CN102634583A - Fluorescent quantitative polymerase chain reaction (PCR) qualitative detection method for pantholops hodgsonii cashmere and products thereof - Google Patents
Fluorescent quantitative polymerase chain reaction (PCR) qualitative detection method for pantholops hodgsonii cashmere and products thereof Download PDFInfo
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- CN102634583A CN102634583A CN2012101137375A CN201210113737A CN102634583A CN 102634583 A CN102634583 A CN 102634583A CN 2012101137375 A CN2012101137375 A CN 2012101137375A CN 201210113737 A CN201210113737 A CN 201210113737A CN 102634583 A CN102634583 A CN 102634583A
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Abstract
The invention discloses a fluorescent polymerase chain reaction (PCR) qualitative detection method for pantholops hodgsonii cashmere and products thereof. The method comprises the following steps of: 1, performing PCR amplification by using DNA (Deoxyribose Nuclei Acid) of the pantholops hodgsonii cashmere as a template; and 2, detecting fluorescent signals of the amplification product to determine the pantholops hodgsonii cashmere, wherein a reaction system for PCR amplification contains specific primer pairs and probes for amplifying the DNA of pantholops hodgsonii cashmere. The fluorescent PCR qualitative detection method for the pantholops hodgsonii cashmere and the products thereof is good in specificity and high in sensitivity, and has a very positive effect of protecting pantholops hodgsonii and fighting illegal smuggling activities.
Description
Technical field
The invention belongs to the PCR detection range, specifically, is the quantitative fluorescent PCR qualitative checking method about a kind of chiru cashmere and goods thereof.
Background technology
Tibetan antelope (Pantholops hodgsonii) is one of distinctive rare species of China, belongs to animals under first-class state protection, is put into " CITS ".Tibetan antelope lives in high and cold area, HOh Xil, and whole body is covered with the thick hair of one deck, and its under hair is very soft, the about 10 μ m of diameter, and quality is splendid, and heat retention is good, is called as " shahtoosh ", the king's of Persian cashmere the meaning.Article one, the Shahtoosh tippet of heavy 100-150 gram can pass from a ring easily, is considered to tops, the most expensive fashion, and its price can reach tens thousand of dollars.Tibetan antelope is called " spirit on plateau ", can't collect the fine hair that it comes off under the natural condition.And the such scarf of actual braiding will consume 300-400 gram Tibetan antelope and give birth to suede, the i.e. life of 3-4 Tibetan antelope.Pursuing of world market and ordering about of great number interests just, the life of Tibetan antelope has received serious threat, is meeting with poaching about 70,000 to 100,000 of existing population quantity in a large number in last 20 years of 20th century.
By national wild animal conservation law, kill and smuggle 3 of Tibetan antelopes and promptly belong to important case, should give criminal sanctions from heavily handling.In recent years; The lawless person no longer smuggles whole sheepskin abroad; But take to conceal the gimmick of more hidden complicacies such as carrying secretly, the chiru cashmere is ensconced in other article, or be mixed in cashmere, the wool product; The chiru cashmere is transported to areas such as India, Nepal and processes, sell for American-European market again.
For the discriminating of chiru cashmere, mainly rely on its fiber mode of appearance at present, normally used instrument is provided with opticmicroscope, ESEM.Figure 1A-1D has shown that opticmicroscope amplifies 1400 times chiru wool, chiru cashmere, reaches the vertical form of fiber of cashmere, fine wool.Visible by figure, under identical magnification, the diameter of chiru cashmere is thinner than general cashmere, fine wool, and scale in the form of a ring, and is more sparse, and apparent comparatively similar with cashmere obscured easily.
Therefore, need a kind of better method, can differentiate the method for chiru cashmere and goods thereof rapidly and accurately, thereby protection Tibetan antelope, the illegal smuggling activity of strike are played a positive role.
Summary of the invention
It is a kind of new for the chiru cashmere of DNA detection and the quantitative fluorescent PCR qualitative checking method of goods thereof that primary and foremost purpose of the present invention just is to provide.
Second purpose of the present invention is to provide the fluorescent PCR qualitative detection test kit of a kind of chiru cashmere and goods thereof.
The 3rd purpose of the present invention is to provide a kind of application that is used for detection kit.
