CN112831571A - Double-dye fluorescence PCR (polymerase chain reaction) specific detection system for simultaneously detecting species of acipenser ruthenus and acipenser sibiricus and application thereof - Google Patents
Double-dye fluorescence PCR (polymerase chain reaction) specific detection system for simultaneously detecting species of acipenser ruthenus and acipenser sibiricus and application thereof Download PDFInfo
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Abstract
The invention discloses a fluorescence PCR specificity detection system for simultaneously identifying a flash sturgeon species and a Siberian sturgeon species by using double fluorescent dyes and application thereof. The invention firstly uses two fluorescent dyes to carry out species fluorescent PCR detection, adopts species specificity detection primers of the flash sturgeons and the Siberian sturgeons to simultaneously detect the mitochondrial genes of the flash sturgeons and the Siberian sturgeons through double-dye fluorescent PCR amplification in the same PCR system, and generates two specificity amplification curves after amplification, thereby solving the problems of low detection specificity of the fish species, lower detection limit cycle number, easy confusion of the near-source species, repeated operation and the like. Can ensure the full combination of species DNA and dye, and prevent the conditions of off-target dye and amplification failure. The specific primers and the probes are reasonable in design, good in specificity and high in detection sensitivity.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a double-dye fluorescence PCR specificity detection system for simultaneously detecting flashing sturgeons, Siberian sturgeons species EVA-Green and SybrGreen, and application thereof.
Background
The SybrGreen green fluorescent dye is widely applied to fluorescent PCR amplification, but the dye has a detail inhibition effect on PCR amplification, and considering the particularity of fish DNA detection, namely, a plurality of homologous species and high similarity are provided, and a nonspecific amplification curve is easily generated in 35-40 cycles of PCR reaction of a closely related species, so that the condition is often required to be amplified again. In order to reduce amplification time and prevent secondary amplification, and also in order to reduce problems of dye off-target and single dye amplification failure, it is imperative to develop a method for single detection using two dyes. At present, a fluorescence PCR technology adopting two fluorescent dyes at the same time is not available, and the technology is developed for the first time by the inventor and can be popularized to detection and identification of other species.
EvaGreen is a DNA binding dye used for real-time quantitative pcr (qpcr). The advantages of this dye make it far superior to SYBR Green I. In addition to having similar spectral characteristics, EvaGreen has three main features that distinguish it from SYBR Green I.
EvaGreen is much less inhibitory to PCR than SYBR Green I, so qPCR experiments using EvaGreen can use a fast PCR step.
EvaGreen can be used in higher concentration in the experiment, so that an amplification signal which is much stronger than SYBR Green I is obtained; the defect of 'dye redistribution' is also eliminated by the EvaGreen with higher concentration, so that the EvaGreen can be used for multiplex PCR and high-resolution (high-definition) melting curve analysis (HRM); this assay is being increasingly used for post-PCR genotyping and heteroduplex analysis. SYBR Green I cannot solve the problem of dye redistribution due to low concentration, and cannot be used for multiplex PCR and HRM, because SYBR Green I has inhibition on PCR, requiring that its use concentration must be very low. At the same time, dye redistribution problems may also affect the reliability of conventional melting curves, since low melting DNA strands may not be detectable for this reason.
EvaGreen has excellent stability. Will not be destroyed during normal storage, handling and PCR. The dye in the buffer solution can be safely stored at room temperature or in a refrigerator, and can also be repeatedly frozen and thawed. In contrast, SYBR Green I is unstable and more inhibitory to PCR after degradation.
The multiplex PCR is a method which is developed on the basis of PCR and can detect a plurality of targets and species simultaneously, and has the advantages of high sensitivity, high flux and pollution rate reduction.
