CN107653329A - For identifying Specific PCR primers and method and its application in barking deer is identified of barking deer - Google Patents

Abstract

The invention discloses a specific PCR primer for identifying Elaphodus cephalophus, wherein the specific PCR primer includes primer 1 shown in SEQ ID No: 1, and primer 1 shown in SEQ ID No: 2 Primer 2. The invention also discloses a method for identifying hairy deer by using specific PCR primers, the method comprising: extracting the total DNA of the sample to be tested and performing PCR amplification on the total DNA; performing agarose gel electrophoresis detection on the amplified product ; Wherein, the specific PCR primers are the above-mentioned specific PCR primers, and if a band of a specific length appears in the swimming lane, it is judged that the sample to be tested is a sample from a hairy deer. The invention also discloses an application of the above-mentioned specific PCR primer or identification method in identifying the hairy deer. By designing the above-mentioned primers, the purpose of identifying the crested deer in a simple, feasible, economical and practical way is achieved.

Description

Specific PCR primers and methods for identifying hairy deer and its role in identifying hairy deer application in

technical field

The present invention relates to the field of biological detection, in particular to a specific PCR primer for identifying the hairy deer, a method for identifying the hairy deer using the specific PCR primers, and their application in identifying the hairy deer.

Background technique

The crested deer (Elaphodus cephalophus) is a small to medium-sized herbivorous deer family animal that lives in hilly and mountainous areas. It belongs to Mammalia, Artiodactyla and Cervidae in classification. Hairy deer are mainly distributed in Zhejiang, Fujian, Anhui, Jiangxi, Guangdong, Hunan, Hubei, Sichuan, Yunnan and other places in China. It is recorded in the literature that it is also distributed in northern Myanmar abroad, but no living individuals have been found so far. Therefore, the hairy deer can be considered as a rare deer species native to my country. Like other deer species, the tufted deer species is also heavily affected by illegal poaching and illegal trade, and its population is in a state of continuous decline. The "List of Terrestrial Wild Animals Protected by the State that Are Beneficial or of Important Economic and Scientific Research Value" promulgated in 2000 listed them as protected species. The crested deer is listed as a Vulnerable (VU) species in China. It has become the consensus of the majority of animal protection workers to increase the protection and biological research of the crested deer species.

In the conservation and management of wild animals, accurate species identification based on taxonomy is the primary premise. The results of species identification are often the basis for forestry public security, industry and commerce, border inspection and other administrative law enforcement departments to carry out wildlife resource protection and management work. Traditional morphological classification methods have always been a common method for wild animal identification, but some inherent defects of morphological classification methods have also brought difficulties to species identification, such as phenotypic plasticity and genetic variability, biological sex and developmental stage restrictions, etc. . The practice of identifying deer animals shows that due to the value of poachers’ skins and meat, the samples sent for inspection are fragmented (the types of samples are hair, stumps, bones, skins, and feces, etc., and the sample size is small) , it is impossible to accurately identify the species morphologically.

With the continuous application of Polymerase Chain Reaction (PCR) technology in forensic identification, molecular biology technology represented by DNA sequence analysis has also been widely used in the identification of endangered wild animals.

With the continuous application of Polymerase Chain Reaction (PCR) technology in forensic identification, molecular biology technology represented by DNA sequence analysis has also been widely used in the identification of endangered wild animal species.

Therefore, it is necessary to provide a specific PCR primer that does not require cumbersome steps such as DNA sequencing and sequence comparison, and has no restrictions on the source of the sample, and can quickly and effectively realize the accurate determination of the crested deer species through only one PCR amplification. The problem that the present invention needs to solve urgently.

Contents of the invention

In view of the above-mentioned prior art, the purpose of the present invention is to overcome the limitation of determining the species of the crested deer through morphological characteristics in the prior art, and provide a simple, quick and economical specific PCR primer for identifying the crested deer and a method for identifying the crested deer. methods, and their application in the identification of crested deer.

In order to achieve the above object, the present invention provides a specific PCR primer for identifying hairy deer (Elaphodus cephalophus), said specific PCR primer includes primer 1 shown in SEQ ID No: 1, and primer 1 shown in SEQ ID No: 2 Primer 2 indicated. The specific primers can perform PCR amplification on the target sample, and identify the species to be tested according to the separation result of agarose gel electrophoresis, that is, whether the target amplification product is produced.

