CN116121397A - Reagent for detecting SNP molecular marker combination related to anti-nerve necrosis virus character of groupers - Google Patents
Reagent for detecting SNP molecular marker combination related to anti-nerve necrosis virus character of groupers Download PDFInfo
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Abstract
The application is a divisional application of an invention patent application of which the application date is 12/21/2020 and the application number is 202011520740X, and the invention name is a reagent for detecting SNP molecular marker combinations related to the anti-nerve necrosis virus properties of groupers. The invention discloses a reagent for detecting SNP molecular marker combinations related to the anti-nervous necrosis virus characteristics of groupers, wherein 5 SNP loci related to the anti-nervous necrosis virus characteristics of groupers are found in the invention, and when the genotype of SNP locus 1 is GG, the genotype of SNP locus 2 is AC, the genotype of SNP locus 3 is CC, the genotype of SNP locus 4 is TA, the genotype of SNP locus 5 is TT, the death probability of groupers infected by the nervous necrosis virus is obviously lower than that of individuals with other genotypes. By detecting the SNPs of the Epinephelus akaara, whether the Epinephelus akaara has the nervous necrosis virus resistance property or not can be effectively determined.
Description
The application is a divisional application of an invention patent application of which the application date is 12/21/2020 and the application number is 202011520740X, and the invention name is a reagent for detecting SNP molecular marker combinations related to the anti-nerve necrosis virus properties of groupers.
Technical Field
The present invention relates to the use thereof. The technical field, in particular to a reagent for detecting SNP molecular marker combinations related to the anti-nerve necrosis virus characteristics of groupers.
Background
The red groupers (Epinephelus akaara) are commonly called erythema, and the genus Epinephelus (Epinephelus) of the order Perciformes (Serratia) is mainly distributed in the western North Pacific ocean and produced in the island of Zhoushan in the area from south via Taiwan strait to south sea and North bay. The grouper feed has the characteristics of delicious meat quality and high nutritive value, and is an important grouper culture variety in coastal areas of south China.
The red-spotted grouper nervous necrosis virus (RGNNV) of the Epinephelus is very harmful to adult and young fishes of the Epinephelus, the death rate can reach 100% when serious, huge economic loss is usually caused to the breeding of the Epinephelus, the healthy development of the Epinephelus breeding industry is seriously threatened, however, no effective treatment means is available for the diseases caused by the virus at present, and the prevention and control monitoring are mainly carried out.
Chinese patent CN201610255794.5 discloses a method for detecting garrupa nervous necrosis virus infection based on sandwick ELASA of nucleic acid aptamer; CN201610465941.1 discloses a colloidal gold immunochromatography test paper for detecting the antibody of the nervous necrosis virus of the grouper and application thereof; CN201910873715.0 discloses a colloidal gold test strip for detecting nerve necrosis virus of grouper, and preparation and detection methods thereof; CN200910039534.4 discloses a primer group, a detection method and a rapid diagnostic kit for detecting the nerve necrosis virus of the garrupa; CN03114369.5 discloses a diagnosis kit and a detection method for grouper virus nerve necrosis virus gene, but lacks a method for detecting grouper nerve necrosis virus resistance.
Genome-wide association analysis (Genome-wide association study, GWAS) refers to a strategy technique of widely screening a large number of single nucleotide polymorphism (single nucleotide ploymorphism, SNP) molecular markers in the whole Genome of a plurality of individuals of a sample, obtaining genotypes, performing association analysis by using the SNP genotypes and the phenotypic traits of the sample, and screening key mutation sites capable of affecting the phenotypic traits by statistical analysis. With the development of genomic research and sequencing technology, researchers have discovered and identified a number of genetic variations associated with phenotypic traits through this technology. In recent years, the method is widely applied to screening and identifying important economic character related genes of aquatic economic animals.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a reagent for detecting SNP molecular marker combinations related to the anti-nerve necrosis virus characteristics of groupers.
The first object of the invention is to provide a reagent for detecting any one or more molecular markers in SNP molecular marker combinations related to the anti-nerve necrosis virus characteristics of groupers.
A second object of the present invention is to provide a primer for detecting the SNP site 1.
A third object of the present invention is to provide a primer for detecting said SNP site 2.
