Seed identification method based on T7 endonuclease I

Abstract

The invention discloses a species identification method based on T7 endonuclease I, and belongs to the technical field of molecular biological detection. Designing an upstream primer and a downstream primer according to a known conserved DNA sequence of a known standard substance; by utilizing the upstream primer and the downstream primer, respectively amplifying the target DNA of the standard substance and the sample to be detected by adopting PCR (Polymerase Chain Reaction); mixing the PCR amplification product of the standard substance and the PCR amplification product of the sample to be detected in proportion, and performing denaturation and annealing treatment; magnetic beads are added to adsorb target DNA, and the fluorescence value 1 is measured; adding a T7 endonuclease I solution into the PCR amplification product added with the magnetic beads, treating for 30-120 minutes, carrying out magnetic separation to remove the magnetic beads, and measuring a fluorescence value 2; and judging whether the to-be-detected sample is consistent with the standard variety or not according to the ratio of the fluorescence value 2 to the fluorescence value 1. The species identification method disclosed by the invention is high in applicability, does not need to predict genetic information of an unknown sample, is short in detection time, and has the advantages of simple process, short consumed time and high efficiency.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology detection, and specifically relates to a species identification method based on T7 endonuclease I. Background Art

[0002] At present, the methods for species identification mainly include barcode sequencing method, fluorescence quantitative PCR method, and characteristic peptide liquid chromatography-mass spectrometry method. The barcode sequencing method uses PCR to amplify the conserved sequence of mitochondrial CO1 gene, obtains its sequence by sequencing, and conducts sequence alignment for species origin analysis. The fluorescence quantitative PCR method compares and analyzes the differential DNA sequences of different species, designs primers and probes based on the differential sequences, and conducts fluorescence quantitative PCR to analyze species. The characteristic peptide liquid chromatography-mass spectrometry method uses proteomics technology to discover the differential polypeptides of different species, and develops a liquid chromatography-mass spectrometry method based on the differential polypeptides to analyze species. The barcode sequencing method needs to entrust a sequencing company for analysis, and the analysis period is about 7 days. The fluorescence quantitative PCR method and the characteristic peptide liquid chromatography-mass spectrometry method require prior knowledge of the differential DNA and polypeptide sequences between species. The above methods are all difficult to achieve rapid analysis of the species of unknown samples. Summary of the Invention

[0003] In view of the technical problems existing in the prior art, the present invention provides a species identification method based on T7 endonuclease I. Two primers are designed for the conserved DNA sequence related to the species of the sample, and are respectively labeled with biotin and fluorescence. The conserved DNA sequence related to the species of the unknown sample and the reference sample is amplified by PCR. The two DNAs are hybridized and immobilized on streptavidin magnetic beads. If the DNA sequences of the unknown sample and the reference sample are inconsistent, heteroduplexes will be formed. T7 endonuclease I can recognize and cleave the incompletely paired heterologous DNA duplexes. By monitoring the fluorescence signal of the DNA cleaved from the magnetic beads into the liquid, cleavage indicates that the conserved DNA sequences related to the species of the unknown sample and the reference sample are inconsistent and belong to different species, thereby realizing the identification of the species of animals, plants, and microorganisms.

[0004] The present invention is realized through the following technical solutions: A species identification method based on T7 endonuclease I, comprising the following steps: (1) Primer design: Design an upstream primer and a downstream primer according to the known conserved DNA sequence of the known standard. (2) PCR amplification reaction: Using the upstream primer 1 and the downstream primer 2 designed in step (1), respectively amplify the target DNA of the standard and the sample to be detected by PCR. (3) Hybridization: Mix the PCR amplification products of the standard and the sample to be detected in step (2) in proportion, and perform denaturation and annealing treatments. (4) Magnetic bead adsorption of target DNA: Add magnetic beads to the PCR amplification product after annealing treatment in step (3) to adsorb the target DNA, and measure the fluorescence value 1; (5) T7 endonuclease I digestion: Add T7 endonuclease I solution to the PCR amplification product with magnetic beads added in step (4), incubate for 30 - 120 min, remove the magnetic beads by magnetic separation, and measure the fluorescence value 2; (6) Result determination: Determine whether the species of the sample to be detected is consistent with that of the standard according to the ratio of fluorescence value 2 to fluorescence value 1.

