CN106399521A - Multiple-PCR primer combination for authenticating sika deer and product genders thereof through one-step method, authentication method of multiple-PCR primer combination and kit - Google Patents

Abstract

The invention provides a multiple-PCR primer combination for authenticating sika deer and product genders thereof through a one-step method. The multiple-PCR primer combination is composed of a first primer pair and a second primer pair; the first primer pair is composed of an upstream primer with a base sequence shown in SEQ ID No.1 and a downstream primer with a base sequence shown in SEQ ID No.2; the second primer pair is composed of an upstream primer with a base sequence shown in SEQ ID No.3 and a downstream primer with a base sequence shown in SEQ ID No.4. The primer combination is good in specificity and high in amplification efficiency, can be well applied to authentication of the sika deer and the product genders thereof, avoids unnecessary economic losses for consumers, lays a good foundation for an authentication method of the sika deer and the product genders thereof, fills up relevant technological blanks and has pioneering significance.

Description

Primer combination of multiplex PCR for one-step identification of the sex of sika deer and its products and its Identification method, kit

technical field

The invention relates to the field of gender identification of sika deer and its products, in particular to a multiplex PCR primer combination for one-step identification of the sex of sika deer and its products, an identification method and a kit.

Background technique

Deer products refer to some tissues and organs of deer, mainly including: deer antler, deer heart, deer kidney, deer fetus, deer tendon, deer penis, deer tail, venison and deer blood, etc. Since most deer products are precious Chinese medicinal materials, The price is expensive, so it is common to see fake and inferior products pretending to be expensive deer products in the market.

In particular, compared with other deer products, sika deer products have more significant health functions. The main health functions are: strengthening Yuanyang, replenishing qi and blood, benefiting essence, and strengthening bones and muscles. Enhance human immunity, improve the body's working ability, improve sleep and diet, reduce muscle fatigue, promote children's growth and development, blood circulation and wound healing. Tonify the kidney and strengthen yang, tonify deficiency and generate essence, treat fatigue due to fatigue, insufficiency of essence and blood, irregular menstruation, blood deficiency, cold blood, uterine deficiency and cold, metrorrhagia and vaginal discharge, long-term infertility, etc., treat fatigue and emaciation, spirit Fatigue, dizziness, deafness, dark eyes, sore waist and knees, impotence, ejaculation, etc. Therefore, sika deer and its products are deeply favored by consumers.

However, due to the different genders of sika deer and their products, the price is also very different. The price of male sika deer products on the market is higher than that of female sika deer products. However, in order to maximize profits, some businesses often use female sika deer products. To pretend to be male sika deer products for sale, so that many consumers suffer economic losses, and there is no more general method for identifying the gender of sika deer and its products in the prior art.

In view of the existence of the above problems, the present invention is proposed.

Contents of the invention

The first object of the present invention is to provide a primer combination for multiple PCR for one-step identification of the sex of sika deer and its products. The primer combination has good specificity and high amplification efficiency, and can be well applied to the sex of sika deer and its products. The identification avoids unnecessary economic losses for consumers, lays a good foundation for the identification method of the sex of sika deer and its products, fills the gap in related technologies, and has pioneering significance.

The second object of the present invention is to provide a one-step identification method for identifying the sex of sika deer and its products. The identification method is easy to operate and has high identification efficiency, and the identification method has high sensitivity, specificity, accuracy and application It can be well used in the gender identification of sika deer and its products, which makes up for the technical gap in the gender identification of sika deer and its products. This identification method is very suitable for wide-scale popularization and application.

The third object of the present invention is to provide a kit including the above primer combination, which is easy to use and can extract reagents through corresponding detection methods.

In order to realize the above-mentioned purpose of the present invention, special adopt following technical scheme:

The invention provides a primer combination for multiplex PCR for one-step identification of the sex of sika deer and its products, consisting of a first primer pair and a second primer pair, the first primer pair is represented by SEQ ID No.1 The upstream primer of the base sequence has the downstream primer of the base sequence shown in SEQ ID No.2, and the second primer pair is composed of the upstream primer with the base sequence shown in SEQ ID No.3 and has SEQ ID No.4 The downstream primer composition of the base sequence shown.

