Composition, kit and method for detecting Atlantic mackerels and three types of mackerels and application

Abstract

The invention discloses a composition, a kit and a method for detecting Atlantic mackerels and three types of mackerels and application, belongs to the technical field of nucleic acid detection of marine fish meat products, and particularly relates to a method for detecting components of the Atlantic mackerels and the three types of mackerels (Japanese mackerels, Australian mackerels and Keli mackerels) by using single and double real-time fluorescent PCR (Polymerase Chain Reaction). The invention also relates to a specific nucleotide primer pair and probe composition for the method, a kit containing the primer pair and the probe, and a method for detecting the fish components by using the kit. When the composition and the kit provided by the invention are used for real-time fluorescent PCR detection, rapid detection of components of Atlantic mackerel and three types of mackerel (Japanese mackerel, Australian mackerel and Keli mackerel) in food can be realized. The method has the advantages of high specificity, high sensitivity, small mass fraction error, simplicity in operation and short time consumption.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid detection of marine fish products, and specifically relates to a method for detecting components of Atlantic mackerel and three types of mackerel (Japanese mackerel, Australian mackerel, and Collie's mackerel), a composition of specific nucleotide primer pairs and probes used in the method, and a kit containing the composition. Background Art

[0002] Mackerel is a very important economic fish species worldwide. It belongs to the order Perciformes, family Scombrida, and genus Scomber. This genus includes four species of fish species, namely, Atlantic mackerel (Scomber scombrus), Japanese mackerel (Scomber japonicus), Australian mackerel (Scomber australasicus), and Collie mackerel (Scombercolias). Atlantic mackerel, also known as Norwegian mackerel, bluefish, and Norwegian mackerel, is mainly distributed in the coastal waters of the North Atlantic. It is a warm-water, pelagic migratory fish. Atlantic mackerel is rich in nutrients, rich in DHA and EPA; due to its relatively low price and high nutritional value, Atlantic mackerel is widely welcomed by consumers (Wu Jiling et al. Application of sorting method in screening of deodorization process of Atlantic mackerel. Food Research and Development. 2020, Vol. 41 (Issue 23), pp. 73-79.). Japanese mackerel, also known as Spanish mackerel, Spanish mackerel, white-bellied mackerel, Japanese mackerel, blue mackerel, etc., is widely distributed in the Pacific and Indian Oceans, with a small part distributed in the North Atlantic. Australian mackerel, also known as Australian mackerel, flower-bellied mackerel, and sesame mackerel, is widely distributed in the Pacific. Japanese mackerel and Australian mackerel are important economic fish in the northwest Pacific region. Collie's mackerel is similar in appearance to Atlantic mackerel and is a pelagic fish. It is mainly distributed in the warm waters of the Atlantic Ocean near the equator and the temperate waters of the Mediterranean Sea. It is of great significance in the Atlantic and Mediterranean fisheries. The price of Atlantic mackerel is significantly higher than that of the other three mackerels. In the European market, the average price of Atlantic mackerel is about 1.24 euros per kilogram, while the average price of Japanese mackerel, Australian mackerel, and Collie's mackerel is about 0.58 euros per kilogram (2016). These four mackerels look very similar. After being processed, peeled, or made into canned products, consumers cannot distinguish the authenticity.

[0003] Compared with common PCR technology and DNA barcode technology, the method of detecting the authenticity of fish meat based on real-time fluorescence PCR (polymerase chain reaction) technology does not require electrophoresis, purification, sequencing, comparison and uncapping of nucleic acid amplification products, and has the advantages of preventing pollution, low cost, short time consumption and simple operation. However, the method of using real-time fluorescence PCR technology to detect the components of Atlantic mackerel and the method of using a set of primers and probes to detect the components of three types of mackerel (Japanese mackerel, Australian mackerel and Collie mackerel) have not been reported. Summary of the invention

[0004] In order to solve the above practical problems, the purpose of the present invention is to use Taqman MGB probe real-time fluorescence PCR technology to simply, quickly, specifically and sensitively detect the components of Atlantic mackerel and three types of mackerel (Japanese mackerel, Australian mackerel, and Collie's mackerel).

[0005] The invention designs specific nucleotide primer pairs and probes based on the mitochondrial cytb gene of Atlantic mackerel by analyzing the genetic background of Atlantic mackerel and three mackerels (Japanese mackerel, Australian mackerel and Colley mackerel). A group of specific universal nucleotide primer pairs and probes are designed based on the common sequences of the cytb genes of Japanese mackerel, Australian mackerel and Colley mackerel, as well as the sequences that are different from the cytb genes of other species.

[0006] The present invention is realized by the following technical scheme: the composition 1 for detecting Atlantic mackerel components of the present invention comprises an Atlantic mackerel specific nucleotide primer pair: an upstream primer F: 5'-TGTTATCTTAGCGGCAGCGG-3' (SEQ ID No. 1), a downstream primer R: 5'-GGAGGTAAGGCCTATAAGGAGA-3' (SEQ ID No. 2), a specific probe P: 5'-(FAM)CAAAATCTCGTTYCATCCGT-3' (MGB) (SEQ ID No. 3), and an amplified sequence length: 163 bp. The 5' end of the specific probe is connected to a fluorescent reporter group FAM, and the 3' end is connected to a fluorescent quencher group MGB.

