CN110468227A - A kind of primer, probe and the method for the specific detection mung bean anaphylactogen based on TaqMan probe real-time fluorescence PCR - Google Patents
A kind of primer, probe and the method for the specific detection mung bean anaphylactogen based on TaqMan probe real-time fluorescence PCR Download PDFInfo
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- CN110468227A CN110468227A CN201910754888.0A CN201910754888A CN110468227A CN 110468227 A CN110468227 A CN 110468227A CN 201910754888 A CN201910754888 A CN 201910754888A CN 110468227 A CN110468227 A CN 110468227A
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Abstract
Primer, probe and the method for the present invention provides a kind of specific detection mung bean anaphylactogen based on TaqMan probe real-time fluorescence PCR, the nucleotide sequence LvD-QF of its upstream primer is as shown in SED ID NO:1 in sequence table, and the nucleotide sequence LvD-QR of downstream primer is as shown in SEQ ID NO:2 in sequence table.The nucleotide sequence LvD-P of probe is as shown in SEQ ID NO:3 in sequence table.Method of the present invention is not in the case where calculating DNA extraction process time-consuming, real-time fluorescence quantitative PCR time-consuming 80min, so the measurement work of mung bean allergen content in food and agricultural products can be completed in detection method of the invention within 1.5h, there are the advantages such as accurate, quick, easy to operate.
Description
Technical field
The invention belongs to field of biotechnology, more particularly, to a kind of based on the special of TaqMan probe real-time fluorescence PCR
Property detection mung bean anaphylactogen primer, probe and method.
Background technique
In China, mung bean is mainly processing raw material for many food, although it is not main allergen, also there is mung bean mistake
Quick report.And with the continuous development of molecular biology, the identification of mung bean anaphylactogen is carried out using polymerase chain reaction (PCR),
It is no longer technical problem.Zhao Zhonglin etc. is proposed general in " foundation of mung bean ingredient PCR method for detecting specificity in food "
The method of logical PCR specific detection mung bean.The method is mainly used for food Adulteration detection, is detected using real time fluorescent PCR method
Mung bean allergy ultimate constituent there is no report.And regular-PCR technology in the prior art is compared with real-time fluorescent PCR technology, sensitivity is low, inspection
The survey period is long, can use in experimentation toxic reagent (EB), unfavorable to experimenter's health.
Summary of the invention
In view of this, the present invention is directed to propose a kind of specific detection mung bean based on TaqMan probe real-time fluorescence PCR
Primer, probe and the method for anaphylactogen have filled up the domestic technological gap using real-time fluorescence PCR detection mung bean anaphylactogen, together
When improve the sensitivity that mung bean in food detects, shorten the detection cycle of mung bean detection, avoid toxic reagent to experiment
The harm of personnel health.
In order to achieve the above objectives, the technical scheme of the present invention is realized as follows:
A kind of primer of the specific detection mung bean anaphylactogen based on TaqMan probe real-time fluorescence PCR, upstream primer
Nucleotide sequence LvD-QF be 5 '-TGTTTGCCTCATGCTATTATTGATC-3 ', such as SED ID NO:1 institute in sequence table
Show, the nucleotide sequence LvD-QR of downstream primer is 5 '-TCTCAATCCTGTTTGTACTACCTTCAG-3 ', in sequence table
Shown in SEQ ID NO:2.
A kind of probe of the specific detection mung bean anaphylactogen based on TaqMan probe real-time fluorescence PCR, nucleotides sequence
Arrange LvD-P are as follows: 5 '-FAM-AAGAAAACAAATGCTGCAATGCAACCTCA-TAMRA-3 ', such as SED ID NO:3 in sequence table
It is shown.
A method of the specific detection mung bean anaphylactogen based on TaqMan probe real-time fluorescence PCR, using above-mentioned
Primer or probe in detecting mung bean anaphylactogen.
The method of the above-mentioned specific detection mung bean anaphylactogen based on TaqMan probe real-time fluorescence PCR, including walk as follows
It is rapid:
(1) mung bean genomic DNA is extracted as test sample DNA profiling and to dilute spare；
(2) mung bean genomic DNA is subjected to gradient dilution, the concentration of diluted mung bean genomic DNA is respectively 100%,
10%, 1%, 0.1% and 0.01%, and standard curve is established respectively；
(3) above-mentioned primer, probe are used, PCR amplification is carried out to test sample DNA profiling and fluorescence signal detects.
(4) after reaction, according to calibration curve coefficient correlation R2, amplification efficiency E determines whether standard curve conforms to
It asks；
(5) after standard curve meets the requirements, the content of wherein mung bean ingredient is determined according to commercial samples numerical value.
Further, use CTAB method extraction mung bean genomic DNA as test sample DNA profiling in the step (1), it is dilute
It releases spare to 25ng/ μ L.
Further, mung bean genomic DNA is subjected to gradient dilution with salmon sperm dna in the step (2).
