CN107653329B

CN107653329B - Specific PCR primer and method for identifying hairy deer and application of specific PCR primer and method in identifying hairy deer

Info

Publication number: CN107653329B
Application number: CN201711019250.XA
Authority: CN
Inventors: 晏鹏; 李香凝; 王美菊; 李延颖
Original assignee: Anhui Normal University
Current assignee: Anhui Normal University
Priority date: 2017-10-27
Filing date: 2017-10-27
Publication date: 2021-05-14
Anticipated expiration: 2037-10-27
Also published as: CN107653329A

Abstract

The invention discloses a specific PCR primer for identifying fawn (Elaphodus cephalophus), wherein the specific PCR primer comprises a primer sequence shown in SEQ ID No: 1, and the primer 1 shown as SEQ ID No: 2, and 2 is shown in the figure. The invention also discloses a method for identifying the fawn by using the specific PCR primer, which comprises the following steps: extracting total DNA of a sample to be detected and carrying out PCR amplification on the total DNA; carrying out agarose gel electrophoresis detection on the amplified product; wherein, the specific PCR primer is the above specific PCR primer, and if 1 strip with a specific length appears in a lane, the sample to be detected is judged to be the sample from the hairy deer. The invention also discloses application of the specific PCR primer or the identification method in identifying the deer maw. By designing the primers, the aim of identifying the fawn deer is fulfilled, which is simple, convenient, easy, economical and practical.

Description

Specific PCR primer and method for identifying hairy deer and application of specific PCR primer and method in identifying hairy deer

Technical Field

The invention relates to the field of biological detection, in particular to a specific PCR primer for identifying a fawn, a method for identifying the fawn by using the specific PCR primer, and application of the specific PCR primer to identification of the fawn.

Background

Corolla deer (Elaphodus cephalophyus) is a small and medium-sized herbivorous Cervidae animal living in hilly and mountainous regions, and belongs to Mammalia (Mammalia), Artiodactyla (Artiodactyla) and Cervidae (Cervidae) in classification. The hairy deer is mainly distributed in Zhejiang, Fujian, Anhui, Jiangxi, Guangdong, Hunan, Hubei, Sichuan and Yunnan provinces in China. Although the literature describes distribution in northern Burma abroad, no living individuals have been found so far. Therefore, the hairy deer can be regarded as a rare cervidae animal which is particularly produced in China. Like other deer species, the species of corolla deer are also heavily influenced by illegal hunting and illegal trade, with their population numbers in a state of constant decline. The beneficial or important economic and scientific research value of the terrestrial wild animal famous book for national protection issued in 2000 is listed as a protective species, and the red famous book for biological diversity in China-vertebrate newly issued in 2015 is further listed as a Chinese easily-dangerous (VU) species of the hairy deer. The increasing of the protection and biological research on the species of the deer mao has become a consensus of the majority of animal protection workers.

Accurate species identification based on taxonomy is a leading prerequisite in the work of protection and management of wildlife. The result of species identification is often the basis for developing wild animal resource protection and management work by administrative law enforcement departments such as forest public security, industry and commerce, side inspection and the like. The traditional morphological classification method is always a common method for identifying wild animals, but some defects inherent in the morphological classification method also bring difficulties to species identification work, such as phenotypic plasticity and genetic variability, biological sex and developmental stage limitation, and the like. The identification work practice for deer animals shows that the accurate identification of species cannot be realized morphologically because the stealers grab the skin and meat values of the stealers, so that the samples to be detected are crushed (the sample types are hair, stump, bone, skin, excrement and the like, and the sample amount is small).

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

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

Therefore, the invention provides a specific PCR primer which does not need complicated steps such as DNA sequencing and sequence comparison, has no limit to sample sources, and can quickly and effectively realize accurate determination of species of the fawn deer only through one-time PCR amplification, and the problem to be solved by the invention is urgent.

Disclosure of Invention

Aiming at the prior art, the invention aims to overcome the limitation of determining species of the fawn by morphological characteristics in the prior art, and provides a simple, convenient, rapid and economic specific PCR primer for identifying the fawn, a method for identifying the fawn and application of the specific PCR primer and the method in identifying the fawn.

In order to achieve the above objects, the present invention provides a specific PCR primer for identifying fawn deer (elaphus cephalophus), the specific PCR primer comprising the sequence as set forth in SEQ ID No: 1, and the primer 1 shown as SEQ ID No: 2, and 2 is shown in the figure. The specific primer can carry out PCR amplification on a target sample, and the species to be detected is identified according to the result of the agarose gel electrophoresis, namely whether a target amplification product is generated or not.