Typically, each additional nucleotide increases primer specificity by a factor of 4, so that the shortest primer length for most applications is 18 nucleotides. The upper limit of primer length is not very important, mainly related to reaction efficiency.

In the sequence of general primers, the G+C content is generally 40%-60%, and the GC content and Tm value of a pair of primers should be coordinated. The G+C contents of the primers in the primer combination of the present invention are all controlled within an appropriate range, and the bases are randomly distributed, thus having a moderate melting temperature and facilitating amplification.

In a word, the primer combination of the present invention has good specificity and high amplification efficiency, and can be well applied to identify the sex of sika deer and its products. There is no record in the prior art about the combination of primers for multiplex PCR of product gender, and the present invention is still the first. By adopting the combination of primers to identify the sex of sika deer-related products, unpredictable economic losses for consumers can be avoided, which shows that its practical significance is also great.

The present invention also provides a method for one-step identification of the sex of sika deer and its products by using the above primer combination, which specifically includes the following steps:

The DNA of the sample to be tested is extracted as a template, the first primer pair and the second primer pair are used to perform PCR amplification on the DNA, and after the amplification reaction is completed, the PCR product is subjected to electrophoresis analysis.

Preferably, the reaction system of PCR amplification is:

Preferably, the concentration of the first primer pair is controlled between 10-20 μM, and the concentration of the second primer pair is controlled between 10-20 μM.

Taq DNA polymerase has good thermal stability and can maintain high catalytic activity under the high temperature conditions of PCR cycles. dNTP is the raw material for DNA amplification, and the premix also contains Mg ²⁺ , which belongs to Taq DNA polymerase The concentration of the coenzyme directly affects the activity and specificity of the enzyme.

The present invention relates to the amount of each component in the same system or proportioning, which should be regarded as the limitation of the proportional relationship between the relevant components, rather than the limitation of the absolute value of the respective amounts. The proportional relationship between the components can be appropriately Change.

Preferably, in the above-mentioned method for identifying the sex of sika deer and its products, the PCR amplification reaction program is 94-95°C pre-denaturation for 2-3 minutes; 94-95°C denaturation for 2-3 minutes, 55-60°C annealing for 20-30s, 72- Extend at 75°C for 40-60s, and after a total of 35-45 cycles, extend at 72-75°C for 6-8min.

With the gradual progress of the reaction, the enzyme will be gradually inactivated, dNTP and other raw materials will be gradually consumed, and the amplification of some non-specific products will increase accordingly. Therefore although along with the increase of reaction cycle number, product can increase, cycle number should still not be too much, thereby the present invention is limited to 35-45 cycle.

Preferably, when performing electrophoresis analysis, the analysis method for the result is: if a band of 335-340bp appears in the DNA of the sample to be tested, it is female; if a band of 335-340bp and 460-470bp appears simultaneously, it is male. It can be observed at a glance through the electrophoresis graph, the operation is very convenient, and the identification method of the present invention can be well operated without the guidance of a professional, which is convenient and quick.

Wherein, it should be noted that, because all male and female deer all contain the gene fragment of the band of 335-340bp, so the first pair of primers is used to amplify the gene fragment of the band of 335-340bp, and the second pair of primers It is the gene fragment used to amplify the 460-470bp band.

Preferably, the above-mentioned electrophoresis analysis adopts an agarose gel electrophoresis analysis method, and the agarose concentration is controlled between 1.5-2%;

Preferably, the electrophoresis voltage is controlled at a constant voltage of 120-130V, and the electrophoresis time is 20-30min;

Preferably, before the electrophoresis analysis, the agarose gel and the TAE buffer are mixed according to a mass volume ratio of (1.5-2):100.

In addition, it is better to heat the mixture of agarose gel and TAE buffer solution in a microwave oven for a period of time to completely melt the agarose. The heating time is about 2 minutes. Be careful not to cause the solution to boil.

