CN106755536B - Specific PCR primer for identifying hyena and method for identifying hyena by using specific PCR primer - Google Patents

Specific PCR primer for identifying hyena and method for identifying hyena by using specific PCR primer Download PDF

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CN106755536B
CN106755536B CN201710119279.9A CN201710119279A CN106755536B CN 106755536 B CN106755536 B CN 106755536B CN 201710119279 A CN201710119279 A CN 201710119279A CN 106755536 B CN106755536 B CN 106755536B
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sample
hyena
specific pcr
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pcr primer
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CN106755536A (en
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晏鹏
李延颖
王美菊
吴孝兵
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Anhui Normal University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms

Abstract

The invention discloses a specific PCR primer and a method for identifying hyena (Capricornis sumatraensis) by using the same, wherein the method comprises the following steps: 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); in the agarose gel electrophoresis image obtained in the step 3), if 1 strip with a specific length appears in the lane, the sample to be detected is judged to be the sample from hyena, and if 1 strip does not appear in the lane, the sample to be detected is judged not to be the sample from hyena. The DNA template in the method can be extracted from animal hair, stump, bone, skin and excrement, so that the sampling difficulty is greatly reduced, and the method can simply, conveniently and quickly identify the hyena, thereby achieving the aim of simply, conveniently, economically and practically identifying the hyena.

