CN104862311A - Real-time fluorescence quantification PCR primer for detecting Chinese alligator based on Taqman probe, kit and method - Google Patents
Real-time fluorescence quantification PCR primer for detecting Chinese alligator based on Taqman probe, kit and method Download PDFInfo
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- CN104862311A CN104862311A CN201510223933.1A CN201510223933A CN104862311A CN 104862311 A CN104862311 A CN 104862311A CN 201510223933 A CN201510223933 A CN 201510223933A CN 104862311 A CN104862311 A CN 104862311A
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Abstract
The invention discloses a real-time fluorescence quantification PCR primer for detecting Chinese alligator based on a Taqman probe, a kit and a method. The detection method and the kit are simple and rapid in operation, high in specificity, sensitive and reliable in detection effect; the lowest detection concentration is 1x101 copy/miu l. Leather products on the market can be rapidly detected, and the application is extremely convenient; for introducing a specific amplification primer and a fluorescence probe, the sensitivity and specificity for detection are greatly enhanced; and thereby, the detection method avoids the shortcomings that the specificity of the other detecting method is low, and cannot judge if the product contains components of Chinese alligator. Based on above advantages, the kit is suitable for popularizing and applying to wild animal protection organizations in all levels, market supervision department and various exit and entry agencies; besides, the kit is wide in application prospect.
Description
Technical field
The invention belongs to field of molecular biotechnology, be specifically related to a kind of PCR kit for fluorescence quantitative and detection method of specific detection Chinese alligator composition.
Background technology
Chinese alligator (Alligator sinensis) or be called Chinese alligator (tu ó) is the distinctive a kind of crocodile of China, is one of minimum in the world crocodile kind.It is ancient, is again very rare, the endangered in the world Reptilia of quantity on hand.Because it lives in the Yangtze valley, therefore claim " Chinese alligator ".With it Chinese alligator, many features of previous dinosaurs Reptilia can also be found so far.So people claim Chinese alligator to be " living fossil ".Therefore, Chinese alligator studies the ups and downs of Reptilia in ancient times and the evolution of research palaeogeology and biology for people, all significant.Chinese alligator belongs to Reptilia Crocodilia alligator section animal, and there is 4 genus 8 kinds in this section, only Chinese alligator in state-owned distribution.
Chinese alligator whole body is all precious, and its meat was once the delicacies in feast, and its skin is the upper good material manufacturing high-grade leather goods, and it also has very high pharmaceutical use, therefore suffers that the unrest of people is caught and indiscriminately slaughters.Again because Chinese alligator is a kind of flesh-eater, can dig cave on Xu Di and burrow, therefore peasant was once used as it as pest, had seen and had had just catched and killed.Chinese alligator has been in the condition almost will died out.
For this reason, Chinese alligator was classified as animals under first-class state protection in 1972 by the Chinese government, and it lists in and lays special stress on protecting animal register, embargo by endangered species of wild fauna and flora international trade pact in 1973.1981, estimate according to expert, the Chinese alligator of field survivorship only had 300-500 bar, and these species may be died out in 10 years.China is classified as Chinese alligator as animals under first-class state protection, forbids to catch and kill.In order to enable the race of this precious animal continue, China also in Anhui, zhejiang and other places establishes the wilderness area of Chinese alligator and propagates field artificially.
In order to more effectively protect Chinese alligator, need to monitor the leathercraft on market.Therefore, this patent sets up a kind of Chinese alligator goods real-time fluorescence quantitative PCR specificity identification method based on taqman probe.
Real-time RT-PCR (real time RT-PCR, RRT-PCR) is also known as TaqMan PCR.It is quick, responsive, special that RRT-PCR detects bird flu, because it not use only Auele Specific Primer, and have employed specific probe, make the specificity of the method higher than normal PCR, greatly reduce the false positive results that non-specific amplification causes.The fluorescent RT-PCR technology of totally-enclosed reaction is by computer recording and analyzes the fluorescent signal produced in PCR process, achieve pcr amplification process Real-Time Monitoring, without the need to electrophoresis detection, the Aerosol Pollution of PCR primer and EB are thoroughly stopped to the pollution of environment.Application One step RT-PCR and fluorescent hydrolysis probe, for detecting the bird flu of A type, its susceptibility is very high, can reach 10 fg(about 100) target RNA molecule.
