CN114107521A - Primer probe composition and kit for detecting pure Siberian sturgeon nuclear gene and application of primer probe composition and kit - Google Patents
Primer probe composition and kit for detecting pure Siberian sturgeon nuclear gene and application of primer probe composition and kit Download PDFInfo
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Abstract
The invention discloses a primer probe composition for detecting a pure Siberian sturgeon nuclear gene, a kit and application thereof. The invention firstly discloses a primer probe composition for detecting a Siberian sturgeon nuclear gene, which comprises a primer probe I for detecting the Siberian sturgeon nuclear gene BGS and a primer II for detecting a Siberian sturgeon nuclear gene microsatellite SX37, wherein the sequences are shown as SEQ ID NO. 1-6. The invention also discloses a method for identifying pure Siberian sturgeon based on the primer probe composition. The method is quick, sensitive and specific, can be used for germplasm identification of the Siberian sturgeon, can be applied to identification outlets of pure sturgeons, and has wide application prospect.
Description
Technical Field
The invention belongs to the field of sturgeon germplasm identification and sturgeon source component detection, and particularly relates to a primer probe composition for detecting pure Siberian sturgeon nuclear genes, a kit and application thereof.
Background
Commercial sturgeon culture in China is brought about from the nineties of the twentieth century, full artificial propagation technologies of nearly 10 main sturgeons such as Siberian sturgeons, paddlefish, small-bodied sturgeons, acipenser schrenckii, Russian sturgeons, large hybrids (huso x schneisser sturgeons), huso, Chinese sturgeons, Yangtze sturgeons and the like are successively broken through, sturgeon culture sturgeons and caviar processing have been developed into industries, and the sturgeon culture yield is the first country in the world and sturgeon caviar processing is exported to the great country. Along with the rapid development of sturgeon breeding industry, the sturgeon germplasm resources are mixed and the quality and the quantity of produced offspring seeds are reduced to some extent due to the random hybridization of unknown parent fish germplasm in a plurality of sturgeon breeding farms, so that the overall development of the sturgeon industry is restricted. How to protect the wild population of sturgeons, and simultaneously, reasonably and regularly carry out sturgeon culture, genetic breeding and sturgeon industry healthy development are the key points of current research.
Therefore, the identification of the sturgeon variety is very important, and on one hand, endangered wild sturgeon species can be protected; in addition, the strain of the sturgeon can be determined by identifying the parents and is used for guiding the crossbreeding, breeding and production of the sturgeon; meanwhile, the variety identification is carried out on commercial fishes, detection identification proofs can be provided in the domestic circulation link, and the healthy development of the sturgeon breeding industry is better supervised and standardized. In recent years, the export trade of Siberian sturgeon is increased rapidly in China, and considerable economic benefit is obtained. However, due to the regulation limit of CITES convention and the phenomenon of sturgeon germplasm hybridization in China, the export trade of pure Siberian sturgeons is influenced, and serious economic loss is caused to domestic breeding enterprises. Therefore, a pure Siberian sturgeon identification method needs to be established to guide actual work, be applied to domestic sturgeon germplasm identification, breeding transaction and import and export trade, and protect and promote the healthy and sustainable development of the domestic sturgeon industry.
Currently, species identification methods are widely studied by specific PCR methods established for the nuclear genes cytochrome B (Cytb), cytochrome oxidase subunit I (CO I), DNA control region (D-LOOP), and the like. The microsatellite marker is a common molecular marker in population genetics research, has the advantages of co-dominance, high resolution, high repetition rate and high reliability, and is a most widely used nuclear genome marker in aquatic product research. The germplasm identification of the sturgeon can be developed from two levels of mitochondrial genes and nuclear genomes respectively, and is an effective way for comprehensively and accurately detecting the source of the male parent and the female parent of the hybrid sturgeon. However, since sturgeons of the order acipenser are of various species and the genetic sequences of different species of sturgeons have high homology, 10 pairs of microsatellite primers are usually used in combination with software analysis to identify sturgeons of different species in the literature published at home and abroad at present. The methods have the advantages of complex primers, complex operation and long time consumption, also need professional molecular biology software, have higher requirement on the professional degree of detection personnel, are not suitable for port base laboratories, and cannot meet the requirement of rapid customs clearance of customs ports.
In recent years, simplified genome sequencing technology (RAD-Seq) is more and more widely applied and can obtain genome information at lower cost, and because genetic hybridization exists widely in Acipenseridae species, male parent genes need to be considered in germplasm identification of Acipenser sinensis, namely nuclear gene identification, so that RAD is used for analyzing nuclear genome information of Acipenser siberian and other Acipensers, and detection and identification are carried out on the nuclear genes of the Acipenser siberian by combining TaqMan fluorescence PCR detection technology and microsatellite SYBR GREE I fluorescence PCR detection technology, so that the method can be used for germplasm identification of pure Acipenser sturgeons and has wide application prospect.
Disclosure of Invention
In view of the above, the present invention provides a primer probe composition, a kit and applications thereof for detecting pure Siberian sturgeon nuclear genes, aiming at the defects of the prior art. Comprises a pair of primer sequences, an MGB probe sequence and a pair of microsatellite primer sequences. Compared with the traditional common TaqMan probe, the MGB probe shortens the length of the probe and improves the signal to noise ratio and the resolution of qPCR. The microsatellite primer is fully verified to have strong specificity. The invention mainly solves the technical problems through the following technical scheme.
In one aspect, the invention provides a primer probe composition for detecting pure Siberian sturgeon nuclear genes, which comprises a primer probe I for detecting the Siberian sturgeon nuclear genes BGS and a primer II for detecting Siberian sturgeon nuclear gene microsatellite SX 37; the primer probe I comprises a primer pair consisting of a single-stranded DNA molecule shown in SEQ ID NO.1 and a single-stranded DNA molecule shown in SEQ ID NO.2, and TaqMan-MGB probes shown in SEQ ID NO.3 and SEQ ID NO. 4; the primer II comprises a primer pair consisting of a single-stranded DNA molecule shown in SEQ ID NO.5 and a single-stranded DNA molecule shown in SEQ ID NO. 6.
Preferably, the 5 'end of the TaqMan-MGB probe is marked with a fluorescence reporter group, and the 3' end of the TaqMan-MGB probe is marked with an MGB fluorescence-free quenching group.
