CN104059978A - Multi-polymerase chain reaction (PCR) primer and method for identifying clouded leopard and application of primer or method in identification of clouded leopard - Google Patents
Multi-polymerase chain reaction (PCR) primer and method for identifying clouded leopard and application of primer or method in identification of clouded leopard Download PDFInfo
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Abstract
The invention discloses a multi-polymerase chain reaction (PCR) primer for identifying a clouded leopard. The multi-PCR primer comprises primer pairs 1 shown in SEQ ID No:1 and SEQ ID No:2, and primer pairs 2 shown in SEQ ID No:3 and SEQ ID No:4. The invention also discloses a method for identifying the clouded leopard by using multi-PCR. The method comprises the following steps: extracting total deoxyribonucleic acid (DNA) of a to-be-tested sample and carrying out PCR amplification on the total DNA; and carrying out agarose gel electrophoresis detection on the amplified product, wherein the multi-PCR primer is the multi-PCR primer, and judging the to-be-tested sample to be the sample from the clouded leopard if two stripes appear in a lane. The invention also discloses an application of the multi-PCR primer or identification method in identification of the clouded leopard. The target of simply, feasibly, economically and practically identifying the clouded leopard is achieved by designing two pairs of primers.
Description
Technical field
The present invention relates to field of biological detection, particularly, relate to a kind of multiple PCR primer for the identification of clouded leopard and use described multiple PCR primer to identify the method for clouded leopard, and their application in identifying clouded leopard.
Background technology
Based on taxonomic accurate species, identify it is the prerequisite of human cognitive nature and Sustainable development.It is the sustainability utilization of biological multifarious protection, species resource, and the new product development of biogenetic derivation etc. provides knowledge and theory basis.For the evaluation of species, traditional method is mainly to adopt morphological evidence to carry out.But some defects of the method have also caused the limitation of animals on the brink of extinction protection work, such as phenotypical plasticity and hereditary mutability, cannot distinguish and hiddenly deposit taxon, be subject to biological sex and etap restriction etc.Putting into practice in work of the conservation of wildlife, some censorship animal samples of originating not clear are imperfect and sample size is less often, are difficult to be identified from morphology, as hair, deformed limb, corrupt tissue, bone, hide and ight soil etc.But; according to the regulation of the wild animal conservation law > > of the < < People's Republic of China (PRC); species information is the important legal argument of the measurement of penalty accurately, is directly involved in suspect's personal power and the strict law enforcement problem of administrative department.
Clouded leopard (Neofelis nebulosa) is the feline of Mammalia (Mammalia) Carnivora (Carnivora) cat family (Felidae) Neofelis (Neofelis).The dark-coloured speckle of clouded leopard health both sides tool cloud form, body is about 70～110 centimetres, and tail is about 70～90 centimetres, and approximately 16～40 kilograms of body weight are Pantherinae reckling.Clouded leopard is in Chinese distribution north limit in Qinling Mountains in Shaanxi, SOUTH OF GANSU, and to Chayu, Tibet and other places, south terminates in Hainan Province, to the east of Zhejiang Province and Taiwan Province.China has two subspecies: nominate subspecies (Neofelis nebulosa nebulosa), and China clouded leopard is all nominate subspecies; Taiwan subspecies (Neofelis nebulosa brachyurus), now become extinct.Clouded leopard is as the top carnivore in food web, and predation many animals in occupation of a plurality of ranks, is also the key species that maintains ecosystem function and species diversity level in trophic level sequence.The same with other feline of the world, clouded leopard is subject to the extensive threat of the factors such as distribution density is low, scope of activity large, the decline of prey quantity, poaching equally.In China, clouded leopard has been listed in country-level focused protection wildlife.In the whole world; also be put into easily danger (VU) level species of World Conservation Union (IUCN); also by < < CITS > > (CITES), listed in appendix I species, clouded leopard and species product thereof are strictly prohibited international business's trade simultaneously.Therefore, in actual conservation of wildlife work, to clouded leopard species and correlated product thereof carry out science, species identify it is the prerequisite of carrying out every law-enforcing work exactly.