Caviar, goose liver and truffle are called the three delicacies in the world, which respect the 'crown of king' in the delicacies. The caviar is expensive and is called natural gift, "black gold" and so on. According to the definition of food and agricultural organization of the united nations, roe products only derived from fishes of the order sturgeon can be called caviar, and other roe products are classified as "caviar substitutes". At present, 26 kinds of sturgeons are available all over the world, and only sturgeon eggs such as Siberian sturgeons and sparkling sturgeons can be used for preparing the well-known precious caviar. Among them, the glittering sturgeon caviar is the most common high-grade caviar nowadays, high-grade restaurants are generally prepared with the glittering sturgeon caviar, and the siberian sturgeon caviar is a product with higher cost performance. Siberian sturgeon caviar is prepared from Siberian sturgeon roe, is the variety with the largest market supply in international caviar at present, and is mostly gray and brown in color. The prices of the two caviar products are different by several times, but the appearance difference is small, and the caviar products are difficult to distinguish from the appearance.
Sturgeons of the genus acipenser of the family Acipenseridae are distributed in the sea of the Liriosea, the Suyahai, the Black sea and the Aegean sea and in rivers among people. Due to over fishing, river dam building, environmental pollution and the like, the roe sauce price of the flash sturgeon is high and cannot stay, and the price of the roe sauce of the flash sturgeon in European and American markets is 4000 dollars/kg. The caviar is frequently used as a popular caviar with higher cost performance and is used as a fake. Moscow department laboratories have identified flash sturgeon caviar from China as counterfeit products from other species. It has also been reported that artificial glittering sturgeon caviar is manufactured by processing with chemicals, pigments and flavorants. Meanwhile, experts indicate that the fake caviar is very difficult to distinguish, and the taste and the particle size of the fake caviar can highly imitate the real glittering sturgeon caviar. Therefore, a specific and sensitive identification method for the species of the acipenser ruthenus and the acipenser baeri is needed, so that the identification and source tracing of products such as two kinds of caviar and the like are needed.
Disclosure of Invention
The invention aims to overcome the defects and provide an application method of a double-dye fluorescence PCR system for simultaneously detecting the species EVA-Green and SybrGreen of the acipenser ruthenus and the acipenser sibiricus. The invention can provide two amplification curves aiming at a single species, and simultaneously solves the problems of low detection specificity, low detection limit cycle number, easy confusion of near-source species, repeated operation and the like of fish species. And the trace DNA of the automatic sample is detected, and two sturgeon species and caviar product sources can be identified simultaneously. The detection sensitivity is high, the method is rapid and easy to operate.
The technical scheme adopted by the invention for realizing the purpose is as follows: a double-dye fluorescent PCR specificity detection system for simultaneously detecting flashing sturgeon and Siberian sturgeon species EVA-Green and SybrGreen carries out PCR amplification on the flashing sturgeon and the Siberian sturgeon species by using two dyes EVA-Green and SybrGreen in one-time PCR reaction.
With respect to the above-mentioned technical solution, further, the system further comprises the following primer sequences: the primer sequence of the flash sturgeon species is shown as SEQ ID NO.1 and SEQ ID NO. 2; the primer sequence of the Siberian sturgeon species is shown as SEQ ID NO.1 and SEQ ID NO. 2; namely, the same species has two amplification curves, wherein one amplification curve is generated by EVA-Green fluorescence, and the other amplification curve is generated by SybrGreen fluorescence;
the primer sequences of the flash sturgeons are shown in the table 1:
TABLE 1 primer sequences for Acipenser flaccida
| Upstream primer | 5’-CTCCCAATAATACCAGCCGTG-3’ | SEQ ID NO.1 |
| Downstream primer | 5’-TATGCTACGGCTACTTCCAGTAGA-3’ | SEQ ID NO.2 |
The primer sequences of the species including the acipenser baerii are shown in table 2:
TABLE 2 Siberian sturgeon species primer sequences
With respect to the above-described technical solution, further, the system further comprises the following reagents: PCR buffer solution, dNTPs, Hotstart Taq enzyme, a DNA template, EVA-Green fluorescent dye and SybrGreen fluorescent dye.