[0005] Further, when the ratio of fluorescence value 2 to fluorescence value 1 in step (6) ≥ 0.1, the species of the sample to be detected is inconsistent with that of the standard; when the ratio of fluorescence value 2 to fluorescence value 1 < 0.1, the species of the sample to be detected is consistent with that of the standard.

[0006] Further, the 5' end of primer 1 described in step (1) is biotinylated, and the 5' end of primer 2 is modified with Tamra fluorescence labeling.

[0007] Further, the PCR system for PCR amplification in step (2) is 50 μl, including 25 μl of 2x EasyTaqTM PCR SuperMix, 1 μl of 10 pmol / L primer 1, 1 μl of 10 pmol / L primer 2, 2 μl of DNA template, and 21 μl of water.

[0008] Further, the PCR amplification conditions in step (2) are: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 S, annealing at 62°C for 30 S, extension at 72°C for 30 S, for a total of 35 cycles, and then extension at 72°C for 10 min.

[0009] Further, the concentration ratio of the standard PCR product to the PCR product of the sample to be detected in step (3) is 1:1.

[0010] Further, the denaturation treatment conditions in step (3) are denaturation at 95°C for 3 min; the annealing treatment conditions are annealing at 62°C for 30 S, down to 4°C.

[0011] Further, the magnetic beads in step (4) are streptavidin magnetic beads, the diameter of the magnetic beads is 5 nm, and the molar ratio of the hybridized PCR product to streptavidin on the magnetic beads is 1:4 - 7.

[0012] Further, the amount of T7 endonuclease I added in step (5) is 50 IU.

[0013] T7 Endonuclease I is a highly specific DNA cleavage enzyme that precisely cleaves heteroduplex regions by recognizing local structural distortions in double-stranded DNA caused by mismatches, insertions / deletions, or heterologous base stacking. The present invention utilizes the property of T7 Endonuclease I to recognize and cleave incompletely paired DNA. First, for gene regions that are conserved among species and polymorphic (such as ITS, COI, 16S rRNA, and STR), universal primer pairs are designed to perform PCR amplification on unknown samples and reference samples. The amplification products of the two are mixed, hybridized through high-temperature denaturation and gradient renaturation. If there are species differences in the sequences, heteroduplexes containing mismatch sites will be formed. T7 Endonuclease I will cleave the mismatch regions, and the completely matched homologous duplexes will remain intact, thereby determining whether the unknown sample and the reference sample are of the same species.

[0014] The beneficial effects achieved by the present invention are as follows: (1) It has strong applicability and can identify the species of all animals, plants, and microorganisms; (2) It is not necessary to know the genetic information of the unknown sample in advance; (3) The total time for PCR amplification of the conserved gene of the sample and detection by T7 Endonuclease I digestion is about 3 hours. There is no need to entrust a sequencing company for detection, and it has the advantages of simple process, short time consumption, and high efficiency. Detailed implementation manners

[0015] The specific details of the present invention are further elaborated to fully understand the present invention. The terms used in the description of the present invention are only used to illustrate the advantages and characteristics of the present invention and are not intended to limit the present invention.

[0016] Unless otherwise defined, all professional and scientific terms used in the present invention have the same meaning as understood by those skilled in the technical field of the present invention. Without special instructions, the drugs or reagents used in the present invention are used according to the product instructions or the conventional usage methods in the relevant field. Now, the technical solutions of the present invention are further described according to the specific implementation manners.