A fourth object of the present invention is to provide a primer for detecting said SNP site 3.
A fifth object of the present invention is to provide a primer for detecting said SNP site 4.
A sixth object of the present invention is to provide a primer for detecting said SNP site 5.
The seventh object of the invention is to provide the reagent, or the application of any one or more of the primers in preparing a grouper nervous necrosis virus resistance detection kit.
The seventh object of the invention is to provide a grouper nervous necrosis virus resistance detection kit.
An eighth object of the present invention is to provide the use of one or more of the reagents, the primers, or the kit for the resistance of grouper to nervous necrosis virus.
In order to achieve the above object, the present invention is realized by the following means:
the invention claims a reagent for detecting any one or more molecular markers in SNP molecular marker combinations related to the anti-nerve necrosis virus characteristics of groupers, which is used for detecting genotypes of SNP locus 1, SNP locus 2, SNP locus 3, SNP locus 4 and/or SNP locus 5;
the SNP locus 1 is positioned at 301bp from the 5' end of a nucleotide sequence shown in SEQ ID NO.1, and when the genotype of a sample is GG, the death probability of the sample after infection of the nervous necrosis virus is obviously lower than that of individuals with other genotypes;
the SNP locus 2 is positioned at 300bp from the 5' end of a nucleotide sequence shown in SEQ ID NO.2, and when the genotype of a sample is AC, the death probability of the sample after infection of the nervous necrosis virus is obviously lower than that of individuals with other genotypes;
the SNP locus 3 is positioned at 301bp from the 5' end of a nucleotide sequence shown in SEQ ID NO.3, and when the genotype of a sample is CC, the death probability of the sample after infection of the nervous necrosis virus is obviously lower than that of individuals with other genotypes;
the SNP locus 4 is positioned at 300bp from the 5' end of a nucleotide sequence shown in SEQ ID NO.4, and when the genotype of a sample is TA, the death probability of the sample after infection of the nervous necrosis virus is obviously lower than that of individuals with other genotypes;
the SNP locus 5 is positioned at 301bp from the 5' end of the nucleotide sequence shown in SEQ ID NO.5, and when the genotype of the sample is TT, the death probability of the sample after infection of the nervous necrosis virus is obviously lower than that of individuals with other genotypes.
Preferably, the groupers are Epinephelus akaara.
Preferably, the reagent is a primer.
The invention also claims the following primers:
the primer for detecting the SNP locus 1 according to claim 1, wherein the nucleotide sequence of the primer is shown as SEQ ID NO. 6-7.
The primer for detecting the SNP locus 2 according to claim 1, wherein the nucleotide sequence of the primer is shown as SEQ ID NO. 8-9.
The primer for detecting the SNP locus 3 according to claim 1, wherein the nucleotide sequence of the primer is shown as SEQ ID NO. 10-11.
The primer for detecting the SNP locus 4 according to claim 1, wherein the nucleotide sequence of the primer is shown as SEQ ID NO. 12-13.
The primer for detecting the SNP locus 5 according to claim 1, wherein the nucleotide sequence of the primer is shown as SEQ ID NO. 14-15.
Furthermore, the invention claims the application of the reagent, or any one or more of the primers in preparing a grouper nervous necrosis virus resistance detection kit.
The invention also discloses a grouper nervous necrosis virus resistance detection kit which comprises the reagent.
Preferably, any one or several of the primers for detecting SNP loci 1 to 5 are included.
More preferably, primers for detecting SNP sites 1 to 5 are included.
Even more preferably, the primers with the acid sequences shown in SEQ ID No. 6-15 are included.
Most preferably, the grouper nervous necrosis virus resistance detection kit comprises primers with nucleotide sequences shown in SEQ ID NO. 6-15 and PCR reagents.
The application method comprises the following steps: the using method of the kit comprises the following steps:
(1) Extracting genome DNA of the to-be-detected Epinephelus akaara;
(2) Respectively carrying out PCR amplification by taking the DNA obtained in the step (1) as a template and taking nucleotide sequences shown in SEQ ID NO. 6-7, 8-9, 10-11, 12-13 and 14-15 as primers to obtain PCR amplification products;
(3) Sequencing the PCR amplification product obtained in the step (2), and determining genotypes of SNP loci 1-5 of the to-be-detected Epinephelus akaara;
(4) Determining whether the Epinephelus akaara to be detected is resistant to the nervous necrosis virus according to the genotypes of the SNP loci 1-5 determined in the step (3):
The invention also claims the use of one or more of said reagents, said primers, or said kit for the resistance of grouper to nervous necrosis virus.