Further, the total volume of pcr amplification reaction is 25 μ L, amplification reaction system are as follows: Takara in the step (3)
12.5 μ L of Premix Ex Taq, 10 μm of ol/L upstream primers and each 1.0 μ L of 10 μm of ol/L downstream primers, 10 μm of ol/L probes
0.5 μ L, 2 μ L of test sample DNA profiling, distilled water supply 25 μ L；PCR response procedures are as follows: 95 DEG C of denaturation 10min；95 DEG C of denaturation
15s, 60 DEG C of annealing extend 60s, are collected simultaneously fluorescence signal, carry out 45 circulations altogether.
Further, the satisfactory condition of step (4) standard curve are as follows: coefficient R2>=0.98, amplification
The range of efficiency E is 90%≤E≤105%.
Compared with the existing technology, the specific detection mung bean of the present invention based on TaqMan probe real-time fluorescence PCR
Primer, probe and the method for anaphylactogen have the advantage that
Method of the invention is not in the case where calculating DNA extraction process time-consuming, real-time fluorescence quantitative PCR time-consuming 80min,
So the measurement work of mung bean allergen content in food and agricultural products can be completed in detection method of the invention within 1.5h,
With the advantages such as accurate, quick, easy to operate.In addition, method of the invention has filled up domestic green using real-time fluorescence PCR detection
The technological gap of beans anaphylactogen, while the sensitivity that mung bean detects in food is improved, the detection cycle of mung bean detection is shortened,
Avoid harm of the toxic reagent to experimenter's health.
Detailed description of the invention
Fig. 1 is the primer obtained using mung bean and screening, the canonical plotting of probe combinations foundation；From left to right successively
Are as follows: 1,100% mung bean；2,10% mung bean；3,1% mung bean；4,0.1% mung bean；5,0.01% mung bean.
Fig. 2 is the test sample testing result figure of mung bean content known to 4 parts.It is followed successively by 1 from left to right: test sample 1；2: surveying
Test agent 2；3: test sample 3；4: test sample 4.
Specific embodiment
In addition to being defined, technical term used in following embodiment has universal with those skilled in the art of the invention
The identical meanings of understanding.Test reagent used in following embodiment is unless otherwise specified conventional biochemical reagent；It is described
Experimental method is unless otherwise specified conventional method.
Below with reference to examples and drawings, the present invention will be described in detail.
(1) reagent: the Takara Premix Ex Taq solution of TaKaRa company production；By Suzhou Jin Weizhi biotechnology
The primer and TaqMan probe of Co., Ltd's synthesis；
(2) primer, probe sequence table are as follows:
| Title | Sequence 5 ' -3 ' |
| LvD-QF | TGTTTGCCTCATGCTATTATTGATC |
| LvD-QR | TCTCAATCCTGTTTGTACTACCTTCAG |
| LvD-P | FAM-AAGAAAACAAATGCTGCAATGCAACCTCA-TAMRA |
(3) sampling and DNA are extracted
Using 4 parts of samples of known mung bean percentage composition as test sample, (concentration of the mung bean percentage composition of 4 parts of test samples is
Test sample 1:10%, test sample 2:10%, test sample 3:0.1% and test sample 4:0%), use Plant Genome
DNA extraction kit extracts genomic DNA.
(4) standard curve is established
Mung bean genomic DNA is subjected to gradient dilution with salmon sperm dna, DNA concentration is respectively 100%, 10%, 1%,
0.1% and 0.01%, establish standard curve.
(5) real-time fluorescence quantitative PCR expands
Using above-mentioned primer, probe, PCR amplification carried out to test sample DNA profiling, pcr amplification reaction therein it is total
Volume is 20 μ L, amplification reaction system are as follows: 12.5 μ L of Takara Premix Ex Taq, 10 μm of ol/L upstream and downstream primers
Each 1.0 μ L, 10 μm of 0.5 μ L of ol/L probe, 2 μ L of test sample DNA profiling, distilled water supply 25 μ L；PCR response procedures are as follows: 95 DEG C
It is denaturalized 10min；95 DEG C of denaturation 15s, 60 DEG C of annealing extend 60s, are collected simultaneously fluorescence signal, carry out 45 circulations altogether.
(6) result judges
The slope of standard curve is -3.345, wherein coefficient R2=1, amplification efficiency E=99.046% are complied fully with
Coefficient R2>=0.98, the requirement of amplification efficiency E (90%≤E%≤105%).Meanwhile to the 4 of known mung bean percentage composition
Part test sample has carried out relative quantification test.The relative quantification result of this 4 parts of test samples is respectively 10%, 10%, 0.1% and
0%, this is corresponding with the percentage composition of mung bean when being pre-mixed.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (8)
1. a kind of primer of the specific detection mung bean anaphylactogen based on TaqMan probe real-time fluorescence PCR, it is characterised in that: its
The nucleotide sequence LvD-QF of upstream primer is as shown in SED ID N O:1 in sequence table, the nucleotide sequence LvD- of downstream primer
QR is as shown in SEQ ID NO:2 in sequence table.
2. a kind of probe of the specific detection mung bean anaphylactogen based on TaqMan probe real-time fluorescence PCR, it is characterised in that: institute
The nucleotide sequence LvD-P of probe is stated as shown in SED ID NO:3 in sequence table.
