Composition, kit and method for detecting mitochondrial gene of sea eel

Abstract

The invention discloses a composition, a kit and a method for detecting a mitochondrial gene of sea eel. A primer probe composition targeting the D-LOOP gene of the sea eel mitochondria is designed, and the nucleotide sequences of the primer probe composition correspond to SEQ ID No.1-3. On the basis, the invention further constructs and optimizes a specific real-time fluorescent PCR detection method for the D-LOOP gene of the sea eel mitochondria. According to the method, a part of sequence of the D-LOOP gene of the mitochondria of the sea eel is taken as a target region, a primer and a TaqMan-MGB probe are designed in a targeted manner, and finally a set of rapid, sensitive and high-specificity detection system is established. The detection system can be effectively applied to adulteration and germplasm identification work of the sea eel, and has a very wide industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid detection technology for marine fish products, specifically relating to a method for rapid detection of eel components using real-time fluorescence PCR technology, a composition for the method, and a kit containing the composition. Background Technology

[0002] The conger eel (Muraenesox cinereus) belongs to the order Anguilliformes, family Anguillidae, and genus Muraenesox. It is a warm-water, near-bottom-dwelling fish. Its geographical distribution covers the Red Sea in East Africa, the northern Indian Ocean, and the coastal areas of Myanmar and Malaysia in the Northwest Pacific. In my country, the East China Sea is its main production area. This fish has thick, delicate, and delicious flesh with a high fat content and excellent gel-forming ability, making it an important raw material for producing high-end surimi products.

[0003] In recent years, the fish paste market has been plagued by problems such as adulteration and counterfeiting. For example, some so-called "seafood balls" are made solely from ordinary fish paste and artificial flavorings, containing almost no seafood. Many imitation seafood products are also made from low-quality fish paste mixed with flour, seriously harming consumers' rights. Meanwhile, with the continued growth in international demand for conger eels, the scale of conger eel farming in coastal areas of Fujian and Zhejiang provinces in my country has expanded rapidly. However, different eel species are quite similar in appearance, especially in the juvenile stage, making accurate differentiation by appearance alone difficult. To avoid the problems of adulteration or genetic confusion in conger eel products, establishing a molecular marker-based conger eel species identification technology is particularly necessary.

[0004] Real-time fluorescence PCR technology can be used to identify the authenticity of fish meat, allowing direct detection of target components through fluorescence signal analysis. Compared to traditional PCR and DNA barcoding techniques, this method eliminates the need for electrophoresis, purification, sequencing, and sequence alignment of amplified products, while also avoiding the need for opening the container. This offers significant advantages in reducing contamination risks, lowering detection costs, shortening experimental time, and simplifying procedures. Currently, no research has been reported on the use of real-time fluorescence PCR technology for detecting components in eels. Summary of the Invention

[0005] To address the aforementioned practical problems, this invention, based on the molecular genetic characteristics of moray eels, designs a specific primer and probe combination by analyzing their mitochondrial D-LOOP gene. This technical solution utilizes real-time fluorescence PCR technology to establish an efficient and specific method for detecting moray eel components.

[0006] This invention is achieved through the following technical solution: The composition for detecting components in moray eels comprises a moray eel-specific nucleotide primer pair: upstream primer F: 5'-CGACGACATGGATAGATCTCCG-3' (SEQ ID No. 1), downstream primer R: 5'-CGTTCGCGACGATTCATTGG-3' (SEQ ID No. 2), and specific probe P: 5'-(FAM)AGAGTGGATACTAGCACGCAGA-3'(MGB) (SEQ ID No. 3), with an amplified sequence length of 140 bp. The specific probe has a fluorescent reporter group FAM attached to its 5' end and a fluorescent quencher group MGB attached to its 3' end.

[0007] The present invention provides a kit for detecting components in moray eels, comprising the above-mentioned composition. Further, the kit also includes 19 μL of real-time fluorescence PCR reaction mixture, comprising the following components: 10 μL of 2×PCR buffer, 1 μL each of 10 μmol / L primers SEQ ID No. 1 and SEQ ID No. 2, 0.5 μL of 10 μmol / L probe SEQ ID No. 3, and ddH2O added to 19 μL.

[0008] The kit also includes a positive control, a negative control, and a blank control. The positive control is a moray eel genome extract, the negative control is an equal volume of genome extracts from other species that do not contain moray eel components, and the blank control is an equal volume of ddH2O to replace template DNA.

[0009] The method for detecting the components of moray eel includes the following steps.

[0010] (1) Extract genomic DNA from the sample to be tested.

[0011] (2) Real-time fluorescence PCR system: Add 1 μL of extracted sample DNA to the real-time fluorescence PCR reaction mixture.