[0017] Example 1 Detection limit (1) Primer design: According to the COI sequence of Deinagkistrodon acutus, upstream primer 1 and downstream primer 2 are designed. The primer sequences are as follows: Upstream primer 1: Biotin-modified 5'-GGTCAACAAATCATAAAGATATTGG-3' (SEQ ID NO.1), Downstream primer 2: Tamra-modified -5'-TAAACTTCAGGGTGACCAAAAAATCA-3' (SEQ ID NO.2); (2)PCR amplification reaction: Use the genomic DNA of Deinagkistrodon acutus as a template to perform PCR amplification of the COl fragment (a total of 12 samples); the PCR system is 50 μl, containing 25 μl of 2x EasyTaqTM PCR SuperMix, 1 μl each of 10 pmol / L upstream and downstream primers, 2 μl of DNA template, and 21 μl of sterile water; add all components into a 200 μl PCR tube, mix well by instantaneous centrifugation, and place it in a PCR instrument for amplification. The amplification conditions are pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 62°C for 30 s, extension at 72°C for 30 s, for a total of 35 cycles, and then extension at 72°C for 10 min; (3)Hybridization: Measure the concentration of the PCR product. Add 2 PCR amplification products at a 1:1 concentration to a centrifuge tube until it reaches 100 μl, denature at 95°C for 3 min, anneal at 62°C for 30 s, and cool down to 4°C; (4)Magnetic bead adsorption of target DNA: Add 10 μl of streptavidin magnetic beads (100 μg, the diameter of the magnetic beads is 5 nm, and the molar ratio of the hybridized PCR product to streptavidin on the magnetic beads is 1:5), mix well, for 3 min, perform magnetic separation, remove the aqueous phase, and separate the magnetic beads; add 100 μl of water, wash the magnetic beads, perform magnetic separation, and remove the liquid; repeat 2 times, add water to 100 μl and mix well, measure the fluorescence value at an excitation wavelength of 555 nm and an emission wavelength of 575 nm to obtain fluorescence value 1, perform magnetic separation, and remove the aqueous phase; (5)Digestion with T7 endonuclease I: Add 10 μl of 10X T7 endonuclease I buffer to the centrifuge tube containing magnetic beads, add 50 IU of T7 endonuclease I (10 IU / ul), make up the volume to 100 μl with water, react at 37°C for 30 min, perform magnetic separation of the magnetic beads, aspirate the supernatant, measure the fluorescence value at an excitation wavelength of 555 nm and an emission wavelength of 575 nm to obtain fluorescence value 2; (6)According to the ratio of fluorescence value 2 to fluorescence value 1 shown in the table, 6 samples with consistent sequences were detected. The results are shown in Table 1. The average value of the ratio of fluorescence value 2 to fluorescence value 1 is 0.015, and the standard deviation (SD) is 0.005. According to the conventional calculation method, the detection limit is the average value plus 3 times the standard deviation, that is, 0.03. Considering the actual application requirements, the detection limit is relaxed to 0.1; Table 1 Test results of Example 1 。

