A kit and method for identifying multiple fish-derived ingredients

Abstract

The present invention discloses a kit and method for identifying fish-derived ingredients, specifically a method for identifying fish-derived ingredients using a visualized gene membrane chip. The kit comprises a gene membrane chip, a mixed primer, a multiplex PCR premix, and a hybridization color development reagent. The gene membrane chip is a nylon membrane carrier on which internal reference genes, and probes specific for Atlantic salmon, rainbow trout, sablefish, large yellow croaker, salmon, Atlantic cod, pufferfish, anglerfish, silver pomfret, tilapia, and small yellow croaker are sequentially fixed. The mixed primer is a multiplex primer used for PCR amplification of the above 11 fish species. After DNA extraction and multiplex PCR amplification, the sample is hybridized with the gene membrane chip probes, and the fish species is determined based on chemical color development. The method designed by the present invention has the advantages of high throughput and strong specificity, while being simple to operate and cost-effective.

Description

Technical Field

[0001] The present invention relates to a kit and method for identifying fish-derived ingredients, and in particular to a method for identifying fish-derived ingredients by utilizing a visualized gene membrane chip. Background Art

[0002] Aquatic products are delicious and rich in protein, amino acids, unsaturated fatty acids, various vitamins, and minerals such as calcium, phosphorus, potassium, and iodine. They are also low in fat and easily digestible and absorbed, making them considered one of the most nutritionally balanced natural foods for humans. In recent years, with the development of the social economy and the improvement of people's living standards, the demand for and consumption of aquatic products has been increasing, with fish and their products being the most important.

[0003] However, mislabeling, counterfeiting, and adulteration are widespread in the global fish market, with examples including dyed white croaker and yellow croaker passed off as yellow croaker, tilapia as sea bream, oilfish as cod, and rainbow trout as Atlantic salmon. At the 2019 Global Seafood Conference, Professor Alan Reill of University College Dublin, citing data from the European Commission's Food Fraud Management System (AAC-FF), pointed out that seafood adulteration ranks as the leading food fraud problem in Europe. This is due to the wide variety of fish species, some of which share similar morphology and tissue structure, making them difficult to distinguish, leading to frequent mislabeling and counterfeiting. Furthermore, varying quality, coupled with supply and demand imbalances caused by natural factors, creates significant price discrepancies. Driven by profit, unscrupulous vendors engage in counterfeiting and adulteration, particularly in processed fish products such as fillets, surimi, fish balls, fish sausages, fish fillets, dried fish, shark fins, fish maws, and canned fish, which are particularly vulnerable to counterfeiting.

[0004] Counterfeit and substandard aquatic products, particularly fish and their products, not only disrupt market order, negatively impact public image, and harm the legitimate rights and interests of consumers, but also pose potential safety risks. For example, dyeing yellow croaker involves the use of banned dyes, which poses serious risks to human health. Impersonating freshwater-farmed rainbow trout as deep-sea salmon significantly increases the risk of parasitic infections when consumed raw. Oily fish disguised as cod, due to its high wax ester content, is difficult for the human body to digest and absorb, leading to symptoms such as diarrhea and gastrointestinal cramps. Market regulation is urgently needed to address the current irregularities in the industry. Therefore, accurate and reliable methods for tracing and identifying fish-derived ingredients are crucial. Numerous methods have been developed for authenticating fish products, but molecular biological testing offers significant advantages. DNA, as genetic material, is the foundation of biological growth and development and possesses a stable structure, making it suitable for processed products. However, different molecular biology methods also have their own advantages and disadvantages. The ordinary PCR method does not have high requirements for instruments, but requires subsequent electrophoresis, which is relatively cumbersome; the fluorescent quantitative PCR method has high sensitivity and good specificity, but the cost is relatively high. It is limited by the number of fluorescent channels of the instrument itself and cannot detect more than 6 targets; isothermal amplification has high requirements for primer design; restriction fragment length polymorphism analysis is cumbersome to operate; DNA barcode identification technology has the advantage of high-throughput analysis, but involves sequence determination and is relatively expensive; gene chips are characterized by high throughput, rapidness and convenience, and are particularly suitable for identifying adulteration of fish products with unpredictable ingredients. Summary of the Invention

[0005] The purpose of the present invention is to address the deficiencies in the existing technology and provide a kit and method for identifying fish-derived ingredients, which can simultaneously screen 11 fish-derived ingredients. It not only has the advantages of high throughput, fast speed and low cost, but also is simple to operate and easy to use.