Finally, the DNA extraction method of the sample to be tested is generally carried out as follows:

1) To process the material, take 30 mg of deer product, smash it into a cell suspension, centrifuge at 10,000 rpm for 1 min, pour off the supernatant, add 200 μL of buffer GA, and oscillate until completely suspended;

2) Add 20 μL of Proteinase K solution, mix well, place at 56°C until the tissue dissolves, and briefly centrifuge to remove water droplets on the inner wall of the tube cap;

3) Add 200 μL buffer GB, mix thoroughly by inverting, place at 70°C for 10 minutes, the solution should become clear, and briefly centrifuge to remove water droplets on the inner wall of the tube cap;

4) Add 200 μL of absolute ethanol, shake and mix well for 15 seconds, at this time flocculent precipitation may appear, briefly centrifuge to remove water droplets on the inner wall of the tube cap;

5) Add the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3, centrifuge at 12000 rpm for 30 sec, and pour off the waste liquid;

6) Add 500 μL buffer solution GD to the adsorption column, centrifuge at 12000 rpm for 30 sec, and pour off the waste liquid;

7) Add 600 μL of buffer solution PW to the adsorption column, centrifuge at 12000 rpm for 30 sec, and discard the waste liquid;

8) Repeat operation step 7);

9) Centrifuge at 12000rpm for 2min, pour off the waste liquid, and place the adsorption column at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material;

10) Transfer the adsorption column into a clean centrifuge tube, add 50-200 μL eluent TE dropwise to the middle part of the adsorption membrane, and place it at room temperature for 2-5 minutes;

11) Centrifuge at 12000rpm for 2min, and collect the solution into a centrifuge tube;

12) DNA can be stored at 2-8°C, and if it needs to be stored for a long time, it can be placed at -20°C.

According to the above operation steps, the DNA extraction of deer products can be completed as a template.

Preferably, the embodiment of the present invention also includes a kit for identifying the sex of sika deer and its products using the above primer combination. The kit is reasonably prepared, simple to prepare, objective and accurate in judging the results, and can be used well. And the kit can be used for early identification of the sex of sika deer besides being used. The method of early identification of the sex of sika deer is: when the deer fetus is not born, the peripheral blood of the doe can be used to detect the sex of the fetus, so that through the early sex Detection, by knowing the sex of the sika deer in advance, is conducive to the development of other follow-up work.

Compared with prior art, the beneficial effect of the present invention is:

(1) The present invention is used for the one-step identification multiplex PCR primer combination of sika deer and its product sex, and specificity is good, and amplification efficiency is high, can be well applied to the identification of sika deer and its product sex, has avoided consumer's misuse The necessary economic loss has laid a good foundation for the identification method of the sex of sika deer and its products, filled the relevant technical gap, and has pioneering significance;

(2) The identification method of the present invention is easy to operate and has high identification efficiency, and the identification method has high sensitivity, specificity, accuracy and applicability, and can be well used for the identification of the sex of sika deer and its products;

(3) The accuracy rate of the identification method of the present invention can reach 100%, which has strong practical significance, is scientific and effective, and maintains a good reputation for cracking down on unscrupulous merchants who use female sika deer products to pretend to be male sika deer products for sale. market order has a more positive effect.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art.

Fig. 1 is a scanning diagram of the results of agarose gel electrophoresis in Example 2 of the present invention.

detailed description

Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only for illustrating the present invention, and should not be considered as limiting the scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

Example 1

A method for identifying the sex of sika deer and its products, comprising the steps of:

1) Extraction of template DNA;

2) using the first primer pair and the second primer pair to carry out PCR amplification on the DNA extracted in step 1) as a template;

3) Analyzing the PCR product after the amplification reaction in step 2) by electrophoresis.

