CN101974522B - Primer, probe, kit and method for identifying authenticity of cervus elaphus linnaeus - Google Patents

Abstract

The invention relates to specific oligonucleotide primers and probes for authenticity identification of red deer antler. The invention also relates to a real-time fluorescent PCR detection method for identifying the authenticity of the red deer antler, and the method includes using the specific oligonucleotide primers and probes of the invention. The invention also relates to a PCR detection kit for identifying the authenticity of the red deer antler, and the kit includes the specific oligonucleotide primers and probes of the invention. The invention also relates to the application of the specific oligonucleotide primer and probe of the invention in identifying the authenticity of the red deer antler. Using the real-time fluorescent PCR detection method of the invention, the authenticity of the red deer antler can be identified simply, quickly, specifically and sensitively.

Description

Primers, probes, kits and methods for authenticity identification of red deer antler

technical field

The present invention belongs to the field of biotechnology, specifically, the present invention relates to specific oligonucleotide primers and probes for identifying the authenticity of red deer antler, a real-time fluorescent PCR detection method for identifying the authenticity of red deer antler, and the Application of specific oligonucleotide primers and probes or kit in identifying authenticity of red deer antler.

Background technique

Antler (Cornu Cervi Pantotrichum) is the young antlers of deer (Cervus nippon Temminck) or red deer (Cervus elaphus Linnaeus) stags, which are not ossified and densely grown, and are commonly called flower antler and red antler. Deer antler was first recorded in "Shen Nong's Materia Medica" and listed as a middle-grade product. It has the functions of strengthening kidney yang, benefiting essence and blood, and strengthening bones and muscles. It is one of the precious animal medicines in my country. There are many types and specifications on the market. Due to the difference in the original animals, there are two types: flower antler and red antler; It is also divided into saddle, Ergang, Sancha, lotus and so on.

There are many kinds of deer, but those with medicinal value mainly refer to sika deer and red deer. The red deer is a large deer second only to the moose. It is named for its shape like a horse. The types of red deer mainly include Norwegian red deer, Barbary red deer, Corsican red deer, West African red deer, Central European red deer, Scottish red deer, Spanish red deer, Eastern European red deer, Great Summer red deer, Tarim red deer, Kashmir red deer, Tibetan red deer, white-armed deer.

Due to the high price of red deer, some lawbreakers seek huge profits by making false ones, which not only endangers the interests of consumers, but also has a serious impact on the construction of a harmonious market order. Slices made of counterfeit products are often sold in the market. It is mainly fakes made of the horns of roe deer (Capreolus capreolus), reindeer (Alces alces), reindeer (Rangifer tarandus), and other animals' bones or animal glue embedded in animal skin and then cut.

In order to protect the interests of consumers and a good market order, many scholars at home and abroad have studied a variety of methods to identify the authenticity of velvet antler. Commonly used methods are character identification, microscopic identification, thin-layer chromatography identification, spectral identification, ultraviolet spectroscopy identification, etc. However, traditional detection methods are susceptible to the influence of chemical components contained in velvet antlers of different varieties and origins. Due to the increasing level and means of adulteration, the adulteration components are becoming more and more complex, which seriously limits the traditional detection methods. application space.

Molecular biology technology, with its convenience, accuracy, rapidity and simplicity, has become a research hotspot for domestic and foreign scholars in recent years. It provides a true and reliable basis for proving the authenticity of food, and provides a basis for the identification of food types and product traceability. Fresh blood has been injected into food authentication research. However, up to now, the application of molecular biology technology in the identification of counterfeit Chinese herbal velvet antler is still in the preliminary research stage.

At present, there are few reports at home and abroad that can quickly, simply, specifically and sensitively identify the method of Chinese herbal velvet antler.

Therefore, there is a need in this field for a fast, specific, and highly sensitive method for identifying the authenticity of the Chinese herbal antler, so as to identify the authenticity of the Chinese herbal antler.

Contents of the invention

One object of the present invention is to provide specific oligonucleotide primers and probes for rapid identification of red deer antler.

Another object of the present invention is to provide a real-time fluorescent PCR detection method for quickly identifying the authenticity of the red deer antler.

Another object of the present invention is to provide a kit for quickly identifying the authenticity of the red deer antler.

