CN106701907A - Primer, probe, method and kit for detecting cassava-derived ingredients - Google Patents
Primer, probe, method and kit for detecting cassava-derived ingredients Download PDFInfo
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Abstract
The invention relates to an oligonucleotide primer and a probe for detecting cassava-derived ingredients. The invention further relates to a real-time fluorescence PCR detection method for detecting the cassava-derived ingredients. According to the method, the specific oligonucleotide primer and the probe aiming at the cassava-derived ingredient are used. The invention further relates to a real-time fluorescence PCR detection kit used for rapidly detecting the cassava-derived ingredients, and the kit comprises the specific oligonucleotide primer and the probe used for real-time fluorescence PCR detection for the cassava-derived ingredients. The invention further relates to applications of the specific oligonucleotide primer and the probe aiming at the cassava-derived ingredients in detecting the cassava-derived ingredients. With the adoption of the real-time fluorescence PCR detection method and the kit provided by the invention, whether the samples including starch, vermicelli noodles, bean vermicelli, cakes and the like contain the cassava-derived ingredients or not can be specifically, sensitively and accurately detected.
Description
Technical field
The invention belongs to biological technical field, specifically, Oligonucleolide primers and probe the present invention relates to be used for the detection of cassava derived component, real-time fluorescence PCR detection method for determining cassava derived component, the application of the specific oligonucleotide primer and probe of real-time fluorescence PCR assay kit and cassava derived component for quick detection cassava derived component in cassava derived component is detected.
Background technology
Cassava is a kind of various barren soils of adaptation, easily plantation, tropical crops of small investment, and its content of starch is high, have the laudatory title of " king of starch ".Tapioca is the second largest purposes for producing cassava.The potato starch that China mainly eats including starch from sweet potato, farina, tapioca etc., wherein, the price highest of starch from sweet potato, the price of tapioca is minimum.Sensory properties, physical and chemical indexes due to variety classes starch granules etc. are close, and consumer is difficult identification, therefore, the tapioca of the conventional low price of illegal retailer pretends to be starch from sweet potato to sell, and trys to gain exorbitant profit, and the interests to consumer cause damage.
In recent years, domestic and foreign scholars have carried out the research in terms of Starches discriminating using technological means such as infrared spectrum, ESEM, peptide fingerprinting spectrums.Such as, the potato adulterated in lotus root starch, Ipomoea batatas, cassava and cornstarch etc. can effectively be differentiated using infrared spectrum technology；With ESEM and stable carbon isotope than method analysis farina and otherness of the cornstarch on particle nanotopographical, can accurately differentiate adulterated cornstarch in farina.Peptide fingerprinting composes the difference in area between by analyzing different starch, can distinguish starch from sweet potato, tapioca and farina.
But, the above method is easily influenceed by factors such as kind, the place of production, harvest seasons, in addition, also easily limited by the problems such as sampling uniformity and representativeness, and the instrument and equipment price for relying on is costly, it is difficult to be promoted in common lab.Real-time fluorescence PCR technology identifies species real property by analyzing the difference on species DNA level, as a result accurately, it is reliable, and do not influenceed by environmental factor etc., instrument popularity rate is high, in an increasingly wide range of applications in terms of food real property discriminating at present.
At present, there is not been reported both at home and abroad can quickly, method and kit easy, special and that delicately detect cassava derived component in the samples such as starch, vermicelli, bean vermicelli, cake.
Therefore, this area needs a kind of detection method of quick, specific good, sensitivity cassava derived component high, carries out the detection of cassava derived component in the samples such as starch, vermicelli, bean vermicelli, cake..
The content of the invention
It is an advantage of the invention to provide the specific oligonucleotide primer and probe of accurate detection cassava derived component.
It is a further object of the invention to provide the real-time fluorescence PCR detection method of Accurate Determining cassava derived component.
It is a further object of the invention to provide the real-time fluorescence PCR assay kit of Accurate Determining cassava derived component.
It is of the invention further an object is that, there is provided application of the specific oligonucleotide primer and probe of cassava derived component in cassava derived component in accurate detection sample.