The quantitative fluorescent PCR qualitative checking method of chiru cashmere of the present invention and goods thereof may further comprise the steps:
The first step is a template with the DNA of chiru cashmere, carries out pcr amplification;
In second step, the fluorescent signal of detection amplified production is to determine whether the being chiru cashmere;
It is characterized in that, the Auele Specific Primer that the reaction system that is used for pcr amplification contains amplification chiru cashmere DNA to and probe.
Wherein, the right sequence of said Auele Specific Primer is shown in SEQ ID NO.1 and 2; The sequence of said specific probe is shown in SEQ ID NO.3.
According to a preferred embodiment of the invention, the condition of said pcr amplification is following:
Reaction system is: ABI Taqman universal PCR master mix (2 *) 10 μ l, dna profiling 4 μ l, primer 1.6 μ l, probe 2 μ l, ddH
2O 2.4 μ l;
The condition of reaction is: 95 ℃, and 5min; 95 ℃, 15s, 58 ℃, 45s, 40 circulations.
The detection kit of chiru cashmere provided by the invention and goods thereof contains following reagent:
(a) Auele Specific Primer of amplification chiru cashmere DNA is right;
(b) specific probe of chiru cashmere DNA.
According to the present invention, said test kit can also comprise:
(c) marker.
Wherein, the right sequence of the Auele Specific Primer of said amplification chiru cashmere DNA is shown in SEQ IDNO:1 and 2; The sequence of the specific probe of said amplification chiru cashmere DNA is shown in SEQ IDNO:3.
The detection kit of chiru cashmere of the present invention and goods thereof can be used for fluorescence qualitative detection chiru cashmere and goods thereof.
The Auele Specific Primer of the chiru cashmere real-time fluorescence PCR of design of the present invention amplification to probe and the detection kit processed thereof; Not only specificity is good to be used to detect chiru cashmere and goods thereof; And highly sensitive, have very positive effect for protection Tibetan antelope, the illegal smuggling activity of strike.
Description of drawings
Figure 1A～D has shown that respectively opticmicroscope amplifies 1400 times chiru wool, chiru cashmere, cashmere, and fiber vertical form of fine wool.
Fig. 2 is the synoptic diagram of the optimum result of PCR reaction system.
Fig. 3 has shown the blast result of Tibetan antelope primer probe sequence and goat sequence.
Fig. 4 has shown the blast result of Tibetan antelope primer probe sequence and sheep sequence.
Fig. 5 has shown the real-time fluorescence PCR result of chiru cashmere.
Fig. 6 has shown the real-time fluorescence PCR detection case behind 10 times of gradient dilutions, from top to bottom is followed successively by 1,10,10
2, 10
3With 10
4
Fig. 7 has shown the real-time fluorescence PCR result's of 10 times of gradient dilutions linear relationship.
Fig. 8 has shown the chiru cashmere detection case of different proportionings, from top to bottom is followed successively by 100%, 10% and 1%.
Fig. 9 is the part sequencer map of real-time fluorescence PCR product.
Embodiment
The present invention passes through comparison Tibetan antelope (Pantholops hodgsonii), goat (Capra hircus), reaches the sequence of the plastosome 12SrRNA gene of sheep (Ovis aries); Choose suitable sequence and carried out the primer of real-time fluorescence PCR amplification and the design of probe, and the PCR reaction system has been carried out preferably.On this basis, respectively the primer of real-time fluorescence PCR amplification is verified with specificity and the sensitivity of probe again that the result shows, the primer that the real-time fluorescence PCR that the present invention designs increases is to not only specificity is good with probe, and highly sensitive.
Below in conjunction with specific embodiment, the quantitative fluorescent PCR qualitative checking method of chiru cashmere of the present invention and goods thereof is done further explain.Should be understood that following examples only are used to the present invention is described but not are used to limit scope of the present invention.
The primer and the probe design of embodiment 1, the amplification of chiru cashmere real-time fluorescence PCR
Relatively the sequence of Tibetan antelope (Pantholops hodgsonii), goat (Capra hircus), sheep (Ovisaries) plastosome 12SrRNA gene is chosen suitable sequence and is carried out design of primers.