[0006] To achieve the above-mentioned object, the present invention provides a kit for identifying multiple fish-derived ingredients, comprising a gene membrane chip, a mixed primer, a multiplex PCR premix, and a hybridization color development reagent. The gene membrane chip is a negatively charged nylon membrane on which eukaryotic reference genes, Atlantic salmon, rainbow trout, sablefish, large yellow croaker, salmon, Atlantic cod, pufferfish, anglerfish, silver pomfret, tilapia, and small yellow croaker specific probes (SEQ ID NOs. 1-12) are sequentially immobilized. The probe sequences are shown in the table below.

[0007]

[0008]

[0009] The 5' end was modified with NH2 C6. The mixed primer sequences (SEQ ID NO. 13-36) and the terminal modifications are shown in the table below:

[0010]

[0011] As a further improvement of the above scheme, the cross-color development reagent includes deactivation solution, deactivation cleaning solution, hybridization solution, hybridization cleaning solution, enzyme incubation solution, incubation cleaning solution 1, incubation cleaning solution 2, alkaline phosphatase labeled streptavidin and alkaline phosphatase labeled chemical display substrate.

[0012] As a further improvement of the above scheme, the deactivation solution includes 100 mmol / L NaOH, the deactivation cleaning solution includes 2×SSPE and 0.1% SDS, the hybridization solution includes 2×SSPE and 0.1% SDS, the hybridization cleaning solution includes 2×SSPE and 0.5% SDS, the enzyme incubation solution includes 2×SSPE and 0.5% SDS, the incubation cleaning solution 1 includes 2×SSPE and 0.5% SDS, the incubation cleaning solution 2 includes 2×SSPE, and the alkaline phosphatase labeling chemical display substrate is 5-bromo-4-chloro-3-indole-phosphate / nitrotetrazole chloride nitro blue and BCIP-NBT.

[0013] A method for identifying multiple fish-derived ingredients comprises the following steps:

[0014] a. Extract DNA from fish samples to be tested;

[0015] b. The extracted DNA was added to the same reaction tube and the universal primers in any one of the kits according to claim 1-3 were used for multiplex PCR amplification reaction;

[0016] c. Denaturing and hybridizing the target test solution obtained by PCR amplification in step b with the gene membrane chip probe;

[0017] d. The internal reference gene must be displayed and the blank must not show color, otherwise the test result will be invalid. The presence of the component is determined based on whether the corresponding fish-derived probe position shows color. If it does, the fish-derived component has been detected; if it does not, the fish-derived component has not been detected.

[0018] As a further improvement of the above scheme, when the reaction system of the amplification reaction is a 25 μL system, the reaction system includes: 12.5 μL of multiplex PCR premix, 3 μL of upstream and downstream primers with a concentration of 100 μmol, 5 μL of template DNA, and ddH2O is added to 25 μL. When the reaction system of the amplification reaction is a 50 μL system, the reaction system includes: 25 μL of fluorescent quantitative PCR reaction solution, 6 μL of upstream and downstream primers with a concentration of 100 μmol each, 1-5 μL of template DNA is added, and ddH2O is supplemented to 50 μL.

[0019] As a further improvement of the above scheme, the reaction parameters of the PCR amplification are:

[0020] Step 1: denaturation at 95°C for 10 min;

[0021] Step 2: denaturation at 95°C for 15 seconds, annealing at 58°C, and extension at 72°C for 60 seconds, for 35-40 cycles;

[0022] Step 3: Extend at 72°C for 10 min and store at 4°C.

[0023] As a further improvement of the above scheme, the product denaturation in step c is to denature the PCR product at 95°C for 5 minutes and then immediately place it on ice for later use. Then, 20 μL of PCR reaction solution is added to 200 μL of hybridization solution to prepare a hybridization system solution, and alkaline phosphatase-labeled streptavidin is added to the enzyme incubation solution at a ratio of 1:2000 to prepare an enzyme incubation system solution.

[0024] As a further improvement of the above scheme, the hybridization reaction in step c comprises:

[0025] Deactivation: 1 mL of deactivation solution, 37°C, 8 min;

[0026] Deactivation cleaning: 1 mL of deactivation cleaning solution, 60°C, 5 min;

[0027] Hybridization: Add hybridization system solution, 45℃, 10min;

[0028] Hybridization wash twice: add 1 mL of hybridization wash solution, incubate at 52°C for 3 min;

[0029] Enzyme incubation labeling: add 1 mL of enzyme incubation system solution, 42°C, 10 min;

[0030] Enzyme labeling cleaning I: add 1.1 mL of incubation cleaning solution and incubate at 42°C for 3 min.