3. a kind of method of the specific detection mung bean anaphylactogen based on TaqMan probe real-time fluorescence PCR, it is characterised in that: answer
With the primer or probe in detecting mung bean anaphylactogen in claim 1-2.
4. the method for the specific detection mung bean anaphylactogen as claimed in claim 3 based on TaqMan probe real-time fluorescence PCR,
It is characterized in that: including the following steps:
(1) mung bean genomic DNA is extracted as test sample DNA profiling and to dilute spare；
(2) mung bean genomic DNA is subjected to gradient dilution, the concentration of diluted mung bean genomic DNA is respectively 100%, 10%,
1%, 0.1% and 0.01%, and standard curve is established respectively；
(3) above-mentioned primer, probe are used, PCR amplification is carried out to test sample DNA profiling and fluorescence signal detects.
(4) after reaction, according to calibration curve coefficient correlation R2, amplification efficiency E determines whether standard curve meets the requirements；
(5) after standard curve meets the requirements, the content of wherein mung bean ingredient is determined according to commercial samples numerical value.
5. the side of the specific detection mung bean anaphylactogen according to claim 4 based on TaqMan probe real-time fluorescence PCR
Method, it is characterised in that: CTAB method is used to extract mung bean genomic DNA as test sample DNA profiling, dilution in the step (1)
It is spare to 25ng/ μ L.
6. the side of the specific detection mung bean anaphylactogen according to claim 4 based on TaqMan probe real-time fluorescence PCR
Method, it is characterised in that: mung bean genomic DNA is subjected to gradient dilution with salmon sperm dna in the step (2).
7. the side of the specific detection mung bean anaphylactogen according to claim 4 based on TaqMan probe real-time fluorescence PCR
Method, it is characterised in that: the total volume of pcr amplification reaction is 25 μ L, amplification reaction system are as follows: Takara in the step (3)
Premix Ex Taq12.5 μ L, 10 μm of ol/L upstream primers and each 1.0 μ L of 10 μm of ol/L downstream primers, 10 μm of ol/L probes 0.5
μ L, 2 μ L of test sample DNA profiling, distilled water supply 25 μ L；PCR response procedures are as follows: 95 DEG C of denaturation 10min；95 DEG C of denaturation 15s, 60
DEG C annealing extend 60s, be collected simultaneously fluorescence signal, altogether carry out 45 circulation.
8. the side of the specific detection mung bean anaphylactogen according to claim 4 based on TaqMan probe real-time fluorescence PCR
Method, it is characterised in that: the satisfactory condition of step (4) standard curve are as follows: coefficient R2>=0.98, amplification efficiency
The range of E is 90%≤E≤105%.
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Citations (3)
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| CN105132549A (en) * | 2015-08-31 | 2015-12-09 | 中国农业科学院植物保护研究所 | Vigna radiata specific PCR (polymerase chain reaction) primer pair and method for detecting vigna radiata in phytophagous insect bodies |
| CN105463091A (en) * | 2015-12-17 | 2016-04-06 | 安徽出入境检验检疫局检验检疫技术中心 | PCR primer group for detecting green bean cake components and detection method using primer group |
| CN107385067A (en) * | 2017-08-17 | 2017-11-24 | 天津市农业质量标准与检测技术研究所 | A kind of method of the bulbus fritillariae cirrhosae species specificity detection based on TaqMan probe Real-Time Fluorescent Quantitative PCR Technique |
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- 2019-08-15 CN CN201910754888.0A patent/CN110468227A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105132549A (en) * | 2015-08-31 | 2015-12-09 | 中国农业科学院植物保护研究所 | Vigna radiata specific PCR (polymerase chain reaction) primer pair and method for detecting vigna radiata in phytophagous insect bodies |
| CN105463091A (en) * | 2015-12-17 | 2016-04-06 | 安徽出入境检验检疫局检验检疫技术中心 | PCR primer group for detecting green bean cake components and detection method using primer group |
| CN107385067A (en) * | 2017-08-17 | 2017-11-24 | 天津市农业质量标准与检测技术研究所 | A kind of method of the bulbus fritillariae cirrhosae species specificity detection based on TaqMan probe Real-Time Fluorescent Quantitative PCR Technique |
Non-Patent Citations (4)
| Title |
|---|
| K CHATTOPADHYAY等: "PCR-based Characterization of Mung Bean (Vigna radiata) Genotypes from Indian Subcontinent at Intra- and Inter-Specific Level", 《J. PLANT BIOCHEMISTRY & BIOTECHNOLOGY》 * |
| 叶伯平等: "《职业点菜师》", 30 September 2006, 中国轻工业出版社 * |
| 国家认证认可监督管理委员会: "《国家认监委实验室能力验证技术报告汇编》", 31 October 2007, 中国计量出版社 * |
| 赵仲麟等: "食品中绿豆成分PCR特异性检测方法的建立", 《河南农业大学学报》 * |
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Application publication date: 20191119 |