For the above technical solution, further, the composition comprises, per 20 μ L of the reaction system: containing Mg 2+2. mu.L of 10 XPCR buffer; 2. mu.L of 10 XdNTPs; 0.2 muL of Hotstart Taq enzyme at a concentration of 5 units/. mu.L; the concentration of the primers of the upstream and downstream of the flash sturgeon is 5 mu mol/L, and the concentration of the primers of the upstream and downstream of the flash sturgeon is 0.5 mu L respectively; the concentration of the upstream primer and the downstream primer of the Siberian sturgeon is 5 mu mol/L, and the concentration of the downstream primer and the upstream primer are respectively 0.5 mu L; 100ng of DNA template; 1 mu L of each of EVA-Green fluorescent dye or SybrGreen fluorescent dye; the balance of double distilled water.
In general, the reaction system of the composition is as follows in table 3:
TABLE 3 reaction systems of the double-dye fluorescent PCR specificity detection system for the species of acipenser iridipes, acipenser sibiricus EVA-Green, SybrGreen
For the technical solution described above, further, the reaction parameters of the dual-dye fluorescence PCR specific detection system for simultaneously detecting the shining sturgeon species, the siberian sturgeon species EVA-Green, and the SybrGreen are as follows in table 4:
table 4 reaction parameters for simultaneous detection of double-dye fluorescent PCR specificity detection systems for acipenser flash, acipenser sibiricus species EVA-Green, and SybrGreen
The application of the double-dye fluorescence PCR specificity detection system for simultaneously detecting the iridescent sturgeons, Siberian sturgeons species EVA-Green and SybrGreen is characterized in that: simultaneously detecting a double-dye fluorescence PCR specificity detection system of the flash sturgeon species, the Siberian sturgeon species EVA-Green and the SybrGreen by using the flash sturgeon species; respectively configuring a reaction system for fish components of two species of the flash sturgeon and the Siberian sturgeon by using EVA-Green fluorescent dye or SybrGreen fluorescent dye; the specific detection of the double-dye fluorescence PCR is carried out simultaneously in the same PCR reaction. Respectively configuring a reaction system for fish components of two species of the flash sturgeon and the Siberian sturgeon by using EVA-Green fluorescent dye or SybrGreen fluorescent dye; the double-dye fluorescence PCR specific detection for simultaneously detecting the species of the flash sturgeons and the Siberian sturgeons, namely EVA-Green and SybrGreen, is carried out in the same PCR reaction, and the identification detection can be respectively carried out on the species of the flash sturgeons and the Siberian sturgeons.
Further, the double-dye fluorescence PCR specific detection method for simultaneously detecting the species of the flash sturgeons and the species of the Siberian sturgeons comprises the following steps: the method comprises the steps of simultaneously detecting CoxI genes of the flash sturgeons, the Siberian sturgeons species EVA-Green and the SybrGreen double-dye fluorescent PCR specific detection composition, carrying out PCR reaction by using Hotstart Taq DNA polymerase, respectively generating two specific amplification curves in the same species in a positive sample, wherein one specific amplification curve is EVA-Green fluorescent generation, the other specific amplification curve is SybrGreen fluorescent generation, fluorescent dye can be combined with renatured DNA to emit fluorescence, and obtaining the specific amplification curve after signal collection treatment. Meanwhile, corresponding curves of two species can be distinguished through different TM values of melting curves, so that specific identification is carried out on the species components of the flash sturgeons and the Siberian sturgeons.