On the other hand, the invention also provides application of the primer probe composition for detecting the pure Siberian sturgeon nuclear gene in germplasm identification of Siberian sturgeon or detection of Siberian sturgeon derived components.
In another aspect, the invention also provides application of the primer probe composition for detecting the pure Siberian sturgeon nuclear gene in preparation of a reagent or a kit for detecting the Siberian sturgeon nuclear gene.
In another aspect, the invention also provides a kit for detecting the Siberian sturgeon nuclear gene, and the effective component of the kit is a primer probe composition for detecting the pure Siberian sturgeon nuclear gene.
Preferably, the kit further comprises reagents for fluorescent quantitative PCR reaction.
In another aspect, the present invention also provides a method for identifying pure Siberian sturgeons, comprising the following steps:
1) extracting DNA in a sample to be detected;
2) taking the DNA extracted in the step 1) as a template, and carrying out two-step fluorescence PCR amplification reaction by using the primer probe composition of claim 1 or 2 to obtain an amplification curve, a Ct value and a melting curve;
3) and (3) carrying out comprehensive result judgment according to the amplification curve, the Ct value and the melting curve, wherein the specific judgment method comprises the following steps:
when the BGS nuclear gene is detected, two detection channels are used for reading data, for example, only the FAM detection channel with 530nm of characteristic amplification curve is generated, and Cp/Ct value is less than or equal to 30.0, while the ROX detection channel with 601nm of no Cp/Ct value and no amplification curve, the male parent nuclear gene and the female parent nuclear gene of the sample source animal can be judged to be from Siberian sturgeon, Russian sturgeon or sturgeon, namely the source animal is Siberian sturgeon, Russian sturgeon or sturgeon pure breed or hybrid between the Siberian sturgeon, the Russian sturgeon or the sturgeon;
if only ROX detection channel single positive or double detection channel positive appears, at least one of the male parent nuclear genes or the female parent nuclear genes of the sample source animal is judged not to come from Siberian sturgeon, Russian sturgeon or sturgeon, namely the source animal cannot be a pure Siberian sturgeon;
performing microsatellite SX37 nuclear gene detection on the sample with FAM detection channel single positive, and if the melting curve of the sample has a characteristic peak pattern at 78 +/-0.5 ℃, determining that at least one of the male parent nuclear genes or the female parent nuclear genes of the animal from the sample is from sturgeon schrenki or sturgeon Russian, namely the animal from the sample is not pure Siberian sturgeon; if the melting curve of the sample does not have a characteristic peak pattern at 78 +/-0.5 ℃, the nuclear genes of the sample can be judged to be from the Siberian sturgeons, namely the source animal is the pure Siberian sturgeons.
Preferably, the sample to be detected is sturgeon fin ray tissue and food or cosmetic containing sturgeon-derived components.
The positive progress effects of the invention are as follows:
the invention relates to a method for obtaining pure Siberian sturgeon identification by taking Siberian sturgeon nuclear gene as a target region design primer and TaqMan-MGB double probe, and combining with a microsatellite primer group to establish and optimize, wherein the method has the following advantages:
1) and (3) fast: the method monitors the PCR product in real time, and the result can be obtained after the PCR is finished;
2) sensitivity: the method has high sensitivity, and the detection result of the method on the plasmid DNA which is serially diluted shows that the detection sensitivity aiming at the BGS nuclear gene can reach 2 multiplied by 10-8ng; the detection sensitivity aiming at the microsatellite SX37 nuclear gene can reach 2 multiplied by 10-8ng。
3) Specifically: the specific primer and the MGB labeled probe are adopted and combined with the specific microsatellite primer group, so that the Siberian sturgeon nuclear gene sequence can be effectively identified.
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FIG. 1 shows the detection limit results of the fluorescent quantitative PCR detection of plasmid DNA containing pure Siberian sturgeon nuclear gene BGS using primers for detecting the Siberian sturgeon nuclear gene BGS and MGB probe No. 1;
FIG. 2 shows the detection limit results of the fluorescent quantitative PCR detection of plasmid DNA containing pure Acipenser baerii nuclear gene BGS using primers for detecting the Siberian Acipenser baeri nuclear gene BGS and MGB probe No. 2;
FIG. 3 shows the linear regression results of the fluorescent quantitative PCR detection of plasmid DNA containing pure Siberian sturgeon nuclear gene BGS using primers for detecting the Siberian sturgeon nuclear gene BGS and MGB probe No. 1;
FIG. 4 shows the linear regression results of the fluorescent quantitative PCR detection of plasmid DNA containing pure Acipenser baerii nuclear gene BGS using primers for detecting the Siberian Acipenser baeri nuclear gene BGS and MGB probe No. 2;
FIG. 5 shows the detection limit results of SYBR Green I fluorescent quantitative PCR detection of DNA containing a pure Acipenser schrenki SX37 plasmid using primers for detecting the microsatellite SX37 nuclear gene;
FIG. 6 shows the results of linear regression of SYBR Green I fluorescent quantitative PCR assays using primers for the detection of the microsatellite SX37 nuclear gene on DNA containing the pure Acipenser schrenki SX37 plasmid;
FIG. 7 shows the results of five pure and hybrid sturgeons specificity tests using primers for detecting the nuclear gene BGS of Acipenser baerii and dual MGB probes;
FIG. 8 shows the results of specific assays for the detection of Acipenser schrenki, Acipenser russiamensis and Acipenser siberian using primers for the detection of the microsatellite nuclear gene SX37 and SYBR Green I fluorescent PCR method;
FIG. 9 shows the result of the BGS nuclear gene fluorescence PCR assay of Siberian sturgeon from clinical samples, a variety of sturgeons, using the pure species Siberian sturgeon assay;
FIG. 10 shows the results of the BGS nuclear gene prescreening of Siberian sturgeons from clinical samples, a variety of sturgeons, using the pure Siberian sturgeon identification method followed by the identification using SYBR Green I fluorescence PCR detection.
Detailed Description
A primer probe composition for detecting pure Siberian sturgeon nuclear genes comprises a primer probe I for detecting Siberian sturgeon nuclear genes BGS and a primer II for detecting Siberian sturgeon nuclear gene microsatellite SX 37; the primer probe I comprises a primer pair consisting of a single-stranded DNA molecule shown in SEQ ID NO.1 and a single-stranded DNA molecule shown in SEQ ID NO.2, and TaqMan-MGB probes shown in SEQ ID NO.3 and SEQ ID NO. 4; the primer II comprises a primer pair consisting of a single-stranded DNA molecule shown in SEQ ID NO.5 and a single-stranded DNA molecule shown in SEQ ID NO. 6.