For foregoing invention purpose, the present invention provides following technical scheme：
The present inventor is according to cassavag3pdhGene（Glyceraldehyde-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase gene）Devise can specificity differentiate the Oligonucleolide primers pair and probe of cassava derived component, one section shorter of cassava specific gene fragment can be gone out by efficient specific amplified from sample DNA.An embodiment of the invention, the present invention is provided to the specific oligonucleotide primer pair and fluorescence labeling probe of real time fluorescent PCR method detection cassava derived component, the primer pair and probe are basesg3pdhGene order in different plant species have otherness the characteristics of and design.The primer pair is made up of sense primer and anti-sense primer, and the sense primer is Cassava-F：TGGTTCTTGATCTGCGTGAG（SEQ
ID No.1）, the anti-sense primer is Cassava-R：TGGGAGAACCTTTCCAACAG（SEQ ID No.2）；The probe is Cassava-P：TGCTGTTGCAACTGCTTAGG（SEQ
ID No.3）, a fluorescent quenching group TAMAR is connected with 3 ' ends of probe, 5 ' ends are connected with a fluorescent reporter group FAM.In one embodiment, the cassava specific detection composition that the present invention is provided, the composition includes specific oligonucleotide primer pair and probe.In a preferred embodiment, the invention provides the composition for real time fluorescent PCR method qualitative detection cassava derived component, the composition includes cassava specific oligonucleotide primer pair and probe, to being made up of sense primer and anti-sense primer, the base sequence of the sense primer is SEQ to wherein described cassava specific primer
ID No.1, the base sequence of the anti-sense primer is SEQ ID No.2；The base sequence of the probe is SEQ ID No.3, and a fluorescent quenching group TAMAR is connected with 3 ' ends of probe, and 5 ' ends are connected with a fluorescent reporter group FAM.
Another embodiment of the invention, the present invention provides the real-time fluorescence PCR detection method of cassava derived component, and methods described includes that the primer pair and probe are bases using the specific oligonucleotide primer pair and probe for cassava derived componentg3pdhGene order in different plant species have otherness the characteristics of and design.In one embodiment, in the real-time fluorescence PCR qualitative checking method of cassava derived component of the invention, the cassava specific oligonucleotide primer pair for being used is made up of sense primer and anti-sense primer, the base sequence of the sense primer is SEQ ID No.1, and the base sequence of the anti-sense primer is SEQ ID No.2；The base sequence of the probe is SEQ
ID No.3, a fluorescent quenching group TAMAR is connected with 3 ' ends of probe, and 5 ' ends are connected with a fluorescent reporter group FAM.In one embodiment, described PCR amplification conditions are 95 DEG C, 10 min；95 DEG C of 15 s, 60 DEG C of 1 min, totally 40 circulations.
Another embodiment of the invention, the present invention is provided to the kit of cassava derived component in accurate detection sample, the kit includes the present invention for the specific oligonucleotide primer pair and probe and operation instructions of real time fluorescent PCR method detection cassava derived component.In kit preferred embodiment of the invention, cassava derived component qualitative detection specific oligonucleotide primer pair of the invention is basisg3pdhGene order in different plant species have otherness the characteristics of and design.In one embodiment, the cassava specific oligonucleotide primer pair of the kit is made up of sense primer and anti-sense primer, and the base sequence of the sense primer is SEQ
ID No.1, the base sequence of the anti-sense primer is SEQ ID No.2；The base sequence of the kit middle probe is SEQ ID No.3, and a fluorescent quenching group TAMAR is connected with 3 ' ends of probe, and 5 ' ends are connected with a fluorescent reporter group FAM.Another embodiment of the invention, the present invention is provided to the kit of accurate qualitative detection cassava derived component, the kit includes the specific oligonucleotide primer pair and probe and operation instructions for differentiating cassava derived component for real time fluorescent PCR method of the invention.In a preferred embodiment of kit, cassava specific oligonucleotide primer pair SEQ is included in the kit
ID No.1, SEQ ID No.2 and cassava specific probe SEQ ID No.3, in cassavag3pdh3 ' ends of gene probe are connected with a fluorescent quenching group TAMAR, and 5 ' ends are connected with a fluorescent reporter group FAM.In preferred embodiments, the operation instructions of the kit include the description to the real-time fluorescent PCR amplification condition for quick detection cassava derived component.In a preferred embodiment, the PCR amplification conditions for being given in the specification of the kit are 95 DEG C, 10 min；95 DEG C of 15 s, 60 DEG C of 1 min, totally 40 circulations.In a specific embodiment, the present invention also includes reference substance for the kit of cassava derived component qualitative detection.Preferably, reference substance includes negative controls and positive reference substance.In one embodiment, negative control is Ipomoea batatas DNA.