[0007] The composition 2 for detecting components of three kinds of mackerel (Japanese mackerel, Australian mackerel, and Collie's mackerel) of the present invention comprises Japanese mackerel, Australian mackerel, and Collie's mackerel specific universal nucleotide primer pairs: upstream primer F: 5'-GCAGTCCCATAYRTHGGHAC-3' (SEQID No.4), downstream primer R: 5'-TAGYAGGTGAAGAATTGTTGCY-3' (SEQ ID No.5), specific universal probe P: 5'-(Hex)CCACCCTYACCCGRTTCTTY-3' (MGB) (SEQ ID No.6), and amplified sequence length: 141 bp. The 5' end of the specific universal probe is connected to a fluorescent reporter group Hex, and the 3' end is connected to a fluorescent quencher group MGB.

[0008] The composition 3 of the present invention is used for simultaneously detecting components of Atlantic mackerel and three types of mackerel (Japanese mackerel, Australian mackerel, and Collie's mackerel), comprising specific nucleotide primer pairs SEQ ID No.1 and SEQ ID No.2 and specific universal nucleotide primer pairs SEQID No.4 and SEQ ID No.5, as well as specific probes SEQ ID No.3 and specific universal probes SEQ ID No.6, wherein the 5' end of the SEQID No.3 sequence is connected to a fluorescent reporter group FAM, and the 3' end is connected to a fluorescent quencher group MGB, and the 5' end of the SEQ ID No.6 sequence is connected to a fluorescent reporter group Hex, and the 3' end is connected to a fluorescent quencher group MGB.

[0009] The kit 1 for detecting Atlantic mackerel components of the present invention comprises the above-mentioned composition 1. Further, the kit 1 also comprises 18 μL of PCR amplification reaction solution, comprising the following components: 10×PCR buffer: 2 μL, 10 μmol / L dNTP: 1 μL, 5 U / μL Taq DNA polymerase: 0.5 μL, 1 μmol / L primers SEQ ID No.1 and SEQ ID No.2: 1 μL each, 1 μmol / L probe SEQ ID No.3: 0.5 μL, and ddH2O is supplemented to 18 μL.

[0010] The kit 2 for detecting components of three mackerels (Japanese mackerel, Australian mackerel, and Collie mackerel) of the present invention comprises the above-mentioned composition 2. Further, the kit 2 also comprises 18 μL of PCR amplification reaction solution, comprising the following components: 10×PCR buffer: 2 μL, 10 μmol / L dNTP: 1 μL, 5 U / μL Taq DNA polymerase: 0.5 μL, 1 μmol / L primers SEQ ID No.4 and SEQ ID No.5: 1 μL each, 1 μmol / L probe SEQ ID No.6: 0.5 μL, and ddH2O is supplemented to 18 μL.

[0011] The kit 3 for simultaneously detecting components of Atlantic mackerel and three types of mackerel (Japanese mackerel, Australian mackerel, and Collie mackerel) of the present invention comprises the above-mentioned composition 3. Further, the kit 3 also comprises 18 μL of PCR amplification reaction solution, comprising the following components: 10×PCR buffer: 2 μL, 10 μmol / L dNTP: 1 μL, 5 U / μL Taq DNA polymerase: 0.5 μL, 1 μmol / L primers SEQ ID No.1, SEQ ID No.2, SEQ ID No.4, and SEQ ID No.5: 1 μL each, 1 μmol / L probes SEQ ID No.5 and SEQ ID No.6: 0.5 μL each, and ddH2O is supplemented to 18 μL.

[0012] The kit also includes a positive control, a negative control and a blank control. The positive control is an Atlantic mackerel genome extract and a Japanese mackerel genome extract (or a cloning vector with the Australian mackerel cytb gene, a cloning vector with the Collie mackerel cytb gene), the negative control is a genome extract of other species with the same volume but not containing Atlantic mackerel, Japanese mackerel, Australian mackerel and Collie mackerel components, and the blank control is a template DNA replaced by ddH2O with the same volume.

[0013] The method for testing the composition of Atlantic mackerel and three types of mackerel (Japanese mackerel, Australian mackerel, and Collie's mackerel) includes the following steps.

[0014] (1) DNA extraction from samples: Take about 10 mg (9.90-10.10 mg) of sample that has been dried at 105 °C and ground into dry powder using a blender. Follow the steps of a commercial DNA extraction kit and dissolve it in 100 μL TAE Buffer or ddH2O.

[0015] (2) Real-time fluorescence PCR system: Real-time fluorescence PCR system for detecting the components of Atlantic mackerel: add 2 μL of the extracted sample DNA to kit 1; real-time fluorescence PCR system for detecting the components of three types of mackerel (Japanese mackerel, Australian mackerel, and Collie's mackerel): add 2 μL of the extracted sample DNA to kit 2; real-time fluorescence PCR system for simultaneously detecting the components of Atlantic mackerel and three types of mackerel (Japanese mackerel, Australian mackerel, and Collie's mackerel): add 2 μL of the extracted sample DNA to kit 3.

[0016] (3) PCR reaction parameters: pre-denaturation at 95 °C for 30 s; 95 °C for 15 s, 58 °C for 30 s, 40 cycles.

[0017] When detecting the components of Atlantic mackerel, the FAM channel of the real-time fluorescence PCR instrument needs to be opened. When detecting the components of three types of mackerel (Japanese mackerel, Australian mackerel, and Collie mackerel), the Hex channel of the real-time fluorescence PCR instrument needs to be opened. When detecting the components of Atlantic mackerel and three types of mackerel (Japanese mackerel, Australian mackerel, and Collie mackerel) at the same time, both the FAM channel and the Hex channel of the real-time fluorescence PCR instrument need to be opened.

[0018] The quality control standard for judging the amplification results is: if one of the following conditions is not met, the experiment is considered invalid: blank control: no fluorescence curve, and Ct value>38 or no value; negative control: no fluorescence curve, and Ct value>38 or no value; positive control: fluorescence curve appears, and Ct value<30.