According to the invention, a specific sequence of the Siberian sturgeon nuclear gene BGS is screened out by a simplified genome sequencing (RAD-Seq) technology to be used as a target region, and primer and double-probe design is carried out on the basis of multiple sequence comparison of different individual Siberian sturgeons and different species of sturgeons. The length of the primers is about 20 bases, the GC content is 50-60%, secondary structures and repeatability do not exist in the primers, complementary sequences do not exist between the primers and in the primers, and the difference of melting temperature (Tm value) between the primers is less than 5 ℃. The length of the probe is about 20 bases, the Tm value is about 5 ℃ higher than that of the primer, and the double probes are modified by MGB and marked with fluorescent groups. And then a SYBR Green I fluorescent PCR method is designed according to the specificity of the microsatellite primer SX 37. Wherein, the 5 'end of the TaqMan-MGB probe is marked with a fluorescence reporter group, and the 3' end is marked with an MGB non-fluorescence quenching group.
The invention also provides application of the primer probe composition for detecting the pure Siberian sturgeon nuclear gene in germplasm identification of Siberian sturgeon or detection of Siberian sturgeon derived components and application in preparation of a reagent or a kit for detecting the Siberian sturgeon nuclear gene.
The invention also provides a kit for detecting the Siberian sturgeon nuclear gene, and the effective component of the kit is the primer probe composition for identifying the pure Siberian sturgeon nuclear gene. Further, the kit also comprises other reagents for fluorescent quantitative PCR reaction, such as fluorescent PCR reaction solution, negative control, positive control, nuclease-free water and the like, and the fluorescent PCR reaction solution can comprise PCR buffer solution, MgCl2, dNTP and Taq DNA polymerase. Preferably, the negative control may be a tissue sample that does not contain a constituent of acipenser baerii origin, for example a chicken tissue suspension made up of 20% by mass in 0.01mol/l ph7.2 PBS buffered saline.
Preferably, the nuclear gene BGS positive control is divided into two types, one type is a BGS nuclear gene capable of detecting Siberian sturgeon/Russian sturgeon/Shih sturgeon, and the BGS nuclear gene is a plasmid DNA containing a target amplification gene: the artificially synthesized Siberian sturgeon nuclear gene BGS with the length of 135bp is cloned between Sac I and Not I sites of pBluescript II SK + vector to obtain the geneNamed pBSK-BGS 135. The plasmid concentration was 105 copies/. mu.L. The other is a BGS nuclear gene which can detect sturgeons of other species except Siberian sturgeons/Russian sturgeons/Shih sturgeons and is a plasmid DNA containing a target amplification gene: the artificially synthesized microcystin nuclear gene BGS with the length of 135bp is cloned between Sac I and Not I sites of a pBluescript II SK + vector to obtain the gene, and the gene is named pBSK-RD 135. Plasmid concentration of 105copies/μL。
The 135bp sequence of the target gene inserted into the positive quality control product pBSK-BGS135 is as follows:
GCGGCCACTC TATTAACCCC TTCCCACCTG GAAAATGGCA AAACAAAAGA GGACAGATCC ATCACCACAA AACAACAAAA CATATGGAGC AAAACATACA CTCCATATTA AGCAGAAGAT GTGCAGTGTG AACTT(SEQ ID NO.7)
135bp sequences of target genes inserted into the positive quality control product pBSK-RD135 are as follows:
GCGGCCACTC TATTAACCCC TTCCCACCTG GAAAATGGCA AAACAAAAGA GGACAGATCC ATCACCACAA AACAACAAAA CATATGGCAC AAAACATACA CTCCATATTA AGCAGAAGAT GTGCAGTGTG AACTT(SEQ ID NO.8)
preferably, the microsatellite SX37 nuclear gene positive control can be plasmid DNA containing an amplification gene of interest: the artificially synthesized microsatellite SX37 nuclear gene with the length of 122bp is cloned between Sac I and Not I sites of a pBluescript II SK + vector to obtain the microsatellite SX37 nuclear gene, and the microsatellite SX37 nuclear gene is named as pBSK-SG 37. Plasmid concentration of 105copies/μL。
The 122bp sequence of the target gene inserted into the positive quality control product pBSK-SG37 is as follows:
TAGGAATCGC ATCAGCAGTG GAGCAGAATG AAAGACAAAT TTTGCATTGA GAAAAGACAA ATCATTATTT ATTTACAAAG GTAGCGCTAC CTTTGTAAAT AATGTGAACT CGCGGTTTTT AC(SEQ ID NO.9)
the invention also provides a method for identifying the pure Siberian sturgeon (a TaqMan-MGB probe dual fluorescence PCR detection method is combined with a SYBR Green I fluorescence PCR detection method), which comprises the following steps:
1) extracting DNA in a sample to be detected;
2) taking the DNA extracted in the step 1) as a template, and carrying out two-step fluorescence PCR amplification reaction by using the primer probe composition of claim 1 or 2 to obtain an amplification curve, a Ct value and a melting curve;
3) and (3) carrying out comprehensive result judgment according to the amplification curve, the Ct value and the melting curve, wherein the specific judgment method comprises the following steps:
when the BGS nuclear gene is detected, two detection channels are used for reading data, for example, only the FAM detection channel with 530nm of characteristic amplification curve is generated, and Cp/Ct value is less than or equal to 30.0, while the ROX detection channel with 601nm of no Cp/Ct value and no amplification curve, the male parent nuclear gene and the female parent nuclear gene of the sample source animal can be judged to be from Siberian sturgeon, Russian sturgeon or sturgeon, namely the source animal is Siberian sturgeon, Russian sturgeon or sturgeon pure breed or hybrid between the Siberian sturgeon, the Russian sturgeon or the sturgeon;
if only ROX detection channel single positive or double detection channel positive appears, at least one of the male parent nuclear genes or the female parent nuclear genes of the sample source animal is judged not to come from Siberian sturgeon, Russian sturgeon or sturgeon, namely the source animal cannot be a pure Siberian sturgeon;
performing microsatellite SX37 nuclear gene detection on the sample with FAM detection channel single positive, and if the melting curve of the sample has a characteristic peak pattern at 78 +/-0.5 ℃, determining that at least one of the male parent nuclear genes or the female parent nuclear genes of the animal from the sample is from sturgeon schrenki or sturgeon Russian, namely the animal from the sample is not pure Siberian sturgeon; if the melting curve of the sample does not have a characteristic peak pattern at 78 +/-0.5 ℃, the nuclear genes of the sample can be judged to be from the Siberian sturgeons, namely the source animal is the pure Siberian sturgeons.