[0018] Example 2 Deinagkistrodon acutus ( Bothrops atrox ) species identification (1) Primer design: According to the COI sequence of Deinagkistrodon acutus, upstream primer 1 and downstream primer 2 were designed. The primer sequences were as follows: Upstream primer 1: Biotin-modified 5'-GGTCAACAAATCATAAAGATATTGG-3' (SEQ ID NO.3), Downstream primer 2: Tamra-modified-5'-TAAACTTCAGGGTGACCAAAAAATCA-3' (SEQ ID NO.4); (2) PCR amplification reaction: Using the genomic DNA of 4 kinds of snake venoms (Deinagkistrodon halys Gloydius blomhoffi siniticus、 Deinagkistrodon acutus Deinagkistrodon acutus、 Vipera russelli Daboia siamensis and Deinagkistrodon acutus Bothrops ) and the genomic DNA of the standard Deinagkistrodon acutus ( Bothrops atrox ) as templates, PCR amplification of the COl fragment was carried out; The PCR system was 50 μl, containing 25 μl of 2x EasyTaqTM PCR SuperMix, 1 μl each of 10 pmol / L upstream and downstream primers, 2 μl of DNA template, and 21 μl of sterile water; All components were added to a 200 μl PCR tube, centrifuged briefly to mix evenly, and placed in a PCR instrument for amplification. The amplification conditions were pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 62°C for 30 s, extension at 72°C for 30 s, for a total of 35 cycles, and then extension at 72°C for 10 min; (3) Hybridization: Measure the concentration of the PCR product. Add the PCR amplification products of the standard and unknown samples at a 1:1 concentration to a centrifuge tube to 100 μl, denature at 95°C for 3 min, anneal at 62°C for 30 s, and cool down to 4°C; (4) Magnetic bead adsorption of target DNA: Add 10 μl of streptavidin magnetic beads (100 μg, magnetic bead diameter is 5 nm, the molar ratio of the hybridized PCR product to streptavidin on the magnetic beads is 1:5), mix evenly, for 3 min, magnetic separation, remove the aqueous phase, and separate the magnetic beads; Add 100 μl of water, wash the magnetic beads, magnetic separation, and remove the liquid; Repeat 2 times, add water to 100 μl and mix evenly, measure the fluorescence value at an excitation wavelength of 555 nm and an emission wavelength of 575 nm to obtain fluorescence value 1, magnetic separation, and remove the aqueous phase; (5) T7 endonuclease I digestion: Add 10 μl of 10X T7 endonuclease I buffer to the centrifuge tube containing magnetic beads, add 50 IU of T7 endonuclease I (10 IU / ul), make up the volume to 100 μl with water, react at 37°C for 30 min, magnetic separate the magnetic beads, aspirate the supernatant, measure the fluorescence value at an excitation wavelength of 555 nm and an emission wavelength of 575 nm to obtain fluorescence value 2; (6)The detection results are shown in Table 2. Whether the sample to be detected and the standard variety belong to the same species is judged according to the ratio of fluorescence value 2 to fluorescence value 1; when the ratio of fluorescence value 2 to fluorescence value 1 ≥ 0.1, the sample to be detected and the standard variety do not belong to the same species; when the ratio of fluorescence value 2 to fluorescence value 1 < 0.1, the sample to be detected and the standard variety belong to the same species.

[0019] In step (1), the ratios of fluorescence value 2 to fluorescence value 1 of the 4 samples are shown in Table 1 below. When the ratio of fluorescence value 2 to fluorescence value 1 ≥ 0.1, the sample to be detected and the standard variety do not belong to the same species; when the ratio of fluorescence value 2 to fluorescence value 1 < 0.1, the sample to be detected and the standard variety belong to the same species. Table 2 Detection Results of Different Snake Venom Samples .

