CN102839212B - Mango derived component fast screening reagent kit and detection method in food and beverage - Google Patents
Mango derived component fast screening reagent kit and detection method in food and beverage Download PDFInfo
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- CN102839212B CN102839212B CN201210268574.8A CN201210268574A CN102839212B CN 102839212 B CN102839212 B CN 102839212B CN 201210268574 A CN201210268574 A CN 201210268574A CN 102839212 B CN102839212 B CN 102839212B
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- 235000014826 Mangifera indica Nutrition 0.000 title claims abstract description 54
- 235000004936 Bromus mango Nutrition 0.000 title claims abstract description 52
- 235000009184 Spondias indica Nutrition 0.000 title claims abstract description 52
- 238000001514 detection method Methods 0.000 title claims abstract description 19
- 235000013305 food Nutrition 0.000 title abstract description 18
- 235000013361 beverage Nutrition 0.000 title abstract description 11
- 239000003153 chemical reaction reagent Substances 0.000 title abstract description 4
- 238000012216 screening Methods 0.000 title abstract description 4
- 240000007228 Mangifera indica Species 0.000 title description 3
- 241001093152 Mangifera Species 0.000 claims abstract description 53
- 239000000523 sample Substances 0.000 claims description 25
- 239000000243 solution Substances 0.000 claims description 17
- 238000006243 chemical reaction Methods 0.000 claims description 15
- 230000003321 amplification Effects 0.000 claims description 11
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 11
- 239000013641 positive control Substances 0.000 claims description 7
- 238000012408 PCR amplification Methods 0.000 claims description 6
- 230000000692 anti-sense effect Effects 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 4
- 230000001186 cumulative effect Effects 0.000 claims description 4
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 2
- 230000000295 complement effect Effects 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 10
- 239000000203 mixture Substances 0.000 abstract description 10
- 108090000623 proteins and genes Proteins 0.000 abstract description 7
- 238000005516 engineering process Methods 0.000 abstract description 4
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- 235000013399 edible fruits Nutrition 0.000 description 10
- 239000000047 product Substances 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 6
- 230000002068 genetic effect Effects 0.000 description 5
- 238000012545 processing Methods 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 206010020751 Hypersensitivity Diseases 0.000 description 3
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- 230000004087 circulation Effects 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 239000012154 double-distilled water Substances 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 241000208223 Anacardiaceae Species 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 235000009508 confectionery Nutrition 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
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Abstract
The invention belongs to the fast screening reagent kit and detection technique of mango derived component in food and beverage, specifically using the ITS2 gene fragments of real-time fluorescence PCR technology for detection mango.The present invention for mango derived component uses real-time fluorescence PCR technology, rapidly, sensitively, specifically detects and includes gene, so as to screen in food and beverage whether derived component containing mango.The fast screening reagent kit and detection technique are used to detect that anaphylactogen mango composition may to be contained in biased sample, can be used for detecting in sample whether do not contain mango composition.This method is quick, high specificity, high sensitivity, easy to operate, reproducible.
Description
Technical field
The invention belongs to plant derived component mango DNA detection techniques in food and beverage, specifically using real-time
Fluorescence PCR technology is quickly screened by mango progress in food and beverage and the method for detection, extraction of the method including DNA,
Real-time fluorescent PCR amplification and result judgement.Its advantage is quick, high specificity, high sensitivity, easy to operate, reproducible.
Background technology
Mango mango, scientific name Mangifera indica Linn., alias:Mango, vast and boundless fruit, vexed fruit, honey are hoped, hope fruit, face
Fruit and Buddhist nunnery POLO fruit, belong to Anacardiaceae (Anacardiaceae) Mangifera.Not only pulp is abundant sweet for mango, and contributes to U.S.
Hold beauty treatment and fat-reducing, the reputation for having " tropical fruit king " claims.The whole world there are about individual Different Cultivars of Mangifera indica more than 1000, and mango is often processed
Into mango juice, food, ice cream, Yoghourt, sugar etc..Sweet and dilitious mango meat is the tropical fruit (tree) being loved by people.Though mango
It is so delicious nutritious, but because its property is damp and hot, more foods are not only unhelpful to be harmful on the contrary, and especially some people have allergy to mango
Shape, therefore mango is also one of common fruit anaphylactogen.In recent years because the patient of mango allergy is more and more, there is aphtha,
Fruit rash, oral cavity fiber crops itch, and there will be severe the symptoms such as asthma and anaphylactic shock.Mango food processing product are being processed place
In after reason, the identifiable morphological feature of mango is often lost, is made troubles for mango food authenticity.With mango product and beverage
Constantly expand, illegal manufacturer is cost-effective, is adulterated using food addition raw material, pretends to be pure natural fruit juice manufacture fruit
Processed goods, upset processing market.In order to protect public's rights and interests, the detection method for establishing reliable mango is extremely urgent.