In the invention, genome extraction is not particularly limited, and can be extracted by adopting a traditional phenol-chloroform method or adopting a kit, and the specific embodiment of the invention is that the kit is used for extraction, and the kit is simple and convenient to operate and rapid and has high quality of extracted DNA.
In addition, the method for detecting the genotype of the Epinephelus akaara individuals to be detected is not particularly limited, and the techniques such as time-of-flight mass spectrometry, sequencing, chip, single-strand conformation polymorphism polymerase chain reaction, restriction fragment length polymorphism polymerase chain reaction and the like can be used for detecting SNP.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, 5 SNP loci related to the anti-nervous necrosis virus characteristics of the Epinephelus akaara are found, and when the genotype of the SNP locus 1 is GG, the genotype of the SNP locus 2 is AC, the genotype of the SNP locus 3 is CC, the genotype of the SNP locus 4 is TA, the genotype of the SNP locus 5 is TT, the death probability of the Epinephelus akaara infected with the nervous necrosis virus is obviously lower than that of individuals with other genotypes. Therefore, by detecting the SNPs described above, it is possible to efficiently determine whether or not it has a nervous necrosis virus resistance trait. Therefore, the SNP marker is closely related to the susceptibility of the Epinephelus akaara to the nervous necrosis virus, and the selection of individuals with susceptibility genotypes in parent breeding is favorable for improving the capability of offspring to resist the nervous necrosis disease.
The primer pair of the SNP locus can be used for effectively detecting the genotype of the Epinephelus akaara to be detected, and the result can be used for judging whether the Epinephelus akaara to be detected has resistance to the nervous necrosis virus or not on one hand and providing reference for parent selection on the other hand. Therefore, the SNP locus primer can be used for detecting the genotype of the Epinephelus akaara and judging whether the individual has resistance to the nervous necrosis virus, can be effectively used for the molecular marker assisted selection breeding of the Epinephelus akaara and quickens the breeding process of the Epinephelus akaara good disease-resistant variety.
The invention also provides a kit for detecting the SNPs mark, which can effectively detect the susceptibility of the Epinephelus akaara to the nervous necrosis virus and is used for molecular marker assisted breeding of the Epinephelus akaara.
Drawings
FIG. 1 is a diagram showing an electrophoresis detection of PCR amplification using the primer of SNP site 1.
FIG. 2 is a diagram showing an electrophoresis detection of PCR amplification using primers for SNP site 2.
FIG. 3 is an electrophoresis detection chart of PCR amplification using the primer of SNP site 3.
FIG. 4 is a diagram showing an electrophoresis detection of PCR amplification using the primers of SNP site 4.
FIG. 5 is an electrophoresis detection chart of PCR amplification using the primer of SNP site 5.
Detailed Description
The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration 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 of SNPs of the PPAR-delta Gene of Epinephelus akaara
1. Experimental method
(1) Epinephelus akaara sample source
The sample to be tested is obtained from 2 month-old Epinephelus akaara artificially bred in the same batch in the small-path Epinephelus akaara farm, and healthy fish fries 300 tails with the weight of about 50 g/tail are randomly selected.
Injecting half-lethal concentration (1×10) into Epinephelus akaara by intraperitoneal injection 7 TCID 50 Per mL) the nervous necrosis virus culture broth, the dead individuals and the individuals with typical onset symptoms are susceptible individuals within 3 days, and the individuals which survive after 10 days and have no obvious onset symptoms are resistant individuals.
Randomly selecting 50 anti-susceptibility individuals (anti-susceptibility groups) and 50 susceptibility individuals (susceptibility groups), cutting fin bars of the anti-susceptibility individuals, and storing the fin bars in absolute ethyl alcohol for extracting genome DNA.