[0019] Result determination: When the Ct value is ≤38, it indicates that the sample is positive; when the Ct value is >38 or no value is found, it indicates that the sample is negative.

[0020] In the real-time fluorescence PCR detection method described above, the sample DNA preparation method can not only use a commercially available DNA extraction kit, but also use the CTAB method, alkaline lysis method, etc.

[0021] When detecting Atlantic mackerel components, the sensitivity of Atlantic mackerel genomic DNA can reach 10 -2 ng, the mass fraction sensitivity can reach 1%; when detecting the components of three mackerel (Japanese mackerel, Australian mackerel, Collie mackerel), the genomic DNA sensitivity of three mackerel (Japanese mackerel, Australian mackerel, Collie mackerel) can reach 10 -2 ng, and the mass fraction sensitivity can reach 0.01%; when the components of Atlantic mackerel and three kinds of mackerel (Japanese mackerel, Australian mackerel, and Collie's mackerel) are detected simultaneously, the genomic DNA sensitivity of Atlantic mackerel and three kinds of mackerel (Japanese mackerel, Australian mackerel, and Collie's mackerel) can reach 10 -2 ng, when Atlantic mackerel and Japanese mackerel (or Australian mackerel, Colley's mackerel) were mixed at different mass fractions, the mass fraction sensitivity of Atlantic mackerel was 90%, and the mass fraction sensitivity of Japanese mackerel (or Australian mackerel, Colley's mackerel) was 1%; when Atlantic mackerel and Japanese mackerel (or Australian mackerel, Colley's mackerel) were mixed at the same mass fraction, Atlantic mackerel could not be detected, and the mass fraction sensitivity of Japanese mackerel (or Australian mackerel, Colley's mackerel) was 0.01%.

[0022] (4) Quantitative detection of Atlantic mackerel mass fraction: Atlantic mackerel and other fish samples were placed in an electric constant temperature forced air drying oven, dried at 105°C, and ground into dry powder using a blender. The sample to be tested and other fish dry powder were mixed in different proportions according to dry weight, and then placed in a blender for thorough grinding and mixing to prepare dry powders with mass fractions of 100%, 80%, 60%, 40%, 20%, 10%, 1%, 0.1%, 0.01%, and 0.001%, respectively. About 10 mg (9.90-10.10 mg) of the sample was weighed using a 1 / 10,000 electronic balance. 2 μL of the extracted DNA was added to Kit 1 and tested according to the single-plex PCR reaction procedure for detecting Atlantic mackerel components. Four replicates were performed for each concentration. The logarithm of the mass of the dry powder of the sample to be tested was used as the horizontal axis, and the corresponding average Ct value was used as the vertical axis to draw a standard curve. The correlation coefficient R 2 It must be greater than 0.9, otherwise the standard curve needs to be redone.

[0023] Take the sample to be tested and perform the single-plex real-time fluorescence PCR detection method (1) to (3) for detecting Atlantic mackerel components. Perform 4 parallel replicates for each sample. Substitute the average Ct value of the sample to be tested into the standard curve to obtain the mass of the sample to be tested and further calculate the mass score.

[0024] (5) Quantitative detection of mass fraction of Japanese mackerel, Australian mackerel and Collie's mackerel: Take Japanese mackerel (or Australian mackerel, Collie's mackerel) and other fish samples, place them in an electric constant temperature forced air drying oven, dry them at 105°C, grind them into dry powder with a blender, mix the sample to be tested and other fish dry powders in different proportions according to dry weight, and then mix them in a blender to make dry powders with mass fractions of 100%, 80%, 60%, 40%, 20%, 10%, 1%, 0.1%, 0.01% and 0.001%, respectively. Use a 1 / 10,000 electronic balance to weigh about 10 mg (9.90-10.10 mg) of the sample, add 2 μL of the extracted DNA into Kit 2, and detect the components of the three mackerel (Japanese mackerel, Australian mackerel and Collie's mackerel) according to the single-plex PCR reaction procedure. Perform four parallel replicates for each concentration. Use the logarithm of the mass of the dry powder of the sample to be tested as the horizontal axis and the corresponding average Ct value as the vertical axis to draw a standard curve. The correlation coefficient R 2 It must be greater than 0.9, otherwise the standard curve needs to be redone.

[0025] Take the sample to be tested and perform the single-plex real-time fluorescence PCR detection method (1) to (3) for detecting the components of three types of mackerel (Japanese mackerel, Australian mackerel, and Collie mackerel). Make 4 parallel replicates for each sample, and bring the average Ct value of the sample to be tested into the standard curve to obtain the mass of the sample to be tested and further calculate the mass score.

