CN102618652A - PCR (Polymerase chain reaction) kit for rapidly detecting Tarim wapiti-derived deer's products and preparation method for PCR kit - Google Patents

Abstract

The invention discloses a PCR kit for rapidly detecting Tarim wapiti-derived deer's products and a preparation method for the PCR kit. Specific molecule fragments of mitochondria DNA of a Tarim wapiti are amplified through adopting a PCR technology to prepare the kit, and the kit is used for detecting whether a sample contains the Tarim wapiti-derived compositions and detecting a mixed sample. The invention has the characteristics that the operation is simple, the sensitivity is high, and the speed is high.

Description

Red deer source, rapid detection Tarim Basin property deer product P CR test kit and preparation method

Technical field:

The present invention discloses red deer source, a kind of rapid detection Tarim Basin property deer product P CR test kit, particularly detects the composition that whether contains Tarim Basin red deer product in the deer product.Be used for quick and precisely discerning the deer product, belong to the biological detection reagent kit technical field.

Background technology:

The deer product is some tissue or organs of deer, mainly includes: pilose antler, the deer heart, deer kidney, deer tire, deer's sinew, deer whip, deer's tail, venison and deer blood etc.Because the deer product is rare traditional Chinese medicine mostly, cost an arm and a leg, so see repeatly on the market with low-quality goods and pretend to be famous and precious deer product.

At present, traditional method is adopted in the evaluation of deer product mostly, mainly comprises macroscopical identification, microscopical identification and physics and chemistry evaluation.Though traditional authentication method is simple, quick, cost is low, influences the factor a lot (like human factor, environmental factors, trophic factor) of traditional authentication method, thereby can not judge the situation of poor quality of deer product exactly.Such as in the course of processing of deer product, its proterties, microstructure, physico-chemical property etc. usually change, and utilize traditional authentication method to identify that erroneous judgement may appear in the deer product.Yet molecular assay method (based on the method for DNA) does not receive the influence of these factors, and has characteristics such as accuracy is big, highly sensitive, good stability, highly versatile.

Summary of the invention:

The present invention discloses red deer source, a kind of rapid detection Tarim Basin property deer product P CR test kit, has solved the problem that whether contains Tarim Basin red deer composition in the deer product.

The present invention also provides the preparation method of the PCR test kit of above-mentioned rapid detection deer product, adopts single step reaction to differentiate the Tarim Basin red deer derived component in the deer product through a PCR method reaction system.

The PCR test kit of red deer source, rapid detection provided by the invention Tarim Basin property deer product is characterized in that:

Comprise DNA extraction liquid, PCR reaction solution and negative standard substance:

Described DNA extraction liquid:

(1) low salt buffer (pH 7.6-7.8): 12-13 part Tris alkali (Tutofusin tris), 32-35 part Na ₂EDTA (EDTA Disodium), 7-8 part MgCl ₂, 4-5 part NaCl, surplus part is water;

(2) cell pyrolysis liquid: 4-6 part SDS sodium lauryl sulphate, 4-6 part Triton X-100 (polyoxyethylene glycol is to the iso-octyl phenyl ether), 1000 parts of low salt buffers;

Described PCR reaction solution:

DdH2O, 10 * PCR Buffer, dNTPs, upstream primer, downstream primer, Taq enzyme;

Concrete primer sequence is:

Upstream primer: 5 '-CTCAACATCCATTTTACATCTTACATTA-3 '

Downstream primer: 5 '-CATGGTAATTAAGCTCGTGATCTA-3 '

Negative standard substance: be aseptic double-distilled water.

The preparation method of the PCR test kit of red deer source, rapid detection according to the invention Tarim Basin property deer product may further comprise the steps:

1) DNA extraction liquid

(1) low salt buffer (pH 7.6-7.8): 12-13 part Tris alkali (Tutofusin tris), 32-35 part Na ₂EDTA (EDTA Disodium), 7-8 part MgCl ₂, 4-5 part NaCl, surplus part is water;

(2) cell pyrolysis liquid: 4-6 part SDS sodium lauryl sulphate, 4-6 part Triton X-100 (polyoxyethylene glycol is to the iso-octyl phenyl ether), 1000 parts of low salt buffers;

2) PCR reaction solution.