Further embodiment of the invention, the present invention provides the application for the specific oligonucleotide primer and probe of real time fluorescent PCR method detection cassava derived component in cassava derived component in detecting food of the invention.In a preferred embodiment, the present invention provides the specific oligonucleotide primer pair SEQ of cassava derived component in food starch
ID No.1, SEQ ID No.2 and specific probe SEQ ID No.3, in cassavag3pdh3 ' ends of gene probe are connected with a fluorescent quenching group TAMAR, and 5 ' ends are connected with a fluorescent reporter group FAM.In another embodiment, the present invention also provides application of the kit of the invention in qualitative detection cassava derived component.Preferably, in above-mentioned application of the invention, the kit includes cassava specific oligonucleotide primer pair of the invention and probe.It is furthermore preferred that in above-mentioned application of the invention, the kit includes the application of cassava specific oligonucleotide primer pair of the invention and probe in cassava derived component in detecting sample.
The present invention is to detect basis with cassava DNA, according tog3pdhThe characteristics of gene order has otherness in different plant species, comparison analyzes cassavag3pdhGene order.According to these primers, using the cassava derived component in the legal detection sample of real-time fluorescence PCR.
Real-time fluorescence quantitative PCR is on the basis of conventional PCR method, add the probe or fluorescent dye of fluorescence labeling, with the accumulation of PCR primer, the fluorescence signal enhancing that probe or dyestuff send, and fluorescence monitoring system can receive fluorescence signal, a DNA chain is often produced, just there is a fluorescence molecule to be formed, the accumulation and PCR primer for realizing fluorescence signal form Complete Synchronization.Therefore with the whole PCR courses of reaction of monitor in real time, and the initial copy number of testing sample can be finally detected, so as to can detect contained cassava derived component in food starch to be measured.
Real-time fluorescence PCR detection method of the invention is detected using complete stopped pipe, without PCR post processings, it is to avoid cross pollution and false positive.The method of the present invention has dexterously used the DNA efficient amplifications of round pcr, the specificity of nucleic acid hybridization and the quick and sensitiveness of detection technique of fluorescence, has the advantages that simple to operate, time saving and energy saving, reliable results and accurate sensitive.The kit being made according to primer sequence of the invention, for the qualitative detection of such product, has the advantages that sensitivity high, high specificity, result is reliable and stable and avoids cross pollution from causing false positive.Using PCR detection method of the invention and PCR detection kit, can be used for qualitative detection, be suitable for detection of cassava derived component in the samples such as starch on domestic and international market, vermicelli, bean vermicelli, cake etc. the characteristics of its is simple, quick, special and sensitive.
Brief description of the drawings
Fig. 1 is shown the result of real-time fluorescence PCR specific detection cassava, wherein using specific oligonucleotide primer pair SEQ ID No.1 and SEQ ID No.2 and SEQ
ID No.3 are detected, wherein baseline top is the amplification curve of cassava sample, and baseline lower section is Ipomoea batatas, taro, Chinese yam, potato, lotus rhizome, corn, wheat, barley, rye, rice, buckwheat, sorghum, the seed of Job's tears, millet, mung bean, rde bean, cowpea, French beans, Kidney bean, broad bean, soya bean and blank（Aseptic double-distilled water）.
Fig. 2 is that the absolute sensitivity of real-time fluorescence PCR specific detection cassava composition is evaluated, by 10 times of gradient dilutions of cassava DNA solution, 10 ng/ μ L, 1ng/ μ L, 0.1ng/ μ L, 0.01ng/ μ L, 0.001ng/ μ L, 0.0001ng/ μ L and blank are followed successively by（Aseptic double-distilled water）.
Fig. 3 is that the relative sensitivity of real-time fluorescence PCR specific detection cassava composition is evaluated, and tapioca and starch from sweet potato are mixed, and the mass ratio of tapioca and starch from sweet potato is respectively 100%, 50%, 10%, 5%, 1%, 0.1%, 0.01%.
Fig. 4 is the result that the real time fluorescent PCR method set up using the present invention is detected to commercial samples.More than baseline it is positive control（Cassava DNA）, tapioca 1#, tapioca 2# and starch from sweet potato 3#, baseline position be other starch samples and blank（Aseptic double-distilled water）.