[0026] The present invention has the following beneficial effects: (1) The present invention establishes a method for rapidly detecting the components of Atlantic mackerel and three types of mackerel (Japanese mackerel, Australian mackerel, and Collie's mackerel) by using single and double real-time fluorescence PCR technology, which has strong specificity and high sensitivity, and can meet the identification requirements of using three types of mackerel (Japanese mackerel, Australian mackerel, and Collie's mackerel) with lower prices to impersonate Atlantic mackerel with higher prices. (2) The present invention adopts real-time fluorescence PCR technology, and the nucleic acid amplification product can be directly monitored by fluorescence signal, thereby realizing the rapid detection of the components of Atlantic mackerel and three types of mackerel (Japanese mackerel, Australian mackerel, and Collie's mackerel). Compared with ordinary PCR technology and DNA barcode technology, there is no need to electrophoresing, purify, sequence, and compare the nucleic acid amplification product, and it has the advantages of preventing pollution, low cost, short time consumption, simple operation, etc. The reaction only takes 1 hour, saving time and cost; the double real-time fluorescence PCR can simultaneously detect the components of Atlantic mackerel and three types of mackerel (Japanese mackerel, Australian mackerel, and Collie's mackerel) with only one reaction, and the components of three types of mackerel (Japanese mackerel, Australian mackerel, and Collie's mackerel) with lower prices can be detected by using a set of primers and probes, which is simple and efficient to operate. (3) The present invention analyzes Atlantic mackerel and Japanese mackerel samples with different mass fractions, and the logarithm of the mass and the average Ct value are linearly correlated, with a correlation coefficient R 2 The Ct values ​​were all greater than 0.99, the mass fraction error of Atlantic mackerel was less than or equal to ±5.9%, and the mass fraction error of Japanese mackerel was less than or equal to ±6.6%, indicating that the Ct value can be used to determine whether other meats or impurities are adulterated in the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the specific test curve for Atlantic mackerel; Figure 1 The numbers in the table represent: 1. Atlantic mackerel; 2. Japanese mackerel; 3. cloning vector pUC57 carrying cytb gene of Australian mackerel; 4. cloning vector pUC57 carrying cytb gene of Collie mackerel; 5. Atlantic salmon; 6. Chinook salmon; 7. Coho salmon; 8. Sockeye salmon; 9. Humpback salmon; 10. Rainbow trout; 11. Atlantic cod; 12. Sablefish; 13. Pacific cod; 14. Pangasius; 15. Chinese sturgeon; 16. Nile tilapia; 17. Atlantic bluefin tuna; 18. Bigeye tuna; 19. Bonito; 20. Chinese Spanish mackerel; 21. Blue-spot Spanish mackerel; 22. Large yellow croaker; 23. Small yellow croaker; 24. Crucian carp; 25. Grass carp; 26. Blank control.

[0028] Figure 2 The specific test curves are for three types of mackerel (Japanese mackerel, Australian mackerel, and Collie mackerel); Figure 2The numbers in the table represent: 1. Atlantic mackerel; 2. Japanese mackerel; 3. cloning vector pUC57 carrying cytb gene of Australian mackerel; 4. cloning vector pUC57 carrying cytb gene of Collie mackerel; 5. Atlantic salmon; 6. Chinook salmon; 7. Coho salmon; 8. Sockeye salmon; 9. Humpback salmon; 10. Rainbow trout; 11. Atlantic cod; 12. Sablefish; 13. Pacific cod; 14. Pangasius; 15. Chinese sturgeon; 16. Nile tilapia; 17. Atlantic bluefin tuna; 18. Bigeye tuna; 19. Bonito; 20. Chinese Spanish mackerel; 21. Blue-spot Spanish mackerel; 22. Large yellow croaker; 23. Small yellow croaker; 24. Crucian carp; 25. Grass carp; 26. Blank control.

[0029] Figure 3 This is the sensitivity test curve of Atlantic mackerel real-time fluorescence PCR genome.

[0030] Figure 4 This is the sensitivity test curve of the Japanese mackerel real-time fluorescence PCR genome.

[0031] Figure 5 is the fluorescence curve of Atlantic mackerel mass fraction; Figure 5 The numbers in the table represent the mass of Atlantic mackerel dry powder; 1: 10.03 mg; 2: 7.96 mg; 3: 6.05 mg; 4: 4.06 mg; 5: 2.014 mg; 6: 1.005 mg; 7: 0.1002 mg; 8: 0.00998 mg.

[0032] Figure 6 This is the standard curve of Atlantic mackerel mass fraction.

[0033] Figure 7 Quantitative experimental curve for mass fraction of Atlantic mackerel; Figure 7 The numbers in the figure represent: the average Ct value of the samples to be tested; 1: 27.7625; 2: 28.2625; 3: 28.8875; 4: 29.3825; 5: 29.8175; 6: 30.825; 7: 31.9025; 8: 36.3325; 9: no numerical value for Ct value.

[0034] Figure 8 is the fluorescence curve of Japanese mackerel mass fraction; Figure 8 The numbers in the table represent the mass of Japanese mackerel dry powder; 1: 10.05 mg; 2: 8.04 mg; 3: 6.006 mg; 4: 3.996 mg; 5: 2.008 mg; 6: 1.006 mg; 7: 0.0995 mg; 8: 0.00997 mg; 9: 0.001003 mg; 10: 0.0001004 mg.

[0035] Fig. 9 This is the standard curve of the mass fraction of Japanese mackerel.

[0036] Fig.10 This is the quantitative experimental curve of the mass fraction of Japanese mackerel; Fig.10 The numbers in the figure represent: the average Ct value of the samples to be tested; 1: 21.47; 2: 21.6275; 3: 22.075; 4: 22.51; 5: 22.6325; 6: 24.575; 7: 25.075; 8: 28.94; 9: 32.4175; 10: 35.5025; 11: no numerical value for the Ct value.

[0037] Fig.11 It is the specific test curve of dual real-time fluorescence PCR; Fig.11 The numbers in the table represent: 1. Atlantic mackerel; 2. Japanese mackerel; 3. cloning vector pUC57 carrying cytb gene of Australian mackerel; 4. cloning vector pUC57 carrying cytb gene of Collie mackerel; 5. Atlantic salmon; 6. Chinook salmon; 7. Coho salmon; 8. Sockeye salmon; 9. Humpback salmon; 10. Rainbow trout; 11. Atlantic cod; 12. Sablefish; 13. Pacific cod; 14. Pangasius; 15. Chinese sturgeon; 16. Nile tilapia; 17. Atlantic bluefin tuna; 18. Bigeye tuna; 19. Bonito; 20. Chinese Spanish mackerel; 21. Blue-spot Spanish mackerel; 22. Large yellow croaker; 23. Small yellow croaker; 24. Crucian carp; 25. Grass carp; 26. Blank control.

