Dual real-time fluorescence PCR primer group, kit and method for identifying Atlantic salmon and rainbow trout simultaneously

[0041] Example 1 A dual real-time fluorescent PCR primer set and probe for simultaneous identification of Atlantic salmon and rainbow trout

[0042] The primer set of the present invention can simultaneously detect Atlantic salmon and rainbow trout. The primer set of the present invention includes F1 and R1, F2 and R2, and its base sequence is shown below.

[0043] F1: 5'-agcagaactc agccagcct-3' (SEQ ID NO: 1),

[0044] R1: 5'-aaaggaggga gggagaagtc aa-3' (SEQ ID NO: 2),

[0045] P1: 5'-ccttctggga gatgacc-3' (SEQ ID NO: 3);

[0046] F2: 5'-accattatta acataaaacc tccag-3' (SEQ ID NO: 4),

[0047] R2: 5'-gtaatgcctg ctgccagga-3' (SEQ ID NO: 5),

[0048] P2: 5'-cgtttgagcc gtgcta-3' (SEQ ID NO: 6);

[0049] Above F1 and R1 are Atlantic salmon-specific primers, and P1 is Atlantic salmon-specific probe. The 5'end of P1 was modified with a fluorescent group FAM, and the 3'end of P1 was modified with a non-fluorescent quenching group MGB. F2 and R2 are rainbow trout-specific primers, and P2 is rainbow trout-specific probe. The 5'end of P2 is modified by the fluorescent group VIC, and the 3'end of P2 is modified by the non-fluorescent quenching group MGB.

[0050] Example 2 A dual real-time fluorescent PCR method for simultaneous identification of Atlantic salmon and rainbow trout

[0051] In this embodiment, four experimental groups are set up. Add 50ng Atlantic salmon DNA to the tube of experimental group 1, 50ng rainbow trout DNA to the tube of experimental group 2, and 25ng Atlantic salmon DNA and 25ng rainbow trout DNA to the tube of experimental group 3. Add ddH to 4 2 O serves as a negative control.

[0052] 1) Extract the sample DNA; use sterile scissors to weigh the sample into a 2 mL sterile centrifuge tube, use a commercial animal tissue DNA extraction kit for DNA extraction, and use a nucleic acid protein analyzer to determine the concentration of the extracted DNA.

[0053] 2) Using DNA as a template, using the above F1 and R1, F2 and R2 as primers, and P1 and P2 as probes, perform dual real-time fluorescent PCR amplification;

[0054] Among them, the dual real-time fluorescent PCR amplification system is: 2×Premix Ex Taq 12.5μL, F1 final concentration is 0.1μM, R1 final concentration is 0.2μM, P1 final concentration is 0.14μM, F2 final concentration is 0.2μM, R2 is final concentration The concentration is 0.3μM, the final concentration of P2 is 0.13μM, the amount of template DNA added is 25-100ng, supplemented with ddH 2 O to 25μL system. Among them, 2×Premix Ex Taq is a product of Takara, and the article number is RR390A.

[0055] Among them, the double real-time fluorescent PCR amplification program is: 95°C pre-denaturation for 3 minutes, 94°C denaturation for 10s, 63°C annealing and extension for 20s, 40 cycles.

[0056] 3) Judgment of the result: After the reaction is over, the result is judged according to the Ct value (there is an amplification curve and the Ct value<30 is judged as positive), that is, if primer F1, R1 and probe P1 can amplify a logarithmic amplification curve , And Ct value <30, then it is judged as positive for Atlantic salmon; if primer F2, R2 and probe P2 can amplify a logarithmic amplification curve, and Ct value is less than 30, then it is judged as positive for rainbow trout. Specifically, it is divided into the following 4 situations:

[0057] If Atlantic salmon has a fluorescence logarithmic amplification curve, and the Ct value is less than 30 (that is, F1, R1, and P1 have a fluorescence logarithmic amplification curve, and the Ct value is less than 30); at the same time, rainbow trout Ct value ≥ 30 (that is, F2, R2 and P2 have no amplification curve or the Ct value of the amplification curve is ≥30); the sample is positive for Atlantic salmon and negative for rainbow trout;

[0058] If rainbow trout has a fluorescence logarithmic amplification curve, and the Ct value is less than 30 (that is, F2, R2 and P2 have fluorescence logarithmic amplification curves, and the Ct value is less than 30); at the same time, the Atlantic salmon has a Ct value ≥ 30 (that is, F1, R1 and P1 have no amplification curve or the Ct value of the amplification curve is greater than or equal to 30); the sample is negative for Atlantic salmon and positive for rainbow trout;