Whether mango derived component, still neither one quick decision method are mixed with food.Most of consumer demand
Judge the method containing mango derived component, food processing of a few consumers due to being free of mango composition to mango allergy demand
Product.The quality of life of consumer needs detection method really up to the mark and according to ensureing.
The content of the invention
The purpose of the present invention is to design a group-specific primers for mango ITS2 genes, establishes one kind in food and beverage
In quickly screen and detect mango derived component real-time fluorescence PCR detection method, existed with overcoming based on formalness detection method
Limitation in food processing products detection, the method for providing quick detection for mango Product checking in food and beverage.
The present invention cardinal principle be:One group of primer pair and probe that can specifically identify mango sequence is designed, it is perfect
Experimental system and reaction condition, target DNA accelerated accumulation is made to 10 by real-time fluorescence PCR reaction9~1010Copy, by fluorescence
Amplification curve, Ct values differentiate amplification.
The ripe experimental method established in the present invention can ensure to detect mango source property in the fabricated products such as food, beverage
Composition.This method eliminates the processes such as agarose electrophoresis, directly can carry out interpretation with fluorescent PCR observation amplification curve, simply
And the unit of a lines is quickly detected especially suitable for some.
The kit of quick detection mango derived component includes two kinds of pcr amplification reaction liquid, positive control dna characteristic portions
Point;
Reaction system cumulative volume is 25 μ L, wherein containing:Sample DNA (10 μ g/mL-100 μ g/mL) 2 μ L, mango primer pair
(10 μm of ol/L) each 0.5 μ L, 1 μ L, Taq archaeal dna polymerase (5U/ μ L) of mango probe (10 μm of ol/L) 0.5 μ l, dNTP (10 μ
Mol/L) the μ L of 2 μ L, 10 × PCR buffer solution 2.5, water complement to the μ L of cumulative volume 25.
Also the real-time fluorescent PCR amplification system of commercialization, μ L of reaction premixed liquid Real time Mix 10,10 μ can be used
μ L of mol/L sense primers 0.5,10 μm of μ L of ol/L anti-sense primers 0.5,10 μm of μ L of ol/L probes 1 and the μ l of template composition 2, add ddH2O
(sterilizing distilled water) complements to 25 μ l.
The primer and probe of real-time fluorescent PCR amplification detection of the present invention, its sequence are as follows:
(1) sense primer:5’-TCTGAGTTCTCGGTGACGCTTTC-3’;
(2) anti-sense primer:5’-CCGGTCTCTAGGGTCGAAGAGC-3’;
(3) probe sequence:5’-FAM-ATCCTGTCGTGCGGTTGCGTTCTCC-TAMRA-3’.
In actual applications, the volume ratio of sense primer, anti-sense primer and probe is:1∶1∶2.
Positive control dna is the DNA extract solutions containing mango derived component.
The method of mango composition in real-time PCR detection food processing products, comprise the following steps (1)-(3) successively:
(1) measuring samples DNA extraction
A. 0.1g samples are weighed, are gone in 1.5mL centrifuge tubes.The lysis buffer of 600 μ L preheatings is added, is gently mixed
Afterwards, 65 DEG C of water-bath insulation 10min;
B. isometric μ L of phenol/chloroform 600 are added in pipe, abundant mixing of turning upside down, extract 2min;
C.12000g 5min is centrifuged, draws supernatant into a new centrifuge tube, addition and the isometric precipitated liquid of supernatant, is mixed
It is even, after room temperature 10min, 12000g centrifugation 5min, supernatant is removed, retains precipitation;
D. 60 μ L RNases are added in precipitation, after 37 DEG C are placed 2min, are fully mixed it with pipette tips, in 37 DEG C of dissolvings
Precipitate, 300 μ L buffer solutions are added after 5min, mixing 10 times of turning upside down;
E. centrifugal column is taken out, centrifugal column is placed on 1 2mL sleeve pipe, solution is added in centrifugal column, is placed
2min;
F. centrifugal column and 2mL sleeve pipes one are reinstated into 8000g centrifugation 30sec, discards solution in sleeve pipe, added in centrifugal column
200 μ L washing lotions, 8000g centrifugation 30sec, discard solution;Repeat this step once;
G. 200 μ L70% ethanol are added in centrifugal column, 8000g centrifugation 30sec, discard solution;Repeat this step once;
H.12000g 30sec is centrifuged, removes trace residue solution in centrifugal column;
I. centrifugal column is placed in a new 1.5mL centrifuge tube, it is slow to add 50 μ L elutions in centrifugal column bottom center
Fliud flushing, after 37 DEG C are placed 2min, 12000g centrifugations 30sec.Solution in centrifuge tube is the template that can be used as PCR reactions.