[0038] Fig.12 This is the double real-time fluorescence PCR quality score sensitivity test curve 1; Fig.12 The numbers in the figure represent the mass fractions of Atlantic mackerel and Japanese mackerel respectively; 1.99%: 1%; 2. 90%: 10%; 3. 80%: 20%; 4. 60%: 40%; 5. 50%: 50%.

[0039] Fig.13 This is the dual real-time fluorescence PCR quality score sensitivity test curve 2; Fig.13 The numbers in the figure represent the mass fractions of Atlantic mackerel and Japanese mackerel respectively; 6.40%: 60%; 7.20%: 80%; 8.10%: 90%; 9.1%: 99%; 10.0.1%: 99.9%.

[0040] Fig.14 This is the dual real-time fluorescence PCR quality score sensitivity test curve 3; Fig.14The numbers in the figure represent the mass fractions of Atlantic mackerel and Japanese mackerel respectively; 11. 20%: 20%; 12. 10%: 10%; 13. 1%: 1%; 14. 0.1%: 0.1%; 15. 0.01%: 0.01%; 16. 0.001%: 0.001%. DETAILED DESCRIPTION

[0041] The present invention is further described with reference to the embodiments.

[0042] Example 1 Atlantic mackerel specificity test: Using the composition 1, kit 1 and methods (1), (2) and (3) for detecting Atlantic mackerel components described in the specification, genomic DNA of Atlantic mackerel, Japanese mackerel, Atlantic salmon, Chinook salmon, Coho salmon, Sockeye salmon, Humpback salmon, Rainbow trout, Atlantic cod, Sablefish, Pacific cod, Pangasius, Chinese sturgeon, Nile tilapia, Atlantic bluefin tuna, bigeye tuna, bonito, Chinese Spanish mackerel, bluespot Spanish mackerel, large yellow croaker, small yellow croaker, crucian carp and grass carp, and cloning vector pUC57 carrying the Australian mackerel cytb gene and cloning vector pUC57 carrying the Collie mackerel cytb gene were used as templates, and the DNA concentration of the above samples was determined using an ultra-micro spectrophotometer. The DNA concentration was diluted to 5 ng / μL, and pure water was used as a template as a blank control to perform real-time fluorescence PCR to verify the specificity of the primers and probes used to detect Atlantic mackerel components.

[0043] Test results such as Figure 1 As shown, the Atlantic mackerel genomic DNA showed a fluorescence curve with a Ct value of 21.64, while no fluorescence signals were detected in other samples and blank controls, indicating that the primers and probes used to detect Atlantic mackerel components have good specificity.

[0044] Example 2 Specificity test for three types of mackerel (Japanese mackerel, Australian mackerel, and Collie's mackerel): Composition 2, kit 2, and methods (1), (2), and (3) for detecting components of three types of mackerel (Japanese mackerel, Australian mackerel, and Collie's mackerel) described in the specification were used, respectively, with genomic DNA of Atlantic mackerel, Japanese mackerel, Atlantic salmon, Chinook salmon, Coho salmon, Red salmon, Humpback salmon, Rainbow trout, Atlantic cod, Sablefish, Pacific cod, Pangasius, Chinese sturgeon, Nile tilapia, Atlantic bluefin tuna, Bigeye tuna, Bonito, Chinese Spanish mackerel, Blue-spot Spanish mackerel, Large yellow croaker, Small yellow croaker, Crucian carp, and Grass carp, as well as cloning vector pUC57 carrying the Australian mackerel cytb gene and cloning vector pUC57 carrying the Collie's mackerel cytb gene as templates, and the DNA concentration of the above samples was measured using an ultra-micro spectrophotometer, and the DNA concentration was diluted to 5 ng / μL, and pure water was used as the template as the blank control, and real-time fluorescence PCR was performed to verify the specificity of the primers and probes used to detect the components of three mackerel species (Japanese mackerel, Australian mackerel, and Collie's mackerel).

[0045] Test results such as Figure 2 As shown, Japanese mackerel genomic DNA, cloning vector pUC57 carrying Australian mackerel cytb gene, and cloning vector pUC57 carrying Colley mackerel cytb gene all showed fluorescence curves, and the Ct values ​​were 21.53, 7.54, and 6.17, respectively. No fluorescence signals were detected in other samples and blank controls, indicating that the primers and probes used to detect the components of the three mackerels (Japanese mackerel, Australian mackerel, and Colley mackerel) have good specificity.

[0046] Example 3 Real-time fluorescence PCR genome sensitivity test: Using the composition 1, kit 1 and methods (1), (2) and (3) for detecting Atlantic mackerel components described in the specification, the concentration of Atlantic mackerel DNA was determined using an ultra-micro spectrophotometer, and the DNA was diluted 10 times with ultrapure water. The DNA template amount was selected to be 50 ng / μL, 5 ng / μL, 5×10 -1 ng / μL, 5×10 -2 ng / μL, 5×10 -3 ng / μL, 5×10 -4 ng / μL, 5×10 -5 There were 7 dilution levels (ng / μL) and three replicates for each concentration for real-time fluorescence PCR detection.