Another object of the present invention is to provide the application of the specific oligonucleotide primers and probes of the present invention in identifying the authenticity of red deer antler.

For the purpose of the above invention, the present invention provides the following technical solutions:

According to one embodiment of the present invention, the present invention provides a pair of specific oligonucleotide primers and probes for identifying the authenticity of red deer antler. The present invention is based on the DNA of red deer, and according to the characteristic that the D-loop (Displacement loop, D-Loop) region sequence of mitochondrial DNA (mtDNA) has differences in different species of cervidae, the mitochondrial DNA D-Loop of red deer is cloned. loop sequence. Primers and probes were designed according to these sequences, and real-time fluorescent PCR method was used to detect the red deer antler components in the samples.

The oligonucleotide primer pair and probe of the present invention are designed according to the characteristics of differences in mitochondrial DNA D-ring regions of different deer species. In one embodiment, the primer pair is composed of an upstream primer and a downstream primer, the upstream primer is Marlu-F: AACACGTGATATAACCTTATGCGC (SEQ ID No.1), and the downstream primer is Marlu-R: TATGTCCTATACACTAACTCATGTGC (SEQ ID No .2); the probe is Marlu-P: TGTGCTAGAACACGCATGTATAACAGCAC (SEQ ID No.3), a fluorescent quenching group BHQ1 is connected to the 3' end of the probe, and a fluorescent reporter group FAM is connected to the 5' end . Using the combination of the primer pair and the probe of the present invention, compared with other primer pairs, the target fragment can still be amplified specifically and sensitively in the case of a very small sample amount.

According to another embodiment of the present invention, the present invention provides a real-time fluorescent PCR detection method for identifying the authenticity of red deer antler, the method includes using a pair of specific oligonucleotide primers and probes for red deer antler, and the primer pair is It is designed according to the characteristics of the differences in the mitochondrial DNA D-loop region sequences of different cervidae species. In one embodiment, in the real-time fluorescent PCR detection method for identifying the authenticity of red deer antler of the present invention, the specific oligonucleotide primer pair used is composed of an upstream primer and a downstream primer, and the base sequence of the upstream primer is It is SEQ ID No.1, the base sequence of the downstream primer is SEQ ID No.2; the base sequence of the probe is SEQ ID No.3, and a fluorescence quencher is connected to the 3' end of the probe The 5' end of the group BHQ1 is connected with a fluorescent reporter group FAM. The inventors of the present invention have determined the specific oligonucleotide primer pair and probe of the present invention through a large amount of screening work, and established a stable PCR system to specifically and sensitively identify the authenticity of the red deer antler.

In one embodiment, the red antler real-time fluorescent PCR detection method of the present invention further includes the step of extracting the total DNA of the antler sample. In one embodiment, in the DNA extraction step, the extraction quality of the total DNA of the sample is tested by detecting the sequence of the D-loop region of the mitochondrial DNA of cervidae. In a preferred embodiment, the universal primer pair for detecting the mitochondrial DNA D-ring region sequence is made up of an upstream primer and a downstream primer, and the base sequence of the upstream primer is Luke-F: CATACGCAATYCTACGATCAATTCC (SEQ ID No.4), The base sequence of the downstream primer is Luke-R: GCTACTARGATTCAGAATAGGCATTG (SEQ ID No.5); the base sequence of the probe is Luke-P: TGGTCGGAATATYATGCTGCGTTGTTTGG (SEQ ID No.6), at the 3' of the probe There is a fluorescent quenching group BHQ2 connected to the end, and a fluorescent reporter group TAMRA connected to the 5' end. The PCR amplification conditions are 95° C., 10 min; 95° C., 15 s; 60° C., 1 min, 40 cycles.