Description

Specific PCR primer for identifying hyena and method for identifying hyena by using specific PCR primer
Technical Field
The invention relates to the field of biological detection, in particular to a specific PCR primer and a method for identifying hyena and application thereof in identifying hyena.
Background
The plant of antelope hyena (Capricorn sumatraensis) is a herbivorous animal widely distributed in the east China woodland, also called Sumengao and Miniang sheep, and belongs to Mammalia, Artiodactyla and Bovidae in classification. The antelope hyena is a typical forest inhabiting beast, is one of typical beasts in tropical and subtropical regions in the southeast of China, and is mainly used in coniferous and broad mixed forests, coniferous forests or rocky mixed forests with the elevation of 1000-4400 m. As a large-scale forest field beasts, the peel and the flesh of the hyena have great economic value, so illegal hunting and trade of the hyena have been important influencing factors influencing the population health development. In terms of species protection, hyena angustifolia is listed in the red directory of endangered species (near-risk level) of national class II key protection animals and the world natural protection alliance (IUCN). Internationally, the International trade convention for endangered species of wild animals and plants (CITES) is further included in the appendix I species protection list, and the international exchanges and trades are strictly controlled.
Accurate species identification based on taxonomy is a necessary prerequisite for human cognition in nature and sustainable development. 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 primates, 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. Particularly, in the practical identification work of the species of hyena in recent years, it has been found that the porigo hyena samples which are often inspected by value extraction of skin and meat are all hair, stump, bone, skin and feces, and are not intact individuals, and the sample amount is small, so that the porigo hyena samples are almost unidentifiable from the morphology. This reflects that in real-world work, species identification methods based solely on morphological characteristics have difficulty meeting the practical requirements of hyena protection work.
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. The main objective of this study was to achieve species-specific molecular identification of hyena based on site-specific PCR methods. According to the method, by utilizing the species specific primers of the hyena, only 1 PCR amplification is needed, the source identification of the sample can be completed by judging and reading the target band of the electropherogram, and the method has the advantages of high timeliness (less than 3 hours), low cost and simplicity and convenience in operation, can be widely applied to the field of population genetics research by taking a stool sample as a research object, greatly enriches the protective biology research means of the hyena, and has great popularization value.
Therefore, the present invention provides a specific PCR primer that does not require complicated steps such as DNA sequencing and sequence comparison, has no limitation on sample sources, and can rapidly and effectively achieve accurate determination of species of hyaline through one-time PCR amplification, which is a problem to be solved by the present invention.
Disclosure of Invention
In view of the above prior art, the present invention aims to overcome the limitations of the prior art in determining species of hyena angustifolia by morphological characteristics, or the problems of time and labor consumption and high cost of the conventional methods for determining species by measuring DNA in hair, stump, bone, hide and feces, and to provide a specific PCR primer for identifying hyena angustifolia and a method for identifying hyena angustifolia that are simple, fast, economical, and their applications in identifying hyena angustifolia.
In order to achieve the above object, the present invention provides a specific PCR primer for identifying hyena, wherein the specific PCR primer comprises the nucleotide sequence shown in SEQ ID No: 1 and SEQ ID No: 2, and (b) a primer set shown in (2).
The invention also provides a method for identifying hyena angustifolia by using the specific PCR primer, wherein the method comprises the following steps:
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, the sample to be detected is judged to be the sample from hyena, and if no strip appears in the lane, the sample to be detected is not the sample from hyena.
According to the invention, by designing a group of specific PCR primers and utilizing the specific PCR primer amplification method, whether the species is the hyena can be judged by strip display in an agarose gel electrophoresis chart through one-time conventional PCR amplification on a sample to be detected, and the used DNA template can be extracted from hair, stump, bone, skin and excrement of an animal, so that the sampling difficulty is greatly reduced, and the method can simply, conveniently and quickly identify the hyena, thereby achieving the purpose of identifying the hyena simply, conveniently, economically and practically.
Additional features and advantages of the invention will be set forth in the detailed description 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 a photograph of glycogel electrophoresis of the amplification products of examples 1 to 5 and comparative examples 1 to 15.
Detailed Description
The following detailed description of embodiments of the invention refers to the accompanying drawings. 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 invention provides a specific PCR primer for identifying hyena anguenaria (Capricornis sumatraensis), wherein the specific PCR primer comprises a primer sequence shown in SEQ ID No: 1 and SEQ ID No: 2, and (b) a primer set shown in (2).
The invention also provides a method for identifying hyena angustifolia by using the specific PCR primer, wherein the method comprises the following steps:
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, the sample to be detected is judged to be the sample from hyena, and if no strip appears in the lane, the sample to be detected is not the sample from hyena.
The length of the DNA fragment contained in 1 band appearing in the present invention was 420 bp.
In the invention, in order to achieve better PCR amplification effect, make the obtained agarose gel electrophoresis image more clear and visible and facilitate better determination of experimental results, the dosage of each primer is 7.5-15 × 10 based on 30 μ L of PCR amplification system-6pmol (concentration of each primer is 0.25-0.5 pmol/L); the amount of DNA template used was 45-50 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.
The annealing process in the PCR amplification process 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 in the PCR amplification process is 55-60 ℃, and the annealing time is 25-40 s. In addition, the setting of the parameters such as denaturation condition, extension condition and cycle number in PCR amplification are all conventional in the art, and the detailed description of the present invention is omitted here.
The present invention will be described in detail below by way of examples.
It is to be noted that the primers are synthesized by Shanghai Biotechnology engineering Co., Ltd and the concentration of the primers 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 commercially available product from Transgene, proteinase K is a commercially available product from Merck, the DNA purification kit is a commercially available product from Tiangen, and the rest of the chemical reagents used are conventional commercially available analytical reagents.
In fig. 1, lane 1 is from a boar sample, lane 2 is from a garland snake sample, lane 3 is from a hyigone sample, lane 4 is from a chamois sample, lane 5 is from a garland snake sample, lane 6 is from a hyigone sample, lane 7 is from a fagopyrum sample, lane 8 is from a swizzle sample, lane 9 is from a sika sample, lane 10 is from a hyigone sample, lane 11 is from an anggi andrias amurensis sample, lane 12 is from a forest musk deer sample, lane 13 is from a white \\/40519 sample, lane 14 is from a hyigone sample, lane 15 is from a black chamois sample, lane 16 is from a white neck tail pheasant sample, lane 17 is from a large green rana frog sample, lane 18 is from a macaque sample, lane 19 is from a hyena sample, lane 20 is from a tibetana macaque sample, lane C is a blank control, lane M is a DL2000 molecular marker.
Example 1