The present invention also provides a method for identifying Elaphodus cephalophus using specific PCR, the method comprising: (1) extracting the total DNA of the sample to be tested; (2) performing PCR on the total DNA using specific PCR primers Amplify; (3) get the product that obtains in step (2) and do agarose gel electrophoresis detection; Wherein, described specificity PCR primer is the specificity PCR primer described in claim 1; Wherein, in step (3 ) in the agarose gel electrophoresis graph obtained, if a band appears in the swimming lane containing the product obtained in step (2), then it is judged that the sample to be tested is a sample from the hairy deer, if it contains the product obtained in step (2) If no band appears in the swimming lane of the product, it is judged that the sample to be tested is not a sample from the hairy deer.

In the present invention, the length of the DNA fragment contained in one band that appeared was 240 bp.

In the present invention, in order to achieve a better PCR amplification effect, make the obtained agarose gel electrophoresis pattern more clearly visible, and facilitate better determination of the experimental results, in the PCR amplification process, a 30 μL PCR amplification system As a basis, the concentration of the primer is preferably 0.25-0.5 pmol/L.

The PCR amplification system refers to all the reagents and materials added in the PCR amplification process including primers, Taq enzyme, dNTPMix, DNA template (total DNA), Mg ²⁺ and buffer.

In addition, in the present invention, in order to achieve a better PCR amplification effect, the agarose gel electrophoresis pattern obtained is more clearly visible, which is convenient to better determine the experimental results. In step (2), the total DNA used for PCR amplification is The dosage is 45-55ng.

Wherein, the DNA polymerase and buffer used for PCR amplification can be conventional DNA polymerase and buffer in the art, and the specific dosage can refer to the detailed description in the product manual. The present invention will not be described in detail here.

In the present invention, in order to achieve a better PCR amplification effect, the obtained agarose gel electrophoresis pattern is more clearly visible, which is convenient for better determining the experimental results. Preferably, the PCR amplification process includes 28-33 cycles, A single cycle includes the steps of denaturation, annealing and extension.

The annealing process in the cyclic reaction can be an annealing method and annealing parameters conventionally used in the art. In the present invention, in order to obtain a better amplification effect, the annealing temperature is 55-65°C, and the annealing time is 25-40s .

The denaturation process in the cycle reaction can be the denaturation method and denaturation parameters routinely used in the field. In the present invention, in order to obtain better amplification effect, the denaturation temperature is 93-95°C.

For the extension mode and extension parameters in the cycle reaction, in the present invention, in order to obtain a better amplification effect, the extension temperature is 70-73°C.

In addition, in order to ensure better amplification effect, the amplification process also includes pre-denaturation at 93-95°C for 5 minutes before the first cycle, annealing temperature at 58°C, and extension at 71-73°C for 10 minutes after the last cycle.

The present invention also provides an application in identifying Elaphodus cephalophus according to the above-mentioned specific PCR primer or the above-mentioned identification method.

In the present invention, by designing specific PCR primers and utilizing the method of specific PCR amplification, the sample to be tested can be amplified by a conventional PCR to determine whether the species is a crested deer through the band display in the agarose gel electrophoresis graph. Moreover, the DNA template used can be extracted from animal fur, muscle tissue, and viscera, which greatly reduces the difficulty of sampling, and the method can easily and quickly identify the crested deer, thereby achieving the purpose of identifying the crested deer in a simple, feasible, economical and practical manner. The whole detection process takes no more than 3 hours in total, and the operation process is simple, which has wide application prospects. After being developed into a kit, the primers and identification methods involved in the present invention can be widely used in the identification of the crested deer species and population detection research, and have great promotion value.

The advantages of the present invention will be further described in detail in the detailed description below.

Description of drawings

The accompanying drawings are used to provide a further understanding of the present invention, and constitute a part of the description, together with the following specific embodiments, are used to explain the present invention, but do not constitute a limitation to the present invention. In the attached picture:

Figure 1 is an agarose gel electrophoresis diagram of the amplification products of Examples 1-4 and Comparative Examples 1-11.