Quantitative fluorescent PCR product size to Tibetan antelope plastosome 12SrRNA gene is 76bp.The sequence of primer and probe is following:
chiruF：5’-CCCTCCTCAAATAAACATAATGCA-3’；(SEQ?ID?NO.1)
chiruR：5’-TTGTTACGACTTGTCTCCTCTCATG-3’；(SEQ?ID?NO.2)
Pchiru：FAM-TTAAATATACTTACACGCACCCAC-MGB。(SEQ?ID?NO.3)
Designed simultaneously Tibetan antelope, goat, sheep total pass through primer, the product size is 88bp.The sequence of primer and probe is following:
GsyF：5’-GGGTTTACGACCTCGATGTTG-3’；(SEQ?ID?NO.4)
GsyR：5’-ACGTAGGACTTTAATCGTTGAACAAA-3’；(SEQ?ID?NO.5)
Pgsy：FAM-ACCGCTATCAAAGGTT-MGB。(SEQ?ID?NO.6)
The optimization of embodiment 2, PCR reaction system
Primer concentration is 10 μ M, and concentration and probe concentration is 2 μ M.Reaction system is removed ABI Taqman universal PCR master mix (2 *) 10 μ l, and beyond the dna profiling 4 μ l, other compositions are as shown in table 1.
The condition of reaction is: 95 ℃, and 5min; 95 ℃, 15s, 58 ℃, 45s, 40 circulations.
(the unit: μ l) of the consumption of other compositions in table 1, the optimizing reaction system
| System | Primer | | ddH2O | |
| 1 | 0.4 | 1 | 4.6 | |
| 2 | 0.8 | 1 | 4.2 | |
| 3 | 1.2 | 1 | 3.8 | |
| 4 | 1.6 | 1 | 3.4 | |
| 5 | 0.4 | 2 | 3.6 | |
| 6 | 0.8 | 2 | 3.2 | |
| 7 | 1.2 | 2 | 2.8 | |
| 8 | 1.6 | 2 | 2.4 |
Results of optimization is as shown in Figure 2.Result by Fig. 2 is visible, the fluorescence signal intensity of system 8, and the Ct value is minimum, so select the real-time fluorescence PCR reaction system of system 8 for amplification chiru cashmere composition, promptly the primer final concentration is 0.8 μ M, the probe final concentration is 0.2 μ M.
The primer of embodiment 3, the amplification of chiru cashmere composition real-time fluorescence PCR and probe specificity comparison and checking
3.1, the primer and the probe specificity comparison of chiru cashmere composition real-time fluorescence PCR amplification
Through comparison, the similarity of 12SrRNA gene of Tibetan antelope and the 12SrRNA gene of goat and sheep is 93%, with the similarity of sheep 12SrRNA gene be 93%.
Fig. 3 has shown the blast result of Tibetan antelope primer probe sequence and goat sequence.
Fig. 4 has shown the blast result of Tibetan antelope primer probe sequence and sheep sequence.
3.2, the primer and the probe specificity experimental verification of chiru cashmere real-time fluorescence PCR amplification
Get chiru cashmere, cashmere, continuous mountain hair 5mg respectively, with progema hair extraction agent box extracting DNA, 70 μ l elutriant eluted dnas.Primer concentration is 10 μ M, and concentration and probe concentration is 2 μ M.Respectively with ddH
2O, chiru cashmere, cashmere, continuous mountain hair DNA are that template is carried out the fluorescent PCR amplification.Annealing temperature is 58 ℃ of 45s.The primer final concentration is 0.8 μ M, and the probe final concentration is 0.2 μ M, reaction system such as table 2.
Table 2, yak primer probe specificity confirmatory experiment reaction system (unit: μ l)
| TV (μ l) | Mix | Primer | Probe | DNA | ddH 2O | |
| 1 | 20 | 10 | 1.6 | 2 | 4(chiru) | 1.4 |
| 2 | 20 | 10 | 1.6 | 2 | 4(goat) | 1.4 |
| 3 | 20 | 10 | 1.6 | 2 | 4(sheep) | 1.4 |
| 4 | 20 | 10 | 1.6 | 2 | 4(ddH2O) | 1.4 |
The result of checking is as shown in Figure 5.Result by Fig. 5 is visible, and the primer of the chiru cashmere real-time fluorescence PCR amplification of the present invention's design and the specificity of probe are very good; Sheep's wool DNA has faint response value, and the Ct value belongs to non-specific amplification near 40, does not influence qualitative judgement.
Embodiment 4, sensitivity detect
The DNA of the chiru cashmere of extracting gained is carried out 10 times of gradient dilutions (1,10,10
2, 10
3, 10
4), carry out the real-time fluorescence PCR amplification with optimizing good reaction system (system 8 of embodiment 2), to carry out the mensuration of sensitivity, the result is as shown in Figure 6; Simultaneously the dilution amplification of difference is carried out linear analysis, figure is shown in Figure 7 as a result.