In the invention, the sample to be detected can be sturgeon fin rays and other tissues, food or cosmetics possibly containing sturgeon components and the like. In order to improve the efficiency of nucleic acid extraction, especially when the sample to be detected is food or cosmetic possibly containing sturgeon components, pretreatment is required before extracting DNA.
In the invention, the reaction system of the Siberian sturgeon nuclear gene BGS fluorescence PCR amplification reaction is shown as the following table:
preferably, the reaction conditions of the fluorescent quantitative PCR amplification reaction are: 95 ℃/3 min; 35 cycles of 95 ℃/15s, 58 ℃/15s, 72 ℃/30 s; fluorescence collection was set at the 72 ℃ annealing extension stage.
In the invention, the reaction system of the Siberian sturgeon nuclear gene microsatellite SX37-SYBR Green I fluorescent PCR amplification reaction is shown as the following table:
| components | Final concentration |
| SX 37-sense primer (10. mu. mol/L) | 0.3μmol/L |
| SX 37-antisense primer (10. mu. mol/L) | 0.3μmol/ |
| 10 XPCR buffer (containing Mg)2+) | 1 XPCR buffer |
| dNTP(2.5mM) | 0.2mmol/L |
| TaqDNApolymerase | 1.25U |
| DNA template | 2.0～5.0μL |
| SYBRGreenI working solution | 0.5 |
| Adding DEPC water to the |
25 |
Preferably, the reaction conditions of the microsatellite SX37-SYBR Green I fluorescent PCR amplification reaction are as follows: firstly, 95 ℃/3 min; 95 ℃/30s, 55 ℃/30s, 72 ℃/30s, 40 cycles, fluorescence collection and 72 ℃ annealing extension stage; analysis of melting curve, heating from 70 deg.C to 95 deg.C, collecting fluorescence at 0.1 deg.C/s or 0.15 deg.C/s interval, and analyzing the melting curve.
The present invention will be described in detail with reference to examples for better understanding the objects, technical solutions and advantages of the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1 establishment of a Rapid identification method for pure Siberian sturgeon
Firstly, the step one: method for detecting and identifying Siberian sturgeon nuclear gene BGS
1. Primer probe sequence for detecting Siberian sturgeon nuclear gene BGS
The Siberian sturgeon nuclear gene BGS specific sequence is selected as a target region, and primer and double-probe design is carried out on the basis of comparison of multiple sequences of different Siberian sturgeons and comparison of sequences of sturgeons of different species. The length of the primers is about 20 bases, the GC content is 50-60%, secondary structures and repeatability do not exist in the primers, complementary sequences do not exist between the primers and in the primers, and the difference of melting temperature (Tm value) between the primers is less than 5 ℃. The length of the probe is about 20 bases, the Tm value is about 5 ℃ higher than that of the primer, and the double probes are modified by MGB and marked with fluorescent groups.
The nucleotide sequences of the specific primer pair and the TaqMan-MGB double probe are as follows:
primer pairs and TaqMan dual probes (primer probe I) for detecting different sturgeon BGS nuclear genes:
1)5’-GCGGCCACTCTATTAACCC-3'(SEQ ID NO.1)
2)5’-AAGTTCACACTGCACATCTTC-3’(SEQ ID NO.2)
3)5’-[FAM]-CATATGGAGCAAAACATACAC-[MGB]-3’(SEQ ID NO.3)
4)5’-[ROX]-CATATGGCACAAAACATACAC-[MGB]-3’(SEQ ID NO.4)
the primer pair for detecting the Siberian sturgeon nuclear gene BGS consists of single-stranded DNA molecules shown by SEQ ID NO.1 and SEQ ID NO.2, and is a sense primer and an antisense primer for detecting the Siberian sturgeon nuclear gene BGS respectively;
the MGB probe No.1 for detecting the Siberian sturgeon nuclear gene BGS consists of a single-stranded DNA molecule shown in SEQ ID NO.3, and is a probe for detecting the Siberian sturgeon/Russian sturgeon/Shisha sturgeon nuclear gene BGS;
the MGB probe No.2 for detecting the Siberian sturgeon nuclear gene BGS is composed of a single-stranded DNA molecule shown in SEQ ID NO.4, and is a probe for detecting the Siberian sturgeon/Russian sturgeon/Shi sturgeon nuclear gene BGS of other sturgeons except the Siberian sturgeon.
2. Method for detecting Siberian sturgeon nuclear gene BGS
The detection method for detecting the Siberian sturgeon nuclear gene is established by adopting a TaqMan-MGB probe dual fluorescent quantitative PCR detection technology as follows: extracting DNA in a sample to be detected, taking the DNA as a template, performing double fluorescence PCR amplification reaction by adopting the primer probe set in the first step to obtain an amplification curve and a Ct value, and judging whether the sample to be detected contains the Siberian sturgeon nuclear gene or not according to the amplification curve and the Ct value, wherein the method specifically comprises the following steps:
firstly, pretreatment and DNA extraction of a sample to be detected
The food or cosmetic containing sturgeon-derived ingredients is pretreated and DNA extracted according to the following method:
food or cosmetic sample: samples were taken at multiple points and 100mg samples were added to 200. mu.L of buffer GA (from the DNA extraction kit) and shaken to thoroughly suspend. Add 50. mu.L of protease K solution, mix well, digest overnight at 37 ℃ and take the supernatant to extract total DNA using Tiangen TIANAmp blood/cell/tissue genomic DNA extraction kit (spin column, catalog: DP304) as specified.
For other blood and tissue, total DNA was extracted using Tiangen TIANAmp blood/cell/tissue genomic DNA extraction kit (centrifugal column type, catalog number: DP304) as instructed.
And secondly, performing double fluorescent quantitative PCR amplification by using the DNA of the sample to be detected extracted in the step I as a template to obtain an amplification curve.