The specific primer disclosed by the invention is reasonable in design, good in specificity and high in detection sensitivity, is used for species detection of the acipenser ruthenus and the acipenser sibiricus, and can be used for simultaneously carrying out two fluorescence amplification curve detection on two target species. The results of detecting the animal components of the flash sturgeons and the Siberian sturgeons by adopting the method show that the CoxI genes are simultaneously detected by using the specificity detection primers of the flash sturgeons and the Siberian sturgeons, and the two specificity amplification curves (one is generated by EVA-Green fluorescence and the other is generated by SybrGreen fluorescence) can be respectively generated by carrying out double-dye fluorescence PCR amplification on the flash sturgeons, the Siberian sturgeons and the Siberian sturgeons, wherein the EVA-Green amplification curves have high fluorescence values and better sensitivity and detectability; the SybrGreen amplification curve has better repeatability, and can perform double verification on the EVA-Green amplification curve. Through the simultaneous detection of the species EVA-Green and SybrGreen double-dye fluorescence PCR analysis of the flash sturgeons and the Siberian sturgeons, the species components of the flash sturgeons and the Siberian sturgeons can be accurately identified, the specificity is good, secondary detection is not needed, and a large amount of detection time is saved.
Drawings
FIG. 1 is a diagram of the detection result of the fluorescent PCR specificity of the double dyes for simultaneously detecting the species EVA-Green and SybrGreen of the acipenser ruthenicus and the Siberian sturgeon, wherein: 1. siberian sturgeon EVA-Green, 2, flash sturgeon EVA-Green, 3, Siberian sturgeon SYBRGreen, 4, flash sturgeon SYBRGreen, 5, huso, 6, acipenser baeri, 7, acipenser schrencki, 8, Acipenser russiamensis, 9, Acipenser dabryanus, 10, a negative control, 11, a blank control.
FIG. 2 is a diagram of the detection result of the fluorescent PCR specificity of the double dyes for simultaneously detecting the species EVA-Green and SybrGreen of the acipenser ruthenicus and the Siberian sturgeon, wherein: 1. siberian sturgeon EVA-Green, 2, flash sturgeon EVA-Green, 3, siberian sturgeon SYBRGreen, 4, flash sturgeon SYBRGreen, 5, pacific pollack, 6, pollack, 7, haddock, 8, pollack, 9, whitlock, 10, halibut, 11, turbot, 12, flounder, 13, turbot, 14, yellowtail, 15, glossogyne, 16, pacific herring, 17, atlantic salmon, 18, mahogany, 19, redfish, 20, hexagrammos otakii, 21, large yellow croaker, 22, chinese perch, 23, atlantic, 24, bluefin tuna, 25, striped tetranychus, 26, bluered spot, 27, blackspot, 28, shark, 29, swamp, 31, swamp, and gabonito negative controls, 31, and gabonito negative controls.
FIG. 3 is a graph of the results of a simulated sensitivity addition test for a flash sturgeon, in which: 1. 10% amplification result; 2. 1% amplification result; 3. 0.1% amplification result; 4. 0.01% amplification result; 5. negative control; 6. blank control.
FIG. 4 is a graph of the results of a simulated Siberian sturgeon add sensitivity test in which: 1. 10% amplification result; 2. 1% amplification result; 3. 0.1% amplification result; 4. 0.01% amplification result; 5. negative control; 6. blank control.
Detailed Description
The present invention will be described in detail below with reference to the drawings and examples, but the present invention is not limited to the specific examples.
The double-dye fluorescence PCR specificity detection system for simultaneously detecting the flash sturgeons and the Siberian sturgeon species EVA-Green and SybrGreen, which is utilized in the embodiment, comprises sequences of primers of the flash sturgeons and the Siberian sturgeons.