According to another embodiment of the present invention, the present invention provides a kit for quickly identifying the authenticity of red deer antler, said kit comprising the specific oligonucleotide used for real-time fluorescent PCR method of the present invention to identify the authenticity of red deer antler Primer pairs as well as probes and instructions for use. In a preferred embodiment of the kit of the present invention, the pair of specific oligonucleotide primers of the present invention is designed according to the characteristics of the differences in the mitochondrial DNA D-loop region sequences of different cervidae species. In a preferred embodiment, the specific oligonucleotide primer pair that comprises in the kit is made up of upstream primer and downstream primer, and the base sequence of described upstream primer is SEQ ID No.1, and described downstream primer The base sequence of the probe is SEQ ID No.2; the base sequence of the probe contained in the kit is SEQ ID No.3, and a fluorescent quenching group BHQ1 is attached to the 3' end of the probe, and the 5' A fluorescent reporter group FAM is attached to the end. In a preferred embodiment, the kit further includes reagents for sample DNA extraction and reagents for PCR reaction. In a preferred embodiment, the instructions for use of the kit include a description of the PCR amplification conditions used to quickly identify the authenticity of the red deer antler. In a preferred embodiment, the PCR amplification conditions given in the instructions of the kit are: 95° C., 10 min; 95° C., 15 s; 60° C., 1 min, 40 cycles.

According to yet another embodiment of the present invention, the present invention provides the application of the specific oligonucleotide primer pair and probe of the present invention in identifying the authenticity of red deer antler. In a preferred embodiment according to the application of the present invention, the specific oligonucleotide primer pair is made up of an upstream primer and a downstream primer, the base sequence of the upstream primer is SEQ ID No.1, and the base sequence of the downstream primer is The base sequence is SEQ ID No.2; the base sequence of the probe is SEQ ID No.3, a fluorescent quencher group BHQ1 is connected to the 3' end of the probe, and a fluorescent reporter group is connected to the 5' end Regiment FAM. In another embodiment, the present invention also provides the application of the kit of the present invention in identifying the authenticity of red deer antler. Preferably, in the above-mentioned application of the present invention, the kit comprises the specific oligonucleotide primer pair and probe of the present invention.

The present invention uses the DNA of red deer antler as the detection basis, and according to the characteristics of differences in the mitochondrial DNA D-ring region sequences in different cervidae species, clones the red deer mitochondrial DNA D-ring region sequences, and designs primers and probes based on these sequences, Using real-time fluorescent PCR method to identify the authenticity of red deer antler.

Real-time fluorescent quantitative PCR is based on the conventional PCR method, adding fluorescently labeled probes or fluorescent dyes. With the accumulation of PCR products, the fluorescent signals emitted by the probes or dyes are enhanced, and the fluorescent monitoring system can receive the fluorescent signals. That is, every time a DNA strand is produced, a fluorescent molecule is formed, realizing the complete synchronization of the accumulation of fluorescent signals and the formation of PCR products. Therefore, the whole PCR reaction process can be monitored in real time, and the initial copy number of the sample to be tested can be finally detected, so that the components of the red deer antler to be tested can be detected.

The real-time fluorescent PCR detection method of the present invention adopts completely closed-tube detection, does not need PCR post-processing, and avoids cross-contamination and false positives. The method of the invention skillfully utilizes the high-efficiency DNA amplification of PCR technology, the specificity of nucleic acid hybridization, and the rapidity and sensitivity of fluorescence detection technology, and has the advantages of simple operation, time-saving and labor-saving, reliable, accurate and sensitive results, and the like. The real-time fluorescent PCR detection method of the present invention is simple, rapid, specific and sensitive, and is suitable for identifying the authenticity of red deer antler components in domestic and foreign markets.

Description of drawings

Fig. 1 is the real-time fluorescent PCR result showing the DNA extraction effect of the sample to be tested, wherein the universal primers of cervidae are used to detect SEQ ID No.4 and SEQ ID No.5 and probe SEQ ID No.6, wherein the sample of the fluorescence curve 1. Sika deer (1); 2. Sika deer (2); 3. Sika deer (3); 4. Sika deer (4); 5. Sika deer (5); 6. Sika deer (6); 7. Sika deer (7) ;8. Sika deer (8); 9. Sika deer (9); 10. Sika deer (10); 11. Sika deer (11); 12. Sika deer (12); 13. Sika deer (13); 14. Sika deer (14); 15. Sika deer (15); 16. Sika deer (16); 17. Sika deer (17); 18. Sika deer (18); 19. Sika deer (19); 20. Sika deer (20); 21. Sika deer (21); 22 .Sika deer (22); 23. Sika deer (23); 24. Red deer (1); 25. Red deer (2); 26. Red deer (3); 27. Red deer (4); 28. Red deer ( 5); 29. Red deer (6); 30. Red deer (7); 31. Fallow deer (1); 32. Fallow deer (2); 33. Blank control (ddH ₂ O).