(1) design of primers: the Mitochondrial DNA D-loop of red deer the zone in the Tarim Basin, design a pair of Auele Specific Primer, comprise a upstream primer and a downstream primer.Sequence is following:

Upstream primer: 5 '-CTCAACATCCATTTTACATCTTACATTA-3 '

Downstream primer: 5 '-CATGGTAATTAAGCTCGTGATCTA-3 '

(2) PCR reaction solution:

DdH2O 9.3ul, 10 * PCR Buffer 1.5ul, dNTPs (100 uM) 2.0ul, upstream primer (20 uM) 0.5ul, downstream primer (20 uM) 0.5ul, Taq enzyme (5U/ul) 0.2ul.

(3) negative standard substance: be aseptic double-distilled water.

The method of use that red deer source, Tarim Basin of the present invention property deer product P CR detects:

(1) from sample to be checked, extracts genomic dna;

(2) preparation PCR reaction system:

DNA 1ul and the negative control got in the step (1) join in the PCR reaction solution, increase with the PCR appearance;

(3) set response procedures and carry out pcr amplification, response procedures is all following:

94 ℃ of 5min; 94 ℃ of 30s, 62 ℃ of 30s, 72 ℃ of 30s, 72 ℃ of 5min, 30 circulations of increasing.

(4) get 5 μ l PCR products after reaction finishes and analyze observations under the uv lamp at 1% agarose gel electrophoresis.

The result judges: if amplify the fragment that length is 272bp, explain and contain Tarim Basin red deer derived components in the test sample.

Positively effect of the present invention is:Adopt the special molecular fragment of the Mitochondrial DNA of round pcr amplification Tarim Basin red deer, the generate a reagent box is used for detecting test sample and whether contains Tarim Basin red deer derived components, and can detect biased sample; Have simple to operate, highly sensitive, characteristics such as speed is fast.

Description of drawings

Fig. 1 is a specificity between the kind of primer;

Fig. 2 is the interior versatility of the kind of primer.

Embodiment

Below in conjunction with the practical implementation instance, further set forth the present invention:

Embodiment 1: the composition of test kit and preparation

1) preparation of DNA extraction liquid

(1) low salt buffer (pH 7.6): 800ml dissolved in distilled water 1.21gTris alkali (Tutofusin tris), 3.38g Na ₂EDTA (EDTA Disodium), 0.76g MgCl ₂, 0.47gNaCl.Add concentrated hydrochloric acid and transfer pH to desirable value, adding distil water is settled to 1L.

(2) cell pyrolysis liquid: add 1g SDS (sodium lauryl sulphate), 1ml Triton X-100 (polyoxyethylene glycol is to the iso-octyl phenyl ether) in the 200ml low salt buffer.

Process for extracting: 200ul anticoagulation and 400ul low salt buffer join in the 1.5ml centrifuge tube, and fully the centrifugal 10min of 1000RPM behind the mixing abandons supernatant; Add the 500ul low salt buffer, behind the mixing under room temperature the centrifugal 5min of 1000RPM, repeat once; Careful sucking-off supernatant adds the 300ul cell pyrolysis liquid, behind the mixing in 65 ℃ of water-bath 10min; Add the saturated NaCl of 75ul, fully the centrifugal 5min of 12000RPM behind the mixing transfers to supernatant in the new 1.5ml centrifuge tube; Add 2 times of volume absolute ethyl alcohols, in the centrifugal 2min of 10000RPM, abandon supernatant behind the abundant mixing; Add 500ul ice bath 70% ethanol, put upside down mixing and abandon supernatant in the centrifugal 1min of 10000RPM in the back for several times; Dry 5min in 65 ℃ of baking ovens dissolves 15min in 65 ℃ of water-baths behind the adding 100ul ddH2O.