[0012] (3) Real-time fluorescence PCR amplification conditions: 95 ℃ pre-denaturation for 30 s; 95 ℃ for 15 s, 60 ℃ for 60 s, 40 cycles.

[0013] The quality control criteria for judging the amplification results are as follows: if any of the following conditions are 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.

[0014] Result interpretation: When the Ct value is ≤38, the sample is positive; when the Ct value is >38 or there is no value, the sample is negative.

[0015] In the real-time fluorescence PCR detection method described above, the sample DNA preparation method can not only use commercially available DNA extraction kits, but also the CTAB method, alkaline lysis method, etc.

[0016] In this detection method, DNA extraction from the sample can be performed using a variety of mature technologies: commercially available standardized DNA extraction kits can be used, as well as conventional laboratory extraction methods, including but not limited to CTAB (hexadecyltrimethylammonium bromide) extraction and alkaline lysis extraction.

[0017] The technical advantages of this invention are mainly reflected in the following aspects: (1) This invention establishes a method for rapid detection of eel components using real-time fluorescence PCR technology, which can meet the detection requirements for adulteration and germplasm identification of eels. (2) Convenient operation and pollution prevention: The entire detection process takes only 60 minutes. The closed tube detection mode is adopted. After the reaction is completed, there is no need to open the lid, which eliminates the traditional electrophoresis, sequencing and other subsequent processing steps. The results can be interpreted by collecting fluorescence signals through the instrument, avoiding aerosol pollution. (3) High specificity: The specific primers and probes of this invention can specifically detect eel components. Brown eel (Muraenesox bagio), which belongs to the same genus as eel and is closely related to it, and other eels cannot be detected. (4) High sensitivity: The sensitivity of genomic DNA can reach 10. -3 The mass fraction sensitivity can reach 0.01%. Attached Figure Description

[0018] Figure 1 This is a result of a specific test.

[0019] Figure 2 This is a comparison diagram of the sequencing results of the amplified products.

[0020] Figure 3 Results of genome sensitivity testing; Figure 3 The numbers in the text represent: 1:10 2 ng; 2: 10 ng; 3: 1ng; 4: 10 -1 ng; 5:10 -2 ng; 6:10 -3 ng; 7:10 -4 ng; 8: Blank control.

[0021] Figure 4 This represents the linear relationship between the logarithm of genomic DNA content and the Ct value.

[0022] Figure 5 The results are from the mass fraction sensitivity test. Figure 5The numbers in the table represent: 1: 100%; 2: 10%; 3: 1%; 4: 0.1%; 5: 0.01%; 6: 0.001%; ​​7: 0.0001%; ​​8: blank control.

[0023] Figure 6 This represents the linear relationship between the logarithm of the mass fraction and the Ct value.

[0024] Figure 7 This is the result of a repeatability test. Detailed Implementation

[0025] The present invention will be further described in conjunction with the embodiments.

[0026] Example 1: Specificity Test: Genomic DNA from various fish species, including moray eel, Japanese eel, American eel, conger eel, spotted eel, rainbow trout, Atlantic mackerel, Japanese mackerel, tongue sole, Atlantic cod, sable scad, Pacific cod, basa fish, Chinese sturgeon, Nile tilapia, Atlantic bluefin tuna, bigeye tuna, skipjack tuna, Chinese mackerel, blue-spotted mackerel, large yellow croaker, and small yellow croaker, along with the cloning vector pUC57 carrying the brown moray eel D-LOOP gene (NC_013614.1), was measured using an ultra-micro spectrophotometer. The concentration of moray eel genomic DNA was adjusted to 10 ng / μL, and the concentration of DNA from the remaining samples was uniformly adjusted to 100 ng / μL. 1 μL of the sample DNA solution was added as a template to the real-time fluorescence PCR reaction mixture, with an ultrapure water blank control included. Real-time fluorescence PCR was performed to verify the specificity of primers and probes used to detect components of moray eels, and the amplification products were sequenced. The sequencing results were compared with those in NCBI.

[0027] Specificity verification results showed that ( Figure 1 The genomic DNA of the moray eel showed a fluorescence curve with a Ct value of 19.56, while no fluorescence signal was detected in other samples and the blank control, indicating that the primers and probes used to detect moray eel components have good specificity. The sequencing sequences of the amplified products from the positive samples were BLAST-aligned with those of the moray eel mitochondrial gene sequence using NCBI, showing 100% similarity. Figure 2 As shown, the designed primer and probe combination demonstrates a high degree of specificity in recognizing components of moray eels. This result confirms that the proposed detection method can effectively distinguish moray eels from other closely related species, meeting practical detection needs.