Specific embodiment
The present invention is further illustrated by way of embodiment, but the present invention is not limited only to following examples.
Embodiment
1
The present embodiment is to carry out specificity and sensitivity evaluation to the primer pair and probe of cassava by following experiment.
By detectiong3pdhGene order, it may be determined that the specificity and detection sensitivity of cassava primer combination of probe.Reaction system is：Fast
Start Universal PCR Master Mix 12.5 μL；The μ L of probe (10 μM) 0.5；Each 0.5 μ L of upstream and downstream primer (10 μM)；The μ L of template DNA 5；Plus ddH2O to cumulative volume be 25 μ L.Response procedures are 95 DEG C of 10 min；95℃ 15 s；60 DEG C of 1 min, 40 circulations.
The primer and probe sequence of the detection cassava for being used be：
Primer sequence is SEQ ID No.1 and SEQ ID No.2, and probe sequence is SEQ
ID No.3,3 ' ends are connected with a fluorescent quenching group TAMAR, and 5 ' ends are connected with a fluorescent reporter group FAM.
The detection key instrument for being used：
Micropipettor (10 μ L, 100 μ L, 1000 μ L, Eppendorf), quantitative real time PCR Instrument (ABI 7500, Applied
Biosystems), high speed tabletop centrifuge (Pico17 Thermo) etc..
Detection main agents：
Chloroform, isopropanol are purchased from the logical company of Beijing six directions respectively；CTAB lysates (20 g/L CTAB, 1.4
Mol/L NaCl, 0.1 mol/L Tris, 0.02 mol/L Na2- EDTA), CTAB precipitated liquids (5 g/L CTAB, 0.04 mol/L NaCl), 1.2
Mol/L NaCl are this experiment and voluntarily prepare；Fast Start Universal Probe Master Mix
(Rox) it is purchased from Roche Holding Ag；Primer and probe are synthesized by Shanghai Ying Jun bio tech ltd.
Detection key step：
1 DNA is extracted
Detection sample：(1) 28 kinds of samples such as cassava, Ipomoea batatas, taro, Chinese yam, potato, lotus rhizome, corn, wheat, oat, barley, rye, rice, buckwheat, sorghum, the seed of Job's tears, millet, mung bean, rde bean, cowpea, pea, French beans, Kidney bean, broad bean, soya bean, jujube, apricot, lily, olive are used for specificity analysis；(2) the cassava DNA solution sterilized water of extraction is diluted to the concentration of 10 ng/ μ L, 1ng/ μ L, 0.1ng/ μ L, 0.01ng/ μ L, 0.001ng/ μ L, 0.0001ng/ μ L respectively, the absolute sensitivity for analyzing primer combination of probe；(3) tapioca and starch from sweet potato are mixed, the mass ratio of tapioca and starch from sweet potato is respectively 50%, 10%, 5%, 1%, 0.1%, 0.01%, to determine the relative sensitivity that cassava specific primer probe is combined.
Weigh 0.1 g sample powders a to cleaning 2.0
In mL centrifuge tubes, 1.5 mL CTAB lysates, 65 DEG C of 1 h are added, a phase turns upside down mixing several times；The min of 8000 rpm 15, in taking 1 mL supernatants to 1 mL centrifuge tube of cleaning 2.0, add 700 μ L chloroforms, acutely mix 30 s, the min of 14500 rpm 10, take in 650 μ L of supernatant liquid to clean 2.0 mL centrifuge tubes respectively, add 1300 μ L CTAB precipitated liquids, 30 s are acutely mixed, 1 h is stored at room temperature；The min of 14500 rpm 10, abandon supernatant, add the M NaCl of 350 μ L 1.2, and acutely 30 s of vibration, add 350 μ L chloroforms, acutely mix 30 s, the min of 14500 rpm 10；The μ L of supernatant 320 are taken respectively, 0.8 times of volume isopropanol is added, and after mixing, -20 DEG C of 1 h, the min of 14500 rpm 20 abandon supernatant, add the ethanol of 500 μ L 70%, and after mixing, 14500 rpm 20min abandon supernatant, dry in the air to air-drying, and add 100 μ L ddH2O dissolves, and 4 DEG C store for future use.