[0047] The composition 2, kit 2 and methods (1), (2) and (3) for detecting components of three mackerels (Japanese mackerel, Australian mackerel and Collie mackerel) described in the manual were used to determine the concentration of Japanese mackerel DNA using an ultra-micro spectrophotometer. The DNA was diluted 10 times with ultrapure water, and the DNA template amounts were selected to be 50 ng / μL, 5 ng / μL, 5×10-1 ng / μL, 5×10 -2 ng / μL, 5×10 -3 ng / μL, 5×10 -4 ng / μL, 5×10 -5 There were 7 dilution levels (ng / μL) and three replicates for each concentration for real-time fluorescence PCR.

[0048] The Ct values ​​of each dilution level are shown in Table 1. Figure 3 , Figure 4 As shown, when the DNA amount of Atlantic mackerel is ≥10 -2 ng, and the Ct values ​​were all less than 38. The logarithmic value of the DNA amount was linearly correlated with the average Ct value, and the correlation coefficient was R 2 =0.9986, the standard curve was y=-4.417x+26.096, and the amplification effect was good; the standard deviation of the Ct value of each concentration was between 0.024 and 0.34, and the relative standard deviation was between 0.092 and 0.98, which had good repeatability.

[0049] When the DNA amount of Japanese mackerel is ≥10 -2 ng, and the Ct values ​​were all less than 38. The logarithmic value of the DNA amount was linearly correlated with the average Ct value, and the correlation coefficient was R 2 =0.9988, the standard curve is y=-3.997x+25.172, the amplification effect is good; the standard deviation of the Ct value of each concentration is between 0.068~0.10, and the relative standard deviation is between 0.26~0.99, which has good repeatability. The amount of DNA is 10 -3 When ng was 0.5, only one fluorescence curve appeared, and the Ct value was 37.20, indicating that the repeatability of this concentration did not meet the amplification requirements.

[0050] Table 1 Real-time fluorescence PCR sensitivity test .

[0051] Example 4 Quantitative detection of mass fraction of Atlantic mackerel: Take Atlantic mackerel and Nile tilapia samples, place them in an electric constant temperature blast drying oven, dry them at 105 ° C, and grind them into dry powder in a blender. Mix the Atlantic mackerel and Nile tilapia dry powders in different proportions according to the dry weight, and then place them in a blender to fully grind and mix. Use an electronic balance to weigh about 10 mg (9.90-10.10 mg) of the sample. The weighed mass, mass fraction of Atlantic mackerel dry powder and mass of Atlantic mackerel dry powder are (10.03 mg, 100%, 10.03 mg), (10.02 mg, 79.4%, 7.96 mg), (10.01 mg, 60.4%, 6.05 mg), (10.02 mg, 40.5%, 4.06 mg), (10.07 mg, 20%, 2.014 mg), (10.05 mg, 10%, 1.005 mg), (10.02 According to the composition 1, kit 1 and methods (1), (2) and (3) for detecting Atlantic mackerel components, four parallel replicates were performed for each concentration; the logarithm of the mass of Atlantic mackerel dry powder was used as the horizontal axis and the average Ct value was used as the vertical axis to make a standard curve.

[0052] When the mass fraction of Atlantic mackerel was ≥1%, fluorescence curves appeared and the Ct values ​​were all less than 38. The Ct values ​​of Atlantic mackerel powder used for the standard curve are shown in Table 2 and Figure 5 The standard deviation of Ct values ​​at each concentration was between 0.064 and 0.20, and the relative standard deviation was between 0.23 and 0.62, which showed good repeatability.

[0053] Table 2 Standard curve of mass fraction of Atlantic mackerel .

[0054] The logarithmic values ​​of the mass of Atlantic mackerel dry powder are 10.03 mg, 7.96 mg, 6.05 mg, 4.06 mg, 2.014 mg, 1.005 mg, and 0.1002 mg as the abscissa and the corresponding average Ct value as the ordinate to make a standard curve. The standard curve is y=-4.2431x+32.123, R²=0.9963, such as Figure 6 shown.

[0055] Take about 10 mg (9.90-10.10 mg) of Atlantic mackerel dry powder of different mass fractions, and perform four parallel replicates for each concentration according to the composition 1, kit 1 and methods (1), (2) and (3) for detecting Atlantic mackerel components; the average Ct value of the sample to be tested ( Figure 7 ) was substituted into the formula y = -4.2431x + 32.123 to obtain the mass and mass fraction of Atlantic mackerel in the measured samples, which were compared with the actual mass fractions. As shown in Table 3, all errors were ≤ ± 5.9%.

[0056] Table 3 Quantitative test of mass fraction of Atlantic mackerel .

[0057] Example 5 Quantitative detection of the mass fraction of Japanese mackerel: Take Japanese mackerel and Nile tilapia samples, place them in an electric constant temperature blast drying oven, dry them at 105 ° C, and grind them into dry powder in a blender. The Japanese mackerel and Nile tilapia dry powders are mixed in different proportions according to the dry weight, and then placed in a blender for thorough grinding and mixing. Use an electronic balance to weigh about 10 mg (9.90-10.10 mg) of the sample. The weighed mass, the mass fraction of the Japanese mackerel dry powder and the mass of the Japanese mackerel dry powder are (10.05 mg, 100%, 10.05 mg), (10.05 mg, 80%, 8.04 mg), (10.01 mg, 60%, 6.006 mg), (9.99 mg, 40%, 3.996 mg), (10.04 mg, 20%, 2.008 mg), (10.06 mg, 10%, 1.006 mg), (9.95 mg, 1%, 0.0995 mg), (9.97 mg, 0.1%, 0.00997 mg), (10.03 mg, 0.01%, 0.001003 mg), (10.04 mg, 0.001%, 0.0001004 mg), according to the composition 2, kit 2 and method (1), (2), (3) for detecting the components of three mackerels (Japanese mackerel, Australian mackerel, Collie mackerel), four parallel replicates were performed for each concentration; the logarithm of the mass of Japanese mackerel dry powder was used as the horizontal axis and the average Ct value was used as the vertical axis to make a standard curve.