The primer sequence of the flash sturgeon is as follows:
an upstream primer: 5'-CTCCCAATAATACCAGCCGTG-3', respectively; SEQ ID NO.1
A downstream primer: 5'-TATGCTACGGCTACTTCCAGTAGA-3', respectively; SEQ ID NO.2
The primer sequence of the Siberian sturgeon species is as follows:
an upstream primer: 5'-taaacccaactctggtaaatatg-3', respectively; SEQ ID NO.3
A downstream primer: 5'-tttgggtatgaaccctgttaa-3', respectively; SEQ ID NO.4
The double-dye fluorescence PCR specificity detection system for simultaneously detecting the iridescent sturgeons, the Siberian sturgeons species EVA-Green and the SybrGreen also comprises a reaction system consisting of other reagents, and the reaction system is as follows 6:
TABLE 6 reaction system for simultaneous detection of FISHR, Siberian sturgeon species EVA-Green, SybrGreen double-dye fluorescence PCR specificity detection systems
TABLE 7
The reaction parameters of the double-dye fluorescent PCR specificity detection system for the iridescent sturgeons, Siberian sturgeons EVA-Green and SybrGreen are as follows in Table 8:
TABLE 8 reaction parameters of the dual-dye fluorescent PCR specificity detection system for acipenser iridipes, acipenser sibiricus species EVA-Green, SybrGreen
The application of the double-dye fluorescence PCR specificity detection system for the flashing sturgeon and the Siberian sturgeon species EVA-Green and the SybrGreen is characterized in that the double-dye fluorescence PCR specificity detection system for the flashing sturgeon and the Siberian sturgeon species is utilized, and the specific steps are as follows:
1. synthesizing species specificity detection primers of the flash sturgeons and the Siberian sturgeons: the synthetic flash sturgeon species-specific detection primer 2 pairs were synthesized by baobaozha, and the sequences thereof (see table 5):
2. extracting DNA of Siberian sturgeons and acipenser ruthenus: DNA extraction kit (purchased from Takara Bio Inc.) is adopted to extract template DNA of pacific flash sturgeon and Siberian sturgeon, and a micro-spectrophotometer is used to detect the concentration of the extracted template DNA of pacific flash sturgeon to be 100 ng.
3. Simultaneously detecting CoxI genes of the acipenser ruber and the acipenser ruber through double-dye fluorescent PCR amplification of the species EVA-Green and the SybrGreen: preparing a 25 muL PCR reaction system (shown in the table 3) for simultaneously detecting the flash sturgeon species, the Siberian sturgeon species EVA-Green and the SybrGreen double-dye fluorescence PCR specificity detection system, and setting reaction parameters (shown in the table 4) for simultaneously detecting the flash sturgeon species, the Siberian sturgeon species EVA-Green and the SybrGreen double-dye fluorescence PCR specificity detection system.
Specificity test 1: SYBR-Green and EVA-Green fluorescent dyes are respectively used, and a Siberian sturgeon and flash sturgeon specific primer dual-fluorescence PCR method is used for detecting sturgeon species to obtain a specific amplification curve (shown in figure 1), but no amplification curve appears in other species of the same order. Therefore, the method can accurately identify the ingredients of the Siberian sturgeons and the acipenser ruthenus and has good specificity.
Specificity test 2: the Siberian sturgeon and the flash sturgeon species are used for simultaneously detecting the flash sturgeon, the Siberian sturgeon species EVA-Green and the SybrGreen double-dye fluorescence PCR specificity detection system is used for carrying out marine fish species specificity detection, specific amplification curves generated by the Siberian sturgeon and the flash sturgeon are obtained, and no amplification curve exists in other species. Therefore, the method can accurately identify the ingredients of the Siberian sturgeons and the acipenser ruthenus and has good specificity (as shown in figure 2).
Simulating addition sensitivity test of the flash sturgeon: crucian powder is used as a substrate for an addition test. Mixing the samples of the flash sturgeon meat and the crucian meat powder according to the proportion (100%, 10%, 1%, 0.1%, 0.01% and 0.001%), extracting mixed sample DNA with different concentrations, and using the mixed sample DNA as a sensitivity detection template. The addition test results show that the crucian powder is used as a matrix of a primary processed product, flash sturgeon meat is respectively added, the DNA of an added sample is extracted, and the detection sensitivity can stably reach 0.01 percent through the fluorescent PCR detection (as shown in figure 3).