2 real-time PCR detection the primers and probe
Primer sequence is SEQ ID No.1 and SEQ ID No.2；
Probe sequence is SEQ ID No.3, and 3 ' ends are connected with a fluorescent quenching group TAMAR, and 5 ' ends are connected with a fluorescent reporter group FAM.
3 real-time fluorescence PCR reaction systems：
Fast Start Universal PCR Master Mix 12.5 μL
Probe (10 μM)
0.5μL
Sense primer (10 μM)
0.5μL
Anti-sense primer (10 μM)
0.5μL
The μ L of template DNA 5
Plus ddH2O to cumulative volume be 25 μ L
Note：Corresponding blank (replacing DNA profiling with the ultra-pure water for preparing reaction system, whether detection reagent is contaminated) is set up in each PCR detections；
4 real-time fluorescence PCR response parameters：
95℃ 10 min
95℃ 15
s
60℃ 1
min
40 circulations.
Note：Different instruments should make the appropriate adjustments each reagents of PCR and response parameter.
As shown in figure 1, using real-time fluorescence PCR specific detection cassavag3pdhDuring gene order, in addition to there is typical S types amplification curve in cassava sample, other samples：28 kinds of samples such as Ipomoea batatas, taro, Chinese yam, potato, lotus rhizome, corn, wheat, oat, barley, rye, rice, buckwheat, sorghum, the seed of Job's tears, millet, mung bean, rde bean, cowpea, pea, French beans, Kidney bean, broad bean, soya bean, jujube, apricot, lily, olive and blank (ddH2O) do not occur amplification curve, absolutely prove that the primed probe of this experimental design is special to cassava sample.
To determine the absolute sense limit of cassava specific primer probe combination, the cassava DNA solution sterilized water of extraction is diluted to 10 respectively
Ng/ μ L, 1ng/ μ L, 0.1ng/ μ L, 0.01ng/ μ L, 0.001ng/ μ L, the concentration of 0.0001ng/ μ L, carry out real-time fluorescent PCR amplification, as a result as shown in Figure 2 by above-mentioned condition respectively.Cassava DNA concentration has specific amplification curve when being 10 ng/ μ L, 1 ng/ μ L, 0.1 ng/ μ L, 0.01 ng/ μ L, 0.001 ng/ μ L, and concentration is down to when below 0.001 ng/ μ L, occurs without specific amplification curve.Test result indicate that the content that the real-time fluorescence PCR detection method set up can detect cassava composition is 0.001
ng/μL。
Tapioca and starch from sweet potato are mixed, the mass ratio of tapioca and starch from sweet potato is set to be respectively 50%, 10%, 5%, 1%, 0.1%, 0.01%, real-time fluorescent PCR amplification is carried out by above-mentioned condition respectively, to determine the relative sensitivity (Fig. 3) that cassava specific primer probe is combined.Experimental result illustrates that the relative sensitivity of the method detection cassava composition is 0.1%.
Embodiment
2
The present embodiment provides the kit of cassava derived component in accurate detection sample.The kit includes that the present invention differentiates the specific oligonucleotide primer pair and probe and operation instructions of cassava derived component for real time fluorescent PCR method.The kit includes primer pair SEQ ID No.1, SEQ ID No.2 and probe SEQ
ID No.3,3 ' ends of probe are connected with a fluorescent quenching group TAMAR, and 5 ' ends are connected with a fluorescent reporter group FAM, PCR amplification conditions are given in the operation instructions, and the condition is 95 DEG C of 10 min；95℃ 15 s；60 DEG C of 1 min, 40 circulations.For different instruments, response parameter makes the appropriate adjustments.
To ensure that the method set up has feasibility, 7 parts of commercial samples, including 2 parts of tapioca are chosen（1# and 2#）, 3 parts of starch from sweet potato（1#、2#、3#）, 2 parts of lotus root starch（1# and 2#）Deng, it is identical with the method described in embodiment 1, real-time PCR detection is carried out, wherein using aseptic double-distilled water as kit blank product, using cassava DNA as kit positive reference substance.
As shown in figure 4, using real-time PCR detection cassavag3pdhDuring gene order, cassava DNA and 2 parts of tapioca samples and 1 part of starch from sweet potato（3#）There is typical fluorescent amplification curve more than baseline, other samples and blank expansion curve show that the method energy effective detection goes out cassava composition in baseline position.
