阅读说明：本技术 鉴别鱼类源性成分的引物及鉴定方法 (Primer and method for identifying fish-derived components ) 是由 王守伟 许随根 李家鹏 李金春 熊苏玥 韦忆萱 刘睿茜 杨君娜 陈曦 曲超 乔晓 于 2020-06-23 设计创作，主要内容包括：本发明公开了鉴别鱼类源性成分的引物及鉴定方法。本发明提供三对特异性引物(SEQ ID NO:1-6),基于多重实时荧光PCR熔解曲线鉴别鱼类或鱼肉制品中是否含有蓝鳍金枪鱼、银鳕鱼、油鱼三种动物源性成分。本方法具有检测精准、特异性强、灵敏度高、简便快速的优点,能满足大批量、快速鉴别鱼类或鱼肉制品中是否含有蓝鳍金枪鱼、银鳕鱼、油鱼源性成分的要求,具有巨大市场需求和广阔的应用前景。(The invention discloses a primer and an identification method for identifying fish-derived components. The invention provides three pairs of specific primers (SEQ ID NO:1-6), which are used for identifying whether three animal-derived components including bluefin tuna, pollack and oil fish exist in fish or fish products based on a multiple real-time fluorescence PCR melting curve. The method has the advantages of accurate detection, strong specificity, high sensitivity, simplicity, convenience and rapidness, can meet the requirement of rapidly identifying whether the bluefin tuna, pollack and oil fish derived components are contained in the fish or fish products in a large scale, and has huge market requirements and wide application prospects.)

1. The primer for identifying the source component of the bluefin tuna (Thunnus thynnus) is characterized by comprising a forward primer shown as SEQ ID NO. 1 and a reverse primer shown as SEQ ID NO. 2.

2. The primer for identifying the source component of the pollack (Anoplopoma fimbria) is characterized by comprising a forward primer shown as SEQ ID NO. 3 and a reverse primer shown as SEQ ID NO. 4.

3. The primer for identifying the source component of the oil fish (Lepidocybium flavobrunneum) is characterized by comprising a forward primer shown as SEQ ID NO. 5 and a reverse primer shown as SEQ ID NO. 6.

4. A primer combination for identifying fish-derived components, wherein the fish is at least two selected from the group consisting of bluefin tuna, pollack and oil fish;

the primers for identifying the origin components of tuna, pollack and oil fish are shown in SEQ ID NO. 1-2, SEQ ID NO. 3-4 and SEQ ID NO. 5-6 respectively.

5. The primer combination according to claim 4, which consists of the primers shown in SEQ ID NOS 1-2, 3-4 and 5-6.

6. A detection reagent or kit comprising the primer of any one of claims 1 to 3 or the primer combination of claim 4 or 5.

7. A method for identifying a fish-derived component, wherein the fish is at least one selected from the group consisting of tuna bluefin, pollack, and sashimi, and the method comprises: performing multiplex PCR detection on a sample to be detected by using the primer combination of claim 5.

8. The method of claim 7, comprising:

1) extracting DNA of the fish or fish product to be detected;

2) taking DNA as a template, performing multiplex real-time fluorescent PCR amplification by using primers shown in SEQ ID NO. 1-6, and drawing a melting curve;

3) and judging the result according to the melting curve.

9. The method of claim 8, wherein the multiplex real-time fluorescent PCR uses a reaction system comprising:

wherein, the sequences of the primer F and the primer R of the tuna are shown as SEQ ID NO. 1-2, the sequences of the primer F and the primer R of the pollock are shown as SEQ ID NO. 3-4, and the sequences of the primer F and the primer R of the oil fish are shown as SEQ ID NO. 5-6; and/or

The reaction conditions of the multiplex real-time fluorescent PCR are as follows: 5min at 95 ℃; 35 cycles of 10-15 s at 95 ℃ and 45-60 s at 55-60 ℃; and/or

The melting curve was prepared under the following conditions: heating to 95 ℃ at the speed of 0.02-0.05 ℃/s for 1min at the temperature of 95 ℃ and 1min at the temperature of 65-70 ℃.

10. The method according to claim 8 or 9, wherein the determination result is determined based on the number and position of peaks in the melting curve, and specifically comprises: detecting the source components of the bluefin tuna when a melting peak appears within the temperature range of 73.9-76.4 ℃; detecting the cod-origin component when a melting peak appears within the temperature range of 77.5-79.8 ℃; detecting the oily fish-derived component when a melting peak appears within the temperature range of 81.8-83.1 ℃.

Technical Field

The invention belongs to the technical field of food detection, and particularly relates to a primer and an identification method for identifying fish-derived components.