2) PCR reaction solution

(1) design of primers: according to the animal in deer family plastosome whole genome sequence of having announced in the GeneBank DB; After adopting Bioedit 7.0.9.0 software to carry out sequence alignment; According to having conservative property in planting, having specific principle between planting and select mitochondrial D-loop zone, utilize the Auele Specific Primer of Oligo 6.0 software design Tarim Basin red deers.Comprising one downstream primer, a upstream primer.Primer is to having specificity between kind of interior versatility and kind.Sequence is following:

Upstream primer: 5 '-CTCAACATCCATTTTACATCTTACATTA-3 '

Downstream primer: 5 '-CATGGTAATTAAGCTCGTGATCTA-3 '

(2) PCR reaction solution:

DdH2O 9.3ul, 10 * PCR Buffer 1.5ul, dNTPs (100 uM) 2.0ul, upstream primer (20 uM) 0.5ul, downstream primer (20 uM) 0.5ul, Taq enzyme (5U/ul) 0.2ul.

(3) negative standard substance: be aseptic double-distilled water.

The foundation of embodiment 2:PCR optimum reaction condition.

For a PCR reaction, most important reaction conditions is exactly an annealing temperature, therefore sets the top condition of PCR reaction from this aspect.Specificity and the interior generally adopted principle of kind between the setting of optimum reaction condition at first should be followed and plant, the temperature when selecting expanding effect the most desirable then on this basis is set at the optimal reaction temperature of primer.

1) extraction of DNA

From blood, extracted the genomic dna of Tarim Basin red deer according to the method for embodiment 1.

2) preparation of PCR reaction system:

The DNA 1ul that extracts in the step 1) is joined in the PCR reaction solution among the embodiment 1, constitute the reaction system of 15ul.

3) setting of PCR reaction optimum annealing temperature.

The gradient scope of annealing temperature is 54-68 ℃, whenever establishes a gradient at a distance from 2 ℃, is reflected on the Eppendorf AG instrument and increases.

Program is following:

94 ℃ of 5min; 94 ℃ of 30s, 54-68 ℃ of 30s, 72 ℃ of 30s, 72 ℃ of 5min, 30 circulations of increasing.

Get 5 μ l PCR products after reaction finishes and in 1% sepharose, carry out electrophoresis detection, write down experimental result in the gel imaging system, confirm the optimum annealing temperature of every pair of primer according to the brightness of band in the gel.

4) establishment of best PCR reaction conditions.

Final definite best PCR reaction conditions is:

94 ℃ of 5min; 94 ℃ of 30s, 62 ℃ of 30s, 72 ℃ of 30s, 72 ℃ of 5min, 30 circulations of increasing.

Embodiment 3: the specificity test of test kit

1) proves that primer has specificity between kind.

(A) To prove the present invention has a species-specific primers, in accordance with the method of Example 1 was extracted deer, red deer, Tarim red deer, red deer, reindeer, deer, Eld's deer, white-lipped deer, elk, fallow deer, pigs , cattle, sheep, chicken genomic DNA, a DNA extraction system for preparing a PCR reaction, sterile water as a negative control.

(2) preparation of PCR reaction system: the DNA 1ul that gets step (1) extraction joins in the PCR reaction solution among the embodiment 1, constitutes the 15ul system.

(3) according to response procedures: 94 ℃ of 5min; 94 ℃ of 30s, 62 ℃ of 30s, 72 ℃ of 30s, 72 ℃ of 5min, 30 circulations of increasing.Carry out pcr amplification.

(4) The results shown in Figure 1: N: deer; C: red deer; Y: Tarim red deer; E: red deer; R: Reindeer; 1: Sambar; 2: Eld; 3: white-lipped deer; 4: elk; 5 : Fallow deer; 6: Pig; 7: Cattle; 8: Sheep; 9: chicken; 10: negative control; M: Marker? I (600,500,400,300,200,100? bp);

In all species, genomic DNA PCR reaction results, only the Tarim Malu Ji genomic DNA fragment was amplified 272bp specificity, and deer, red deer, red deer, reindeer, deer, Eld's deer, white-lipped deer, elk, fallow deer , pigs, cattle, sheep, chicken genomic DNA was negative, indicating that the present invention is a species-specific primers.