And (2) taking the DNA of the sample to be detected obtained in the step (i) as a template, taking a tissue sample without sturgeon components as a negative control and taking plasmid DNA containing a corresponding target amplification gene as a positive control, and respectively carrying out fluorescence quantitative PCR amplification reaction by adopting a primer probe in the step (I) to obtain an amplification curve and a Ct value.
The sturgeon component-free sample of the negative control is a tissue suspension which is prepared by 0.01mol/L PBS (phosphate buffered saline) with the pH value of 7.2 and contains no sturgeon component and has the mass percentage of 20%.
The positive control is plasmid DNA containing a target amplification gene, and the positive control comprises the following components:
the No.1 probe is used for detecting positive control of Siberian sturgeon/Russian sturgeon/Acipenser schrencki nuclear gene BGS: directly synthesizing a Siberian sturgeon nuclear gene BGS which is shown as SEQ ID NO.7 and has the length of 135bp, cloning the gene BGS between Sac I and Not I sites of a pBluescript II SK + vector, and after sequencing confirmation, naming the gene BGS as pBSK-BGS135 as the mother solution of the Siberian sturgeon nuclear gene BGS fluorescence PCR positive control product.
The No.2 probe is used for detecting positive control of nuclear genes BGS of other sturgeons except the Siberian sturgeons/Russian sturgeons/Acipenser schrenki: directly synthesizing a microcystin nuclear gene BGS with the length of 135bp shown in SEQ ID NO.8, cloning the microcystin nuclear gene BGS between Sac I and Not I sites of a pBluescript II SK + vector, and after sequencing confirmation, naming the microcystin nuclear gene BGS as pBSK-RD135 which is the mother liquor of the fluorescent PCR positive control product of other sturgeon nuclear genes BGS.
The reaction system of the double fluorescent quantitative PCR amplification reaction comprises the following steps: by gradient modification of Mg2+Concentration (1.5 mmol/L-6.0 mmol/L) and primer probe concentration (primer concentration 0.2. mu. mol/L-1.0. mu. mol/L, probe concentration 0.1. mu. mol/L-0.5. mu. mol/L) for PCR amplificationThe increased reaction system is optimized, and the final reaction system is a 25 mu L system, and the formula is shown in Table 1.
TABLE 1 formulation of the reaction System
Wherein, 10 XPCR buffer, 25mmol/L MgCl2Purchased from Promega corporation; dNTPs were purchased from TaKaRa, primers and probes, synthesized by Shanghai Biotechnology, Inc., where the composition of 10 XPCR buffer was: 500mmol/L KCl, 100mmol/L Tris-HCl (pH9.0, 25 ℃), 1.0% Triton X-100.
Taq DNA polymerase 5U/. mu.L, purchased from Promega;
DEPC water, twice distilled with purified water, adding DEPC to final concentration of 0.1%, stirring at 37 deg.C for 12hr, 15lbf/in2(1.034 × 10)5Pa) autoclave sterilization for 15 minutes.
The reaction conditions of the fluorescent quantitative PCR amplification reaction are as follows: 95 ℃/3 min; 35 cycles of 95 ℃/15s, 58 ℃/15s, 72 ℃/30 s; fluorescence collection was set at the 72 ℃ annealing extension stage.
Thirdly, judging the result according to the amplification curve and the Ct value
When two detection channels are used for reading data, if only an FAM detection channel (530nm) has a characteristic amplification curve, and the Cp/Ct value is less than or equal to 30.0, and the ROX detection channel (601nm) has no Cp/Ct value and no amplification curve, the male parent nuclear gene and the female parent nuclear gene of the animal from the sample can be judged to be from Siberian sturgeon, Russian sturgeon or Shi sturgeon, namely the animal from the sample is the Siberian sturgeon, the Russian sturgeon or the Shi sturgeon pure species or the hybrid between the three.
If only ROX detection channel single positive or double detection channel positive appears, at least one of the male parent nuclear gene or the female parent nuclear gene of the sample source animal is judged not to come from Siberian sturgeon, Russian sturgeon or Shih sturgeon, namely the source animal cannot be pure Siberian sturgeon.
Step two, step two: method for eliminating sturgeon and acipenser ruthensis
1. Microsatellite primer sequences for detecting sturgeon schrenki and sturgeon Russian
Microsatellite primers SX37 were used to detect Acipenser schrenki (S) and Acipenser russian (G), SX37 primer pair:
1)5’-TAGGAATCGCATCAGCAGTG-3'(SEQ ID NO.5)
2)5’-GTAAAAACCGCGAGTTCACA-3’(SEQ ID NO.6)
the primer pair for detecting the acipenser schrenki and the acipenser russianus consists of single-stranded DNA molecules shown in SEQ ID NO.5 and SEQ ID NO.6, and is a sense primer and an antisense primer for detecting a microsatellite SX37 nuclear gene respectively.
2. SYBR Green I fluorescence PCR method for detecting sturgeon schoenbergii and sturgeon Russian
The detection method established by adopting the SYBR Green I fluorescence PCR method is as follows: and (3) carrying out SYBR Green I fluorescent PCR amplification reaction on the sample DNA which is possibly from Siberian sturgeon/Russian sturgeon/Shih sturgeon as a BGS nuclear gene detection result by adopting the SX37 primer to obtain a melting curve, and judging whether the sample to be detected contains the Russian sturgeon/Shih sturgeon nuclear gene or not according to the temperature of the melting curve, thereby screening and obtaining pure Siberian sturgeon. The method specifically comprises the following steps:
firstly, pretreatment and DNA extraction of a sample to be detected
The food or cosmetic containing sturgeon-derived ingredients is pretreated and DNA extracted according to the following method:
food or cosmetic sample: samples were taken at multiple points and 100mg samples were added to 200. mu.L of buffer GA (from the DNA extraction kit) and shaken to thoroughly suspend. Add 50. mu.L of protease K solution, mix well, digest overnight at 37 ℃ and take the supernatant to extract total DNA using Tiangen TIANAmp blood/cell/tissue genomic DNA extraction kit (spin column, catalog: DP304) as specified.
For other blood and tissue, total DNA was extracted using Tiangen TIANAmp blood/cell/tissue genomic DNA extraction kit (centrifugal column type, catalog number: DP304) as instructed.