Figure 2 shows the results of real-time fluorescent PCR specific identification of red deer antler, wherein specific oligonucleotide primers are used to detect SEQ ID No.1 and SEQ ID No.2 and probe SEQ ID No.3, wherein the fluorescence curve The samples are 1. Red deer (1); 2. Red deer (2); 3. Red deer (3); 4. Red deer (4); 5. Red deer (5); 6. Red deer (6) ;7. Red deer (7); 8. Sika deer (1); 9. Sika deer (2); 10. Sika deer (3); 11. Sika deer (4); 12. Sika deer (5); 13. Sika deer (6) ;14. Sika deer (7); 15. Sika deer (8); 16. Sika deer (9); 17. Sika deer (10); 18. Sika deer (11); 19. Sika deer (12); 20. Sika deer (13); 21. Sika deer (14); 22. Sika deer (15); 23. Sika deer (16); 24. Sika deer (17); 25. Sika deer (18); 26. Sika deer (19); 27. Sika deer (20); 28 .Sika deer (21); 29. Sika deer (22); 30. Sika deer (23); 31. Fallow deer (1); 32. Fallow deer (2); 33. Blank control (ddH ₂ O).

Figure 3 takes red deer antler as an example to identify the results of the sensitivity of the combination of specific primers and probes for red deer antler, in which 1-7 is the concentration of the DNA solution for real-time fluorescent PCR amplification, which are 10ng/μL, 1ng/μL, μL, 100pg/μL, 10pg/μL, 1pg/μL, 0.1pg/μL and blank control, repeated 3 times.

Figure 4 takes red antler and sika antler as examples to determine the relative detection limit of red antler-specific primers and probe combinations, where 1-5 is the red antler DNA diluted 10 times with sika antler DNA to make its PCR reaction system The red antler DNA content in the red antler was 10ng, 1ng, 100pg, 10pg, 1pg respectively, repeated three times, and then the results were tested.

Figure 5 is the test results of commercially available velvet antler samples, wherein fluorescence curve 1 is the red deer positive control substance, fluorescence curves 2-21 are commercially available red deer antler samples (1)-(20), and fluorescence curves 22-38 are commercially available red deer antler samples in sequence Red deer antler samples (1)-(16) and blank control (sterile water).

Detailed ways

The present invention is further described by means of examples, but the present invention is not limited only to the following examples.

Example 1

The inventors of the present invention cloned and sequenced the mitochondrial DNA D-loop region sequence of red deer for the first time by PCR.

This example is the sequence of the red deer mitochondrial DNA D-loop region obtained by PCR cloning and sequencing.

According to the difference of the mitochondrial DNA D-loop sequence in different cervids, the upstream and downstream primers were designed to amplify the red deer mitochondrial DNA D-loop sequence. The elk PCR product was purified and recovered according to the instructions of the Wizard GelExtraction Kit (Promega, USA). According to the instructions of the TaKaRa pMD19-T Vector kit (TaKaRa, Japan), the purified product was connected to pMD19-T Vector, the connection system was 10 μL, and the reaction components were: pMD19-T Vector 1 μL, PCR product 2 μL, ddH ₂ O 2 μL, Solution I 5μL, set positive and negative controls at the same time. Place the connection system at room temperature (22-37° C.) for 30 minutes and place it on ice immediately after the reaction. Add the ligation product to 50 μL TOP competent cells, flick and mix well, bathe in ice for 30 minutes, heat shock at 42°C for 90 seconds, immediately place on ice for 2 minutes, then add 800 μL of sterilized brain heart infusion, 37°C, 200r /min shaking culture for 1h. Centrifuge at a low speed of 5000r/min for 1min, discard 600μL of supernatant, mix the precipitate, take 100μL of the mixture and spread it on the nutrient agar plate treated with Amp+, X-Gal and IPTG, culture overnight at 37℃ and place in a refrigerator at 4℃ for 4h. Pick a single colony white spot, and culture it overnight at 37°C with shaking at 200r/min in the brain heart infusion solution containing Amp at a final concentration of 200 μg/mL.