[0058] When the mass fraction of Japanese mackerel was ≥0.01%, fluorescence curves appeared and the Ct values ​​were all less than 38. The Ct values ​​of Japanese mackerel powder used for the standard curve are shown in Table 4 and Figure 8 The standard deviation of Ct values ​​at each concentration was between 0.050 and 0.20, and the relative standard deviation was between 0.23 and 0.65, showing good repeatability.

[0059] Table 4 Standard curve of mass fraction of Japanese mackerel .

[0060] The logarithmic values ​​of the masses of Japanese mackerel powder are 10.05 mg, 8.04 mg, 6.0006 mg, 3.996 mg, 2.008 mg, 1.006 mg, 0.0995 mg, 0.00997 mg, and 0.001003 mg as the abscissa, and the corresponding average Ct value is used as the ordinate to make a standard curve. The standard curve is y=-3.6452x+25.053, R²=0.9993, such as Fig. 9 shown.

[0061] About 10 mg (9.90-10.10 mg) of dried powder of Japanese mackerel with different mass fractions were taken, and four parallel replicates were made for each concentration according to the composition 2, kit 2 and methods (1), (2) and (3) for detecting the components of three mackerels (Japanese mackerel, Australian mackerel and Collie mackerel). The average Ct value ( Fig.10 ) was substituted into the formula y = -3.6452x + 25.053 to obtain the mass and mass fraction of Japanese mackerel in the measured samples, which were compared with the actual mass fractions. As shown in Table 5, all errors were ≤ ± 6.6%.

[0062] Table 5 Quantitative test of mass fraction of Japanese mackerel .

[0063] Example 6 Dual real-time fluorescence PCR specificity test: Using the composition 3, kit 3 and methods (1), (2) and (3) described in the specification for simultaneously detecting components of Atlantic mackerel and three types of mackerel (Japanese mackerel, Australian mackerel and Collie's mackerel), the DNA of Atlantic mackerel, Japanese mackerel, Atlantic salmon, Chinook salmon, Coho salmon, Red salmon, Humpback salmon, Rainbow trout, Atlantic cod, Sablefish, Pacific cod, Pangasius, Chinese sturgeon, Nile tilapia, Atlantic bluefin tuna, Bigeye tuna, Bonito, Chinese Spanish mackerel, Blue-spot Spanish mackerel, Large yellow croaker, Small yellow croaker, Crucian carp and Grass carp, and the cloning vector pUC57 carrying the Australian mackerel cytb gene and the cloning vector pUC57 carrying the Collie's mackerel cytb gene were used as templates, and the DNA concentration of the above samples was measured using an ultra-micro spectrophotometer. The DNA concentration was diluted to 5. ng / μL, and pure water was used as the template as the blank control, and real-time fluorescence PCR was performed to verify the specificity of primers and probes for simultaneous detection of components of Atlantic mackerel and three types of mackerel (Japanese mackerel, Australian mackerel, and Collie's mackerel).

[0064] Test results such as Fig.11As shown, the Atlantic mackerel genomic DNA showed a FAM fluorescence curve with a Ct value of 22.17, the Japanese mackerel genomic DNA, the cloning vector pUC57 carrying the Australian mackerel cytb gene, and the cloning vector pUC57 carrying the Collie cytb gene all showed Hex fluorescence curves with Ct values ​​of 22.96, 7.97, and 7.49, respectively, and the other samples and blank controls had no FAM and Hex fluorescence curves and no Ct values, indicating that the primers and probes used to simultaneously detect the components of Atlantic mackerel and three mackerels (Japanese mackerel, Australian mackerel, and Collie mackerel) had good specificity.

[0065] Example 7 Dual real-time fluorescence PCR genome sensitivity test: Using the composition 3, kit 3 and methods (1), (2) and (3) described in the specification for simultaneously detecting components of Atlantic mackerel and three types of mackerel (Japanese mackerel, Australian mackerel and Collie mackerel), the concentrations of DNA were 50 ng / μL, 5 ng / μL and 5×10 -1 ng / μL, 5×10 -2 ng / μL, 5×10 -3 ng / μL, 5×10 -4 ng / μL of Atlantic mackerel and Japanese mackerel, Atlantic mackerel and Japanese mackerel with different DNA concentrations were mixed separately, and three parallel replicates were made for each concentration for dual real-time fluorescence PCR sensitivity detection.

[0066] The average Ct values ​​of different DNA amounts of Atlantic mackerel and Japanese mackerel are shown in Table 6. 2 ng, the detection limit of Japanese mackerel was 10 ng, and all Atlantic mackerel were detected; when the DNA amount of Atlantic mackerel was 10 ng, the detection limit for Japanese mackerel was 1ng, and all Atlantic mackerel were detected; when the amount of Atlantic mackerel DNA was 1 ng, the detection limit for Japanese mackerel is 1 ng, and when the amount of Japanese mackerel DNA is ≤10ng, Atlantic mackerel can be detected; when the amount of Atlantic mackerel DNA is 10 -1 ng, the detection limit for Japanese mackerel is 10 -1 ng, Japanese mackerel DNA ≤ 10 -1 ng, Atlantic mackerel can be detected; when the amount of Atlantic mackerel DNA is 10 -2 ng, the detection limit for Japanese mackerel is 10 -2 ng, Japanese mackerel DNA ≤ 10 -3 ng, Atlantic mackerel can be detected; when the amount of Atlantic mackerel DNA is 10 -3 ng, the detection limit for Japanese mackerel is 10 -2 ng, and no detection was found in Atlantic mackerel.