[0059] If Atlantic salmon has a fluorescence logarithmic amplification curve, and the Ct value is less than 30 (that is, F1, R1 and P1 have fluorescence logarithmic amplification curves, and Ct value is less than 30); rainbow trout has a fluorescence logarithmic amplification curve, and Ct Value <30 (ie F2, R2 and P2 have fluorescence logarithmic amplification curves, and Ct value <30); then the sample is positive for Atlantic salmon and rainbow trout;

[0060] If the Ct value of Atlantic salmon is ≥30; at the same time (that is, F1, R1 and P1 have no amplification curve or the Ct value of the amplification curve is ≥30), and the Ct value of rainbow trout is ≥30 (that is, F2, R2, and P2 have no amplification curve or amplification curve). The Ct value of the increasing curve is ≥30); the sample is negative for Atlantic salmon and negative for rainbow trout.

[0061] The real-time fluorescent PCR amplification patterns of each experimental group in this example are as follows: figure 1 Shown. The identification result of experimental group 1 was positive for Atlantic salmon and negative for rainbow trout; the identification result of experimental group 2 was negative for Atlantic salmon and positive for rainbow trout; the identification result of experimental group 3 was positive for Atlantic salmon and positive for rainbow trout; identification of experimental group 4 The results were negative for Atlantic salmon and negative for rainbow trout.

[0062] This example illustrates that the method provided by the present invention can accurately identify Atlantic salmon and rainbow trout.

[0063] Example 3 Specific detection

[0064] In this example, the DNA of King salmon (Oncorhynchus. Tshawytscha) and Coho salmon (Oncorhynchus. Kisutch), which are common in the Chinese salmon market, was used to verify the specificity of the primers. Set up six experimental groups. Add 50ng of king salmon DNA to the tube of experimental group 1, 50ng of coho salmon DNA to the tube of experimental group 2, and add 25ng of Atlantic salmon DNA and 25ng of king salmon DNA to the tube of experimental group 3. Add 25ng rainbow trout DNA and 25ng king salmon DNA to tube 4, add Atlantic salmon DNA and 25ng coho DNA to experiment group 5, and add rainbow trout DNA and 25ng coho salmon to experiment group 6 DNA.

[0065] 1) Extracting sample DNA: Weigh the sample with sterile scissors and place it in a 2mL sterile centrifuge tube, use a commercial animal tissue DNA extraction kit for DNA extraction, and use a nucleic acid protein analyzer to determine the concentration of the extracted DNA.

[0066] 2) Using DNA as a template, using the above F1 and R1, F2 and R2 as primers, and P1 and P2 as probes, perform dual real-time fluorescent PCR amplification;

[0067] Among them, the dual real-time fluorescent PCR amplification system is: 2×Premix Ex Taq 12.5μL, F1 final concentration is 0.1μM, R1 final concentration is 0.2μM, P1 final concentration is 0.14μM, F2 final concentration is 0.2μM, R2 is final concentration The concentration is 0.3μM, the final concentration of P2 is 0.13μM, the amount of template DNA added is 25-100ng, supplemented with ddH 2 O to 25μL system. Among them, 2×Premix Ex Taq is a product of Takara, and the article number is RR390A.

[0068] Among them, the double real-time fluorescent PCR amplification program is: 95°C pre-denaturation for 3 minutes, 94°C denaturation for 10s, 63°C annealing and extension for 20s, 40 cycles.

[0069] 3) Result judgment: the same as in Example 2.

[0070] The real-time fluorescent PCR amplification patterns of each experimental group are as follows figure 2 Shown. The identification results of experimental group 1 and experimental group 2 are negative for Atlantic salmon and negative for rainbow trout, indicating that the primers of the reaction system of the present invention have good specificity for Atlantic salmon and rainbow trout; the identification results of experimental group 3 and experimental group 5 are Atlantic salmon Positive and negative for rainbow trout, indicating that coho salmon DNA and king salmon DNA will not affect the detection results of the method of the present invention on Atlantic salmon; the identification results of experimental group 4 and experimental group 6 are negative for Atlantic salmon and positive for rainbow trout, indicating that coho salmon DNA And king salmon DNA will not affect the detection result of rainbow trout by the method of the present invention.

[0071] This example shows that the primers and detection methods of the present invention have good specificity and will not be affected by species such as king salmon and coho salmon.