(1) Identification of positive clones: Pick a single white clone and carry out colony PCR reaction. Take a single colony as a template, add water to make up to 25 μL. The reaction program is: 94°C for 5min, 94°C for 30s, 55°C for 30s, 72°C for 1min, 40 cycles. PCR products were identified by agarose gel electrophoresis.

(2) Sequencing: After the positive clone was determined, the colony was picked and cultured in 5 μL of brain heart infusion solution containing a final concentration of 200 μg/mL Amp overnight at 37°C with shaking at 200 r/min. Take 1mL of the bacterial liquid and send it to the biological company for sequencing identification.

The sequence of the mitochondrial DNA D-loop region of red deer obtained by PCR cloning and sequencing is as follows:

AGACTCAAGG AAGAAGCCAT AGCCCCACTA TCAACACCCA

AAGCTGAAGT TCTATTTAAA CTATTCCCTG ACGCTTATTA

ATATAGTTCC ATAAAAATCA AGAACTTTAT CAGTATTAAA

TTTCCAAAAA ATCT-AATAT TTTAATACAG CTTTCTACTC

AACATCCAAT TTACATTTTA CGTCCTACTA ATTACACAGC

AAAACACGTG ATATAACCTT ATGCGCTTGT AGTACATAAA

ATCAATGTGC TAGAACACGC ATGTATAACA GCACATGAG

TTAGTGTATA GGACATATTA TGTATAATAG TACATAAATC

AATGTATTAG GACATATTAT GTATAATAGT ACATTATATT

ATATGCCCCA TGCATAAACC ATGGTTACA (SEQ ID No. 7).

Example 2

In this embodiment, the extraction quality of the total DNA of the sample is tested by using a universal primer pair and a probe.

By detecting the sequence of the mitochondrial DNA D-loop region, the quality of the total DNA extraction from the sample can be tested. Taqman fluorescent probe method: TaqMan technology is a technology for real-time fluorescent quantitative detection of single-tube PCR products. In an ordinary PCR amplification system, a dual fluorescent-labeled probe that is specifically complementary to the target gene sequence is added to utilize the fluorescent signal. Accumulate and monitor the entire PCR process in real time, and finally perform quantitative analysis on the unknown template through the standard curve. Quantitative steps: ① determine the CT value (C represents the cycle number (Cycle), T represents the fluorescence threshold value (Threshold), that is, the number of cycles experienced when the fluorescence signal in each reaction tube reaches the set threshold value; ② use the standard curve Quantitative determination is carried out to unknown sample.After obtaining the CT value of unknown sample, calculate the initial copy number of this sample from standard curve.There is linear relationship between the logarithm of the CT value of each template and the initial copy number of this template, namely The higher the initial copy number, the smaller the CT value. The reaction system is: Mastermix 12.5 μL; probe (10 μM) 0.5 μL; upstream and downstream primers (10 μM) each 0.5 μL; template DNA 5 μL; add ddH ₂ O to the total volume 25 μ L. The reaction program is 95 ° C for 10 min; 95 ° C for 15 s; 60 ° C for 1 min, 40 cycles.

The universal primer pair used in this embodiment to detect the total DNA extraction quality of red deer antler consists of an upstream primer and a downstream primer. The base sequence of the upstream primer is SEQ ID No.4, and the base sequence of the downstream primer is The sequence is SEQ ID No.5; the base sequence of the probe used is SEQ ID No.6, a fluorescent quenching group BHQ2 is connected to the 3' end of the probe, and a fluorescent reporter group is connected to the 5' end TAMRA.

In this embodiment, 7 red deer samples, 23 sika deer samples, and 2 fallow deer samples were detected.

The main detection instruments used:

Micropipettes (10 μL, 100 μL, 1000 μL, Eppendorf), fluorescent quantitative PCR instrument (ABI 7700 Applied Biosystems, USA), high-speed desktop centrifuge (Pico17 Thermo), high-speed pulverizer (IKA-WEARKE GERMANY), nucleic acid and protein analyzer (DYY-6C Beijing Liuyi Instrument Factory), etc.