The present invention also provides a method for identifying fawn deer (elaphus cephalotus) using specific PCR, the method comprising: (1) extracting the total DNA of a sample to be detected; (2) performing PCR amplification on the total DNA by using a specific PCR primer; (3) carrying out agarose gel electrophoresis detection on the product obtained in the step (2); wherein, the specific PCR primer is the specific PCR primer in claim 1; in the agarose gel electrophoresis image obtained in the step (3), if 1 strip appears in the lane containing the product obtained in the step (2), the sample to be detected is judged to be the sample from the deer majors, and if 1 strip does not appear in the lane containing the product obtained in the step (2), the sample to be detected is judged not to be the sample from the deer majors.

In the present invention, the length of the DNA fragment contained in 1 band appeared was 240 bp.

In the present invention, in order to achieve better PCR amplification effect, so that the obtained agarose gel electrophoresis image can be more clearly seen, and the experimental result can be better determined, in the PCR amplification process, the concentration of the primer is preferably 0.25-0.5pmol/L based on 30 μ L of PCR amplification system.

The PCR amplification system comprises a primer, Taq enzyme, dNTPmix, a DNA template (total DNA) and Mg²⁺And buffers and the like for all reagents and raw materials added in the PCR amplification process.

In addition, in the invention, in order to achieve better PCR amplification effect, make the obtained agarose gel electrophoresis picture more clearly visible and facilitate better determination of experimental results, the using amount of the total DNA for PCR amplification in the step (2) is 45-55 ng.

The DNA polymerase and the buffer solution for PCR amplification can be conventional DNA polymerase and buffer solution in the field, and the specific dosage can refer to the detailed description in the product specification. The present invention will not be described in detail herein.

In the present invention, in order to achieve better PCR amplification effect and make the obtained agarose gel electrophoresis image more clearly visible, and facilitate better determination of experimental results, preferably, the PCR amplification process comprises 28-33 cycles, and each cycle comprises the steps of denaturation, annealing and extension.

The annealing process in the cycling reaction can be an annealing mode and an annealing parameter which are conventionally used in the field, and in the invention, in order to obtain a better amplification effect, the annealing temperature is 55-65 ℃, and the annealing time is 25-40 s.

The denaturation process in the cycling reaction can be a denaturation mode and a denaturation parameter which are conventionally used in the field, and in the present invention, the denaturation temperature is 93-95 ℃ for better amplification effect.

The extension mode and extension parameters in the cycling reaction are, in the invention, the extension temperature is 70-73 ℃ in order to obtain better amplification effect.

In addition, in order to ensure that a better amplification effect is obtained, the amplification process also comprises the steps of pre-denaturation at 93-95 ℃ for 5min before the first cycle, annealing at 58 ℃ and re-extension at 71-73 ℃ for 10min after the last cycle.

The invention also provides an application of the specific PCR primer or the identification method in identifying the fawn (Elaphodus cephalotus).

The invention designs the specific PCR primer, utilizes the specific PCR amplification method, can judge whether the species is the deer maw or not through the strip display in the agarose gel electrophoresis picture by one-time conventional PCR amplification of a sample to be detected, and the used DNA template can be extracted from fur, muscle tissue and internal organs of animals, thereby greatly reducing the sampling difficulty. The total time consumption of the whole detection process is not more than 3 hours, and the operation process is simple and has wide application prospect. After the primer and the identification method are developed into the kit, the kit can be widely applied to identification of species of the fawn and population detection research, and has great popularization value.

The advantages of the present invention will be described in further detail in the detailed description section which follows.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 is an agarose gel electrophoresis image of the amplification products of examples 1 to 4 and comparative examples 1 to 11.

Detailed Description

The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

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

It is to be clarified that the primers are synthesized by Shanghai Biotechnology engineering Co., Ltd and the concentration of the primers used is 10pmol/L, the lysis solution may be a DNA lysis solution conventionally used in the art, for example, in the present invention, 50mmol of Tris-HCl (pH8.0), 25mmol of ethylenediaminetetraacetic acid (pH8.0), 100mmol of sodium chloride and 1% by weight of sodium dodecylsulfate are contained per liter of the lysis solution, dNTPMix used in the present invention is a commercial product of Beijing Quanjin Biotechnology Co., Ltd, proteinase K is a commercial product of Merck in the U.S. A., the DNA purification kit is a commercial product of Beijing Tiangen Biochemical Co., Ltd, and the rest of the used chemical reagents are conventional commercial analytical reagents. PCR reactions were performed on a 2720 PCR instrument (ABI, usa); the results of the agarose gel electrophoresis were analyzed and photographed by a HL-2000 type gel imaging system (UVP, USA).