[0020] Example 3 Species identification of flounder ( Pleuronectes platessa ) (1) Primer design: According to the COI sequence of flounder, upstream primer 1 and downstream primer 2 are designed. The primer sequences are as follows: Upstream primer 1 (MI-COI-F): Biotin-5'-TCAC1AATCATAAAGATATCGGCAC-3' (SEQ ID NO.5); Downstream primer 2 (MI-COI-R): Tamra-5'-TAGACTTCTGGGTGGCCAAAGAATCA-3' (SEQ ID NO.6); (2) PCR amplification reaction: Using the genomic DNA of 4 fish samples extracted (respectively yellow croaker Pelteobagrus fulvidraco、 Japanese flounder Paralichthys olivaceus、 tongue sole Cynoglossus robustus and flounder Pleuronectes platessa ) and the genomic DNA of the standard flounder ( Pleuronectes platessa ) as templates for COl fragment PCR amplification; The PCR system is 50 μl, containing 25 μl of 2xEasyTaqTM PCR SuperMix, 1 μl of each of the upstream and downstream primers at 10 pmol / L, 2 μl of DNA template, and 21 μl of sterile water; Add all components to a 200 μl PCR tube, and mix well by instantaneous centrifugation; Put it into a PCR instrument for amplification. The amplification conditions are pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 30 s, annealing at 62 °C for 30 s, extension at 72 °C for 30 s, for a total of 35 cycles, and then extension at 72 °C for 10 min; (3) Hybridization: Measure the concentration of the PCR product. Add the PCR products of the standard and unknown samples at a 1:1 concentration to a centrifuge tube until it reaches 100 μl, denature at 95 °C for 3 min, anneal at 62 °C for 30 s, and cool down to 4 °C; (4) Magnetic bead adsorption of target DNA: Add 10 μl of streptavidin magnetic beads (100 μg, magnetic bead diameter is 5 nm, the molar ratio of the hybridized PCR product to streptavidin on the magnetic beads is 1:5), mix well, for 3 min, perform magnetic separation, remove the aqueous phase, and separate the magnetic beads; add 100 μl of water, wash the magnetic beads, perform magnetic separation, and remove the liquid; repeat 2 times, add water to 100 μl and mix well, measure the fluorescence value at an excitation wavelength of 555 nm and an emission wavelength of 575 nm to obtain fluorescence value 1, perform magnetic separation, and remove the aqueous phase; (5) Digestion with T7 Endonuclease I: Add 10 μl of 10X T7 Endonuclease I buffer to the centrifuge tube containing magnetic beads, add 50 IU of T7 Endonuclease I (10 IU / ul), make up the volume to 100 μl with water, react at 37 °C for 30 min, perform magnetic separation of the magnetic beads, aspirate the supernatant, measure the fluorescence value at an excitation wavelength of 555 nm and an emission wavelength of 575 nm to obtain fluorescence value 2; (6) The detection results are shown in Table 3. Determine whether the sample to be detected and the standard variety belong to the same species according to the ratio of fluorescence value 2 to fluorescence value 1; when the ratio of fluorescence value 2 to fluorescence value 1 ≥ 0.1, the sample to be detected and the standard variety do not belong to the same species; when the ratio of fluorescence value 2 to fluorescence value 1 < 0.1, the sample to be detected and the standard variety belong to the same species; Table 3 Detection results of different fish samples 。

[0021] Example 3 Identification of STR loci of cell lines (1) Primer design: Design upstream primer 1 and downstream primer 2 according to the D10S611 sequence of the Vero cell genome (STR locus). The primer sequences are as follows: Upstream primer 1 (D10S611-F): Biotin- 5'-AGCCCTGTGTGAAAGTTGGA-3'; Downstream primer 2 (D10S611-R): Tamra- 5'-GCTTCTTCTGGAGATGGTGC-3'; (2) PCR amplification reaction: Use the genomic DNA of 2 cell lines extracted (CHO cells and Vero cells respectively) and the genomic DNA of the standard Vero cells as templates for PCR amplification of the COl fragment; The PCR system is 50 μl, containing 25 μl of 2x EasyTaqTM PCR SuperMix, 1 μl each of 10 pmol / L upstream and downstream primers, 2 μl of DNA template, and 21 μl of sterile water; Add all components to a 200 μl PCR tube, centrifuge briefly to mix; Place in a PCR instrument for amplification. The amplification conditions are pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 30 s, annealing at 62 °C for 30 s, extension at 72 °C for 30 s, for a total of 35 cycles, and then extension at 72 °C for 10 min; (3) Hybridization: Determine the concentration of the PCR product. Add the PCR products of the standard and the unknown sample at a 1:1 concentration ratio into a centrifuge tube until it reaches 100 μl. Denature at 95 °C for 3 min, anneal at 62 °C for 30 s, and cool down to 4 °C. (4) Magnetic bead adsorption of target DNA: Add 10 μl of streptavidin magnetic beads (100 μg, magnetic bead diameter is 5 nm, the molar ratio of the hybridized PCR product to streptavidin on the magnetic beads is 1:5), mix well, incubate for 3 min, perform magnetic separation, remove the aqueous phase, and separate the magnetic beads; add 100 μl of water, wash the magnetic beads, perform magnetic separation, and remove the liquid; repeat 2 times, add water to 100 μl and mix well, measure the fluorescence value at an excitation wavelength of 555 nm and an emission wavelength of 575 nm to obtain fluorescence value 1, perform magnetic separation, and remove the aqueous phase; (5) T7 endonuclease I digestion: Add 10 μl of 10X T7 endonuclease I buffer to the centrifuge tube containing magnetic beads, add 50 IU of T7 endonuclease I, make up the volume to 100 μl with water, react at 37 °C for 30 min, perform magnetic separation of the magnetic beads, aspirate the supernatant, measure the fluorescence value at an excitation wavelength of 555 nm and an emission wavelength of 575 nm to obtain fluorescence value 2; (6) The test results are shown in Table 4. Judge whether the species of the sample to be tested and the standard are the same according to the ratio of fluorescence value 2 to fluorescence value 1; if the ratio of fluorescence value 2 to fluorescence value 1 ≥ 0.1, the species of the sample to be tested and the standard are different; if the ratio of fluorescence value 2 to fluorescence value 1 < 0.1, the species of the sample to be tested and the standard are the same; Table 4 Detection Results of Cell Lines 。