Detection of the main reagents:

Chloroform and isopropanol were respectively purchased from Beijing Liuhetong Company; CTAB lysate (20g/L CTAB, 1.4mol/L NaCl, 0.1mol/L Tris, 0.02mol/L Na ₂ -EDTA), CTAB precipitation solution (5g/L LCTAB, 0.04mol/L NaCl), 1.2mol/L NaCl were self-prepared in this experiment; Fast StartUniversal Probe MasterMix (Rox) was purchased from Roche; primers and probes were synthesized by Shanghai Aoke Biotechnology Co., Ltd., etc.

The main steps of detection:

1 DNA extraction

The antler samples to be tested are: 7 parts of red antler, 23 parts of sika antler, and 2 parts of fallow antler.

Weigh 0.1g velvet antler powder into a clean 2.0mL centrifuge tube, add 1.5mL CTAB lysate, 65℃ for 2h, mix several times in between; 8000rpm 15min, take 1mL supernatant into a clean 2.0mL centrifuge tube , add 700μL chloroform, mix vigorously for 30s, 14500rpm 10min, take 650μL supernatant to a clean 2.0mL centrifuge tube, add 1300μL CTAB precipitation solution, mix vigorously for 30s, let stand at room temperature for 1h; 14500rpm 20min, discard the supernatant , add 350μL 1.2M NaCl, shake vigorously for 30s, then add 350μL chloroform, mix vigorously for 30s, 14500rpm for 10min; take 320μL of supernatant, add 0.8 times the volume of isopropanol, after mixing, -20℃ for 1h, 14500rpm for 20min , discard the supernatant, add 500 μL of 70% ethanol, mix well, 14500 rpm for 20 min, discard the supernatant, air dry, add 100 μL ddH ₂ O to dissolve, store at 4°C for later use.

2 Primers and probes used for real-time fluorescent PCR detection

The primer sequences are SEQ ID No.4 and SEQ ID No.5, the probe sequence is SEQ ID No.6, a fluorescent quencher group BHQ2 is attached to the 3' end, and a fluorescent reporter group TAMRA is attached to the 5' end.

3 Real-time fluorescent PCR reaction system:

Fast Start Universal Probe MasterMix (Rox) 12.5μL

Probe (10 μM) 0.5 μL

Upstream primer (10 μM) 0.5 μL

Downstream primer (10 μM) 0.5 μL

Template DNA 5 μL

Add _ddHO to a total volume of 25 μL

Note: Set up a corresponding blank control for each PCR test (replace the DNA template with ultrapure water prepared in the reaction system, and check whether the reagent is contaminated);

4 Real-time fluorescent PCR reaction parameters:

95℃ 10min

95℃ 15s

60℃ 1min

40 loops.

Note: Different instruments should adjust PCR reagents and reaction parameters appropriately.

As shown in Figure 1, when the DNA of the samples to be tested was amplified with the general primers of cervidae, fluorescence amplification curves could appear above the baseline, indicating that the DNA of all the samples to be tested was successfully extracted.

Example 3

In this example, the specificity and sensitivity of the red deer primer pair and probe were verified through the following tests.

The specificity and detection sensitivity of red deer primer and probe combinations can be determined by detecting the mitochondrial DNA D-loop region sequence. The reaction system was: Fast Start Universal Probe MasterMix (Rox) 12.5 μL; probe (10 μM) 0.5 μL; upstream and downstream primers (10 μM) 0.5 μL each; template DNA 5 μL; add ddH ₂ O to a total volume of 25 μL. The reaction program was 95°C for 10 min; 95°C for 15 s; 60°C for 1 min, 40 cycles.

The primers and probe sequences used to identify the authenticity of red deer antler are: the primer sequences are SEQ ID No.1 and SEQ ID No.2, the probe sequence is SEQ ID No.3, and a fluorescence quencher is connected to the 3' end The 5' end of the group BHQ1 is connected with a fluorescent reporter group FAM.

The main detection instruments used:

Micropipettes (10 μL, 100 μL, 1000 μL, Eppendorf), fluorescent quantitative PCR instrument (ABI 7700 Applied Biosystems, USA)), high-speed desktop centrifuge (Pico17 Thermo), high-speed pulverizer (IKA-WEARKE GERMANY), nucleic acid protein analysis Instrument (DYY-6C Beijing Liuyi Instrument Factory), etc.