Summary of the invention
The object of the present invention is to provide a kind of PCR kit for fluorescence quantitative and detection method of specific detection Chinese alligator composition.
The technical solution used in the present invention is:
Detect a real-time fluorescence quantitative PCR primer for Chinese alligator, its sequence is:
AS-F：5’- CTTACACACAACTAAACAGCAACCG -3’（SEQ ID NO.1），
AS-R：5’- GAAGAAGTCTAGGACGAGGGCC -3’（SEQ ID NO.2）。
Detect a real-time fluorescence quantitative PCR test kit for Chinese alligator, this test kit contains primer described above.
Further, mentioned reagent box also containing real-time fluorescence quantitative PCR detection probes, its sequence as:
AS-P：5’- CAACACGCCCCATGTCTCAGCTCC -3’（SEQ ID NO.3）。
Detect a real time fluorescence quantifying PCR method for Chinese alligator, the method comprises the following steps:
1) DNA extraction of testing sample;
2) fluorescent quantitative PCR:
Reaction system: contain in every 25 μ l reaction systems: PCR Mix 20 μ l, Taq enzyme 1 μ l, DNA 4 μ l to be detected; Wherein the formula of every 50mL PCR Mix is as follows:
Reaction system condition: the instrument cycling conditions such as ABI PRISM 7300/7500, MJ Opticon be 94 DEG C 2 minutes, 94 DEG C 15 seconds, 55 DEG C 30 seconds, 40 circulations; LightCycler etc. use the instrument cycling condition of kapillary be 93 DEG C 2 minutes, 93 DEG C 5 seconds, 58 DEG C 45 seconds, totally 40 circulations;
3) interpretation of result:
Positive: detect sample Ct value and be less than or equal to 35.0, and curve to have obvious Exponential growth stage;
Suspicious: detect sample Ct value and be greater than 35.0 and be less than 40.0, repeat once to test, if Ct value is still less than 40.0, and curve has obvious Exponential growth stage, is the positive, otherwise is negative;
Negative: to can't detect sample Ct value or Ct value is 40;
Above-mentioned detection method is used for non-diseases diagnosis and detection.
Further, the concrete operations of above-mentioned testing sample DNA extraction are:
Solid tissue: get the glass homogenizer homogenate of about 0.5g tissue or scissors shreds, add 1.5ml physiological saline and continue grinding, go in the EP pipe of 1.5ml after homogenate, the centrifugal 2min of 10000rpm, get supernatant 200 μ l in the EP pipe of 1.5ml, add 200 μ l nucleic acid extraction liquid and fully shake;
Blood preparation: get 100 μ l blood and add 100 μ l nucleic acid extraction liquid, fully shake, 100 DEG C of water-bath 10min, 13,000rpm centrifugal 5min under 4 DEG C of conditions, Aspirate supernatant is the DNA of extraction.
The invention has the beneficial effects as follows:
The invention provides a kind of PCR kit for fluorescence quantitative and detection method of specific detection Chinese alligator composition, can realize carrying out rapid detection to the leathercraft on market, it is very convenient to apply.Owing to introducing specificity amplification primer and fluorescent probe, the sensitivity of detection and specificity are strengthened significantly, thus avoid the shortcoming that other detection method specificitys are not high, cannot judge whether to contain in goods Chinese alligator composition.Based on above-mentioned advantage, this test kit is adapted at conservation of wildlife tissue at different levels, market surpervision department and various entry and exit mechanisms etc. and applies, and is with a wide range of applications.
Accompanying drawing explanation
Fig. 1 is the sensitivity experiments that the PCR kit for fluorescence quantitative applying specific detection Chinese alligator composition of the present invention detects Chinese alligator, is from left to right followed successively by 1 × 10
8, 1 × 10
7, 1 × 10
6, 1 × 10
5, 1 × 10
4, 1 × 10
3, 1 × 10
2the amplification of the standard substance of copy/μ l;
Fig. 2 is the specificity experiments result that the PCR kit for fluorescence quantitative applying specific detection Chinese alligator composition of the present invention detects Chinese alligator.