On the other hand, owing to being subject to the restriction of the movable reason such as hide, defy capture, the population ecology research of carrying out at present relevant big cat can only depend on indirectly evidence, such as vestige evidence (as footprint, scratch, urine mark and scratch etc.) with to visiting and investigating of local resident etc.But vestige only can provide data qualitatively to wild clouded leopard population, can not realize the quantity dynamic fluctuation of following the tracks of and monitoring Wild population.In recent years, the Fecal DNA analysis that the animal excrement of take are research material has greatly been expanded the scope of non-damage sampling, has enriched the investigative technique that conservation genetics is learned.By the identification to multiple genetic marker in faeces DNA, investigator can carry out deep research to the aspects such as population quantity investigation, field boundary delimitation, the life history, Genetic Constitution of Population and genetic diversity of wildlife.In the beasts population ecology research of carrying out based on faeces DNA, often need investigator first to picking up from a large amount of beasts faecal samples in field, according to excrement type feature, to carry out preliminary differentiation, the ight soil of clouded leopard species is picked out separately standby, this is a very time-consuming preliminary preparation.Meanwhile, due to the morphological features such as excrement type often in the wild and send back in the transportation in laboratory and be easily damaged, bring difficulty to identification and the evaluation of a large amount of faecal samples.If rely on molecule sequence measurement to identify, the economic problems that can cause cost to increase.In addition, have large quantity research and show, less amplified production length (general <500bp) can improve PCR specificity and success ratio for faeces DNA effectively.This requires the special primer amplified production that we develop also must have the feature that sheet degree can not be too large.
The protection that the develop rapidly of the authenticate technology of DNA classification is in recent years species, effective management of wildlife provide powerful instrument.In the Cyt b gene of animal mitochondria DNA (mtDNA) and 12SrRNA gene, exist comparatively fast and compared with the codon site of slow-motion, and the existence of conservative region and Sudden change region makes it can be applicable to animal system to learn research simultaneously.By numerous scholars, thought to carry out phyletic evolution in animal kind and between infraspecific category at present and study one of best molecular genetic marker.
Therefore, the loaded down with trivial details steps such as a kind of order-checking without DNA, sequence alignment are provided, simultaneously unrestricted to sample source, and can only by a pcr amplification, can fast and effeciently to realize the multiple PCR primer of the Accurate Measurement of clouded leopard species be problems that the present invention needs solution badly.
Summary of the invention
For above-mentioned prior art, the object of the invention is to overcome the limitation of by morphological feature, clouded leopard species being measured in prior art, or with ordinary method to DNA in ight soil measure determine species method take time and effort and spend larger problem, a kind of simple and efficient and economic multiple PCR primer for the identification of clouded leopard is provided and identifies the method for clouded leopard, and their application in identifying clouded leopard.
To achieve these goals, the invention provides a kind of multiple PCR primer for the identification of clouded leopard (Neofelis nebulosa), wherein, described multiple PCR primer comprises the primer pair 1 as shown in SEQ ID No:1 and SEQ ID No:2, and the primer pair 2 as shown in SEQ ID No:3 and SEQ ID No:4.
The present invention also provides a kind of method of utilizing identification with multi-plex PCR clouded leopard (Neofelis nebulosa), and wherein, described method comprises:
(1) extract total DNA of testing sample;
(2) adopt multiple PCR primer to carry out pcr amplification to the total DNA extracting in step (1);
(3) get the product obtaining in step (2) and do agarose gel electrophoresis detection;
Wherein, described multiple PCR primer is multiple PCR primer as above,
Wherein, in the agarose gel electrophoresis figure of step (3) gained, if there are 2 bands in the swimming lane containing the product that (2) obtain in steps, judge that described testing sample is for the sample from clouded leopard, if there are not 2 bands in the swimming lane containing the product that (2) obtain in steps, judge that described testing sample is not the sample from clouded leopard.