Result by Fig. 6 and Fig. 7 is visible, and the primer and the probe of the chiru cashmere real-time fluorescence PCR amplification of the present invention's design can detect 10
4The chiru cashmere DNA that doubly dilutes, and different dilution linearity is very good.
To be prone to obscure; The indistinguishable chiru cashmere of microscopy mixes with different proportionings with cashmere; Wherein the content of chiru cashmere is respectively 100%, 10%, 1%, carries out the real-time fluorescence PCR amplification with optimizing good reaction system (system 8 of embodiment 2), and the result is as shown in Figure 8.
The result of Fig. 8 shows, using method of the present invention can detect proportioning is 1% chiru cashmere.
The verity of embodiment 5, the used standard specimen of sequence verification
In view of Tibetan antelope is high-risk species, be China first class of protection animal, the transaction of any Tibetan antelope suede product all is illegal, checking has caused difficulty to sample collection.We are in Tibetan antelope plastosome 12srRNA gene, and it is right for the primer of 368bp to have designed the product size, and its scope has covered the zone of Taqman PCR upstream and downstream primer, and concrete sequence is following:
AnpeF：5’-ATGGGAAGAAATGGGGCTAC-3’；(SEQ?ID?NO.7)
AnpeR：5’-TTTGGTTTTGATTGGAAGG-3’。(SEQ?ID?NO.8)
DNA with the chiru cashmere is a template, and it is carried out pcr amplification.The upstream and downstream primer concentration is 10 μ M, and the condition of reaction is: 95 ℃, and 5min; 95 ℃, 15s, 57 ℃, 45s, 72 ℃, 40 circulations of 30s; 72 ℃, 7min; 4 ℃, termination reaction.Reaction system is as shown in table 3:
Table 3, pcr amplification reaction system (unit: μ l)
| TV | Mix | Forward primer | Reverse primer | DNA | ddH 2O |
| 30 | 15 | 1 | 1 | 4 | 9 |
Amplified production is carried out electrophoresis detection, and the result shows that the gained band is clear single, and size conforms to expection.Through the sample presentation order-checking, it is more that the assorted peak of the primer about 30bp in downstream is removed in the order-checking of being measured, and the sequence of the Tibetan antelope respective regions that remaining sequence and GenBank provide (DQ191826.1, NC_007441.1) in full accord.The sequencing result of Taqman primer probe area is as shown in Figure 9.
In sum, the Auele Specific Primer of the chiru cashmere real-time fluorescence PCR of the present invention design amplification is to not only specificity is good with probe, and highly sensitive, for differentiating chiru cashmere and goods thereof rapidly and accurately a kind of good method is provided.
And; Said Auele Specific Primer is further processed detection kit to adopting method as known in the art with probe, said test kit except said Auele Specific Primer to probe; Can also comprise marker, this is conspicuous for a person skilled in the art.
More than show and described ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; The present invention is not restricted to the described embodiments; That describes in the foregoing description and the specification sheets just explains principle of the present invention; The present invention also has various changes and modifications under the prerequisite that does not break away from spirit and scope of the invention, and these variations and improvement all fall in the scope of the invention that requires protection.The present invention requires protection domain to be defined by appending claims and equivalent thereof.
Claims (10)
1. the quantitative fluorescent PCR qualitative checking method of chiru cashmere and goods thereof may further comprise the steps:
The first step is a template with the DNA of chiru cashmere, carries out pcr amplification;
In second step, the fluorescent signal of detection amplified production is to determine whether the being chiru cashmere;
It is characterized in that, the Auele Specific Primer that the reaction system that is used for pcr amplification contains amplification chiru cashmere DNA to and probe.
2. the method for claim 1 is characterized in that, the right sequence of said Auele Specific Primer is shown in SEQ ID NO.1 and 2.
3. the method for claim 1 is characterized in that, the sequence of said specific probe is shown in SEQ ID NO.3.
4. the method for claim 1 is characterized in that, the condition of said pcr amplification is following:
Reaction system is: ABI Taqman universal PCR master mix (2 *) 10 μ l, dna profiling 4 μ l, primer 1.6 μ l, probe 2 μ l, ddH
2O 2.4 μ l;
The condition of reaction is: 95 ℃, and 5min; 95 ℃, 15s, 58 ℃, 45s, 40 circulations.