Embodiment
Below in conjunction with specific embodiment, the present invention is further illustrated.
embodiment 1 one kinds detects the real-time fluorescence quantitative PCR test kit of Chinese alligator
(1) Auele Specific Primer and probe
According to whole 8 the species Chinese alligators (Alligator sinensis) of alligator section, Mississippi crocodile (Alligator mississippiensis), eyes crocodile (Caiman crocodilus), wide kiss caiman cayman (Caiman latirostris), Paraguay caiman cayman (Caiman yacare), black crocodile (Melanosuchus niger), the ancient crocodile of blunt kiss (Paleosuchus palpebrosus), the nucleotide sequence of the cone ancient crocodile of kiss (Paleosuchus trigonatus), be designed for primer and the probe of specific detection Chinese alligator, its sequence is as follows:
Upstream primer: AS-F:5 '-CTTACACACAACTAAACAGCAACCG-3 ' (SEQ ID NO.1),
Downstream primer: AS-R:5 '-GAAGAAGTCTAGGACGAGGGCC-3 ' (SEQ ID NO.2),
Fluorescent probe: AS-P:5 '-Fam-CAACACGCCCCATGTCTCAGCTCC-3 ' BHQ1(SEQ ID NO.3), expanding fragment length is 82bp.
(2) positive quality control product
1) with Chinese alligator tissue sample for template, carry out pcr amplification, step is as follows:
2) PCR system:
10 × PCR Buffer 5 μ l, TaKaRa Taq(5U/ μ l) each 2.5mM of 1 μ l, dNTP Mixture() 4 μ l, above-mentioned upstream primer AS-F and downstream primer AS-R(10 μM) each 0.5 μ l, the Chinese alligator tissue DNA 0.5 μ l of extraction and RNase free H
2o 39.25 μ l.
3) PCR condition:
According to 94 DEG C of 3 minutes denaturations, 94 DEG C 30 seconds, 60 DEG C 30 seconds, 72 DEG C 45 seconds, totally 35 circulations, 72 DEG C extend 10 minutes.
4) reaction after get 5 μ l pcr amplification products with 2% sepharose detect, the PCR primer of cutting glue purification is connected with pMD-18T carrier, recombinant plasmid pMD-18T-AS coats on culture dish, transformed competence colibacillus DH5 α cell, picking positive bacterium colony extracting plasmid, get product 5 μ l and dilute itself A260nm and A280nm absorbance of survey, calculate its concentration and be converted into absolute copy number, being diluted to 1.0 × 10
7copy/μ l, as the positive quality control product of the quantitative fluorescent PCR of specific detection Chinese alligator composition.
(3)pCR Mix: the formula of every 50mL PCR Mix is as follows:
(4)taq enzyme and negative quality control product (RNase free H
2o).
embodiment 2 one kinds detects the real time fluorescence quantifying PCR method of Chinese alligator
The present embodiment adopts the test kit described in embodiment 1 to carry out corresponding real-time fluorescence quantitative PCR detection.
(1) collection of specimens and pre-treatment:
Present method and test kit are suitable for the blood, tissue, leathercraft, meat product, Secretory product etc. that specimen types comprises Chinese alligator.
Solid sample: get under aseptic condition and organize about 100 ~ 200mg, inserts in the clean EP pipe of 1.5ml, preserves to be checked.
Blood preparation: get 200uL, inserts in the clean EP pipe of 1.5ml, preserves to be checked.
Secretory product: gather under aseptic condition, preserves to be checked.The sample gathered should censorship as early as possible, or is stored in-20 DEG C.
(2) extraction of DNA:
1) solid tissue: get the glass homogenizer homogenate of about 0.5g tissue or scissors shreds, add 1.5ml physiological saline and continue grinding, go in the EP pipe of 1.5ml after homogenate, the centrifugal 2min of 10000rpm, get supernatant 200 μ l in the EP pipe of 1.5ml, add 200 μ l nucleic acid extraction liquid and fully shake;
2) blood preparation: get 100 μ l blood and add 100 μ l nucleic acid extraction liquid, fully shake, 100 DEG C of water-bath 10min.The centrifugal 5min(4 DEG C of 13,000rpm), Aspirate supernatant is the DNA of extraction.
(3) fluorescent quantitative PCR
Each test reaction system is formulated as follows, PCR Mix 20 μ l, Taq enzyme (1U/ μ l) 1 μ l, and brief centrifugation, then adds DNA 4 μ l to be detected; Equally the positive and negative control are set according to above-mentioned system, add positive quality control product or negative quality control product 4 μ l increases.