Background

With the increasing living standard of people, aquatic food is more and more favored by consumers, and especially the consumption demand of tuna and pollack with higher nutritional value is continuously increased. Tuna enjoys international market reputation of high nutritional value, pure nature and no pollution, enjoying the name of "ocean gold", wherein bluefin tuna (Thunnus thynnus) has the elegant name of "the king of tuna"; the bluefin tuna meat is rich in nutritional ingredients such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), tyrosine, taurine, nucleic acid, iron, calcium, potassium, various essential amino acids, vitamin B12 and the like, can promote the development of brains, bones and teeth of children by eating more, and can prevent and assist in treating iron-deficiency anemia; has effects in promoting metabolism, delaying aging, and delaying memory deterioration, and can be used for the prevention and adjuvant treatment of senile dementia, arteriosclerosis, diabetes, hypertension, and aging. The bluefin tuna meat has low heat, is rich in high-quality protein and other nutrient elements, is deeply liked by women, has delicious and sweet meat quality, smooth mouthfeel, rich grease and light red meat color, is the favorite of diners, is also the material of raw fish slices, and has high economic value. With the mass of fishing, bluefin tunas are also becoming more rare and more expensive. The pollack (Anoplopoma fimbria) contains a large amount of unsaturated fatty acid, is rich in protein, multiple essential amino acids, vitamin A, vitamin D, B vitamins, calcium, magnesium, selenium and other nutrient elements, is rich in nutrition, has sweet meat flavor, is easy to digest and is deeply favored by infants. Oily fish (Lepidocybiumflavobrucum) looks like cod, is often used to impersonate cod, but its muscles and viscera are rich in waxy oil, easily cause diarrhea, and harm to health of consumers. Therefore, there is a need to develop a sensitive and specific method for identifying bluefin tuna and pollack and fish products thereof.

Disclosure of Invention

The invention aims to provide a primer and an identification method for identifying fish-derived components.

In order to achieve the object, in a first aspect, the invention provides primers for identifying a source component of Thunnus thynnus, comprising a forward primer shown as SEQ ID NO. 1 and a reverse primer shown as SEQ ID NO. 2.

In a second aspect, the present invention provides primers for identifying pollack (Anoplopoma fimbria) derived components, comprising a forward primer shown as SEQ ID NO. 3 and a reverse primer shown as SEQ ID NO. 4.

In a third aspect, the invention provides a primer for identifying the source component of the oil fish (Lepidocybium flavobrucum), which comprises a forward primer shown as SEQ ID NO. 5 and a reverse primer shown as SEQ ID NO. 6.

In a fourth aspect, the present invention provides a primer combination for identifying fish-derived ingredients, wherein the fish is selected from at least two of tuna bluefin, pollack and oil fish.

Preferably, the primer combination consists of primers shown in SEQ ID NO. 1-2, SEQ ID NO. 3-4 and SEQ ID NO. 5-6.

In a fifth aspect, the invention provides a detection reagent or kit comprising said primer or a combination thereof.

In a sixth aspect, the present invention provides a method for identifying a fish-derived component, the fish being selected from at least one of tuna bluefin, pollack and oil fish, the method comprising: and carrying out multiplex PCR detection on a sample to be detected by using the primer combination.

The specific method comprises the following steps:

1) extracting DNA of the fish or fish product to be detected;

2) taking DNA as a template, performing multiplex real-time fluorescent PCR (multiplex RT-PCR) amplification by using primers shown in SEQ ID NO. 1-6, and drawing a melting curve;

3) and judging the result according to the melting curve. The content of each fish-derived component in the sample can be quantified according to the height value of the melting curve peak, so that the qualitative detection is realized, and meanwhile, the quantitative detection purpose can be achieved.

Preferably, the reaction system used for multiplex real-time fluorescent PCR is:

wherein, the sequences of the primer F and the primer R of the tuna are shown as SEQ ID NO. 1-2, the sequences of the primer F and the primer R of the pollock are shown as SEQ ID NO. 3-4, and the sequences of the primer F and the primer R of the oil fish are shown as SEQ ID NO. 5-6.

Preferably, the reaction conditions of the multiplex real-time fluorescent PCR are: 5min at 95 ℃; 10-15 s at 95 ℃, 45-60 s at 55-60 ℃ and 35 cycles. More preferably, the PCR reaction conditions are: 5min at 95 ℃; 95 ℃ for 10s, 58 ℃ for 45s, 35 cycles.

Preferably, the melting curve is prepared under the following conditions: heating to 95 ℃ at the speed of 0.02-0.05 ℃/s for 1min at the temperature of 95 ℃ and 1min at the temperature of 65-70 ℃. More preferably, the melting curve is prepared under the following conditions: the temperature is raised to 95 ℃ at the rate of 0.02 ℃/s for 1min at 95 ℃ and 1min at 70 ℃.