The present invention also provides a kind of application in identifying clouded leopard (Neofelis nebulosa) according to above-mentioned multiple PCR primer or above-mentioned authentication method.
The present invention is by two groups of PCR primers of design, utilize the method for multiplex PCR amplification, to testing sample, by once conventional pcr amplification, can show whether determine these species is clouded leopard by the band in agarose gel electrophoresis figure, and DNA profiling used can extract from fur, muscle tissue, internal organ or the ight soil of animal, greatly reduce the difficulty of sampling, and the method can be identified clouded leopard quickly and easily, thereby reached the effect of identifying clouded leopard simple and easy to do and economical and practically.
Other features and advantages of the present invention partly in detail are described the embodiment subsequently.
Accompanying drawing explanation
Accompanying drawing is to be used to provide a further understanding of the present invention, and forms a part for specification sheets, is used from explanation the present invention, but is not construed as limiting the invention with embodiment one below.In the accompanying drawings:
Fig. 1 is the sugared gel electrophoresis figure of the amplified production of embodiment 1-3 and comparative example 1-9; Wherein, from distilled water sample, swimming lane 1, from the muscle samples of snow leopard, swimming lane 2, from muscle samples, swimming lane 3 and the swimming lane 4 of leopard, from the muscle samples of clouded leopard, swimming lane 5, from faecal samples, the swimming lane 6 of clouded leopard, muscle samples, the swimming lane 7 of tiger are molecular weight marker from faecal samples, the swimming lane 11 of macaca thibetana from faecal samples, the swimming lane M of yellow weasel from faecal samples, the swimming lane 10 of wild boar from muscle samples, the swimming lane 9 of leopard cat from hide sample, the swimming lane 8 of lynx to swimming lane C northeast.
Embodiment
Below the specific embodiment of the present invention is elaborated.Should be understood that, embodiment described herein only, for description and interpretation the present invention, is not limited to the present invention.
The invention provides a kind of multiple PCR primer for the identification of clouded leopard (Neofelis nebulosa), wherein, described multiple PCR primer comprises the primer pair 1 as shown in SEQ ID No:1 and SEQ ID No:2, and the primer pair 2 as shown in SEQ ID No:3 and SEQ ID No:4.
The present invention also provides a kind of method of utilizing identification with multi-plex PCR clouded leopard (Neofelis nebulosa), and wherein, described method comprises:
(1) extract total DNA of testing sample;
(2) adopt multiple PCR primer to carry out pcr amplification to the total DNA extracting in step (1);
(3) get the product obtaining in step (2) and do agarose gel electrophoresis detection;
Wherein, described multiple PCR primer is multiple PCR primer as above,
Wherein, in the agarose gel electrophoresis figure of step (3) gained, if there are 2 bands in the swimming lane containing the product that (2) obtain in steps, judge that described testing sample is for the sample from clouded leopard, if there are not 2 bands in the swimming lane containing the product that (2) obtain in steps, judge that described testing sample is not the sample from clouded leopard.
In the present invention, in 2 of appearance bands, the length of contained DNA fragmentation is respectively 230bp and 370bp.
In the present invention, in order to reach better pcr amplification effect, make the agarose gel electrophoresis figure obtaining more high-visible, be convenient to determine better experimental result, the pcr amplification system of 30 μ L of take is benchmark, the concentration of every primer is respectively 0.25-0.5pmol/L, and the consumption of DNA profiling is 45-55ng.Wherein, for the polysaccharase of pcr amplification and damping fluid, can be the conventional polysaccharase in this area and damping fluid, what its consumption was concrete can be with reference to the detailed description in specification sheets.In this not go into detail in the present invention.