Wherein the formula of 50mL PCR Mix is as follows:
Each reaction tubes is put into the reactive tank of quantitative PCR instruments, title and the fluorophor kind of each detection are set, reporter group is FAM, quenching group selects none), setting cycling condition: the instrument cycling conditions such as ABI PRISM 7300/7500, MJ Opticon be 94 DEG C 2 minutes, 94 DEG C 15 seconds, 55 DEG C 30 seconds, 40 circulations; LightCycler etc. use the instrument cycling condition of kapillary be 93 DEG C 2 minutes, 93 DEG C 5 seconds, 58 DEG C 45 seconds, totally 40 circulations.
(4) interpretation of result and judgement:
1, interpretation of result condition sets
1) baseline (baseline) is set: be set to 6-15cycle to instruments such as ABI 7300,7500, be set to 6-12cycle to MJ Research Option2.Can suitably adjust baseline as the case may be.
2) threshold value (threshold) is set: the vertex just having exceeded negative control amplification curve (random noise line) with threshold line.
2, result judges
1) positive: detect sample Ct value and be less than or equal to 35.0, and curve to have obvious Exponential growth stage;
2) suspicious: detect sample Ct value and be greater than 35.0 and be less than 40.0, repeat once to test, if Ct value is still less than 40.0, and curve has obvious Exponential growth stage, is the positive, otherwise is negative;
3) negative: to can't detect sample Ct value or Ct value is 40.
The real-time fluorescence quantitative PCR test kit and method that detect Chinese alligator described in above-described embodiment are carried out to the effect detection of a step below.
one, sensitivity experiment:
Above-mentioned positive quality control product (10
7copy/μ l), dilution is 1.0 × 10 successively
6, 1.0 × 10
5, 1.0 × 10
4, 1.0 × 10
3, 1.0 × 10
2, 1.0 × 10
1, 1.0 × 10
0copy/μ l, carries out sensitivity experiment.
The susceptibility that the PCR kit for fluorescence quantitative detecting Chinese alligator composition detects, experimental result is shown in Fig. 1, is from left to right followed successively by 1 × 10
7, 1 × 10
6, 1 × 10
5, 1 × 10
4, 1 × 10
3, 1 × 10
2, 1 × 10
1, 1 × 10
0the amplification of the standard substance of copy/μ l.
Result proved test kit detection sensitivity is: 1.0 × 10
1μ l
-1.
two, specificity experiments:
Domestic Crocodilia only Chinese alligator is a kind of, therefore use the sample of other animals of reptilia to carry out specificity verification to above-mentioned fluorescent quantitative PCR detection method, and sequence verification is carried out to product, result confirms, the inventive method and test kit specificity good, do not occur false positive, the goodness of fit is 100%, and experimental result is shown in Fig. 2.
Detect the specificity that PCR kit for fluorescence quantitative of the present invention detects Chinese alligator, experimental result is shown in Fig. 2, Chinese alligator tissue (Alligator sinensis, gather from Xuancheng Profile, anhui Province) positive, Zaocys tissue (Zaocys dhumnades, pick up from Lushan Mountain), tortoise (Chinemys reevesii, purchased from Shuande, Foshan pet market), Chinese gecko (Gekko chinensis gathers from In Guangzhou Science City Cheung On Est.) and negative quality control product be feminine gender.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Wei Baixin bio tech ltd, <110> Guangzhou
<120> mono-kind is based on the real-time fluorescence quantitative PCR primer of Taqman probe in detecting Chinese alligator, test kit and method
<130>
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 25
<212> DNA
<213> artificial sequence
<400> 1
cttacacaca actaaacagc aaccg 25
<210> 2
<211> 22
<212> DNA
<213> artificial sequence
<400> 2
gaagaagtct aggacgaggg cc 22
<210> 3
<211> 24
<212> DNA
<213> artificial sequence
<400> 3
caacacgccc catgtctcag ctcc 24
Claims (5)
1. detect a real-time fluorescence quantitative PCR primer for Chinese alligator, its sequence is:
AS-F：5’- CTTACACACAACTAAACAGCAACCG -3’（SEQ ID NO.1），
AS-R：5’- GAAGAAGTCTAGGACGAGGGCC -3’（SEQ ID NO.2）。
2. detect a real-time fluorescence quantitative PCR test kit for Chinese alligator, it is characterized in that: this test kit contains primer according to claim 1.