5. the detection kit of chiru cashmere and goods thereof is characterized in that containing following reagent:
(a) Auele Specific Primer of amplification chiru cashmere DNA is right;
(b) specific probe of chiru cashmere DNA.
6. test kit as claimed in claim 5 is characterized in that, said test kit also comprises: (c) marker.
7. test kit as claimed in claim 5 is characterized in that, the right sequence of the Auele Specific Primer of said amplification chiru cashmere DNA is shown in SEQ ID NO:1 and 2.
8. test kit as claimed in claim 5 is characterized in that, the sequence of the specific probe of said amplification chiru cashmere DNA is shown in SEQ ID NO:3.
9. like the application of each described test kit in the claim 5～8, it is characterized in that, be used for fluoroscopic examination chiru cashmere and goods thereof.
10. the Auele Specific Primer and the specific probe of the quantitative fluorescent PCR of chiru cashmere DNA as claimed in claim 1 is characterized in that:
The sequence of the Auele Specific Primer of said chiru cashmere DNA is shown in SEQ ID NO:1 and 2;
The sequence of the specific probe of said chiru cashmere DNA is shown in SEQ ID NO:3.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102424845A (en) * | 2011-12-23 | 2012-04-25 | 北京同仁堂股份有限公司 | Method for detecting saiga tatarica horn ingredients in mixture and primers used in same |
| CN103149210A (en) * | 2013-02-25 | 2013-06-12 | 东华大学 | System and method for detecting fabric cashmere content based on scale graphic features |
| CN104630344A (en) * | 2014-12-29 | 2015-05-20 | 中华人民共和国辽宁出入境检验检疫局 | Primer and probe for real-time fluorescence PCR (polymerase chain reaction) detection of alpaca component |
| CN110501416A (en) * | 2019-09-18 | 2019-11-26 | 上海海关工业品与原材料检测技术中心 | A kind of detection method of chiru cashmere ingredient |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102304579A (en) * | 2011-08-31 | 2012-01-04 | 福建出入境检验检疫局检验检疫技术中心 | Real-time fluorescent polymerase chain reaction (PCR) identification method for cashmere and sheep wool |
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2012
- 2012-04-18 CN CN201210113737.5A patent/CN102634583B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102304579A (en) * | 2011-08-31 | 2012-01-04 | 福建出入境检验检疫局检验检疫技术中心 | Real-time fluorescent polymerase chain reaction (PCR) identification method for cashmere and sheep wool |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102424845A (en) * | 2011-12-23 | 2012-04-25 | 北京同仁堂股份有限公司 | Method for detecting saiga tatarica horn ingredients in mixture and primers used in same |
| CN102424845B (en) * | 2011-12-23 | 2013-05-15 | 北京同仁堂股份有限公司 | Method for detecting saiga tatarica horn ingredients in mixture and primers used in same |
| CN103149210A (en) * | 2013-02-25 | 2013-06-12 | 东华大学 | System and method for detecting fabric cashmere content based on scale graphic features |
| CN103149210B (en) * | 2013-02-25 | 2015-09-30 | 东华大学 | A kind of fabric cashmere content detection system and method based on scale picture and text feature |
| CN104630344A (en) * | 2014-12-29 | 2015-05-20 | 中华人民共和国辽宁出入境检验检疫局 | Primer and probe for real-time fluorescence PCR (polymerase chain reaction) detection of alpaca component |
| CN110501416A (en) * | 2019-09-18 | 2019-11-26 | 上海海关工业品与原材料检测技术中心 | A kind of detection method of chiru cashmere ingredient |
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Inventor after: Fei Jing Inventor after: Sun Meirong Inventor after: Yang Juan Inventor after: Dong Zhenglian Inventor after: Wang Weihua Inventor after: Zhang Xiaoming Inventor after: Yan An Inventor after: Hou Ying Inventor before: Fei Jing Inventor before: Tang Minfeng Inventor before: Yang Juan Inventor before: Dong Zhenglian Inventor before: Wang Weihua Inventor before: Zhang Xiaoming Inventor before: Yan An Inventor before: Hou Ying |
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Free format text: CORRECT: INVENTOR; FROM: FEI JING TANG MINFENG YANG JUAN DONG ZHENGLIAN WANG WEIHUA ZHANG XIAOMING YAN AN HOU YING TO: FEI JING SUN MEIRONG YANG JUAN DONG ZHENGLIAN WANG WEIHUA ZHANG XIAOMING YAN AN HOU YING |
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Granted publication date: 20140312 Termination date: 20190418 |