Annealing process in described pcr amplification process can by this area the routine annealing way and the annealing parameter that use, in the present invention, in order to obtain better expanding effect, the annealing temperature of described pcr amplification process is 55-60 ℃, annealing time is 25-40s.In addition, be the setting of this area routine for the Denaturing in pcr amplification, extension condition and the isoparametric setting of cycle number, in this not go into detail in the present invention.
The present invention also provides a kind of and has identified clouded leopard (Neofelis nebulosa) according to above-mentioned multiple PCR primer or above-mentioned authentication method) in application.
Below will describe the present invention by embodiment.
What need to illustrate is, described primer synthesizes by Shanghai Sheng Gong biotechnology company limited and primer concentration is 10pmol/L, described lysate can be the conventional DNA cleavage liquid using in this area, for example, in the present invention, the Tris-HCl (pH8.0) that contains 50mmol in every liter of described lysate, the ethylenediamine tetraacetic acid (EDTA) of 25mmol (pH8.0), the sodium lauryl sulphate of the sodium-chlor of 100mmol and 1% weight part, the dNTPMix using in the present invention is the commercially available product of Transgene company, Proteinase K is the commercially available product of Merck company, described DNA purification kit is the commercially available product of Tiangen company, all the other chemical reagent that use are conventional commercially available analytical reagent.
Embodiment 1
The testing sample that is numbered 3 in 0.5g table 1 is placed in to centrifuge tube and at centrifuge tube, testing sample is shredded with sterilization scissors, then to the lysate that adds successively 500 μ L in centrifuge tube, the Proteinase K of 10% sodium lauryl sulphate of 30 μ L and the 20mg/ml of 3 μ L, fully mixes rear 56 ℃ of water-baths digestion 12h and clarifies to liquid in pipe.In above-mentioned postdigestive mixture, add Tris-balance phenol 500 μ L, and the 5min that slightly vibrates is placed on centrifugal 10min on the whizzer of 11000r/min, gets the supernatant liquor after centrifugal.Repeat twice of above-mentioned centrifugation step.To adding freezing dehydrated alcohol 1000 μ L in the supernatant liquor finally obtaining after repeated centrifugation and placing at-20 ℃ on the whizzer that 1h is placed on 12000r/min after centrifugal 13min, abandon supernatant liquor, and to the ethanol 800 μ L that add 70% in the precipitation obtaining, and the 0.5min that slightly vibrates is placed on freezing centrifugal 13min on the whizzer of 13000r/min, abandon supernatant liquor.Repeat twice of above-mentioned centrifugation step.The precipitation obtaining is placed in after naturally drying 2.5h on aseptic operating platform and adds wherein TE solution 400 μ L, slightly vibrate and use finger to flick centrifuge tube after at 4 ℃, place the laggard row agarose gel electrophoresis of 3h, obtain total DNA.
To the 10 * PCR buffer that adds 10 μ L in the sample hose of PCR instrument, every each 1 μ L of primer, the dNTPMix of the 2mmol/L of 2 μ L, the Mg of the 25mmol/L of 2 μ L
2+, the Taq enzyme of 1U and total DNA of 50ng, and with distilled water polishing to 30 μ L.PCR reaction conditions is: 95 ℃ of denaturation 5min; Then carry out 30 circulations, this circulation comprises: 95 ℃ of sex change 40s, 58 ℃ of annealing 30s and 72 ℃ of extension 35s; Finally remake 72 ℃ and extend 10min.Under above-mentioned reaction conditions, carry out pcr amplification.DNA after amplification is carried out to agarose gel electrophoresis, and electrophoresis result is (swimming lane is numbered 3) as shown in Figure 1.
Embodiment 2
Method according to embodiment 1 operates, different is, testing sample is numbered 4 (in tables 1), the consumption of described every primer is 0.75 μ L, the consumption of described total DNA is 45ng, described annealing temperature is 55 ℃, and described annealing time is 40s, and electrophoresis result is (swimming lane is numbered 4) as shown in Figure 1.