Secondly, SYBR Green I fluorescence PCR amplification is carried out by taking the DNA of the sample to be detected extracted in the step I as a template to obtain a melting curve
And (2) taking the DNA of the sample to be detected obtained in the step (i) as a template, taking a tissue sample without sturgeon components as a negative control and taking plasmid DNA containing a corresponding target amplification gene as a positive control, and respectively carrying out fluorescence PCR amplification reaction by using the primers in the step (ii) to obtain a melting curve and a melting temperature.
The sturgeon component-free sample of the negative control is a tissue suspension which is prepared by 0.01mol/L PBS (phosphate buffered saline) with the pH value of 7.2 and contains no sturgeon component and has the mass percentage of 20%.
The positive control is plasmid DNA containing a target amplification gene, and the positive control comprises the following components:
positive control for detection of microsatellite SX37 nuclear gene: the sturgeon nuclear gene 37SG containing 122bp sturgeon shown in SEQ ID NO.9 is directly synthesized, cloned between Sac I and Not I sites of a pBluescript II SK + vector, and after sequencing confirmation, named pBSK-SG37 is the mother solution of the microsatellite SX37 fluorescence PCR positive control.
SYBR Green I fluorescent quantitative PCR amplification reaction system: by gradient modification of Mg2+The concentration (1.5 mmol/L-6.0 mmol/L) and the concentration of the primer probe (0.2 mu mol/L-1.0 mu mol/L primer concentration, 0.1 mu mol/L-0.5 mu mol/L probe concentration) optimize the reaction system of PCR amplification, and finally, the reaction system is selected to be 25 mu L system, and the formula is shown in Table 2.
TABLE 2 formulation of the reaction System
| Components | |
| 10 |
1 XPCR buffer |
| 25mmol/LMgCl2 | 2.0mmol/L |
| 2.5mmol/LdNTP | 0.2mmol/L |
| SX37-primer1 | 0.3μmol/L |
| SX37-primer2 | 0.3μmol/L |
| TaqDNA polymerase (5U/. mu.L) | 2.5U |
| DNA template | 2.0～5.0μL |
| Adding DEPC water to the total volume | 25μL |
| SYBRGreenI working solution | 0.5μL |
Wherein, 10 XPCR buffer, 25mmol/L MgCl2Purchased from Promega corporation; dNTPs were purchased from TaKaRa, primers and probes, synthesized by Shanghai Biotechnology, Inc., where the composition of 10 XPCR buffer was: 500mmol/L KCl, 100mmol/L Tris-HCl (pH9.0, 25 ℃), 1.0% Triton X-100.
Taq DNA polymerase 5U/. mu.L, SYBR Green I working solution from Promega;
DEPC water is treated with purified waterDistilling, adding DEPC to final concentration of 0.1%, stirring at 37 deg.C for 12hr, 15lbf/in2(1.034 × 10)5Pa) autoclave sterilization for 15 minutes.
The reaction conditions of the microsatellite SX37-SYBR Green I fluorescent PCR amplification reaction are as follows: 95 ℃/3 min; 95 ℃/30s, 55 ℃/30s, 72 ℃/30s, 40 cycles, fluorescence collection in 72 ℃ annealing extension stage; melting curve analysis, heating from 70 deg.C to 95 deg.C, collecting fluorescence at 0.1 deg.C/s or 0.15 deg.C/s interval, and analyzing the melting curve.
Thirdly, judging the result according to the characteristic peak diagram of the melting curve
When a melting curve of a sample has a characteristic peak pattern at 78 +/-0.5 ℃, at least one of male parent nuclear genes or female parent nuclear genes of a sample source animal can be judged to come from sturgeon schneideriana or sturgeon russia, namely the source animal is not a pure Siberian sturgeon;
if the melting curve of the sample does not have a characteristic peak diagram at 78 +/-0.5 ℃, the nuclear genes of the sample can be judged to be from the Siberian sturgeons, namely the source animal is the pure Siberian sturgeons.
The PCR product was analyzed by agarose gel electrophoresis if necessary, and it was observed whether a specific band (corresponding to a peak characteristic at 78 ℃. + -. 0.5 ℃) appeared at a position of 122 bp.
Third, comprehensive judgment
According to the first step, screening out a sample of which the nuclear gene can be from Siberian sturgeon/Russian sturgeon/Acipenser schrenki; and according to the second step, identifying a sample of which the nuclear gene is probably from the Russian sturgeon/the sturgeon by the microsatellite SX37, and finally screening and identifying by an exclusion method to obtain the pure Siberian sturgeon.
Example 2 sensitivity, specificity, reproducibility test of pure Siberian sturgeon identification method
1. Sensitivity test
Nuclear gene BGS sensitivity test
0.04 ng/mu L plasmid pBSK-BGS135 containing Siberian sturgeon nuclear gene BGS is serially diluted by 10 times, 0.04 ng/mu L plasmid pBSK-RD135 containing microcystin nuclear gene BGS is serially diluted by 10 times, and a detection method constructed by a primer pair and a TaqMan-MGB double probe simultaneously carries out detection on two kinds of diluted plasmidsThe results of the detection of samples with various dilution concentrations of the particles are shown in figures 1 and 2, and the detection limit of the constructed method for detecting the nuclear gene BGS of the Acipenser sibirica reaches 2 multiplied by 10-8ng; the detection limit of the method for detecting nuclear genes BGS of acipenser ruthenus and the like reaches 2 multiplied by 10-8ng。
The Ct value and the amount of plasmid DNA are subjected to linear regression analysis by using software matched with a fluorescent PCR instrument Q5, a standard curve is established, a correlation coefficient is calculated, and the result shows that the standard curve obtained by the detection method for constructing the primer pair for detecting the Siberian sturgeon nuclear gene BGS and the MGB probe No.1 is shown in figure 3, the concentration of the DNA is linearly related to the Ct value measured by the test, and the R value is linear20.995, slope k-3.35, intercept 11.65, PCR efficiency 99.02%; the detection method constructed by using the primer pair for detecting the Siberian sturgeon nuclear gene BGS and the MGB probe No.1 in the example 1 can be used for detecting and quantifying the Siberian sturgeon nuclear DNA.