3. a kind of real-time fluorescence quantitative PCR test kit detecting Chinese alligator according to claim 2, is characterized in that: this test kit also containing real-time fluorescence quantitative PCR detection probes, its sequence as:
AS-P：5’ - CAACACGCCCCATGTCTCAGCTCC -3’（SEQ ID NO.3）。
4. detect a real time fluorescence quantifying PCR method for Chinese alligator, it is characterized in that: the method comprises the following steps:
1) DNA extraction of testing sample;
2) fluorescent quantitative PCR:
Reaction system: contain in every 25 μ l reaction systems: PCR Mix 20 μ l, Taq enzyme 1 μ l, DNA 4 μ l to be detected; Wherein the formula of every 50mL PCR Mix is as follows:
Reaction system condition: the instrument cycling conditions such as ABI PRISM 7300/7500, MJ Opticon be 94 DEG C 2 minutes, 94 DEG C 15 seconds, 55 DEG C 30 seconds, 40 circulations; LightCycler etc. use the instrument cycling condition of kapillary be 93 DEG C 2 minutes, 93 DEG C 5 seconds, 58 DEG C 45 seconds, totally 40 circulations;
3) interpretation of result:
Positive: detect sample Ct value and be less than or equal to 35.0, and curve to have obvious Exponential growth stage;
Suspicious: detect sample Ct value and be greater than 35.0 and be less than 40.0, repeat once to test, if Ct value is still less than 40.0, and curve has obvious Exponential growth stage, is the positive, otherwise is negative;
Negative: to can't detect sample Ct value or Ct value is 40;
Above-mentioned detection method is used for non-diseases diagnosis and detection.
5. method according to claim 4, is characterized in that: the concrete operations of described testing sample DNA extraction are:
Solid tissue: get the glass homogenizer homogenate of about 0.5g tissue or scissors shreds, add 1.5ml physiological saline and continue grinding, go in the EP pipe of 1.5ml after homogenate, the centrifugal 2min of 10000rpm, get supernatant 200 μ l in the EP pipe of 1.5ml, add 200 μ l nucleic acid extraction liquid and fully shake;
Blood preparation: get 100 μ l blood and add 100 μ l nucleic acid extraction liquid, fully shake, 100 DEG C of water-bath 10min, 13,000rpm centrifugal 5min under 4 DEG C of conditions, Aspirate supernatant is the DNA of extraction.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109943650A (en) * | 2019-05-10 | 2019-06-28 | 太原市食品药品检验所 | A kind of real time fluorescent PCR method using the specific primer identification zaocys dhumnade Chinese medicine true and false |
| CN111961736A (en) * | 2020-09-04 | 2020-11-20 | 南京农业大学 | Primer pair for identifying crocodile-derived component in meat product and application thereof |
| CN114292921A (en) * | 2021-12-20 | 2022-04-08 | 南方医科大学 | Method for controlling quality of Jinlong capsule by detecting copy number of gecko specific fragment based on molecular quantitative technology, primer and probe |
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| CN101368205A (en) * | 2008-05-31 | 2009-02-18 | 安徽师范大学 | Chinese alligator microsatellite DNA mark |
| CN104561329A (en) * | 2015-01-16 | 2015-04-29 | 北京市食品安全监控和风险评估中心 | Kit for detecting roe deer original component in food and application of kit |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109943650A (en) * | 2019-05-10 | 2019-06-28 | 太原市食品药品检验所 | A kind of real time fluorescent PCR method using the specific primer identification zaocys dhumnade Chinese medicine true and false |
| CN111961736A (en) * | 2020-09-04 | 2020-11-20 | 南京农业大学 | Primer pair for identifying crocodile-derived component in meat product and application thereof |
| CN114292921A (en) * | 2021-12-20 | 2022-04-08 | 南方医科大学 | Method for controlling quality of Jinlong capsule by detecting copy number of gecko specific fragment based on molecular quantitative technology, primer and probe |
| CN114292921B (en) * | 2021-12-20 | 2023-09-26 | 南方医科大学 | Method for quality control of Jinlong capsules by detecting copy number of Gekko Swinhonis specific fragment based on molecular quantification technology, primer and probe |
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