRNAs in preparing the product for identifying the sex of the Seriola quinqueradia is characterized in that: the sex tag is dre-miR-223, and the sequence of the sex tag is shown in SEQ ID NO: 1.
2. Use of a primer for amplifying the sex tag dre-miR-223 of claim 1 for the preparation of a high body quince sexing product, characterized in that: the primer is a sex label dre-miR-223 quantitative detection primer, the dre-miR-223 quantitative detection primer comprises a forward primer dre-miR-223-F and a reverse primer dre-miR-223-R, and the base sequence of the forward primer dre-miR-223-F is shown as SEQ ID NO:2, the base sequence of the reverse primer dre-miR-223-R is shown as SEQ ID NO: 3.
3. The application of the kit in preparing a high-body Seriola sex identification product is characterized in that: the kit comprises a dre-miR-223 quantitative detection primer, a dre-miR-223 reverse transcription primer, a dre-miR-223-RT, an internal reference gene oni-let-7a quantitative detection primer, a oni-let-7a reverse transcription primer oni-let-7a-RT and RT-PCR and qPCR conventional reagents in claim 2.
4. A use according to claim 3, characterized in that: the base sequence of the reverse transcription primer dre-miR-223-RT is shown in SEQ ID NO:4 is shown in the figure; the quantitative detection primer of the reference gene oni-let-7a comprises a forward primer oni-let-7a-F and a reverse primer oni-let-7a-R, wherein the base sequence of the forward primer oni-let-7a-F is shown as SEQ ID NO:5, the base sequence of the reverse primer oni-let-7a-R is shown as SEQ ID NO:6 is shown in the figure; the base sequence of the oni-let-7a reverse transcription primer oni-let-7a-RT is shown in SEQ ID NO: shown at 7.
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