Detailed ways

Specific embodiments of the present invention will be described in detail below. It should be understood that the specific embodiments described here are only used to illustrate and explain the present invention, not to limit the present invention.

The present invention will be described in detail below by way of examples.

It should be clarified that the primers are all synthesized by Shanghai Sangon Bioengineering Technology Co., Ltd. and the concentration of the primers used is 10 pmol/L. The lysate can be a DNA lysate routinely used in the art. For example, in the present invention, Contain 50mmol of Tris-HCl (pH8.0), 25mmol of ethylenediaminetetraacetic acid (pH8.0), 100mmol of sodium chloride and 1% sodium lauryl sulfate in every liter of said lysate, The dNTPMix used in the present invention is a commercial product of Beijing Quanshijin Biotechnology Co., Ltd., proteinase K is a commercial product of Merck Company in the United States, and the DNA purification kit is a commercial product of Beijing Tiangen Biochemical Company. The chemical reagents used are all commercially available analytical reagents. The PCR reaction was carried out on a 2720 PCR instrument (ABI, USA); the agarose gel electrophoresis test results were analyzed and photographed by a HL-2000 gel imaging system (UVP, USA).

Example 1

The method for identifying the crested deer using specific PCR primers includes:

(1) Extract the total DNA of the sample to be tested:

Place 0.5 g of the sample to be tested numbered EC1 (see Table 1) in a centrifuge tube and cut the sample to be tested in the centrifuge tube with sterile scissors, then add 500 μL of lysate to the centrifuge tube successively, 30 μL of 10% sodium dodecyl sulfate (SDS) and 3 μL of 20 mg/ml proteinase K (PK), so that the concentration of SDS is 0.5%, and the final concentration of PK is 100 μg/ul, mix well and then digest in a water bath at 56 °C 12h until the liquid in the tube is clarified. Add 500 μL of balanced phenol to the above digested mixture, shake slightly for 5 minutes, and then place it in a centrifuge at 11,000 r/min for 10 minutes, and take the supernatant after centrifugation. Repeat the above centrifugation step twice. Add 500 μl of secondary mixture (chloroform: isoamyl alcohol = 24:1), shake slightly for 5 minutes, centrifuge at 11000 rpm for 10 minutes, and transfer the supernatant to a new EP tube. Repeat the above two steps. Add 1,000 μL of frozen absolute ethanol to the supernatant obtained after repeated centrifugation, place it at -20°C for 1 hour, then centrifuge it in a centrifuge at 12,000 r/min for 13 minutes, discard the supernatant, and add 800 μL of 70% ethanol was shaken slightly for 0.5 min, then placed in a centrifuge at 13000 r/min for 13 min, and the supernatant was discarded. Repeat the above centrifugation step twice. Place the obtained precipitate on a sterile operating table to dry naturally for 2.5 hours, then add 300 μL of TE solution to it, shake slightly and flick the centrifuge tube with your fingers, and then place it at 4 °C for 3 hours to obtain the total DNA;

(2) Using specific PCR primers to carry out PCR amplification of the total DNA:

Add 10 μL of 10×PCR buffer, 1 μL of a pair of PCR primers of 10 pmol/L, 2 μL of 2 mmol/L dNTPMix, 2 μL of 25 mmol/L Mg ²⁺ , 1 U of Taq enzyme and 50 ng The total DNA was filled up to 30 μL with double distilled water. The PCR reaction conditions were: pre-denaturation at 95°C for 5 min; then 30 cycles, including: denaturation at 95°C for 40 s, annealing at 60°C for 30 s, and extension at 72°C for 35 s; finally, extension at 72°C for 10 min. Perform PCR amplification under the above reaction conditions;

(3) get the product obtained in step (2) and do agarose gel electrophoresis detection:

The amplified products were detected by electrophoresis on 1% EB-stained agarose gel, kept at a voltage of 5 V/cm for 30 min, observed on an ultraviolet gel imaging system, and photographed for preservation. The electrophoresis results are shown in Figure 1, and the corresponding lane number is 7.