0.5g of the sample to be tested, numbered CS1 in Table 1, was placed in a centrifuge tube and chopped in the centrifuge tube with sterile small scissors. Then, 500. mu.l of lysis solution, 30. mu.l of 10% sodium dodecyl sulfate and 3. mu.l of 20mg/ml proteinase K are sequentially added into a centrifuge tube, and after fully and uniformly mixing, the mixture is digested in a water bath at 56 ℃ for 12 hours until the liquid in the tube is clear. Adding 500 μ l of Tris-balance phenol into the digested mixture, slightly shaking for 5min, centrifuging at 11000rpm for 10min, and collecting the supernatant. The above centrifugation step was repeated twice. Mu.l of a second mixture (chloroform: isoamyl alcohol: 24: 1) was added thereto, the mixture was gently shaken for 5 minutes and then centrifuged in a centrifuge at 11000rpm for 10 minutes, and the centrifuged supernatant was collected. The above centrifugation step was repeated twice. Adding 1000 μ l of frozen absolute ethanol, storing at-20 deg.C for 1h, centrifuging at 12000rpm for 13 min, discarding supernatant, adding 800 μ l of 70% ethanol, slightly shaking for half min, and freeze-centrifuging at 13000rpm for 13 min. The centrifugation step was repeated two more times. The supernatant was discarded, and the obtained precipitate was placed on a sterile operating table and air-dried for 2.5 hours, and then 300. mu.l of TE was added thereto, followed by slight shaking, and the tube was flicked with a finger and dissolved at 4 ℃ for about 3 hours to obtain total DNA.
To the sample tube of the PCR apparatus, 10. mu.l of 10 XPCR buffer, 1. mu.l (10pmol/L) of each primer, 2. mu.l of dNTPmix (2mmol/L), and 2. mu.l of Mg were added2+(25mmol/L), 1U of Taq enzyme and 50ng of total DNA were made up to 30. mu.l with double distilled water. The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, followed by 30 cycles with the following parameters: denaturation at 95 ℃ for 40s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 35s, annealing at 58 ℃, and extension at 72 ℃ for 10min after the last cycle. In order to avoid exogenous pollution, sterile double distilled water is used as a negative control of a template in all the operations of extraction and PCR amplification. The amplification result was detected by 1% agarose gel electrophoresis. The amplified DNA was subjected to agarose gel electrophoresis, and the results of the electrophoresis are shown in FIG. 1 (lane No. 3).
Example 2
The procedure was carried out in the same manner as in example 1 except that the sample to be tested was numbered CS2 (in Table 1), the amount of each primer was 0.75. mu.L, the amount of the total DNA was 45ng, the annealing temperature was 55 ℃ and the annealing time was 40s, and the electrophoresis result was as shown in FIG. 1 (lane number: 6).
Example 3
The procedure was carried out in the same manner as in example 1 except that the sample to be tested was numbered CS3 (in Table 1), the amount of each primer was 0.75. mu.L, the amount of the total DNA was 45ng, the annealing temperature was 55 ℃ and the annealing time was 40s, and the electrophoresis result was as shown in FIG. 1 (lane number: 10).
Example 4
The procedure was carried out in the same manner as in example 1 except that the sample to be tested was numbered CS4 (in Table 1), the amount of each primer was 0.75. mu.L, the amount of the total DNA was 50ng, the annealing temperature was 60 ℃ and the annealing time was 40s, and the electrophoresis result was as shown in FIG. 1 (lane number: 14).
Example 5
The procedure was carried out in the same manner as in example 1 except that the sample to be tested was numbered CS5 (in Table 1), the amount of each primer was 0.75. mu.L, the amount of the total DNA was 50ng, the annealing temperature was 55 ℃ and the annealing time was 40s, and the electrophoresis result was as shown in FIG. 1 (lane number: 19).
Comparative example 1
The procedure was carried out in the same manner as in example 1 except that the sample to be tested was MK (in Table 1) and the result of electrophoresis was as shown in FIG. 1 (lane No. 4).
Comparative example 2
The procedure was carried out in the same manner as in example 1 except that the sample to be tested was numbered CN (in Table 1), and the result of electrophoresis was shown in FIG. 1 (lane number 9).
Comparative example 3
The procedure was carried out in the same manner as in example 1 except that the sample to be tested was numbered HI (in Table 1), and the electrophoresis results were as shown in FIG. 1 (lane number: 8).
Comparative example 4
The procedure was carried out in the same manner as in example 1 except that the sample to be tested was designated as EC (in Table 1), and the results of electrophoresis were shown in FIG. 1 (lane number: 7).
Comparative example 5
The procedure was carried out in the same manner as in example 1 except that the sample to be tested was numbered MB (in Table 1), and the results of electrophoresis were shown in FIG. 1 (lane number: 12).
Comparative example 6
The procedure was carried out in the same manner as in example 1 except that the sample to be tested was identified as MC (Table 1), and the results of electrophoresis were shown in FIG. 1 (lane No. 15).
Comparative example 7
The procedure was carried out in the same manner as in example 1 except that the sample to be tested was designated SS (Table 1), and the results of electrophoresis were shown in FIG. 1 (lane number 1).
Comparative example 8
The procedure was as in example 1 except that the sample to be tested was MM (Table 1) and the electrophoresis results are shown in FIG. 1 (lane 18).
Comparative example 9
The procedure was carried out in the same manner as in example 1 except that the sample to be tested was MT (in Table 1), and the results of electrophoresis were shown in FIG. 1 (lane number 20).
Comparative example 10
The procedure was as in example 1 except that the sample to be tested was numbered SY (in Table 1) and the electrophoresis result was shown in FIG. 1 (lane number 16).
Comparative example 11
The procedure was carried out in the same manner as in example 1 except that the sample to be tested was numbered LN (Table 1), and the results of electrophoresis are shown in FIG. 1 (lane number: 13).
Comparative example 12
The procedure was carried out in the same manner as in example 1 except that the sample to be tested was designated AC (Table 1), and the results of electrophoresis were shown in FIG. 1 (lane number 5).
Comparative example 13
The procedure was carried out in the same manner as in example 1 except that the sample to be tested was numbered BK (Table 1), and the results of electrophoresis were shown in FIG. 1 (lane number 2).
Comparative example 14
The procedure was carried out in the same manner as in example 1 except that the sample to be tested was numbered HA (in Table 1), and the results of electrophoresis were shown in FIG. 1 (lane number: 11).
Comparative example 15
The procedure was carried out in the same manner as in example 1 except that the sample to be tested was identified as OG (Table 1), and the results of electrophoresis were shown in FIG. 1 (lane No. 17).
Example of detection
The agarose gel block containing the target DNA fragment with 1 band obtained was cut with a clean scalpel, purified in a DNA purification kit, and then sent to Shanghai Bioengineering technology Ltd for sequencing.
TABLE 1
Figure GDA0002673122980000101
As can be seen from the sample information in Table 1 and the electrophoresis chart shown in FIG. 1, the invention can determine whether the species is hyena or not through the strip display in the agarose gel electrophoresis chart by designing two PCR primers and performing only one time of conventional PCR amplification on the sample to be detected, the electrophoresis result completely accords with the sample information, the reliability of the identification method is verified, meanwhile, after the sequence of the DNA fragment sent to Shanghai Biotechnology Limited company for sequencing is compared with the sequence of the known hyena species in GenBank database, the obtained comparison result shows that the homology of the sequence of the sequenced DNA fragment and the sequence of the known hyena species is about 100 percent, so the species of the sample to be detected is further verified to be hyena, and in the experiment, the used DNA template can be extracted from fur, muscle tissue, viscera or feces of animals, the difficulty of sampling is greatly reduced, and the method can simply, conveniently and quickly identify the hyena, thereby achieving the effect of identifying the hyena in a simple, convenient, easy, economical and practical way.
The preferred embodiments of the present invention have been described in detail with reference to the accompanying drawings, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications can 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 hyena angustifolia and application thereof in identifying hyena angustifolia
<130> 04422
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<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> Artificial
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<223> The sequence is synthesized.
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gtcaggggat aatcatactc 20
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Claims (4)