Claims

1. A species identification method based on T7 endonuclease I, characterized in that, It includes the following steps: (1) Primer design: Design upstream primer 1 and downstream primer 2 according to the known conserved DNA sequence of the known standard. (2) PCR amplification reaction: Use the upstream primer 1 and downstream primer 2 designed in step (1) to respectively perform PCR amplification on the target DNA of the standard and the sample to be detected. (3) Hybridization: Mix the PCR amplification products of the standard and the sample to be detected in step (2) in proportion, and perform denaturation and annealing treatments. (4) Magnetic bead adsorption of target DNA: Add magnetic beads to the PCR amplification product after annealing treatment in step (3) to adsorb the target DNA, and measure fluorescence value 1. (5) T7 endonuclease I digestion: Add T7 endonuclease I solution to the PCR amplification product after adding magnetic beads in step (4), treat for 30 - 120 min, remove the magnetic beads by magnetic separation, and measure fluorescence value 2. (6) Result determination: Judge whether the species of the sample to be detected and the standard are the same according to the ratio of fluorescence value 2 to fluorescence value 1.

2. The species identification method based on T7 endonuclease I according to claim 1, wherein In step (6), when the ratio of fluorescence value 2 to fluorescence value 1 ≥ 0.1, the species of the sample to be detected and the standard are different; when the ratio of fluorescence value 2 to fluorescence value 1 < 0.1, the species of the sample to be detected and the standard are the same.

3. The species identification method based on T7 endonuclease I according to claim 1, wherein In step (1), the 5' end of primer 1 is modified with biotin, and the 5' end of primer 2 is modified with Tamra fluorescence labeling.

4. The species identification method based on T7 endonuclease I according to claim 1, wherein In step (2), the PCR system for PCR amplification is 50 μl, including 25 μl of 2xEasyTaqTM PCR SuperMix, 1 μl of 10 pmol / L primer 1, 1 μl of 10 pmol / L primer 2, 2 μl of DNA template, and 21 μl of water.

5. The species identification method based on T7 endonuclease I according to claim 1, wherein In step (2), the PCR amplification conditions are: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 S, annealing at 62°C for 30 S, extension at 72°C for 30 S, for a total of 35 cycles, and then extension at 72°C for 10 min.

6. The species identification method based on T7 endonuclease I according to claim 1, wherein, In step (3), the concentration ratio of the PCR product of the standard to the PCR product of the sample to be detected is 1:

1.

7. The species identification method based on T7 endonuclease I according to claim 1, wherein In step (3), the denaturation treatment conditions are denaturation at 95°C for 3 min; the annealing treatment conditions are annealing at 62°C for 30 S, down to 4°C.

8. The species identification method based on T7 endonuclease I according to claim 1, wherein In step (4), the magnetic beads are streptavidin magnetic beads, the diameter of the magnetic beads is 5 nm, and the molar ratio of the hybridized PCR product to streptavidin on the magnetic beads is 1:4 - 7.

9. The species identification method based on T7 endonuclease I according to claim 1, wherein In step (5), the amount of T7 endonuclease I added is 50 IU.