Continuously detecting the fluorescence intensity at the same time; and (3) reducing the temperature to 40 ℃ and keeping the temperature for 60s, and taking the first negative derivative of the fluorescence signal to the temperature as the ordinate and the temperature as the abscissa to obtain a melting peak map. The melting peak of Tm value of each specific amplification product represents a specific fish-derived component.

The method specifically includes, based on the number and position of peaks in the melting curve, determining a result: detecting the source components of the bluefin tuna when a melting peak appears within the temperature range of 73.9-76.4 ℃; detecting the cod-origin component when a melting peak appears within the temperature range of 77.5-79.8 ℃; detecting the oily fish-derived component when a melting peak appears within the temperature range of 81.8-83.1 ℃.

In a seventh aspect, the invention provides the use of the above method in the identification of bluefin tuna, pollack, oleofish-derived ingredients in fish or fish products.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

aiming at the market detection requirements of the bluefin tuna, the pollack and the oil fish, the invention develops a multiplex PCR detection method capable of simultaneously detecting three species of the bluefin tuna, the pollack and the oil fish by using a melting curve analysis method of SYBR Green fluorescent quantitative PCR, realizes synchronous and rapid detection of multiple species, reduces the detection cost, can meet the rapid detection requirements of multiple samples, and has good application prospect.

The method for analyzing the multiple RT-PCR melting curve provided by the invention adopts the fully-closed reaction tube detection and result analysis, does not need subsequent PCR product electrophoresis detection and analysis, simplifies the operation process, reduces the experiment detection cost, improves the detection efficiency, and avoids the cross contamination of samples and the environment.

And thirdly, the multiple RT-PCR reaction provided by the invention has good specificity, ensures that the method can specifically detect species-derived components, and avoids false positive. The multiple RT-PCR melting curve analysis method also has higher sensitivity, and the detection sensitivity of the method for the blue fin tuna, the silver cod and the oil fish-derived ingredients can reach picogram level. The multiple PCR fish detection method provided by the invention has a lower detection limit, and in the fish or fish product to be detected, the detection limit of the bluefin tuna-derived component is 0.5%, the detection limit of the pollack-derived component is 1%, and the detection limit of the oil fish-derived component is 0.5%.

The invention realizes the high-flux multi-species synchronous detection with low cost, greatly reduces the detection cost, improves the detection efficiency and the accuracy, can realize quantitative detection, and is particularly important for strengthening fish market supervision, effectively resisting fish products adulteration and protecting the benefits of consumers and related aquatic product enterprises. The popularization and the application of the detection method provide technical support for guaranteeing the quality of aquatic products, protecting the right of awareness and selection of consumers, maintaining normal social order and the like. The method can be popularized and applied in fishery breeding, food quality safety supervision, international trade food quality detection, raw material guarantee of food processing enterprises and the like, and has huge market demands and application prospects.

Drawings

FIG. 1 is a specific verification result of a proportioning system of part of different primer concentrations in example 1 of the present invention. Wherein, A to F respectively represent melting peak maps of RT-PCR systems with a blue fin tuna primer pair (C-21-1-F/R), a pollack primer pair (C-23-1-F/R) and an oil fish primer pair (C-22-4-F/R) in the concentration ratio of 0.6:2.0:0.8, 1.1:2.0:0.8, 1.0:2.0:1.6, 1.1:2.0:1.5 and 1.1:2.0: 1.1.

FIG. 2 is a melting peak spectrum of 3 fish-derived components identified in fish or fish meat products in the preferred embodiment of the present invention. Wherein, 1: a bluefin tuna-derived component melting peak; 2: melting peaks of the pollack-derived components; 3: melting peak of oil fish-derived component.

FIG. 3 is a graph showing the results of the sensitivity test of the identification method according to the preferred embodiment of the present invention on bluefin tuna or a product of bluefin tuna meat.

FIG. 4 is a graph showing the results of sensitivity tests of the identification method of the preferred embodiment of the present invention on pollack or pollack meat products.

FIG. 5 is a graph showing the results of the sensitivity test of the identification method for oil fish or oil fish meat products in the preferred embodiment of the present invention.

Detailed Description

The invention provides a method for identifying species source components in fish and fish products based on multiple RT-PCR (reverse transcription-polymerase chain reaction) melting curve analysis, in order to make up the defects in the aspect of species source identification in the existing fish or fish products, and particularly provides a multiple RT-PCR melting curve analysis method for quickly identifying 3 species source components of bluefin tuna, pollock and oil fish in the fish or fish products, and provides a technical means for determining the species sources of the tuna, the pollock and the oil fish, thereby ensuring the consistency of food and labels.

The invention also provides a specific primer combination for identifying the animal-derived components in the fish or the fish product.