(2) PCR is expanded
A. μ L of Real time Mix 10,10 μm of μ L of ol/L sense primers 0.5,10 μm of ol/L are added in amplified reaction pipe
μ L of anti-sense primer 0.5,10 μm of μ L of ol/L probes 1, ddH2O (sterilizing distilled water) complements to 25 μ l, mixes;
B. measuring samples DNA2 μ L (about 200ng) are added in amplified reaction pipe, are mixed;
C. enter performing PCR in fluorescent PCR instrument and react 40-45 circulation, program is:
95 DEG C of 3min of pre-degeneration, 40-45 circulation:95 DEG C of 15s, 60 DEG C of 30s.
(3) result detects
After real-time fluorescence PCR reaction terminates, instrument automatically analyzes data, draws Ct values and amplification curve.Totally see curve
Flex point understands that it is obvious for index, and amplification curve entirety collimation is good, and baseline is without the phenomenon that raises up, and side is by Ct value judged results.
Judged according to the Ct values of fluorescence signal, it is positive when the genetic test Ct values of testing sample are more than or equal to 45
Control and blank control result it is normal, then can determine whether not detect mango gene in sample, do not contained in sample mango source property into
Point.
When the structural specificity genetic test Ct values of testing sample are less than or equal to 36, positive control and blank control result
Normally, then can determine whether, to detect mango gene in sample, to contain mango derived component in sample.
It is less than 45 when detected sample structural specificity genetic test Ct values are more than 36, real-time PCR detection should be reformed,
If reforming rear Ct values still less than 45, positive control and blank control result are normal, then can determine whether to detect mango gene in sample;
If rear Ct values are reformed more than 45, and positive control and blank control result are normal, then can determine whether not detect mango base in sample
Cause.
Brief description of the drawings
The ITS2 genetic fragment real-time fluorescence PCR collection of illustrative plates of the multiple kind mango of Fig. 1.
The real-time fluorescent PCR amplification figure and 2 kinds of unknown concentration mango juice beverages of Fig. 2 difference dilution factor platform awns DNA solutions
Amplification figure.
Embodiment
With reference to embodiment, the present invention will be further described.
Embodiment 1
Detected according to following procedure:
(1) extraction to be measured that mango components Sample DNA may be contained
A. 0.1g samples are weighed, are gone in 1.5mL centrifuge tubes.The lysis buffer of 600 μ L preheatings is added, is gently mixed
Afterwards, 65 DEG C of water-bath insulation 10min;
B. isometric μ L of phenol/chloroform 600 are added in pipe, abundant mixing of turning upside down, extract 2min;
C.12000g 5min is centrifuged, draws supernatant into a new centrifuge tube, addition and the isometric precipitated liquid of supernatant, is mixed
It is even, after room temperature 10min, 12000g centrifugation 5min, supernatant is removed, retains precipitation;
D. 60 μ L RNases are added in precipitation, after 37 DEG C are placed 2min, are fully mixed it with pipette tips, in 37 DEG C of dissolvings
Precipitate, 300 μ L buffer solutions are added after 5min, mixing 10 times of turning upside down;
E. centrifugal column is taken out, centrifugal column is placed on 1 2mL sleeve pipe, solution is added in centrifugal column, is placed
2min;
F. centrifugal column and 2mL sleeve pipes one are reinstated into 8000g centrifugation 30sec, discards solution in sleeve pipe, added in centrifugal column
200 μ L washing lotions, 8000g centrifugation 30sec, discard solution;Repeat this step once;
G. 200 μ L70% ethanol are added in centrifugal column, 8000g centrifugation 30sec, discard solution;Repeat this step once;
H.12000g 30sec is centrifuged, removes trace residue solution in centrifugal column;
I. centrifugal column is placed in a new 1.5mL centrifuge tube, it is slow to add 50 μ L elutions in centrifugal column bottom center
Fliud flushing, after 37 DEG C are placed 2min, 12000g centrifugations 30sec.Solution in centrifuge tube is the template that can be used as PCR reactions.