1. A specific PCR primer for identifying hyena angustifolia (Capricorn sumatraensis), which is characterized by consisting of SEQ ID No: 1 and SEQ ID No: 2, and (b) a primer pair shown in the specification.
2. A method for identifying the hyena anguena (Capricornis sumatraensis) by utilizing a specific PCR primer, which is characterized by comprising the following steps:
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, the sample to be detected is judged to be the sample from hyena, and if no strip appears in the lane, the sample to be detected is not the sample from hyena.
3. The method of claim 2, wherein the amount of each of the specific PCR primers is 7.5X 10 based on 30. mu.L of the PCR amplification system-6-15×10-6pmol, 45-50ng of DNA template was used.
4. The method of claim 2 or 3, wherein the PCR amplification process comprises an annealing process, and the annealing process is performed under the conditions: the annealing temperature is 55-60 ℃, and the annealing time is 25-40 s.
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CN103397101A (en) * 2013-08-22 2013-11-20 山东省农业科学院生物技术研究中心 Fluorescently-labeled gene multiplex amplification method for identifying sources of goat, sheep, pig and duck meat simultaneously
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Peng Yan等.DNA-based species identification for faecal samples: An application on the mammalian survey in Mountain Huangshan Scenic Spot.《African Journal of Biotechnology》.2011,第10卷(第57期),第12134-12141等. *
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