(2) Extraction of grouper sample genome DNA
Extraction of genomic DNA Using Trelief TM Animal Genomic DNA Kit (TsingKe) kit, according to the instructions, operates as follows:
1) Preparing a tissue sample: taking a proper amount of grouper fin tissue in a 1.5mL centrifuge tube, and shearing the fin by using sterilized scissors;
2) An activated silica gel film: placing Spin Column in Collection Tube, adding 250 μL Buffer BL, and centrifuging at 12,000g for 1min;
3) Sample digestion: mu.L of Proteinase K was taken into a fresh 1.5mL centrifuge tube and 200. Mu.L of ddH was added 2 The tissue fragments after O dilution are vortexed for 10s, 200 mu L Buffer gA1 is added, vortexed for 10s, and incubated for 1-3 h or overnight at 56 ℃, and the vortexed for 3-5 times;
4) After the incubation is finished, 200 mu L of absolute ethyl alcohol is added, and vortex vibration is carried out for uniform mixing;
5) Transferring all the solution obtained in the step 4) into Spin Column, centrifuging at 12,000g for 1min, and discarding filtrate;
6) Adding 500 μL Buffer PW, centrifuging at 12,000g for 30s, and discarding the filtrate;
7) Repeating step 6) once;
8) Adding 500 mu L of Wash Buffer, centrifuging at 12,000g for 30s, and discarding the filtrate;
9) Throwing the mixture for 2min at 12,000g, discarding the filtrate, putting the Spin Column into a new 1.5mL centrifuge tube, uncovering and airing for 1min;
10 50-100 mu L of TE Buffer preheated to 65 ℃ in advance is added to the center of the adsorption film, and the solution is placed for 2min at room temperature and centrifuged for 2min at 12,000 g;
11 Adding the obtained solution into Spin Column again, and centrifuging at 12,000g for 2min;
12 2. Mu.L of DNA was subjected to electrophoresis and 1. Mu.L was used for determining the DNA concentration, and stored at-20 ℃.
(3) Acquisition of SNPs related to resistance traits of Epinephelus akaara to nervous necrosis virus
Randomly selecting genomic DNA samples of 50 samples of each anti-susceptibility group and susceptibility group of Epinephelus akaara, sending the genomic DNA samples to Guangzhou cloud cable biotechnology company for simplified genomic sequencing, carrying out whole genome association analysis of anti-susceptibility/susceptibility traits,
2. experimental results
Obtaining 5 obvious association sites with highest-log 10 (P) value, namely SNPs sites related to resistance characters of the Epinephelus akaara and the nervous necrosis virus.
(1) The SNP locus 1 is positioned at the 301 th bp position of the nucleotide sequence shown in SEQ ID NO.1 from the 5' end, is G/C allele mutation, and has a genotype GG or GC.
(2) The SNP locus 2 is positioned at 300bp from the 5' end of the nucleotide sequence shown in SEQ ID NO.2, is A/C allele mutation, and has the genotype of AA or AC.
(3) The SNP locus 3 is positioned at the 301 th bp position of the nucleotide sequence shown in SEQ ID NO.3 from the 5' end, is T/C allele mutation, and the genotype is CC or TC.
(4) The locus SNP locus 4 is positioned at 300bp from the 5' end of the nucleotide sequence shown in SEQ ID NO.4, is a T/A allelic process, and has a genotype TT or TA.
(5) The SNP locus 5 is positioned at the 301 th bp position of the nucleotide sequence shown in SEQ ID NO.5 from the 5' end, is T/G allele mutation, and has a genotype TT or TG.
Example 2 PCR-sequencing verification analysis of SNPs loci associated with the resistance Properties of Epinephelus akaara and nervous necrosis Virus
1. Experimental method
1) Primer design
According to the positional information of 5 SNP loci obtained in example 1, the upstream and downstream sequences of these SNP loci are called in the grouper genome database, the sequence information is shown in FIG. 1, and SNP locus primers are designed based on these sequence information, the primer information is as follows:
SNP-1：
upstream primer F:5'-CCTGCTCACTGGACCCTCAC-3' (SEQ ID NO. 6);
the downstream primer R5'-CAGTCCCAAGCCACGAGAATA-3' (SEQ ID NO. 7).