2) prove versatility in the kind of primer.

Primer also must have kind of an interior versatility simultaneously except between planting, having the specificity, therefore versatility in the kind of primer is also assessed.

(1) extracted the genomic dnas of 9 Tarim Basin red deers according to the method for embodiment 1, the DNA of extraction is used to prepare the PCR reaction system, and sterilized water is as negative control.

(2) preparation of PCR reaction system: the DNA 1ul that gets step (1) extraction joins in the PCR reaction solution among the embodiment 1, constitutes the 15ul system.

(3) according to response procedures: 94 ℃ of 5min; 94 ℃ of 30s, 62 ℃ of 30s, 72 ℃ of 30s, 72 ℃ of 5min, 30 circulations of increasing.Carry out pcr amplification.

(4) result such as Fig. 2: Tarim Basin red deer (1-9), 10: negative control; M:Marker I (600,500,400,300,200,100 bp); The genomic dna of 9 Tarim Basin red deers has all amplified the purpose fragment of 272bp.Explain that primer of the present invention has versatility in planting.

Claims (3)

1. the PCR test kit of red deer source, rapid detection Tarim Basin property deer product is characterized in that comprising following primer:

Upstream primer: 5 '-CTCAACATCCATTTTACATCTTACATTA-3 '

Downstream primer: 5 '-CATGGTAATTAAGCTCGTGATCTA-3 '.

2. the PCR test kit of red deer source, the described rapid detection of claim 1 Tarim Basin property deer product is characterized in that comprising DNA extraction liquid, PCR reaction solution and negative standard substance;

Described DNA extraction liquid:

(1) low salt buffer (pH 7.6-7.8): 12-13 part Tris alkali (Tutofusin tris), 32-35 part Na ₂EDTA (EDTA Disodium), 7-8 part MgCl ₂, 4-5 part NaCl, surplus part is water;

(2) cell pyrolysis liquid: 4-6 part SDS sodium lauryl sulphate, 4-6 part Triton X-100 (polyoxyethylene glycol is to the iso-octyl phenyl ether), 1000 parts of low salt buffers;

Described PCR reaction solution:

DdH2O, 10 * PCR Buffer, dNTPs, upstream primer, downstream primer, Taq enzyme;

Concrete primer sequence is:

Upstream primer: 5 '-CTCAACATCCATTTTACATCTTACATTA-3 '

Downstream primer: 5 '-CATGGTAATTAAGCTCGTGATCTA-3 '

Negative standard substance: be aseptic double-distilled water.

3. the preparation method of the PCR test kit of red deer source, the described rapid detection of claim 2 Tarim Basin property deer product may further comprise the steps:

1) DNA extraction liquid

(1) low salt buffer (pH 7.6-7.8): 12-13 part Tris alkali (Tutofusin tris), 32-35 part Na ₂EDTA (EDTA Disodium), 7-8 part MgCl ₂, 4-5 part NaCl, surplus part is water;

(2) cell pyrolysis liquid: 4-6 part SDS sodium lauryl sulphate, 4-6 part Triton X-100 (polyoxyethylene glycol is to the iso-octyl phenyl ether), 1000 parts of low salt buffers;

2) PCR reaction solution;

(1) design of primers: the Mitochondrial DNA D-loop of red deer the zone in the Tarim Basin, design a pair of Auele Specific Primer, comprise a upstream primer and a downstream primer;

Sequence is following:

Upstream primer: 5 '-CTCAACATCCATTTTACATCTTACATTA-3 '

Downstream primer: 5 '-CATGGTAATTAAGCTCGTGATCTA-3 '

(2) PCR reaction solution:

DdH2O 9.3ul, 10 * PCR Buffer 1.5ul, dNTPs (100 uM) 2.0ul, upstream primer (20 uM) 0.5ul, downstream primer (20 uM) 0.5ul, Taq enzyme (5U/ul) 0.2ul;

(3) negative standard substance: be aseptic double-distilled water.

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