The technical scheme of the invention is as follows:

the invention respectively designs 3 pairs of specific primers by taking mitochondrial DNAs of tuna bluefin, cod and oil fish as target sequences: bluefin tuna F/bluefin tuna R, pollack F/pollack R, and oil fish F/oil fish R, wherein the bluefin tuna F/bluefin tuna R can amplify bluefin tuna-derived ingredients; cod F/cod R can amplify cod-derived components; the oily fish F/oily fish R can amplify oily fish-derived components. The Tm values of the amplification products of the specific primers have significant difference and can be identified by melting peak value map analysis. The specific primer sequences are as follows:

the forward specific primers adopted for amplifying the bluefin tuna-derived components are as follows:

bluefin tuna F: 5'-TCAACATTCATTTCGAATTCAAGCG-3' (SEQ ID NO:1)

The reverse specific primers adopted in the amplification of the bluefin tuna-derived components are as follows:

bluefin tuna R: 5'-TTTACGAGATGTCAGAGATGAGGAG-3' (SEQ ID NO:2)

The forward specific primers adopted when the pollack derived component is amplified are as follows:

pollack F: 5'-CCCCCTAAGTACACTAGAGTTTATT-3' (SEQ ID NO:3)

The reverse specific primers adopted when the pollack derived component is amplified are as follows:

pollack R: 5'-GTACTCTTTGCTTGGTATACACTCA-3' (SEQ ID NO:4)

The forward specific primers adopted when the oil fish-derived component is amplified are as follows:

and (3) oily fish F: 5'-AACCTTTACGCTGTTCATATTGG-3' (SEQ ID NO:5)

The reverse specific primers adopted when the oil fish-derived component is amplified are as follows:

and (3) oil fish R: 5'-GGGTTTGTAAGTGATCGTTCCCA-3' (SEQ ID NO:6)

The invention firstly provides a primer combination containing the 3 pairs of specific primer pairs, and the nucleotide sequences of the primer combination are respectively shown as SEQ ID NO. 1-6.

The invention provides a kit or a detection reagent containing the primer combination.

The invention provides application of the primer combination in identifying species-derived components in fish or fish products, wherein the fish is bluefin tuna, pollack and oil fish.

The invention provides application of a kit or a detection reagent containing the primer combination in identifying species-derived components in fish or fish products, wherein the fish is bluefin tuna, pollack and oil fish.

The invention also provides application of the primer combination in guaranteeing fish food safety.

The invention provides a method for identifying species-derived components in fish or fish products, which is characterized in that a primer combination containing nucleotide sequences respectively shown as SEQ ID NO 1-6 is applied, DNA of fish or fish products to be detected is used as a template, an RT-PCR method is used for detection, and the result is judged according to the number and the position of peaks in a melting peak value diagram.

The preparation method of the template comprises the following steps: mixing the fish or the fish product with sterilized and deionized double distilled water in a weight ratio of 1: 3-1: 5, and homogenizing for 5-10 min by a tissue homogenizer at 10000-14000 r/min to prepare tissue homogenate. The total DNA of the sample genome was extracted using a tissue DNA extraction kit.

Further, in the RT-PCR method, 25 μ L of PCR reaction system is adopted, and the RT-PCR method comprises the following components:

the RT-PCR reaction conditions are as follows: 5min at 95 ℃; 10-15 s at 95 ℃, 45-60 s at 55-60 ℃ and 35 cycles.

The melting curve preparation conditions are as follows: heating to 95 ℃ at the speed of 0.02-0.05 ℃/s for 1min at the temperature of 95 ℃ and 1min at the temperature of 65-70 ℃. Continuously detecting the fluorescence intensity at the same time; and (3) reducing the temperature to 40 ℃ and keeping the temperature for 60s, and taking the first negative derivative of the fluorescence signal to the temperature as the ordinate and the temperature as the abscissa to obtain a melting peak map. The melting peak of Tm value of each specific amplification product represents a specific fish-derived component.

A melting peak of a Tm value of each specific amplification product represents a specific fish-derived component, wherein the occurrence of the melting peak at the Tm value within the range of 73.9-76.4 ℃ is detected as a bluefin tuna-derived component; the Tm value is detected as the pollock source component when a melting peak appears within the range of 77.5-79.8 ℃; the melting peak of the Tm value is 81.8-83.1 ℃, and the oil fish-derived component is detected.

The invention provides application of the method in species origin component identification of fish or fish products.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.

The main instrumentation involved in the following examples: a fluorescent quantitative PCR instrument (Roche 480), a refrigerated desktop centrifuge (Eppendorf 5417R, germany), a micropipette (2.5ul, 10ul, 100ul, 1000ul), a homogenizer (OmniPrep, usa), a fluorescent microplate reader (Bio tek synergy H4, usa), and the like. The main reagents are as follows: qiagen DNeasy Blood & Tissue Kit was purchased from Qiagen, SYBR Green I Master premix was purchased from Roche China, Inc., and primer pairs were synthesized by Saimer Feishel technologies.