SNP-2：
Upstream primer F:5'-CTGCCCGTCTGGGAAACTCT-3' (SEQ ID NO. 8);
the downstream primer R5'-AGGAAATCGGCTCTGGTGTT-3' (SEQ ID NO. 9).
SNP-3：
Upstream primer F:5'-CACTTCCCTGCTGTCCTTTG-3' (SEQ ID NO. 10);
the downstream primer R5'-CATCCACCCAGTGCTGAGAC-3' (SEQ ID NO. 11).
SNP-4：
Upstream primer F:5'-GGATGTTGAAAGCCGAGCCT-3' (SEQ ID NO. 12);
the downstream primer R5'-AAACTGAAATCTTCTGCGATG-3' (SEQ ID NO. 13).
SNP-5：
Upstream primer F:5'-GACCTTTCCTTTAATTTCCCTT-3' (SEQ ID NO. 14);
the downstream primer R5'-CTGTGGAGATTCAGGCGGTA-3' (SEQ ID NO. 15).
2) PCR amplification
The genomic DNA of the population sample of example 1 was used as a template, and PCR amplification was performed using the primers for the SNP sites described above, and the PCR reaction system was as follows:
the PCR reaction system is as follows:
the PCR conditions were as follows:
94 ℃ for 4min;94 ℃ for 10s,55 ℃ for 30s,72 ℃ for 2min and 35 cycles; and at 72℃for 10min.
3) SNP typing analysis of sequencing results
The PCR product was sent to TsingKe for sequencing, and the genotype of each SNP site was obtained from the sequencing result. And correlating the typing results with the resistance to the nervous necrosis virus, the anti-nervous necrosis virus correlation analysis was performed using the chi-square test in SPSS 17.0 statistical software.
2. Experimental results
The results of the analysis of the difference between the alleles and genotypes of the SNP loci are shown in Table 1, and P <0.05 shows that the difference is significant.
Table 1: genotype and allele frequency statistical analysis of 5 SNPs loci in anti-susceptibility and susceptible groups
As can be seen from table 1, the genotypes and allele frequencies of the 5 snp sites were significantly different in both the anti-susceptibility group and the susceptibility group (P < 0.05), and the genotypes significantly associated with the anti-susceptibility in the 5 snp sites were: SNP site 1: GG, SNP site 2: AC, SNP site 3: CC, SNP site 4: TA, SNP site 5: TT; genotypes significantly associated with susceptibility are: SNP site 1: GC, SNP site 2: AA, SNP site 3: TC, SNP site 4: TT, SNP site 5: TG. Therefore, the SNPs mark can be used for breeding the disease resistance of the Epinephelus akaara.
Example 3A method for detecting resistance of Epinephelus akaara against nervous necrosis Virus
And (3) detecting 5 SNP loci of the embodiment 2 on the sample of the Epinephelus akaara to be detected, and determining the disease resistance of the Epinephelus akaara to be detected to the nervous necrosis virus.
The method comprises the following steps:
(1) Extracting genome DNA of the to-be-detected Epinephelus akaara;
(2) Respectively carrying out PCR amplification by taking the DNA obtained in the step (1) as a template and taking nucleotide sequences shown in SEQ ID NO. 6-7, 8-9, 10-11, 12-13 and 14-15 as primers to obtain PCR amplification products;
(3) Sequencing the PCR amplification product obtained in the step (2), and determining genotypes of SNP loci 1-5 of the to-be-detected Epinephelus akaara;
(4) Determining whether the Epinephelus akaara to be detected is resistant to the nervous necrosis virus according to the genotypes of the SNP loci 1-5 determined in the step (3):
Example 4A kit for detecting resistance of Epinephelus akaara against nervous necrosis Virus
1. Composition of the composition
The nucleotide sequence is shown as SEQ ID NO. 6-15, and PCR reagent.
2. Application method
Same as in example 3.
It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Claims (6)
1. The reagent for detecting the molecular marker in the SNP molecular marker combination related to the property of the grouper against the nerve necrosis virus is characterized by being used for detecting the genotype of SNP locus 2;
the SNP locus 2 is positioned at 300bp from the 5' end of the nucleotide sequence shown in SEQ ID NO.2, and when the genotype of a sample is AC, the death probability of the sample after infection of the nervous necrosis virus is obviously lower than that of individuals with other genotypes.