Example 2

A method for identifying the sex of sika deer and its products, comprising the steps of:

1. Primer preparation:

The primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., in which:

First primer pair ZFX:

Upstream primer: 5'CCAGTGTTTGCATTGTGACC3';

Downstream primer: 5'TCCTGCCACTGTGTGTCTTC3';

Second primer pair ZFY:

Upstream primer: 5'TACAGCCACAGGAGCCAAAC3';

Downstream primer: 5'AAAAGGCAATCAGCAAAGGA3';

2. Extraction steps of sika deer and its product DNA:

1) Take 30 mg of deer product, crush it into a cell suspension, centrifuge at 10,000 rpm for 1 min, pour off the supernatant, add 200 μL of buffer GA, and oscillate until completely suspended;

2) Add 20 μL of Proteinase K solution, mix well, place at 56°C until the tissue dissolves, and briefly centrifuge to remove water droplets on the inner wall of the tube cap;

3) Add 200 μL buffer GB, mix thoroughly by inverting, place at 70°C for 10 minutes, the solution should become clear, and briefly centrifuge to remove water droplets on the inner wall of the tube cap;

4) Add 200 μL of absolute ethanol, shake and mix well for 15 seconds, at this time flocculent precipitation may appear, briefly centrifuge to remove water droplets on the inner wall of the tube cap;

5) Add the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3, centrifuge at 12000 rpm for 30 sec, and pour off the waste liquid;

6) Add 500 μL buffer solution GD to the adsorption column, centrifuge at 12000 rpm for 30 sec, and pour off the waste liquid;

7) Add 600 μL of buffer solution PW to the adsorption column, centrifuge at 12000 rpm for 30 sec, and discard the waste liquid;

8) Repeat operation step 7);

9) Centrifuge at 12000rpm for 2min, pour off the waste liquid, and place the adsorption column at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material;

10) Transfer the adsorption column into a clean centrifuge tube, add 50-200 μL eluent TE dropwise to the middle part of the adsorption membrane, and place it at room temperature for 2-5 minutes;

11) Centrifuge at 12000rpm for 2min, and collect the solution into a centrifuge tube;

12) DNA can be stored at 2-8°C, and if it needs to be stored for a long time, it can be placed at -20°C.

3. PCR amplification

1) PCR reaction system:

The primer concentration is between 10-20μM;

2) Amplification reaction program:

Pre-denaturation at 94°C for 3min; denaturation at 94°C for 3min, annealing at 60°C for 30s, extension at 72°C for 40s, 45 cycles, and finally extension at 72°C for 6min;

4. Detection by agarose gel electrophoresis

2% agarose gel was used for detection, the voltage was constant at 120V, and the electrophoresis time was 27 minutes.

5. Result analysis and judgment

After the electrophoresis is over, take out the gel and observe the electrophoresis results in the imager. See Figure 1 for the specific electrophoresis results. If a band of 335bp is amplified, it is female. If the bands of 335bp and 460bp appear at the same time, it is male.

Example 3

A method for identifying the sex of sika deer and its products, comprising the steps of:

1. Primer preparation:

The primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., in which:

First primer pair ZFX:

Upstream primer: 5'CCAGTGTTTGCATTGTGACC3';

Downstream primer: 5'TCCTGCCACTGTGTGTCTTC3';

Second primer pair ZFY:

Upstream primer: 5'TACAGCCACAGGAGCCAAAC3';

Downstream primer: 5'AAAAGGCAATCAGCAAAGGA3';

2. Extraction steps of sika deer and its product DNA:

1) Take 30 mg of deer product, crush it into a cell suspension, centrifuge at 10,000 rpm for 1 min, pour off the supernatant, add 200 μL of buffer GA, and oscillate until completely suspended;

2) Add 20 μL of Proteinase K solution, mix well, place at 56°C until the tissue dissolves, and briefly centrifuge to remove water droplets on the inner wall of the tube cap;

3) Add 200 μL buffer GB, mix thoroughly by inverting, place at 70°C for 10 minutes, the solution should become clear, and briefly centrifuge to remove water droplets on the inner wall of the tube cap;