[0031] Enzyme labeling cleaning II 2 times: add 2,1 mL of incubation cleaning solution, 37°C, 3 min;

[0032] Color development with color developing solution: add 1 mL of color developing solution and incubate at 37°C for 10 min.

[0033] Color development and washing twice: add 1 mL of distilled water, 37°C, 1 min,

[0034] Each of the above processes requires the corresponding liquid to be completely removed before entering the next process.

[0035] The beneficial effects of the present invention are as follows: the fish-derived component identification kit and method have high detection throughput, are rapid and efficient, can identify 11 fish-derived components at a time, and are particularly suitable for the identification of unknown fish products; the results are accurate, stable and reliable; the operation is convenient, and the provided kit includes reagents and consumables used in the detection and analysis process, without the need for tedious preparation work; at the same time, the hybridization process can be carried out using automated instruments according to actual conditions, further improving convenience, or it can be operated manually, providing a variety of options; the result interpretation is intuitive and can be directly identified without relying on any instrument. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a dot matrix image of a fish-derived component membrane chip in an embodiment of the present invention;

[0037] Figure 2 This is a graph showing the positive quality control test analysis results for 11 fish species in an embodiment of the present invention;

[0038] Figure 3 This is a diagram showing the results of the large yellow croaker sample detection and analysis in an embodiment of the present invention;

[0039] Figure 4 This is a diagram showing the results of Atlantic salmon sample testing and analysis in an embodiment of the present invention;

[0040] Figure 5 This is a diagram of the results of the manual random mixed sample detection and analysis in an embodiment of the present invention.

[0041] Among them: 1 is Atlantic salmon, 2 is rainbow trout, 3 is sablefish, 4 is large yellow croaker, 5 is salmon, 6 is Atlantic cod, 7 is puffer fish, 8 is monkfish, 9 is silver pomfret, 10 is tilapia, and 11 is small yellow croaker. DETAILED DESCRIPTION

[0042] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field are within the scope of protection of the present invention.

[0043] Example 1

[0044] Analysis of positive quality control tests for 11 fish species

[0045] DNA was extracted from the meat tissues of Atlantic salmon, rainbow trout, sablefish, large yellow croaker, salmon, Atlantic cod, puffer fish, anglerfish, silver pomfret, tilapia, and small yellow croaker using a marine animal tissue genomic DNA extraction kit (TIANGEN). DNA was then mixed in equal proportions. The gene membrane chip dot array images of the above fish are shown in the figure. Figure 1 As shown, the kit of the present invention was used for detection and analysis, and the results were as follows Figure 2 As shown, it can be seen that the blank control did not show color, while the internal reference gene and the 11 fish target probe positions all showed color.

[0046] Example 2

[0047] Detection and analysis of large yellow croaker samples

[0048] DNA was extracted from large yellow croaker and tested using the kit described in this patent. The results are as follows: Figure 3 As shown, it can be seen that the chip result color display position is the internal reference gene and the large yellow croaker probe position, indicating that the large yellow croaker was detected, which is consistent with expectations.

[0049] Example 3

[0050] Atlantic salmon sample testing and analysis

[0051] DNA was extracted from Atlantic salmon and tested using the kit described in this patent. The results are as follows: Figure 4 As shown, it can be seen that the chip result color display position is the internal reference gene and the Atlantic salmon probe position, indicating that Atlantic salmon was detected, which is consistent with expectations.

[0052] Example 4

[0053] The fish meat of silver pomfret and monkfish was randomly mixed, DNA was extracted using a marine animal tissue genomic DNA extraction kit, and the kit described in this patent was used for detection. The results were as follows Figure 5 As shown, it can be seen that the chip results showed the internal reference gene and the positions of the silver pomfret and anglerfish probes, indicating that the silver pomfret components and anglerfish components were detected, which was consistent with expectations.