[0028] Example 2 Genomic DNA Sensitivity Test: In this study, the concentration of eel genomic DNA was accurately determined using an ultra-micro spectrophotometer, and then serially diluted 10-fold with ultrapure water to obtain 7 concentration gradients (10... 2 ng / μL to 10 -4ng / μL). Take 1 μL of each gradient DNA solution as template and add it to the real-time fluorescence PCR reaction mixture. The corresponding final template amounts are 10 ng / μL respectively. 2 ng to 10 -4 ng. An ultrapure water blank control was also set up, with three replicates for each concentration, and real-time fluorescence PCR experiments were performed.

[0029] The results of the genome sensitivity test show that the Ct values ​​for each dilution level are shown in Table 1. Figure 3 As shown, when the amount of DNA is ≥10 -3 Fluorescence curves were observed at all ng, and the Ct values ​​were all less than 38. The logarithm of the DNA amount was linearly correlated with the average Ct value, such as... Figure 4 As shown, the correlation coefficient R 2 =0.9952, the standard curve was y=-3.9226x+23.847, indicating good amplification effect; the standard deviation of Ct values ​​at various concentrations ranged from 0.040 to 0.36, and the relative standard deviation ranged from 0.20 to 0.99. The results show that the detection sensitivity of this method for eel genomic DNA can reach 10. -3 The ng level is well-reproducible.

[0030]

[0031] Example 3: Mass fraction sensitivity test: The muscle tissue of eel and basa fish was dried at 105 °C to constant weight. The samples were pulverized into powder with uniform particle size using a mixer. Gradient mixed samples ranging from 100% to 0.0001% by mass percentage were precisely prepared. 20 (19.90-20.10) mg of the mixed powder was weighed for DNA extraction. The DNA was dissolved in 50 μL of buffer. 1 μL of the DNA extract was used as a template and added to the real-time fluorescence PCR reaction mixture. An ultrapure water blank control was set up. Four parallel replicates were set up for each gradient and the real-time fluorescence PCR experiment was performed.

[0032] The results of the mass fraction sensitivity test show that the Ct values ​​for each mass fraction are shown in Table 2. Figure 5 As shown, fluorescence curves appeared in all mixed samples when the mass fraction of eel was ≥0.01%, and the Ct values ​​were all less than 38; the logarithm of the mass fraction was linearly correlated with the average Ct value, as shown in the figure. Figure 6 As shown, the correlation coefficient R 2=0.9935, the standard curve was y=-3.757x+24.722, indicating good amplification effect; the standard deviation of Ct values ​​for each mass fraction was between 0.029 and 0.48, and the relative standard deviation was between 0.12 and 1.47, indicating that the limit of quantitative detection of eel component in the composite sample by this method can reach 0.01% mass fraction, and it has reliable repeatability. When the mass fraction of eel in the mixed sample was 0.001%, one replicate sample was negative, indicating that the repeatability of this mass fraction did not meet the amplification requirements.

[0033]

[0034] Example 4 Repeatability Test: To verify the repeatability of this detection method, a 0.01% (w / w) mixed sample of conger eel was selected for a repeatability test. 20 (19.90-20.10) mg of mixed dry powder containing 0.01% conger eel was weighed, genomic DNA was extracted, dissolved in 50 μL buffer, and 1 μL of the DNA solution was used as a template in the real-time fluorescence PCR reaction mixture. Eight replicates were set up for the real-time fluorescence PCR experiment.

[0035] Repeatability test results ( Figure 7 The results showed that all eight parallel replicate reactions exhibited fluorescence curves, and the Ct values ​​were all less than 38 (as shown in Table 3), with a standard deviation of 0.27 and a relative standard deviation of 0.82.

[0036]

[0037] This result confirms that the method has excellent repeatability and stability at a mass fraction of 0.01%, meeting the repeatability requirements for detection.

Claims

1. A composition for detecting components in moray eels, characterized in that, It contains the sequences of eel-specific nucleotide primer pairs 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 reagent kit for detecting components in moray eels, characterized in that, The kit comprises the composition of claim 1 and a real-time fluorescence PCR reaction mixture.

3. The real-time fluorescence PCR reaction mixture according to claim 2, characterized in that, The total volume is 19 μL, containing: 10 μL of real-time fluorescence PCR premix (2×), 1 μL each of primers SEQ ID No.1 (10 μmol / L) and SEQ ID No.2 (10 μmol / L), 0.5 μL of probe SEQ ID No.3 (10 μmol / L), and made up to 19 μL with ultrapure water.

4. A method for detecting components in moray eel, characterized in that, The specific steps of the method are as follows: (1) Extract genomic DNA from the sample to be tested; (2) Take 1 μL of the extracted sample DNA and add it to the real-time fluorescence PCR reaction mixture; (3) Real-time fluorescence PCR amplification conditions: 95 ℃ pre-denaturation for 30 s; 95 ℃ denaturation for 15 s, 60 ℃ annealing for 60 s, 40 cycles.

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