2. The reagent of claim 1, wherein the reagent is a primer.
3. The primer for detecting the SNP locus 2 according to claim 1, wherein the nucleotide sequence is shown in SEQ ID NO.8 to 9.
4. The use of any one or more of the reagents of claim 1 or 2, or the primers of claim 3 in the preparation of a grouper nervous necrosis virus resistance detection kit.
5. A grouper nervous necrosis virus resistance detection kit comprising the reagent of claim 1.
6. Use of one or more of the reagents of claim 1, the primers of claim 3 or the kit of claim 5 for the resistance of grouper to nervous necrosis virus.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202211490519.3A CN116121397A (en) | 2020-12-21 | 2020-12-21 | Reagent for detecting SNP molecular marker combination related to anti-nerve necrosis virus character of groupers |
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| CN202011520740.XA CN112501312B (en) | 2020-12-21 | 2020-12-21 | Reagent for detecting SNP molecular marker combination related to anti-nervous necrosis virus traits of grouper |
| CN202211490519.3A CN116121397A (en) | 2020-12-21 | 2020-12-21 | Reagent for detecting SNP molecular marker combination related to anti-nerve necrosis virus character of groupers |
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| CN202211490691.9A Pending CN115896307A (en) | 2020-12-21 | 2020-12-21 | Reagent for detecting SNP molecular marker combination related to anti-nervous necrosis virus character of grouper |
| CN202211490519.3A Pending CN116121397A (en) | 2020-12-21 | 2020-12-21 | Reagent for detecting SNP molecular marker combination related to anti-nerve necrosis virus character of groupers |
| CN202211491304.3A Pending CN115896308A (en) | 2020-12-21 | 2020-12-21 | Reagent for detecting SNP molecular marker combination related to anti-nervous necrosis virus character of grouper |
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| CN202011520740.XA Active CN112501312B (en) | 2020-12-21 | 2020-12-21 | Reagent for detecting SNP molecular marker combination related to anti-nervous necrosis virus traits of grouper |
| CN202211490691.9A Pending CN115896307A (en) | 2020-12-21 | 2020-12-21 | Reagent for detecting SNP molecular marker combination related to anti-nervous necrosis virus character of grouper |
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| CN202211491305.8A Pending CN115896309A (en) | 2020-12-21 | 2020-12-21 | Reagent for detecting SNP molecular marker combination related to anti-nervous necrosis virus character of grouper |
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| CN115896297A (en) * | 2022-05-23 | 2023-04-04 | 集美大学 | SNP marker for anti-nervous necrosis disease virus breeding of Epinephelus akaara and application thereof |
| CN115992265B (en) * | 2023-03-22 | 2023-07-14 | 中山大学 | Grouper whole genome liquid phase chip and application thereof |
| CN116516028B (en) * | 2023-06-27 | 2023-09-15 | 中国海洋大学三亚海洋研究院 | SNP locus related to anti-nervous necrosis virus character of leopard gill-acanthus japonicus and application thereof |
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| US20170039316A1 (en) * | 2003-11-12 | 2017-02-09 | Yuriy Fofanov | Compositions, processes and algorithms for microbial detection |
| KR20170006690A (en) * | 2015-07-09 | 2017-01-18 | 대한민국(관리부서:국립수산과학원) | Management Strategy for Aquaculturing Sevenband Grouper with Resistance to Viral Nervous Necrosis |
| CN106086195A (en) * | 2016-06-28 | 2016-11-09 | 深圳华大基因研究院 | One SNP marker relevant to the cabrilla speed of growth and application thereof |
| CN109852710B (en) * | 2019-04-11 | 2022-06-21 | 深圳华大海洋科技有限公司 | SNP marker related to ammonia tolerance of grouper and application thereof |
| CN110579591A (en) * | 2019-09-17 | 2019-12-17 | 华南农业大学 | Colloidal gold test strip for detecting nervous necrosis virus of grouper and preparation and detection methods thereof |
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| CN112501312A (en) | 2021-03-16 |
| CN115896309A (en) | 2023-04-04 |
| CN112501312B (en) | 2022-12-16 |
| CN115896307A (en) | 2023-04-04 |
| CN115896308A (en) | 2023-04-04 |
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