Detection of the main reagents:

Chloroform and isopropanol were respectively purchased from Beijing Liuhetong Company; CTAB lysate (20g/L CTAB, 1.4mol/L NaCl, 0.1mol/L Tris, 0.02mol/L Na ₂ -EDTA), CTAB precipitation solution (5g/L LCTAB, 0.04mol/L NaCl), 1.2mol/L NaCl were self-prepared in this experiment; Fast StartUniversal Probe MasterMix (Rox) was purchased from Roche; primers and probes were synthesized by Shanghai Aoke Biotechnology Co., Ltd., etc.

The main steps of detection:

1 DNA extraction

Test samples: (1) 7 parts of red deer antler, 23 parts of sika antler, and 2 parts of fallow antler for specific analysis; (2) Taking red deer as an example, the extracted DNA solution was diluted to 10 ng/μL with sterile water , 1ng/μL, 100pg/μL, 10pg/μL, 1pg/μL, and 0.1pg/μL concentrations are used to analyze the absolute detection limit of primer and probe combinations; (3) Taking red deer and sika deer as examples, use sika deer DNA The red deer DNA was diluted 10 times, so that the red deer DNA content in the PCR reaction system was 10ng, 1ng, 100pg, 10pg, 1pg, respectively, so as to determine the relative detection limit of the red deer-specific primer and probe combination.

Weigh 0.1g of sample powder into a clean 2.0mL centrifuge tube, add 1.5mL of CTAB lysate, 65°C for 2h, mix several times in between; 8000rpm for 15min, take 1mL supernatant into a clean 2.0mL centrifuge tube , add 700μL chloroform, mix vigorously for 30s, 14500rpm 10min, take 650μL supernatant to a clean 2.0mL centrifuge tube, add 1300μL CTAB precipitation solution, mix vigorously for 30s, let stand at room temperature for 1h; 14500rpm 20min, discard the supernatant , add 350μL 1.2M NaCl, shake vigorously for 30s, then add 350μL chloroform, mix vigorously for 30s, 14500rpm for 10min; take 320μL of supernatant, add 0.8 times the volume of isopropanol, after mixing, -20℃ for 1h, 14500rpm for 20min , discard the supernatant, add 500 μL of 70% ethanol, mix well, 14500 rpm for 20 min, discard the supernatant, air dry, add 100 μL ddH ₂ O to dissolve, store at 4°C for later use.

2 Primers and probes used for real-time fluorescent PCR detection

The primer sequences are SEQ ID No.1 and SEQ ID No.2;

The probe sequence is SEQ ID No.3, a fluorescent quencher group BHQ1 is attached to the 3' end, and a fluorescent reporter group FAM is attached to the 5' end.

3 Real-time fluorescent PCR reaction system:

Fast Start Universal Probe MasterMix (Rox) 12.5μL

Probe (10 μM) 0.5 μL

Upstream primer (10 μM) 0.5 μL

Downstream primer (10 μM) 0.5 μL

Template DNA 5 μL

Add _ddHO to a total volume of 25 μL

Note: Set up a corresponding blank control for each PCR test (replace the DNA template with ultrapure water prepared in the reaction system, and check whether the reagent is contaminated);

4 Real-time fluorescent PCR reaction parameters:

95℃ 10min

95℃ 15s

60℃ 1min

40 loops.

Note: Different instruments should adjust PCR reagents and reaction parameters appropriately.

As shown in Figure 2, when real-time fluorescent PCR was used to specifically identify the mitochondrial DNA D-loop region sequence of red deer, seven red deer samples showed typical amplification curves, while other samples: 23 sika deer, 2 fallow No amplification curves appeared in deer and blank control (ddH ₂ O), which fully demonstrated that the primer probes designed in this experiment had better specificity for red deer samples.