Example 2

Operate according to the method of Example 1, the difference is that each 0.75 μL of a pair of PCR primers of 10pmol/L, the consumption of described total DNA is 45ng, the PCR amplification process comprises 28 cycles, and the denaturation temperature in a single cycle is 93 ℃, the annealing temperature is 55℃, the annealing time is 25s, and the extension temperature is 70℃. The amplified DNA was detected by electrophoresis on 1% EB-stained agarose gel, maintained at a voltage of 5V/cm for 30min, and observed on an ultraviolet gel imaging system. The electrophoresis result showed that a band appeared at 240bp.

Example 3

Operate according to the method of Example 1, the difference is that each 1.5 μL of a pair of PCR primers of 10pmol/L, the consumption of described total DNA is 55ng, the PCR amplification process comprises 33 cycles, and the denaturation temperature in a single cycle is 95 ℃, the annealing temperature is 65℃, the annealing time is 40s, and the extension temperature is 73℃. The amplified DNA was detected by electrophoresis on 1% EB-stained agarose gel, maintained at a voltage of 5V/cm for 30min, and observed on an ultraviolet gel imaging system. The electrophoresis result showed that a band appeared at 240bp.

Example 4

The operation was carried out according to the method of Example 1, the difference was that the number of the sample to be tested was EC2 (in Table 1), and the electrophoresis results were as shown in Figure 1, and the corresponding swimming lane number was 6.

Comparative example 1

Operate according to the method of Example 1, the difference is that the sample number to be tested is OG (in Table 1), the electrophoresis result is shown in Figure 1, and the corresponding swimming lane number is 1.

Comparative example 2

Operate according to the method of Example 1, the difference is that the sample number to be tested is BK (in Table 1), the electrophoresis result is shown in Figure 1, and the corresponding swimming lane number is 3.

Comparative example 3

Operate according to the method of Example 1, the difference is that the sample number to be tested is SE (in Table 1), the electrophoresis result is shown in Figure 1, and the corresponding swimming lane number is 2.

Comparative example 4

The operation was carried out according to the method of Example 1, the difference was that the number of the sample to be tested was MT (in Table 1), and the electrophoresis results were shown in Figure 1, and the corresponding swimming lane number was 5.

Comparative example 5

Operate according to the method of Example 1, the difference is that the sample number to be tested is SS (in Table 1), the electrophoresis result is shown in Figure 1, and the corresponding swimming lane number is 10.

Comparative example 6

The operation was carried out according to the method of Example 1, the difference was that the number of the sample to be tested was CS (in Table 1), and the electrophoresis results were as shown in Figure 1, and the corresponding swimming lane number was 11.

Comparative example 7

Operate according to the method of Example 1, the difference is that the sample number to be tested is MB (in Table 1), the electrophoresis result is shown in Figure 1, and the corresponding swimming lane number is 4.

Comparative example 8

Operate according to the method of Example 1, the difference is that the sample number to be tested is HI (in Table 1), the electrophoresis result is shown in Figure 1, and the corresponding swimming lane number is 8.

Comparative example 9

Operate according to the method of Example 1, the difference is that the sample number to be tested is CN (in Table 1), the electrophoresis result is shown in Figure 1, and the corresponding swimming lane number is 9.

Comparative example 10

The operation was carried out according to the method of Example 1, the difference was that the number of the sample to be tested was MC (in Table 1), and the electrophoresis result was shown in Figure 1, and the corresponding swimming lane number was 12.

Comparative example 11

The operation was carried out according to the method of Example 1, the difference was that the number of the sample to be tested was MM (in Table 1), and the electrophoresis results were as shown in Figure 1, and the corresponding swimming lane number was 13.

Comparative example 1

In order to avoid external source contamination, sterile double-distilled water was used as the negative control of the template for all operations during the extraction of total DNA from the sample to be tested and the PCR amplification process. The electrophoresis results are shown in Figure 1, and the corresponding lane number is C.

Table 1

As shown in Table 1 and Figure 1, the swimming lanes in Figure 1 are respectively, M: D2000 molecular marker (Tiangen Biochemical); 1st lane: OG; 2nd lane: SE; 3rd lane: BK; 4th lane: MB; 5th lane: MT; Lane 6: EC2; Lane 7: EC1; Lane 8: HI; Lane 9: CN; Lane 10: SS; Lane 11: CS; Lane 12: MC; Lane 13: MM; Lane C: blank control. As can be seen from the sample information in Table 1 and the electrophoresis diagram shown in Figure 1, the present invention can pass through the agarose gel electrophoresis diagram by designing a group of specific PCR primers, and the sample to be tested can be amplified by conventional PCR only once. The band shows that it is determined whether the species is a crested deer, and the electrophoresis results are completely consistent with the sample information, which confirms the reliability of the identification method.