Example 1

The method for identifying the hairy deer by using the specific PCR primer comprises the following steps:

(1) extracting total DNA of a sample to be detected:

0.5g of a sample to be tested which is numbered EC1 in Table 1 (see Table 1) is placed in a centrifuge tube, the sample to be tested is cut into pieces by using a pair of sterilizing scissors in the centrifuge tube, then 500 mu L of lysate, 30 mu L of 10% Sodium Dodecyl Sulfate (SDS) and 3 mu L of 20mg/ml Proteinase K (PK) are sequentially added into the centrifuge tube, the concentration of the SDS is 0.5%, the final concentration of the PK is 100 mu g/ul, and the mixture is fully mixed and digested for 12h in 56 ℃ water bath until the liquid in the tube is clear. Adding 500 μ L of balance phenol into the digested mixture, slightly shaking for 5min, centrifuging for 10min in a 11000r/min centrifuge, and collecting the centrifuged supernatant. The above centrifugation step was repeated twice. Then 500. mu.l of a second mixture (chloroform: isoamyl alcohol: 24: 1) was added thereto, the mixture was gently shaken for 5 minutes, and centrifuged at 11000rpm for 10 minutes, after which the supernatant was transferred to a new EP tube. Repeating the above two steps. Adding 1000 μ L of frozen anhydrous ethanol into the supernatant obtained after repeated centrifugation, standing at-20 deg.C for 1h, centrifuging for 13min in a 12000r/min centrifuge, removing the supernatant, adding 800 μ L of 70% ethanol into the obtained precipitate, slightly shaking for 0.5min, freeze-centrifuging for 13min in a 13000r/min centrifuge, and removing the supernatant. The above centrifugation step was repeated twice. Placing the obtained precipitate on a sterile operating platform, naturally airing for 2.5h, adding 300 mu L of TE solution, slightly oscillating, flicking the centrifugal tube by fingers, and placing at 4 ℃ for 3h to obtain total DNA;

(2) performing PCR amplification on the total DNA by using specific PCR primers:

to the sample tube of the PCR apparatus, 10. mu.L of 10 XPCR buffer, 10pmol/L of a pair of PCR primers, 1. mu.L each, 2. mu.L of 2mmol/L dNTPmix, 2. mu.L of 25mmol/L Mg²⁺1U of Taq enzyme and 50ng of total DNA, and made up to 30. mu.L with double distilled water. The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; then 30 cycles were performed, which included: denaturation at 95 ℃ for 40s, annealing at 60 ℃ for 30s and extension at 72 ℃ for 35 s; finally, extension is carried out for 10min at 72 ℃. Performing PCR amplification under the above reaction conditions;

(3) and (3) carrying out agarose gel electrophoresis detection on the product obtained in the step (2):

and carrying out electrophoresis detection on the amplification product on 1% agarose gel dyed by EB, keeping the voltage of 5V/cm for 30min, observing on an ultraviolet gel imaging system, and carrying out camera shooting and storing. The electrophoresis result is shown in FIG. 1, and the lane number is 7.

Example 2

The procedure was followed as in example 1 except that 0.75. mu.L of each of 10pmol/L of a pair of PCR primers was used, the total DNA was used in an amount of 45ng, and the PCR amplification process consisted of 28 cycles, wherein the denaturation temperature was 93 ℃, the annealing temperature was 55 ℃, the annealing time was 25s, and the extension temperature was 70 ℃. And (3) carrying out electrophoresis detection on the amplified DNA on 1% agarose gel dyed by EB, keeping the voltage of 5V/cm for 30min, observing on an ultraviolet gel imaging system, and displaying that a strip appears at 240bp according to an electrophoresis result.

Example 3

The procedure was as in example 1 except that 1.5. mu.L each of a 10pmol/L pair of PCR primers was used, the total DNA was 55ng, the PCR amplification process consisted of 33 cycles, and the denaturation temperature was 95 ℃, the annealing temperature was 65 ℃, the annealing time was 40s, and the extension temperature was 73 ℃ in each cycle. And (3) carrying out electrophoresis detection on the amplified DNA on 1% agarose gel dyed by EB, keeping the voltage of 5V/cm for 30min, observing on an ultraviolet gel imaging system, and displaying that a strip appears at 240bp according to an electrophoresis result.

Example 4

The procedure was as in example 1 except that the sample to be tested was numbered EC2 (in Table 1), the electrophoresis result was shown in FIG. 1, and the corresponding lane number was 6.

Comparative example 1

The procedure was as in example 1 except that the sample to be tested was OG (Table 1), the electrophoresis result was as shown in FIG. 1, and the corresponding lane was 1.

Comparative example 2

The procedure was as in example 1 except that the sample to be tested was numbered BK (Table 1) and the electrophoresis result was as shown in FIG. 1, corresponding to lane number 3.