Although being described to specific embodiments of the present invention, those skilled in the art will appreciate that can be variously changed to the present invention and modification on the premise of without departing from the scope of the present invention or spirit.Thus, this invention is intended to cover to fall all these changes in appended claims and its range of equivalency with modification.
Claims (5)
1. the specific oligonucleotide primer pair and probe compositions of cassava derived component in real time fluorescent PCR method detection sample are used for, wherein the primer pair is Cassava-F：TGGTTCTTGATCTGCGTGAG, the anti-sense primer is Cassava-R：TGGGAGAACCTTTCCAACAG；The probe is Cassava-P：TGCTGTTGCAACTGCTTAGG.
2. composition according to claim 1, wherein being connected with a fluorescent quenching group TAMAR at 3 ' ends of probe, 5 ' ends are connected with a fluorescent reporter group FAM.
3. the kit of cassava derived component in sample is detected by real time fluorescent PCR method, and the kit includes the primer combination of probe thing and operation instructions described in claim 1-2.
4. the real time fluorescent PCR method of cassava derived component is accurately detected, methods described includes the primer combination of probe thing described in usage right requirement 1-2 and the kit described in claim 3.
5. the application of the cassava derived component in the samples such as detection starch, vermicelli, bean vermicelli, cake of the kit described in the primer combination of probe thing and claim 3 described in claim 1-2.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109929946A (en) * | 2019-03-07 | 2019-06-25 | 广东出入境检验检疫局检验检疫技术中心 | A kind of kit and its application based on droplet type digital pcr quantitative detection cassava ingredient |
| CN110317895A (en) * | 2019-06-19 | 2019-10-11 | 许昌学院 | It is a kind of for detecting the LAMP primer group and its application of sweet potato derived components |
| CN114350831A (en) * | 2020-10-12 | 2022-04-15 | 谱尼测试集团上海有限公司 | Primer probe and method for detecting cocoa-derived components in cocoa powder |
Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002101079A2 (en) * | 2001-06-12 | 2002-12-19 | Pioneer Hi-Bred International, Inc. | Anti-apoptosis genes and methods of use thereof |
| CN1624153A (en) * | 2003-12-05 | 2005-06-08 | 中国科学院遗传与发育生物学研究所 | Laver DNA fingerprint graphic and its forming process and application |
| CN101283105A (en) * | 2005-07-29 | 2008-10-08 | 杂交生物科学私人有限公司 | Identification of genes and their products which promote hybrid vigour or hybrid debility and uses thereof |
| CN101643787A (en) * | 2009-05-15 | 2010-02-10 | 天津出入境检验检疫局动植物与食品检测中心 | Method for detecting allergen filbert component in foods by fluorescent PCR technology |
| CN101792815A (en) * | 2010-03-25 | 2010-08-04 | 中国检验检疫科学研究院 | PCR detecting method of cashew nut allergen and reagent kit |
| CN101974522A (en) * | 2010-11-23 | 2011-02-16 | 中国检验检疫科学研究院 | Primer, probe, kit and method for identifying authenticity of cervus elaphus linnaeus |
| CN101984076A (en) * | 2010-11-25 | 2011-03-09 | 中国检验检疫科学研究院 | Primer, kit and method for differentiating fins of different spieces of sharks by polymerase chain reaction and restriction fragment length polymorphism analysis (PCR-RFLP) |
| CN102344951A (en) * | 2010-07-30 | 2012-02-08 | 中国检验检疫科学研究院 | Primer, method and kit for detecting pear-derived components in sample |
| CN102344953A (en) * | 2010-07-30 | 2012-02-08 | 中国检验检疫科学研究院 | Primer for detecting peach-derived component in sample, method and kit |
| EP2791341A1 (en) * | 2011-12-12 | 2014-10-22 | Chuanxin Sun | Usage of oligonucleotides in plant biology |
| CN104232752A (en) * | 2014-07-01 | 2014-12-24 | 广西出入境检验检疫局检验检疫技术中心 | Real-time fluorescent PCR (polymerase chain reaction) detection method of water chestnut starch and cassava starch |