Test example 1

The agarose gel block containing the target DNA fragment in the obtained band was excised with a clean scalpel, purified in a DNA purification kit, and then sent to Shanghai Sangon Bioengineering Technology Co., Ltd. for sequencing. After comparing the sequence of the DNA fragment sent to Shanghai Sangon Bioengineering Technology Co., Ltd. for sequencing with the sequence of the known species of tufted deer in the GenBank database, the comparison results show that the sequence of the sequenced DNA fragment is consistent with the known The homology of the sequence of the species of the crested deer is more than 96%, so it is further confirmed that the species of the tested sample is the species of the crested deer.

Not only that, in order to ensure the reliability of the experimental results, we sequenced the amplified products of the crested deer samples EC1 and EC2. After using the Blast software of the GenBank database to search and compare, it was found that the sequences of EC1 and EC2 samples had 100% sequence similarity with the homologous fragment sequences of the hairy deer species in GenBank, and their GenBank accession numbers were KU324462, KU324463 and KU324464. This shows that the PCR-specific primers of the present invention can effectively achieve specific amplification of the crested deer species.

In the present invention, the DNA template used can be extracted from the muscle tissue of the animal, which greatly reduces the difficulty of sampling, and the method can quickly and easily identify the hairy deer, thereby achieving the effect of simple, feasible, economical and practical identification of the hairy deer.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention. These simple modifications All belong to the protection scope of the present invention.

In addition, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way if there is no contradiction. The combination method will not be described separately.

In addition, various combinations of different embodiments of the present invention can also be combined arbitrarily, as long as they do not violate the idea of the present invention, they should also be regarded as the disclosed content of the present invention.

SEQUENCE LISTING

<110> Anhui Normal University

<120> Specific PCR primers and methods for identifying the crested deer and its application in identifying the crested deer

<130> 05631

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 20

<212>DNA

<400> 1

tactctacca aatctctccg 20

<210> 2

<211> 27

<212>DNA

<400> 2

aaaagggtaa tgagggctaa gacggtc 27

Claims (10)

1. a kind of specificity PCR primer that is used to identify tufted deer (Elaphodus cephalophus), is characterized in that, described specificity PCR primer comprises as shown in SEQ ID No: 1 primer 1, and as shown in SEQ ID No: 2 Primer 2. 2. A method utilizing specific PCR primers to identify hairy deer, characterized in that the method comprises: (1) Extract the total DNA of the sample to be tested; (2) performing PCR amplification on the total DNA using specific PCR primers; (3) get the product obtained in step (2) and do agarose gel electrophoresis detection; Wherein, the specific PCR primer is the specific PCR primer described in claim 1; Wherein, in the agarose gel electrophoresis graph obtained in step (3), if a band appears in the swimming lane containing the product obtained in step (2), then it is judged that the sample to be tested is a sample from the hairy deer, if it contains If no one band appears in the swimming lane of the product obtained in step (2), it is judged that the sample to be tested is not a sample from a crested deer. 3. The method according to claim 2, wherein the length of the DNA fragment contained in one band that appears is 240 bp. 4. The method according to claim 2 or 3, wherein, during the PCR amplification process, the concentration of the primers is 0.25-0.5 pmol/L based on a 30 μL PCR amplification system. 5. The method according to claim 4, wherein the amount of total DNA used for PCR amplification in step (2) is 45-55ng. 6. The method according to claim 2 or 3, wherein the PCR amplification process includes 28-33 cycles, and a single cycle includes the steps of denaturation, annealing and extension. 7. The method according to claim 6, wherein the annealing temperature is 55-65°C, and the annealing time is 25-40s. 8. The method according to claim 6, wherein the denaturation temperature is 93-95°C. 9. The method of claim 6, wherein the extension temperature is 70-73°C. 10. The application of the specific PCR primer according to claim 1 or the method according to any one of claims 2-9 in identifying the crested deer.