Siberian sturgeon simulated addition sensitivity test: crucian powder is used as a substrate for an addition test. The Siberian sturgeon meat and crucian meat powder samples are mixed according to the proportion (100%, 10%, 1%, 0.1%, 0.01%, 0.001%), and mixed sample DNA with different concentrations is extracted to be used as a sensitivity detection template. The addition test results show that the crucian powder is used as a matrix of a primary processed product, Siberian sturgeon meat is respectively added, the DNA of an added sample is extracted, and the detection sensitivity can stably reach 0.01 percent through the fluorescent PCR detection (as shown in figure 4).
Real-time fluorescence PCR method practical application detection
The results of testing commercially available sturgeon meat and caviar products are shown in table 8.
TABLE 8 detection of the Source Components of commercial Siberian sturgeon, flashed sturgeon preparations
The results show that: siberian sturgeon-derived components are detected in 100% of Siberian sturgeon samples and 100% of Siberian sturgeon caviar samples, and flash sturgeon-derived components are detected in 100% of flash sturgeon samples. Acipenser sibiricus-derived components were detected in 16.7% of huso dauricus caviar sample and 13.2% of acipenser flash caviar sample. The commercial Siberian sturgeon and Siberian sturgeon caviar contain Siberian sturgeon components, and the partially sparkling sturgeon caviar and huso caviar samples do not contain Siberian sturgeon-derived components and have the possibility of adulteration. As seen from a large number of practical application results, the standard method has good applicability for detecting the source components of the flash sturgeons and Siberian sturgeons in the food.
Sequence listing
<110> Dalian customs technology center
<120> double-dye fluorescence PCR specificity detection system for simultaneously detecting species of acipenser ruthenus and acipenser baeri and application thereof
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Claims (7)
1. A double-dye fluorescence PCR specificity detection system for simultaneously detecting flashing sturgeons and Siberian sturgeons species EVA-Green and SybrGreen is characterized in that: in one PCR reaction, EVA-Green and SybrGreen dyes are used to perform PCR amplification on the species of the acipenser ruthenus and the acipenser baeri.
2. The system for simultaneously detecting the specificity of the fluorescent PCR of the iridescent sturgeons, the species of Siberian sturgeons EVA-Green and the species of SybrGreen according to claim 1, which is characterized in that: comprises the following primer sequences: the primer sequences of the flash sturgeon species are shown as SEQ ID NO.1 and SEQ ID NO. 2; the primer sequence of the Siberian sturgeon species is shown as SEQ ID NO.1 and SEQ ID NO. 2.
3. The system for simultaneously detecting the specificity of the fluorescent PCR of the iridescent sturgeons, the species of Siberian sturgeons EVA-Green and the species of SybrGreen according to claim 1, which is characterized in that: the system also comprises the following reagents of PCR buffer solution, dNTPs, Hotstart Taq enzyme, a DNA template, EVA-Green fluorescent dye and SybrGreen fluorescent dye.
4. The system for simultaneously detecting the specificity of the fluorescent PCR of the iridescent sturgeons, the species of Siberian sturgeons EVA-Green and the species of SybrGreen according to claim 1, which is characterized in that: the 20. mu.L reaction system comprises: containing Mg2+2. mu.L of 10 XPCR buffer; 2. mu.L of 10 XdNTPs; 0.2 muL of Hotstart Taq enzyme at a concentration of 5 units/. mu.L; the concentration of the primers of the upstream and downstream of the flash sturgeon is 5 mu mol/L, and the concentration of the primers of the upstream and downstream of the flash sturgeon is 0.5 mu L respectively; the concentration of the upstream primer and the downstream primer of the Siberian sturgeon is 5 mu mol/L, and the concentration of the downstream primer and the upstream primer are respectively 0.5 mu L; 100ng of DNA template; 1 mu L of each of EVA-Green fluorescent dye or SybrGreen fluorescent dye; the balance of double distilled water.