| CN104965030A (en) * | 2015-06-16 | 2015-10-07 | 秦皇岛出入境检验检疫局检验检疫技术中心 | Method for identifying adulteration of tapioca syrup in honey |
| CN105044014A (en) * | 2015-06-24 | 2015-11-11 | 河南工业大学 | Method for detecting low-quality starch-doped potato starch fast |
-
2015
- 2015-11-18 CN CN201510793759.4A patent/CN106701907B/en active Active
Patent Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002101079A2 (en) * | 2001-06-12 | 2002-12-19 | Pioneer Hi-Bred International, Inc. | Anti-apoptosis genes and methods of use thereof |
| CN1624153A (en) * | 2003-12-05 | 2005-06-08 | 中国科学院遗传与发育生物学研究所 | Laver DNA fingerprint graphic and its forming process and application |
| CN101283105A (en) * | 2005-07-29 | 2008-10-08 | 杂交生物科学私人有限公司 | Identification of genes and their products which promote hybrid vigour or hybrid debility and uses thereof |
| CN101643787A (en) * | 2009-05-15 | 2010-02-10 | 天津出入境检验检疫局动植物与食品检测中心 | Method for detecting allergen filbert component in foods by fluorescent PCR technology |
| CN101792815A (en) * | 2010-03-25 | 2010-08-04 | 中国检验检疫科学研究院 | PCR detecting method of cashew nut allergen and reagent kit |
| CN102344953A (en) * | 2010-07-30 | 2012-02-08 | 中国检验检疫科学研究院 | Primer for detecting peach-derived component in sample, method and kit |
| CN102344951A (en) * | 2010-07-30 | 2012-02-08 | 中国检验检疫科学研究院 | Primer, method and kit for detecting pear-derived components in sample |
| CN101974522A (en) * | 2010-11-23 | 2011-02-16 | 中国检验检疫科学研究院 | Primer, probe, kit and method for identifying authenticity of cervus elaphus linnaeus |
| CN101984076A (en) * | 2010-11-25 | 2011-03-09 | 中国检验检疫科学研究院 | Primer, kit and method for differentiating fins of different spieces of sharks by polymerase chain reaction and restriction fragment length polymorphism analysis (PCR-RFLP) |
| EP2791341A1 (en) * | 2011-12-12 | 2014-10-22 | Chuanxin Sun | Usage of oligonucleotides in plant biology |
| CN104232752A (en) * | 2014-07-01 | 2014-12-24 | 广西出入境检验检疫局检验检疫技术中心 | Real-time fluorescent PCR (polymerase chain reaction) detection method of water chestnut starch and cassava starch |
| CN104965030A (en) * | 2015-06-16 | 2015-10-07 | 秦皇岛出入境检验检疫局检验检疫技术中心 | Method for identifying adulteration of tapioca syrup in honey |
| CN105044014A (en) * | 2015-06-24 | 2015-11-11 | 河南工业大学 | Method for detecting low-quality starch-doped potato starch fast |
Non-Patent Citations (5)
| Title |
|---|
| DUPUTIE,A 等: "Manihot esculenta subsp. esculenta partial g3pdh gene for glyceraldehyde-3-phosphate dehydrogenase, isolate 3", 《GENBANK DATABASE》 * |
| JULIANA CHACÓN 等: "Phylogenetic patterns in the genus Manihot (Euphorbiaceae) inferred from analyses of nuclear and chloroplast DNA regions", 《MOLECULAR PHYLOGENETICS AND EVOLUTION》 * |
| OLSEN, KM 等: "Evidence on the origin of cassava: Phylogeography of Manihot esculenta", 《PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA》 * |
| OLSEN, KM 等: "Minisatellite variation in a single-copy nuclear gene: Phylogenetic assessment of repeat length homoplasy and mutational mechanism", 《MOLECULAR BIOLOGY AND EVOLUTION》 * |
| 王汝宝: "运用PCR法对番茄杂交种子纯度和真实性鉴定的研究", 《种子科技》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109929946A (en) * | 2019-03-07 | 2019-06-25 | 广东出入境检验检疫局检验检疫技术中心 | A kind of kit and its application based on droplet type digital pcr quantitative detection cassava ingredient |
| CN110317895A (en) * | 2019-06-19 | 2019-10-11 | 许昌学院 | It is a kind of for detecting the LAMP primer group and its application of sweet potato derived components |
| CN110317895B (en) * | 2019-06-19 | 2023-07-14 | 许昌学院 | LAMP primer group for detecting sweet potato source components and application thereof |
| CN114350831A (en) * | 2020-10-12 | 2022-04-15 | 谱尼测试集团上海有限公司 | Primer probe and method for detecting cocoa-derived components in cocoa powder |
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