[0054] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

[0055] The above-described embodiments are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A kit for identifying multiple fish-derived ingredients, comprising a gene membrane chip, mixed primers, a multiplex PCR premix, and a hybridization color development reagent, characterized in that: The gene membrane chip refers to a negatively charged nylon membrane on which eukaryotic reference genes, Atlantic salmon, rainbow trout, sablefish, large yellow croaker, salmon, Atlantic cod, pufferfish, anglerfish, silver pomfret, tilapia, and small yellow croaker specific probes are sequentially fixed. The probe sequences are shown in the table below: Among them, the 5' end is modified with NH2 C6. The mixed primer sequence and terminal modification are shown in the following table:

2. The multiple fish-derived ingredient identification kit according to claim 1, characterized in that: The hybridization color development reagent includes deactivation solution, deactivation cleaning solution, hybridization solution, hybridization cleaning solution, enzyme incubation solution, incubation cleaning solution 1, incubation cleaning solution 2, alkaline phosphatase labeled streptavidin and alkaline phosphatase labeled chemical display substrate.

3. The multiple fish-derived ingredient identification kit according to claim 2, characterized in that: The deactivation solution includes 100 mmol / L NaOH, the deactivation cleaning solution includes 2×SSPE and 0.1% SDS, the hybridization solution includes 2×SSPE and 0.1% SDS, the hybridization cleaning solution includes 2×SSPE and 0.5% SDS, the enzyme incubation solution includes 2×SSPE and 0.5% SDS, the incubation cleaning solution 1 includes 2×SSPE and 0.5% SDS, and the incubation cleaning solution 2 includes 2×SSPE. The alkaline phosphatase labeling chemical display substrate is 5-bromo-4-chloro-3-indole-phosphate / nitrotetrazole chloride nitro blue and BCIP-NBT.

4. A method for identifying multiple fish-derived ingredients, characterized by: The steps include: a. Extract DNA from fish samples to be tested; b. To the extracted DNA, in the same reaction tube, add the mixed primers in the kit according to any one of claims 1-3 to perform a multiplex PCR amplification reaction; c. Denaturing and hybridizing the target test solution obtained by PCR amplification in step b with the gene membrane chip probe; d. The internal reference gene must be displayed and the blank must not show color, otherwise the test result will be invalid. The presence of the component is determined by whether the corresponding fish-derived probe position shows color. If it does, the fish-derived component is detected. If no color is developed, the fish-derived ingredient was not detected.

5. The method for identifying multiple fish-derived ingredients according to claim 4, wherein: When the reaction system of the amplification reaction is a 25 μL system, the reaction system includes: 12.5 μL of multiplex PCR premix, 3 μL of upstream and downstream primers with a concentration of 100 μmol, 5 μL of template DNA, and ddH2O is added to 25 μL. When the reaction system of the amplification reaction is a 50 μL system, the reaction system includes: 25 μL of fluorescent quantitative PCR reaction solution, 6 μL of upstream and downstream primers with a concentration of 100 μmol each, 1-5 μL of template DNA is added, and ddH2O is added to 50 μL.

6. The method for identifying multiple fish-derived ingredients according to claim 4 or 5, characterized in that: The reaction parameters of the PCR amplification are: Step 1: denaturation at 95°C for 10 min; Step 2: denaturation at 95°C for 15 seconds, annealing at 58°C, and extension at 72°C for 60 seconds, for 35-40 cycles; Step 3: Extend at 72°C for 10 min and store at 4°C.

7. The method for identifying multiple fish-derived ingredients according to claim 4, wherein: The product denaturation in step c is as follows: the PCR product is denatured at 95° C. for 5 minutes and then immediately placed on ice for standby use; 20 μL of PCR reaction solution is added to 200 μL of hybridization solution to prepare a hybridization system solution; alkaline phosphatase-labeled streptavidin is added to the enzyme incubation solution at a ratio of 1:2000 to prepare an enzyme incubation system solution.

8. The method for identifying multiple fish-derived ingredients according to claim 4, wherein: The hybridization reaction in step c comprises: Deactivation: 1 mL of deactivation solution, 37°C, 8 min; Deactivation cleaning: 1 mL of deactivation cleaning solution, 60°C, 5 min; Hybridization: Add hybridization system solution, 45℃, 10min; Hybridization wash twice: add 1 mL of hybridization wash solution, incubate at 52°C for 3 min; Enzyme incubation labeling: add 1 mL of enzyme incubation system solution, 42°C, 10 min; Enzyme labeling cleaning I: add 1.1 mL of incubation cleaning solution and incubate at 42°C for 3 min. Enzyme labeling cleaning II 2 times: add 2,1mL of incubation cleaning solution, 37℃, 3min; Color development with color developing solution: add 1 mL of color developing solution and incubate at 37°C for 10 min. Color development and washing twice: add 1 mL of distilled water, 37°C, 1 min, Each of the above processes requires the corresponding liquid to be completely removed before entering the next process.

Images