4) Add 200 μL of absolute ethanol, shake and mix well for 15 seconds, at this time flocculent precipitation may appear, briefly centrifuge to remove water droplets on the inner wall of the tube cap;

5) Add the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3, centrifuge at 12000 rpm for 30 sec, and pour off the waste liquid;

6) Add 500 μL buffer solution GD to the adsorption column, centrifuge at 12000 rpm for 30 sec, and pour off the waste liquid;

7) Add 600 μL of buffer solution PW to the adsorption column, centrifuge at 12000 rpm for 30 sec, and discard the waste liquid;

8) Repeat operation step 7);

9) Centrifuge at 12000rpm for 2min, pour off the waste liquid, and place the adsorption column at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material;

10) Transfer the adsorption column into a clean centrifuge tube, add 50-200 μL eluent TE dropwise to the middle part of the adsorption membrane, and place it at room temperature for 2-5 minutes;

11) Centrifuge at 12000rpm for 2min, and collect the solution into a centrifuge tube;

12) DNA can be stored at 2-8°C, and if it needs to be stored for a long time, it can be placed at -20°C.

3. PCR amplification

1) PCR reaction system:

The primer concentration is between 10-20μM;

2) Amplification reaction procedure:

Pre-denaturation at 95°C for 2 minutes; denaturation at 95°C for 2 minutes, annealing at 55°C for 20 seconds, extension at 75°C for 1 minute, and 35 cycles, and finally extension at 75°C for 8 minutes;

4. Detection by agarose gel electrophoresis

The agarose gel and TAE buffer were mixed according to the mass volume ratio of 2:100, and the mixture was heated in a microwave oven for 2 min until the agarose was completely melted. Be careful not to boil the solution, add 1 μl of nucleic acid dye, mix carefully to prevent air bubbles, pour into the gel maker with the comb and glue tank already placed, so that the surface is smooth and there are no impurities, bubbles, etc. inside. Place at room temperature or 4°C for 30-40 minutes until the colloid solidifies, remove the comb, and test the prepared 1% agarose gel with a constant voltage of 130V and electrophoresis time of 20 minutes.

5. Result analysis and judgment

After the electrophoresis is over, take out the gel and observe the electrophoresis results in the imager. If a band of 340bp is amplified, it is female. If the bands of 340bp and 470bp appear at the same time, it is male.

Example 4

A method for identifying the sex of sika deer and its products, comprising the steps of:

The 1st, 2nd steps are substantially identical with embodiment 2;

3. PCR amplification

1) PCR reaction system:

The primer concentration is between 10-20μM;

2) Amplification reaction program:

Pre-denaturation at 94°C for 3min; denaturation at 94°C for 3min, annealing at 60°C for 25s, extension at 72°C for 50s, 45 cycles, and finally extension at 72°C for 7min;

4. Detection by agarose gel electrophoresis

The agarose gel and TAE buffer were mixed at a mass volume ratio of 1.5:100, and the mixture was heated in a microwave oven for 2 min until the agarose was completely melted. Be careful not to boil the solution, add 1 μl of nucleic acid dye, mix carefully to prevent air bubbles, pour into the gel maker with the comb and glue tank already placed, so that the surface is smooth and there are no impurities, bubbles, etc. inside. Place at room temperature or 4°C for 30-40 minutes until the colloid solidifies, remove the comb, and test the prepared 1.5% agarose gel with a constant voltage of 125V, and the electrophoresis time is 30 minutes.

5. Result analysis and judgment

After the electrophoresis is over, take out the gel and observe the electrophoresis results in an imager. If a band of 335bp is amplified, it is female, and if bands of 335bp and 460bp appear simultaneously, it is male.