In order to determine the absolute detection limit of red deer-specific primer and probe combinations, taking red deer as an example, the extracted DNA solution was diluted with sterile water to 10 ng/μL, 1 ng/μL, 100 pg/μL, 10 pg/μL, The concentrations of 1pg/μL and 0.1pg/μL were subjected to real-time fluorescent PCR amplification according to the above conditions respectively, and the results are shown in Figure 3. When the red deer DNA concentration was 10ng/μL, 1ng/μL, 100pg/μL, 10pg/μL, 1pg/μL, there were specific amplification curves, but when the concentration dropped below 1pg/μL, no specific amplification curve appeared. The experimental results show that the established real-time fluorescent PCR detection method can detect the content of red deer components to 1pg/μL.

Taking red deer and sika deer as examples, use sika deer DNA to dilute the red deer DNA by 10 times, so that the red deer DNA content in the PCR reaction system is 10ng, 1ng, 100pg, 10pg, 1pg, respectively, and perform real-time fluorescent PCR according to the above conditions Amplified to determine the relative detection limit of red deer-specific primer and probe combinations (see Figure 4 for results). The experimental results showed that the relative detection limit of the method for detecting red deer components was 10 pg.

Example 4

The inventors of the present invention verified the commercially available deer antler samples through the following tests.

Select 36 antler samples (samples provided by our laboratory), including 20 red antler samples and 16 sika antler samples, for real-time fluorescent PCR reaction to determine whether the established real-time fluorescent PCR method is feasible.

As shown in Figure 5, when using real-time fluorescent PCR to detect the mitochondrial DNA D-loop region sequence, the red deer positive control substance and 20 commercial red deer antler samples had typical fluorescence amplification curves above the baseline, while the other 16 sika deer The amplification curves of the antler sample and the blank control were at the baseline position, indicating that the method can effectively detect the components of red deer antler.

While specific embodiments of the present invention have been described, those skilled in the art will recognize that various changes and modifications can be made therein without departing from the scope or spirit of the invention. Accordingly, it is intended that the present invention cover all such changes and modifications that come within the scope of the claims and their equivalents.

Claims (8)

1. the specificity nucleotide primer that is used for the real time fluorescent PCR method discriminating cervus elaphus linnaeus true and false is to reaching probe; Said primer is to designing based on the nucleic acid shown in the SEQ ID No.7 with probe; Wherein said primer is to being made up of upstream primer and downstream primer; The base sequence of said upstream primer is SEQ ID No.1, and the base sequence of said downstream primer is SEQ IDNo.2, and the base sequence of said probe is SEQ ID No.3; 3 ' end at probe is connected with a fluorescent quenching group B HQ1, and 5 ' end is connected with a fluorescence report group FAM.

2. be used for real time fluorescent PCR method and differentiate the test kit of the cervus elaphus linnaeus true and false, said test kit comprises that specific oligonucleotide primer according to claim 1 is to reaching probe.

3. test kit according to claim 2; Said test kit further comprise other universal primer to and probe; Said other universal primer is to being that the upstream primer of SEQ ID No.4 and downstream primer that base sequence is SEQ ID No.5 are formed by base sequence; The base sequence of said probe is SEQ ID No.6, holds at 3 ' of probe to be connected with a fluorescent quenching group B HQ2, and 5 ' end is connected with a fluorescence report group TAMRA.

4. the real-time fluorescence PCR detection method of cervus elaphus linnaeus real and fake discrimination; Wherein said real time fluorescent PCR method is a Taqman fluorescent probe method, and said method comprises uses the described specific oligonucleotide primer of claim 1 to reaching probe or test kit according to claim 2.

5. real-time fluorescence PCR detection method according to claim 4 also comprises the step of extracting the pilose antler sample total DNA.

6. real-time fluorescence PCR detection method according to claim 5; Through using universal primer to reaching the extraction quality of probe in detecting sample DNA; Said universal primer is to being that the upstream primer of SEQ ID No.4 and downstream primer that base sequence is SEQ ID No.5 are formed by base sequence; The base sequence of said probe is SEQ ID No.6, holds at 3 ' of probe to be connected with a fluorescent quenching group B HQ2, and 5 ' end is connected with a fluorescence report group TAMRA.

7. the described specific oligonucleotide primer of claim 1 is to reaching the application of probe in differentiating the cervus elaphus linnaeus true and false.

8. claim 2 or the 3 described test kits application in differentiating the cervus elaphus linnaeus true and false.

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