The Ct value and the amount of plasmid DNA are subjected to linear regression analysis by using software matched with a fluorescent PCR instrument Q5, a standard curve is established, a correlation coefficient is calculated, and the result shows that the standard curve obtained by the detection method for constructing the primer pair for detecting the microcystis sturgeon nuclear gene BGS and the MGB probe No.2 is shown in figure 4, the concentration of the DNA is linearly related to the Ct value measured by the test, and the R value is linear20.998, slope k-3.342, intercept 13.06, PCR efficiency 99.17%; the detection method constructed by using the primer pair for detecting the microcystis sturgeon nuclear gene BGS and the MGB probe No.2 in the example 1 can be used for detecting and quantifying the nuclear DNA of sturgeons of other species except Siberian sturgeons/Russian sturgeons/Acipenser schrenki sturgeons.
② microsatellite SX37 sensitivity test
0.04 ng/mu L plasmid pBSK-SG37 containing Acipenser schrenki nuclear gene SX37 is serially diluted by 10 times, each diluted concentration sample is detected by constructing SYBR Green I fluorescent PCR method by using SX37 primer pair, the result is shown in figure 5, and the detection limit of the constructed method for detecting microsatellite SX37 nuclear gene reaches 2 x 10-8ng。
Using fluorescence PCR instrument Q5 matched software to make linear regression analysis of Ct value and plasmid DNA quantity, establishing standard curve, calculating correlation coefficient, and displaying resultA standard curve obtained by constructing a SYBR Green I fluorescence PCR detection method on a primer pair for detecting a microsatellite SX37 nuclear gene is shown in FIG. 6, the concentration of DNA is linearly related to a Ct value measured by an experiment, and R is21.0, slope k-3.29, intercept 12.77, PCR efficiency 101.19%; the SYBR Green I fluorescence PCR detection method for detecting the microsatellite SX37 nuclear gene in the example 1 can be used for detecting and quantifying the sturgeon nuclear gene DNA.
2. Specificity test
The detection method using the primer pair for detecting the nuclear gene BGS of Siberian sturgeon and the TaqMan-MGB dual probe construction in the first step of example 1 was used to detect the total DNA extracted from Siberian sturgeon (inbred), the total DNA extracted from Russian sturgeon (inbred), the total DNA extracted from acipenser baeri (inbred), the total DNA extracted from Shi sturgeon (inbred), the total DNA extracted from huso dauricus (inbred), the total DNA extracted from the hybrid of Siberian sturgeon and Shi sturgeon, the total DNA extracted from the hybrid of Siberian sturgeon and Russian, the total DNA extracted from huso dauricus and Shi sturgeon, and the like, and the results are shown in FIG. 7, which show that the primer pair for detecting the nuclear gene of Siberian sturgeon and the TaqMan-MGB dual probe construction in the first step of example 1 and the FAM detection method using the single detection channel for specifically detecting the single specificity of Siberian sturgeon and the TaqMan-MGB dual probe construction Sturgeons in sturgeon history and hybrid sturgeons between the three sturgeons; the ROX detection channel is single positive and FAM/ROX double positive, and can specifically detect acipenser parvum, huso dauricus and hybrid acipenser sturgeon containing acipenser parvum or huso dauricus.
The total DNA extracted from Siberian sturgeon (purebred), the total DNA extracted from Russian sturgeon (purebred), the total DNA extracted from Shih sturgeon (purebred), the total DNA extracted from Siberian sturgeon (purebred) and the hybrid variety of Siberian sturgeon and Shih sturgeon, the total DNA extracted from the hybrid variety of Siberian sturgeon and Russian sturgeon, etc. were detected by using the primer pair for detecting the microsatellite SX37 nuclear gene and the constructed SYBR Green I fluorescence PCR detection method in the second step of example 1 (step two of example 1), and the results are shown in FIG. 8, which shows that the primer pair for detecting the microsatellite SX37 nuclear gene and the constructed SYBR Green I fluorescence PCR detection method in the second step two step 1, and characteristic peak patterns appear at 78 ℃. + -. 0.5 ℃, and can specifically detect the Siberian sturgeon and a Russian gene; only pure Siberian sturgeons have no peak pattern characteristic of the melting curve at 78 ℃. + -. 0.5 ℃.
3. Repeatability test
The primer pair for detecting the Siberian sturgeon nuclear gene BGS and the detection method constructed by the TaqMan-MGB dual probe in the first step of the embodiment 1 are used for carrying out repeatability tests on plasmids pBSK-BGS135 and pBSK-RD135 (the positive control for detecting the Siberian sturgeon nuclear gene and the positive control for detecting the acipenser baeri nuclear gene in the first step of the embodiment 1) which are serially diluted in different concentrations, and the results are shown in Table 3, which indicates that the repeatability and the stability of the detection method constructed by the primer pair for detecting the Siberian sturgeon nuclear gene BGS and the TaqMan-MGB dual probe in the first step of the embodiment 1 are good.
TABLE 3 results of the repeatability tests
The results of a repeatability test carried out on plasmid pBSK-SG37 (positive control for detecting the nuclear gene of the microsatellite SX37 in example 1) serially diluted in different concentrations by using the primer pair for detecting the nuclear gene of the microsatellite SX37 in the second step of example 1 and the constructed SYBR Green I fluorescence detection method (the method described in the second step of example 1) are shown in Table 4, which indicates that the primer pair for detecting the nuclear gene of the microsatellite SX37 in the first step of example 1 and the constructed SYBR Green I fluorescence detection method of the invention have good repeatability and stability.
TABLE 4 results of the repeatability tests
Example 3 identification of a pure Siberian sturgeon from a certain customs exported sturgeon
The method described in step one and step two of example 1 was used to test a sample of a baysian sturgeon declared to be an export from a certain customs, identifying that the sturgeon sample was indeed a pure species of baysian sturgeon.