5. A dual dye fluorescence PCR specific assay system for the simultaneous detection of Acipenser iridipenser, Acipenser sibirica species EVA-Green, SybrGreen according to any one of claims 1 to 4 wherein: the reaction parameters of the EVA-Green and SybrGreen double-dye fluorescent PCR specificity detection system are as follows: denaturation at 94 deg.C for 2 min; amplifying at 94 ℃,6s, 60 ℃ and 20 s; cycle number 40; final extension 72 ℃ for 10 min.
6. Use of a dual dye fluorescence PCR specific assay system for the simultaneous detection of Acipenser iridipenser, Acipenser sibirica species EVA-Green, SybrGreen according to any one of claims 1 to 5 wherein: the double-dye fluorescence PCR specificity detection system for simultaneously detecting the species EVA-Green and SybrGreen of the flash sturgeons and the Siberian sturgeons is utilized; respectively configuring a reaction system for fish components of two species of the flash sturgeon and the Siberian sturgeon by using EVA-Green fluorescent dye or SybrGreen fluorescent dye; the specific detection of the double-dye fluorescence PCR is carried out simultaneously in the same PCR reaction.
7. The use of the dual-dye fluorescence PCR specific detection system for simultaneously detecting Acipenser iridipenser, Acipenser sibirica species EVA-Green, SybrGreen according to claim 6, wherein the dual-dye fluorescence PCR specific detection system comprises: the method comprises the steps of amplifying genes of the flash sturgeon and the Siberian sturgeon CoxI through fluorescent PCR, carrying out PCR reaction by using Hotstart Taq DNA polymerase, respectively generating two specific amplification curves in the same species in a positive sample after double-dye fluorescent PCR amplification, wherein one specific amplification curve is generated by EVA-Green fluorescence, the other specific amplification curve is generated by SybrGreen fluorescence, and carrying out specific identification on species components of the flash sturgeon and the Siberian sturgeon according to different TM values of the melting curves of an amplification system.
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|---|---|---|---|---|
| CN113957159A (en) * | 2021-12-15 | 2022-01-21 | 中国海关科学技术研究中心 | Primer probe composition and kit for detecting Siberian sturgeon mitochondrial gene and application of primer probe composition and kit |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103436611A (en) * | 2013-08-20 | 2013-12-11 | 徐鹏 | PCR-RFLP rapid detection method for common sturgeons |
| CN105734142A (en) * | 2016-03-31 | 2016-07-06 | 万超 | Real-time fluorescent PCR (polymerase chain reaction) specific detection system of acipenser ruthenus and application |
| CN110951892A (en) * | 2019-11-25 | 2020-04-03 | 中国水产科学研究院长江水产研究所 | SSR primer pair group, kit, identification method and application for identification of several sturgeon species |
-
2021
- 2021-02-21 CN CN202110194573.2A patent/CN112831571A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103436611A (en) * | 2013-08-20 | 2013-12-11 | 徐鹏 | PCR-RFLP rapid detection method for common sturgeons |
| CN105734142A (en) * | 2016-03-31 | 2016-07-06 | 万超 | Real-time fluorescent PCR (polymerase chain reaction) specific detection system of acipenser ruthenus and application |
| CN110951892A (en) * | 2019-11-25 | 2020-04-03 | 中国水产科学研究院长江水产研究所 | SSR primer pair group, kit, identification method and application for identification of several sturgeon species |
Non-Patent Citations (2)
| Title |
|---|
| RASMUSSEN ET AL.: "Application of DNA-Based Methods to Identify Fish and Seafood Substitution on the Commercial Market", 《COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY》 * |
| 万超等: "实时荧光定量PCR法鉴别俄罗斯鲟鱼子酱分子", 《食品安全质量检测学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113957159A (en) * | 2021-12-15 | 2022-01-21 | 中国海关科学技术研究中心 | Primer probe composition and kit for detecting Siberian sturgeon mitochondrial gene and application of primer probe composition and kit |
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