Comparative example 3

The procedure was as in example 1 except that the sample to be tested was designated SE (Table 1), the electrophoresis result was as shown in FIG. 1, and the corresponding lane was designated 2.

Comparative example 4

The procedure was followed as in example 1 except that the sample to be tested was MT (in Table 1), and the electrophoresis result was shown in FIG. 1, corresponding to lane number 5.

Comparative example 5

The procedure was as in example 1 except that the sample to be tested was designated SS (Table 1), and the electrophoresis result was as shown in FIG. 1, corresponding to lane number 10.

Comparative example 6

The procedure was as in example 1 except that the sample to be tested was designated CS (Table 1), the electrophoresis result was as shown in FIG. 1, and the corresponding lane was designated 11.

Comparative example 7

The procedure was as in example 1 except that the sample to be tested was numbered MB (in Table 1), and the electrophoresis result was shown in FIG. 1, corresponding to lane number 4.

Comparative example 8

The procedure was as in example 1 except that the sample to be tested was numbered HI (in Table 1) and the electrophoresis result was shown in FIG. 1, corresponding to lane number 8.

Comparative example 9

The procedure was as in example 1 except that the sample to be tested was numbered CN (in Table 1), the electrophoresis result was as shown in FIG. 1, and the lane number was 9.

Comparative example 10

The procedure was as in example 1 except that the sample to be tested was numbered MC (in Table 1), the electrophoresis result was as shown in FIG. 1, and the corresponding lane was numbered 12.

Comparative example 11

The procedure was as in example 1 except that the sample to be tested was MM (in Table 1) and the electrophoresis result was as shown in FIG. 1, and the corresponding lane was 13.

Comparative example 1

In order to avoid exogenous pollution, sterile double distilled water is used as a negative control of a template in all operations of extracting total DNA of a sample to be detected and in the PCR amplification process, the electrophoresis result is shown in figure 1, and the number of a corresponding lane is C.

TABLE 1

As shown in table 1 and fig. 1, lanes in fig. 1 are, respectively, M: d2000 molecular marker (tiangen biochemistry); 1, channel: OG; and 2, performing: SE; and 3, performing the following steps: BK; 4, performing channel: MB; and 5, performing: MT; 6, performing the following steps: EC 2; 7, performing channel: EC 1; and 8, performing: HI; 9, performing channel: CN; 10, performing channel: SS; and (11) performing: CS; 12, performing: MC; and 13, performing: MM; and C, channel C: blank control. As can be seen from the sample information in the table 1 and the electrophoresis chart shown in the figure 1, the invention designs 1 group of specific PCR primers, and judges whether the species is the hairy deer or not through the band display in the agarose gel electrophoresis chart by only one-time conventional PCR amplification on the sample to be detected, and the electrophoresis result completely accords with the sample information, thereby confirming the reliability of the identification method.

Detection example 1

The agarose gel block containing the target DNA fragment in the obtained band is cut by a clean scalpel, purified in a DNA purification kit and sent to Shanghai Bioengineering technology Co. Comparing the sequence of the DNA fragment sequenced by Shanghai biological engineering technology Limited company with the sequence of the known species of the fawn deer in GenBank database, and then obtaining a comparison result which shows that the homology of the sequence of the sequenced DNA fragment and the sequence of the known species of the fawn deer is more than 96 percent, thereby further confirming that the species of the sample to be detected is the fawn deer.

Furthermore, to ensure the reliability of the experimental results, we sequenced the amplification products of fawn samples EC1 and EC 2. After Blast software searching and comparison of GenBank database, the sequence similarity of the EC1 and EC2 samples and the existing homologous fragment sequence of the hairy deer species in GenBank is up to 100 percent, and the GenBank accession numbers are KU324462, KU324463 and KU324464 respectively. This shows that the PCR specific primer of the invention can effectively realize specific amplification to the species of the fawn deer.

In the invention, the used DNA template can be extracted from the muscle tissue of the animal, the sampling difficulty is greatly reduced, and the method can simply, conveniently and quickly identify the fawn deer, thereby achieving the effect of simply, conveniently, economically and practically identifying the fawn deer.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

SEQUENCE LISTING

<110> university of teacher's university in Anhui

<120> specific PCR primer and method for identifying hairy deer and application thereof in identifying hairy deer

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Claims

1. A method for identifying hairy deer (A)Elaphodus cephalophus) The specific PCR primer of (1), wherein the specific PCR primer consists of the primer sequence shown as SEQ ID No: 1, and the primer 1 shown as SEQ ID No: 2, and (b) a primer 2 shown in (2).

2. The use of the specific PCR primer of claim 1 for identifying deer maw.