Experimental example 1 The accuracy experiment of gender identification of deer tendon products sold on the market

The deer tendon products produced by Huayi Deer Products Co., Ltd. in Shuangyang District were used for the experiment, including 30 male deer tendon products and 30 female deer tendon products. Experimenter A randomly mixed these deer tendon products, and experimenter B used this product. The method of invention embodiment 3 carries out the detection of product gender to these deer tendon products, and then the statistical accuracy is carried out by the experimenter according to the difference between experimental value and true value, and the result is as follows:

Embodiment 3 accuracy rate Buck tendon products 100% Doe tendon products 100%

Experimental example 2 The accuracy experiment of gender identification of venison products sold on the market

Similarly, the venison products produced by Huayi Deer Products Company in Shuangyang District were used for the experiment, including 30 male venison products and 30 female venison products. Experimenter A randomly mixed these venison products, and experimenter B used The method of the embodiment of the present invention 2 carries out the detection of product gender to these venison products, then carries out statistical accuracy rate according to the difference of experimental value and true value by the experimenter, the result is as follows:

Embodiment 2 Accuracy rate Buck tendon products 100% Doe tendon products 100%

To sum up, the primer combination of the multiplex PCR for identifying the sex of sika deer products by one-step method of the present invention has good specificity and high amplification efficiency, and can be used to identify the sex of sika deer-derived deer products on the market. The accuracy rate can reach 100%, and it is worthy of wide-scale promotion application.

While particular embodiments of the invention have been illustrated and described, it should be appreciated that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims (10)

1. a kind of one-step method identifies that the primer of Cervus nippon Temminck and products thereof the multiplex PCR of sex combines it is characterised in that being drawn by first Thing to and the second primer pair composition, described first primer pair by the forward primer with base sequence shown in SEQ ID No.1, has Shown in SEQ ID No.2, the downstream primer of base sequence forms, and described second primer pair is by having alkali shown in SEQ ID No.3 The forward primer of basic sequence, has the downstream primer composition of base sequence shown in SEQ ID No.4.

2. a kind of one-step method identify Cervus nippon Temminck and products thereof sex method it is characterised in that comprising the steps：

The DNA extracting sample to be checked, as template, enters performing PCR amplification with the first primer pair and the second primer pair, expands to described DNA Increase after reaction terminates and PCR primer carried out electrophoretic analysiss, you can.

3. one-step method according to claim 2 identification Cervus nippon Temminck and products thereof property method for distinguishing is it is characterised in that described PCR amplification reaction system be：

4. one-step method identification Cervus nippon Temminck according to claim 3 and products thereof property method for distinguishing is it is characterised in that first draws The concentration of thing pair controls between 10-20 μM, and the concentration of the second primer pair controls between 10-20 μM.

5. one-step method identification Cervus nippon Temminck according to claim 2 and products thereof property method for distinguishing is it is characterised in that PCR expands Increasing response procedures is：94-95 DEG C of denaturation 2-3min；94-95 DEG C of degeneration 2-3min, 55-60 DEG C of annealing 20-30s, 72-75 DEG C Extend 40-60s, after common 35-45 circulation, 72-75 DEG C of extension 6-8min.

6. one-step method identification Cervus nippon Temminck according to claim 2 and products thereof property method for distinguishing is it is characterised in that carrying out During electrophoretic analysiss, to the analysis method of result it is：If the band that a 335-340bp in the DNA of sample to be checked is female, if The band 335-340bp and 460-470bp simultaneously is male.

7. one-step method identification Cervus nippon Temminck according to claim 2 and products thereof property method for distinguishing is it is characterised in that electrophoresis divides Analysis uses agarose gel electrophoresiies analysis；

Preferably, agarose concentration is 1.5-2%.

8. one-step method according to claim 7 identifies Cervus nippon Temminck and products thereof property method for distinguishing it is characterised in that electrophoresis Voltage controls in 120-130V constant voltage, and electrophoresis time is 20-30min；

Preferably, before carrying out electrophoretic analysiss, agarose gel and TAE buffer are (1.5-2) according to mass volume ratio： 100 are mixed.

9. the multiplex PCR for identifying Cervus nippon Temminck and products thereof sex that a kind of described primer including claim 1 combines Test kit.

10. application in terms of identification Cervus nippon Temminck and products thereof sex for the test kit described in claim 9.