Embodiment 3
Method according to embodiment 1 operates, different is, testing sample is numbered 5 (in tables 1), the consumption of described every primer is 1.25 μ L, the consumption of described total DNA is 55ng, described annealing temperature is 60 ℃, and described annealing time is 25s, and electrophoresis result is (swimming lane is numbered 5) as shown in Figure 1.
Comparative example 1
Using the distilled water that is numbered C in table 1 as total DNA, according to the method for embodiment 1, increase and agarose gel electrophoresis, electrophoresis result is (swimming lane is numbered C) as shown in Figure 1.
Comparative example 2
Method according to embodiment 1 operates, different, and testing sample is numbered 1 (in table 1), and electrophoresis result is (swimming lane is numbered 1) as shown in Figure 1.
Comparative example 3
Method according to embodiment 1 operates, different, and testing sample is numbered 2 (in tables 1), and electrophoresis result is (swimming lane is numbered 2) as shown in Figure 1.
Comparative example 4
Method according to embodiment 2 operates, different, and testing sample is numbered 6 (in tables 1), and electrophoresis result is (swimming lane is numbered 6) as shown in Figure 1.
Comparative example 5
Method according to embodiment 2 operates, different, and testing sample is numbered 7 (in tables 1), and electrophoresis result is (swimming lane is numbered 7) as shown in Figure 1.
Comparative example 6
Method according to embodiment 2 operates, different, and testing sample is numbered 8 (in tables 1), and electrophoresis result is (swimming lane is numbered 8) as shown in Figure 1.
Comparative example 7
Method according to embodiment 3 operates, different, and testing sample is numbered 9 (in tables 1), and electrophoresis result is (swimming lane is numbered 9) as shown in Figure 1.
Comparative example 8
Method according to embodiment 3 operates, different, and testing sample is numbered 10 (in tables 1), and electrophoresis result is (swimming lane is numbered 10) as shown in Figure 1.
Comparative example 9
Method according to embodiment 3 operates, different, and testing sample is numbered 11 (in tables 1), and electrophoresis result is (swimming lane is numbered 11) as shown in Figure 1.
Test example
The sepharose piece that contains target DNA fragment that obtains two bands is cut and after purifying, deliver in DNA purification kit Shanghai Sheng Gong biotechnology company limited to check order with clean scalpel.
Table 1
| Numbering | Species | Sample type | Locality |
| C | Distilled water | --- | --- |
| 1 | Snow leopard (Panthera uncia) | Muscle | Anhui Ningguo |
| 2 | Leopard (Panthera pardus) | Muscle | Anhui Ningguo |
| 3 | Clouded leopard (Neofelis nebulosa) | Muscle | Anhui Ningguo |
| 4 | Clouded leopard (Neofelis nebulosa) | Muscle | Anhui Ningguo |
| 5 | Clouded leopard (Neofelis nebulosa) | Ight soil | Mt. Huang in Anhui |
| 6 | Northeastern tiger (Panthera tigris altaica) | Muscle | Anhui Ningguo |
| 7 | Lynx (Lynx lynx) | Hide | Anhui Ningguo |
| 8 | Leopard cat (Prionailurus bengalensis) | Muscle | Anhui Ningguo |
| 9 | Wild boar (Sus scrofa) | Ight soil | Mt. Huang in Anhui |
| 10 | Macaca thibetana (Macaca thibetana) | Ight soil | Mt. Huang in Anhui |
| 11 | Yellow weasel (Mustela sibirica) | Ight soil | Mt. Huang in Anhui |
Sample message and the electrophorogram shown in Fig. 1 by table 1 can be found out, the present invention is by two groups of PCR primers of design, to testing sample, only by once conventional pcr amplification, can show whether determine these species is clouded leopard by the band in agarose gel electrophoresis figure, and electrophoresis result and sample message meet completely, confirmed the reliability of this authentication method, simultaneously, after the sequence of delivering to clouded leopard species known in the sequence of the DNA fragmentation that Shanghai Sheng Gong biotechnology company limited checks order and database is compared, the comparison result obtaining shows that the sequence of DNA fragmentation of this order-checking and the homology of the sequence of known clouded leopard species are more than 97%, so also further confirmed that the species of this testing sample are clouded leopard, and in this experiment, DNA profiling used can be from the fur of animal, muscle tissue, in internal organ or ight soil, extract, greatly reduce the difficulty of sampling, and the method can be identified clouded leopard quickly and easily, thereby reached the effect of identifying clouded leopard simple and easy to do and economical and practically.