[0067] Table 6 Average Ct values ​​of dual real-time fluorescence PCR sensitivity test .

[0068] Example 8 Dual real-time fluorescence PCR mass fraction sensitivity test: Atlantic mackerel and Japanese mackerel dry powders were mixed at different mass fractions, and four parallel replicates were performed for each concentration according to the composition 3, kit 3 and methods (1), (2) and (3) for simultaneously detecting components of Atlantic mackerel and three types of mackerel (Japanese mackerel, Australian mackerel and Collie mackerel). The Ct values ​​of the mixtures of Atlantic mackerel and Japanese mackerel dry powders at different mass fractions are shown in Tables 7 and Fig.12 , Fig.13 , Fig.14 As shown; when Atlantic mackerel and Japanese mackerel were mixed at different mass fractions, the mass fraction detection limit of Atlantic mackerel was 90%, and the mass fraction detection limit of Japanese mackerel was 1%; when Atlantic mackerel and Japanese mackerel were mixed at the same mass fraction, Atlantic mackerel could not be detected, and the mass fraction detection limit of Japanese mackerel was 0.01%.

[0069] Table 7 Dual real-time fluorescence PCR quality score sensitivity test .

[0070] Example 9 Application of detection of commercially available mackerel products: 10 portions of frozen Norwegian mackerel fillets, 10 portions of frozen Japanese mackerel fillets, 6 portions of canned mackerel, 1 portion of canned mackerel, and 1 portion of grilled mackerel were purchased, and detection was carried out according to the composition 3 for simultaneously detecting the components of Atlantic mackerel and three types of mackerel (Japanese mackerel, Australian mackerel, and Collie mackerel), the kit 3, and the methods (1), (2), and (3); the results are shown in Table 8. Atlantic mackerel components were detected in ten portions of frozen Norwegian mackerel fillets, one portion of canned mackerel, and one portion of grilled mackerel, while Japanese mackerel, Australian mackerel, and Collie mackerel components were not detected; Japanese mackerel, Australian mackerel, and Collie mackerel components were detected in ten portions of frozen Japanese mackerel fillets and six portions of canned mackerel, while Atlantic mackerel components were not detected; no adulteration of common mackerel products was found; the method established by the present invention can be used for the detection of components derived from commercially available mackerel products.

[0071] Table 8 Testing of commercial mackerel products .

Claims

1. A composition 1 for detecting Atlantic mackerel components, characterized in that: It comprises Atlantic mackerel-specific nucleotide primer pair SEQ ID No. 1 and SEQ ID No. 2 and a specific probe SEQ ID No. 3, wherein the 5' end of the specific probe is connected to a fluorescent reporter group FAM, and the 3' end is connected to a fluorescent quencher group MGB.

2. A composition 2 for detecting components of Japanese mackerel, Australian mackerel and Collie mackerel, characterized in that: The invention comprises a pair of universal nucleotide primers specific for Japanese mackerel, Australian mackerel and Colley mackerel, SEQ ID No. 4 and SEQ ID No. 5, and a specific universal probe sequence, SEQ ID No. 6, wherein the 5' end of the specific universal probe is connected to a fluorescent reporter group Hex, and the 3' end is connected to a fluorescent quencher group MGB; the Japanese mackerel component is taken from Japanese mackerel, the Australian mackerel component is taken from a cloning vector carrying the Australian mackerel cytb gene, and the Colley mackerel component is taken from a cloning vector carrying the Colley mackerel cytb gene.

3. A composition 3 for simultaneously detecting components of Atlantic mackerel, Japanese mackerel, Australian mackerel and Collie mackerel, characterized in that: It comprises specific nucleotide primer pairs SEQ ID No.1 and SEQ ID No.2, specific universal nucleotide primer pairs SEQ ID No.4 and SEQ ID No.5, specific probe SEQ ID No.3 and specific universal probe SEQ ID No.6, wherein the 5' end of the SEQ ID No.3 sequence is connected to a fluorescent reporter group FAM, and the 3' end is connected to a fluorescent quencher group MGB, and the 5' end of the SEQ ID No.6 sequence is connected to a fluorescent reporter group Hex, and the 3' end is connected to a fluorescent quencher group MGB.

4. A kit 1 for detecting components of Atlantic mackerel, characterized in that: The kit comprises the composition 1 described in claim 1 and a PCR amplification reaction solution.

5. A kit 2 for detecting components of Japanese mackerel, Australian mackerel and Collie mackerel, characterized in that: The kit comprises the composition 2 described in claim 2 and a PCR amplification reaction solution.

6. A kit 3 for simultaneously detecting components of Atlantic mackerel, Japanese mackerel, Australian mackerel and Collie mackerel, characterized in that: The kit comprises the composition 3 described in claim 3 and a PCR amplification reaction solution.

7. A method for detecting components of Atlantic mackerel, Japanese mackerel, Australian mackerel and Collie mackerel, characterized in that: The method comprises using the composition according to any one of claims 1 to 3 and the kit according to any one of claims 4 to 6.

8. The method according to claim 7, characterized in that It also includes the extraction of sample DNA as well as the real-time fluorescence PCR system and PCR reaction parameters.

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