Detection with DNA extracted from sturgeon to be detected as template
Shearing a small section of sturgeon back fin ray, fully washing with 0.01mol/L PBS (pH 7.4), shearing the fin ray as much as possible with sterile scissors, adding 200 mu L of buffer GA, and oscillating until complete suspension. Add 50. mu.L of protease K solution, mix well, digest overnight at 37 ℃ and take the supernatant and extract total DNA using TIANAmp blood/cell/tissue genomic DNA extraction kit (DP304) as indicated. The primer probe in the first step of the embodiment 1 is used for detecting DNA of the sturgeon fin as a template, DNA extracted from chicken tissues is used as negative control, the detection result of the Siberian sturgeon BGS nuclear gene fluorescence PCR detection method established by the primer pair and the dual MGB probe in the first step of the embodiment 1 is shown in a figure 9, and the result shows that the sample may be Siberian sturgeon/Russian sturgeon/Acipenser sturgeon; the detection result of the SYBR Green I fluorescence PCR detection method established by the SX37 primer in the second step of the embodiment 1 is shown in figure 10, and the result shows that the sample to be detected is pure Siberian sturgeon.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> research center of China customs science and technology
<120> primer probe composition for detecting pure Siberian sturgeon nuclear gene, kit and application thereof
<130> P210399DD1SQ
<160> 9
<170> SIPOSequenceListing 1.0
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gcggccactc tattaaccc 19
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<213> (Artificial sequence)
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aagttcacac tgcacatctt c 21
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catatggagc aaaacataca c 21
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gcggccactc tattaacccc ttcccacctg gaaaatggca aaacaaaaga ggacagatcc 60
atcaccacaa aacaacaaaa catatggagc aaaacataca ctccatatta agcagaagat 120
gtgcagtgtg aactt 135
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<213> (Acipenser parvum nuclear gene BGS of 135bp in length)
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gcggccactc tattaacccc ttcccacctg gaaaatggca aaacaaaaga ggacagatcc 60
atcaccacaa aacaacaaaa catatggcac aaaacataca ctccatatta agcagaagat 120
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taggaatcgc atcagcagtg gagcagaatg aaagacaaat tttgcattga gaaaagacaa 60
atcattattt atttacaaag gtagcgctac ctttgtaaat aatgtgaact cgcggttttt 120
ac 122
Claims (8)
1. The primer probe composition is characterized by comprising a primer probe I for detecting a pure Siberian sturgeon nuclear gene BGS and a primer II for detecting a Siberian sturgeon nuclear gene microsatellite SX 37;
the primer probe I comprises a primer pair consisting of a single-stranded DNA molecule shown in SEQ ID NO.1 and a single-stranded DNA molecule shown in SEQ ID NO.2, and TaqMan-MGB probes shown in SEQ ID NO.3 and SEQ ID NO. 4;
the primer II comprises a primer pair consisting of a single-stranded DNA molecule shown in SEQ ID NO.5 and a single-stranded DNA molecule shown in SEQ ID NO. 6.
2. The primer probe composition for detecting the pure Siberian sturgeon nuclear gene according to claim 1, characterized in that the 5 'end of the TaqMan-MGB probe is labeled with a fluorescent reporter group, and the 3' end is labeled with an MGB non-fluorescent quenching group.
3. The use of the primer probe composition for detecting a pure breed Acipenser baerii nuclear gene according to claim 1 or 2 in germplasm identification or Acipenser baerii derived component detection.
4. Use of the primer probe composition for detecting the pure breed Siberian sturgeon nuclear gene according to claim 1 or 2 for preparing a reagent or a kit for detecting the Siberian sturgeon nuclear gene.
5. A kit for detecting a nuclear gene of acipenser baerii, characterized in that the effective component of the kit is the primer probe composition according to claim 1 or 2.
6. The kit for detecting the nuclear gene of acipenser baerii according to claim 5, characterized in that the kit further comprises reagents for a fluorescent quantitative PCR reaction.
7. A method for identifying pure Siberian sturgeons is characterized by comprising the following steps:
1) extracting DNA in a sample to be detected;
2) taking the DNA extracted in the step 1) as a template, and carrying out two-step fluorescence PCR amplification reaction by using the primer probe composition of claim 1 or 2 to obtain an amplification curve, a Ct value and a melting curve;
3) and (3) carrying out comprehensive result judgment according to the amplification curve, the Ct value and the melting curve, wherein the specific judgment method comprises the following steps:
when the BGS nuclear gene is detected, two detection channels are used for reading data, for example, only the FAM detection channel with 530nm of characteristic amplification curve is generated, and Cp/Ct value is less than or equal to 30.0, while the ROX detection channel with 601nm of no Cp/Ct value and no amplification curve, the male parent nuclear gene and the female parent nuclear gene of the sample source animal can be judged to be from Siberian sturgeon, Russian sturgeon or sturgeon, namely the source animal is Siberian sturgeon, Russian sturgeon or sturgeon pure breed or hybrid between the Siberian sturgeon, the Russian sturgeon or the sturgeon;
if only ROX detection channel single positive or double detection channel positive appears, at least one of the male parent nuclear genes or the female parent nuclear genes of the sample source animal is judged not to come from Siberian sturgeon, Russian sturgeon or sturgeon, namely the source animal cannot be a pure Siberian sturgeon;
performing microsatellite SX37 nuclear gene detection on the sample with FAM detection channel single positive, and if the melting curve of the sample has a characteristic peak pattern at 78 +/-0.5 ℃, determining that at least one of the male parent nuclear genes or the female parent nuclear genes of the animal from the sample is from sturgeon schrenki or sturgeon Russian, namely the animal from the sample is not pure Siberian sturgeon; if the melting curve of the sample does not have a characteristic peak pattern at 78 +/-0.5 ℃, the nuclear genes of the sample can be judged to be from the Siberian sturgeons, namely the source animal is the pure Siberian sturgeons.
8. The method for identifying a pure breed Siberian sturgeon according to claim 7, characterized in that the sample to be tested is the fin-ray tissue of sturgeon and food or cosmetic containing sturgeon-derived components.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105002170A (en) * | 2015-07-31 | 2015-10-28 | 中国长江三峡集团公司 | Amur sturgeon germplasm molecular marker identification kit and application thereof |
| CN112831573A (en) * | 2021-03-22 | 2021-05-25 | 贵州省水产研究所 | SNP primer and detection method for identifying sturgeon germplasm |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105002170A (en) * | 2015-07-31 | 2015-10-28 | 中国长江三峡集团公司 | Amur sturgeon germplasm molecular marker identification kit and application thereof |
| CN112831573A (en) * | 2021-03-22 | 2021-05-25 | 贵州省水产研究所 | SNP primer and detection method for identifying sturgeon germplasm |
Non-Patent Citations (2)
| Title |
|---|
| MILO S HAVELKA等: "A new marker, isolated by ddRAD sequencing, detects Siberian and Russian sturgeon in hybrids", ANIMAL GENETICS, vol. 50, no. 1, pages 115 - 116, XP071536892, DOI: 10.1111/age.12733 * |
| 胡佳等: "施氏鲟、达氏鳇及其杂交子代的分子鉴定", 中国水产科学, vol. 17, no. 01, pages 23 * |
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