More than describe the preferred embodiment of the present invention in detail; but the present invention is not limited to the detail in above-mentioned embodiment, within the scope of technical conceive of the present invention; can carry out multiple simple variant to technical scheme of the present invention, these simple variant all belong to protection scope of the present invention.
It should be noted that in addition, each concrete technical characterictic described in above-mentioned embodiment, in reconcilable situation, can combine by any suitable mode, for fear of unnecessary repetition, the present invention is to the explanation no longer separately of various possible array modes.
In addition, between various embodiment of the present invention, also can carry out arbitrary combination, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (6)
1. the multiple PCR primer for the identification of clouded leopard (Neofelis nebulosa), it is characterized in that, described multiple PCR primer comprises the primer pair 1 as shown in SEQ ID No:1 and SEQ ID No:2, and the primer pair 2 as shown in SEQ ID No:3 and SEQ ID No:4.
2. a method of utilizing identification with multi-plex PCR clouded leopard (Neofelis nebulosa), is characterized in that, described method comprises:
(1) extract total DNA of testing sample;
(2) adopt multiple PCR primer to carry out pcr amplification to the total DNA extracting in step (1);
(3) get the product obtaining in step (2) and do agarose gel electrophoresis detection;
Wherein, described multiple PCR primer is the multiple PCR primer described in claim 1,
Wherein, in the agarose gel electrophoresis figure of step (3) gained, if there are 2 bands in the swimming lane containing the product that (2) obtain in steps, judge that described testing sample is for the sample from clouded leopard, if there are not 2 bands in the swimming lane containing the product that (2) obtain in steps, judge that described testing sample is not the sample from clouded leopard.
3. method according to claim 2, wherein, in 2 bands of appearance, the length of contained DNA fragmentation is respectively 230bp and 370bp.
4. method according to claim 3, wherein, the pcr amplification system of 30 μ L of take is benchmark, and the concentration of every primer is respectively 0.25-0.5pmol/L, and the consumption of DNA profiling is 45-55ng.
5. according to the method described in claim 2 or 4, wherein, the annealing temperature of described pcr amplification process is 55-60 ℃, and annealing time is 25-40s.
6. the application of the method described in any one in identifying clouded leopard (Neofelis nebulosa) in multiple PCR primer according to claim 1 or claim 2-5.
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| CN105803089A (en) * | 2016-05-05 | 2016-07-27 | 安徽师范大学 | Multi-PCR primer for identifying periplaneta americana and identifying method thereof |
| CN106868122A (en) * | 2017-02-16 | 2017-06-20 | 安徽师范大学 | Multiple PCR primer and method and their applications in macaque is identified for identifying macaque |
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| CN101845508A (en) * | 2010-06-12 | 2010-09-29 | 徐君怡 | PCR detection primer, kit and detection method for tiger-derived and leopard-derived DNA |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105803089A (en) * | 2016-05-05 | 2016-07-27 | 安徽师范大学 | Multi-PCR primer for identifying periplaneta americana and identifying method thereof |
| CN106868122A (en) * | 2017-02-16 | 2017-06-20 | 安徽师范大学 | Multiple PCR primer and method and their applications in macaque is identified for identifying macaque |
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