(2) PCR of component beverage containing mango amplifications to be measured
A. μ L of Fluorescence PCR premixed liquid Real time Mix 10,10 μm of ol/L upstreams are added in amplified reaction pipe
μ L of primer 0.5,10 μm of μ L of ol/L anti-sense primers 0.5,10 μm of μ L of ol/L probes 1, ddH2O (sterilizing distilled water) complements to 25 μ l,
Mix;
B. the μ L (about 200ng) of measuring samples DNA 2 are added in amplified reaction pipe, are mixed;
C. performing PCR reaction is entered in the fluorescent PCR instrument of ABI 7900, program is:
95 DEG C of 3min, 45 circulations:95 DEG C of 15s, 60 DEG C of 30s.
(3) result detects
After real-time fluorescence PCR reaction terminates, instrument automatically analyzes data, draws Ct values and amplification curve.Totally see curve
Flex point understands that it is obvious for index, and amplification curve entirety collimation is good, and baseline is without the phenomenon that raises up, by Ct value judged results.
Judged according to the Ct values of fluorescence signal, the genetic test Ct values of testing sample are positive right between 20-36
According to normal with blank control result, it is judged as detecting mango gene in sample.
Claims (1)
1. the kit of quick detection mango derived component, including P C R amplification reaction solutions, positive control D N two kinds of features of A
Property part:
P C R amplification reaction solutions cumulative volume is 25 μ L, wherein containing:10 μ g/mL-100 μ g/mL of sample DNA 2 μ L, 10 μm of ol/L
Mango sense primer has primer each 1 μ L, 10 μm of 1 μ L, 5U/ μ L Taq archaeal dna polymerases of ol/L mango probe 0.5 μ l, 10 μ with
The μ L of 2 μ L, 10 × PCR buffer solutions of mol/L dNTP 2.5, water complement to the μ L of cumulative volume 25;
The primer and probe for the real-time fluorescent PCR amplification detection being related to, its sequence are as follows:
(1) sense primer:5’-TCTGAGTTCTCGGTGACGCTTTC-3’;
(2) anti-sense primer:5’-CCGGTCTCTAGGGTCGAAGAGC-3’;
(3) probe sequence:5’-FAM-ATCCTGTCGTGCGGTTGCGTTCTCC-TAMRA-3’;
Positive control dna is the DNA extract solutions containing mango derived component.
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CN106555000A (en) * | 2016-11-10 | 2017-04-05 | 湖北省食品质量安全监督检验研究院 | A kind of method of plant derived component in plant identification protein beverage |
JP7253947B2 (en) * | 2019-03-22 | 2023-04-07 | 日清食品ホールディングス株式会社 | Primer and mango detection method |
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CN101250582A (en) * | 2008-03-07 | 2008-08-27 | 山东出入境检验检疫局检验检疫技术中心 | Preparation of rapid test kit for fish anaphylactogen in food and detecting method |
CN101921837A (en) * | 2010-05-27 | 2010-12-22 | 山东出入境检验检疫局检验检疫技术中心 | Primer and prober for detecting carrot component in foods and beverages |
CN101921836A (en) * | 2010-05-27 | 2010-12-22 | 山东出入境检验检疫局检验检疫技术中心 | Preparation of kit and method for rapidly detecting carotene components in foods and beverages |
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CN101250582A (en) * | 2008-03-07 | 2008-08-27 | 山东出入境检验检疫局检验检疫技术中心 | Preparation of rapid test kit for fish anaphylactogen in food and detecting method |
CN101921837A (en) * | 2010-05-27 | 2010-12-22 | 山东出入境检验检疫局检验检疫技术中心 | Primer and prober for detecting carrot component in foods and beverages |
CN101921836A (en) * | 2010-05-27 | 2010-12-22 | 山东出入境检验检疫局检验检疫技术中心 | Preparation of kit and method for rapidly detecting carotene components in foods and beverages |
Non-Patent Citations (1)
Title |
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Phylogenetic relationships of Mangifera species revealed by ITS sequences of nuclear ribosomal DNA and a possibility of their hybrid origin;K.Yonemori et al;《Plant Syst. Evol.》;20021231;第231卷;59-75 * |
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