CN107805670A - Real-time fluorescence PCR differentiates composition, kit and the method for raspberry composition - Google Patents
Real-time fluorescence PCR differentiates composition, kit and the method for raspberry composition Download PDFInfo
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- CN107805670A CN107805670A CN201610809885.9A CN201610809885A CN107805670A CN 107805670 A CN107805670 A CN 107805670A CN 201610809885 A CN201610809885 A CN 201610809885A CN 107805670 A CN107805670 A CN 107805670A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The present invention relates to the real time fluorescent PCR method of raspberry composition.The invention further relates to the Oligonucleolide primers probe compositions for methods described.The invention further relates to the real-time fluorescent PCR reagent case for including the composition.Using the present invention composition carry out real-time PCR detection, can by it is simple, quick, special and delicately detection dried fruit, jam, fruit juice（Except juice）Deng raspberry in food（Containing raspberry, black raspberry, yellow raspberry）Derived component.
Description
Technical field
The invention belongs to biological technical field, specifically, the present invention relates to qualitative detection raspberry composition Taqman
Real time fluorescent PCR method, for the Oligonucleolide primers probe compositions of methods described, and include the reagent of the composition
Box.
Background technology
Raspberry is the rose family（Rosaceae）, Rosoideae, rubus（Rubus spp.）, alias：Rubus corchorifolius, cover basin
Son, mountain throw son, spread seedling bubble etc..Fruit colour is various, there is red, black, purple, yellow etc..Fruit of raspberry sweet and sour taste, nutrition
It is abundant, containing 17 kinds of amino acid needed by human and vitamin, benzoaric acid, cupreol and red raspberry acid etc., have anti-
Aging and raising body immunity and other effects, especially Ve and SOD（Superoxide dismutase）Content for fruit most, it is natural super
Superoxide dismutase and vitamin E are fabulous human body street cleaners, can eliminate a large amount of detrimental metabolic materials caused by human body, carry
High body immunity, fundamentally improve the internal environment of human body, reach beauty, beauty treatment, the purpose promoted longevity.Long-term consumption
Raspberry, can effective cardioprotection, prevent hypertension, vascular wall atherosis, cerebrovascular sclerosis rupture etc. cardiovascular and cerebrovascular disease.Tree
The certain kind of berries is widely used, and fruit soft and succulency, color and luster is pleasant, suitable for eating or being processed into beverage, fruit juice, jam, sweet can etc. raw.Food
Relieve summer heat and promote the production of body fluid, eliminating phlegm of quenching the thirst, sober up and refresh oneself, can eat as fruit, can also be used as medicine.
With the fashionable world of the products such as raspberry dried fruit, jam, fruit juice, its abundant nutritive value, medical value promote to set
The adulteration of the certain kind of berries grows in intensity, and some illegal manufacturers use fruit juice adulteration means, is such as played tricks in juice of raspberry content sign,
Make some cheap or even false fruit juice flood market, also the allotment such as saccharin, carbohydrate, pigment and water form etc., seriously endanger
The health of consumer.Need the simple, fast true and false authentication technique of exploitation badly.Its research carried out was concentrated mainly in the past aobvious
On the conventional physiochemical techniques such as micro- discriminating, chromatogram, spectrum, but largely can be by kind, the place of production, harvest season, raw material
The influence of the several factors such as environment, processing conditions, storing manner of packing.With the fast development of Protocols in Molecular Biology, it is based on
DNA various Protocols in Molecular Biologies judge Species composition by detecting species-specific gene fragment, have high specificity,
High sensitivity, it is not easy to be disturbed by environmental condition, the advantages that as a result stablizing, the application in the identification of the small berries such as raspberry is increasingly
Extensively.
The content of the invention
It is an object of the present invention to simply, quickly, specifically and delicately detect food and fruit juice（Except juice）In tree
Certain kind of berries composition, it can effectively prevent from adding the fraud mode of other cheap fruit in the raspberry product such as fruit juice, jam.
For the present inventor according to fruits and vegetables reference gene NADH, the real-time glimmering of fruits and vegetables internal reference can be common to by devising
Light PCR detection method；Using raspberry chloroplaset ndhF genes as target, primer and the spy of energy specific detection raspberry composition are devised
Pin；The bp of target sequence 160, guarantee to detect target plant composition in precipitating from jam, fruit juice etc..
In one aspect of the invention, there is provided be common to the Oligonucleolide primers upstream NADH- of fruits and vegetables composition internal reference
F：5’-GCTGAAGCAGCTACTTTCGAAGTAACA-3’（SEQ ID No.1）, and downstream NADH-R：5’-
AGGAGCCGTGTGAGATGAAAGTCTCA-3’（SEQ ID No.2）.Probe is NADH-P：5’-
TGGAGTGGGAGAGTCAGAGTCGAAAAGAGG-3’（SEQ ID No.3）, a fluorescent quenching is connected with 3 ' ends of probe
Group TAMRA, 5 ' ends are connected with a fluorescent reporter group FAM.The primer can specific recognition fruits and vegetables composition NADH sequences,
The bp of expanding fragment length 138.Specificity extension self-increasing reaction condition is：50℃ 2 min；95 DEG C of min of pre-degeneration 10；95℃ 15
S, 60 DEG C of 1 min, 40 circulations.
In one aspect of the invention, the specific oligonucleotide primer upstream of specific detection raspberry composition is additionally provided
Raspberry-F: 5’- GTTATTCGTTGGAACCGTAGGAA -3’（SEQ ID No.4）, and downstream Raspberry-R：
5’- GACTGAAAAAACTGCATTTGTGA -3’（SEQ ID No.5）.Probe is Raspberry-P: 5’-
ATTATCCAAATTGTTAACCCCGTCCATAAACCT-3’（SEQ ID No.6）, a fluorescence is connected with 3 ' ends of probe
Quenching group TAMRA, 5 ' ends are connected with a fluorescent reporter group FAM.The primer can specific recognition raspberry ndhF sequences,
The bp of expanding fragment length 160.Specificity extension self-increasing reaction condition is：50℃ 2 min；95 DEG C of min of pre-degeneration 10；95℃ 15
S, 60 DEG C of 1 min, 40 circulations.
In another aspect of the present invention, there is provided include the composition of above-mentioned oligonucleotide sequence., the composition bag
Containing selected from following（1）Extremely（2）In any one or more groups of primer and probe sequences：
（1）Fruits and vegetables internal reference gene Oligonucleolide primers are to SEQ ID No.1 ~ SEQ ID No.2 and probe SEQ ID No.3
Sequence；
（2）Raspberry specific oligonucleotide primer pair SEQ ID No.4 and SEQ ID No.5 and probe SEQ ID No.6
Sequence.
In one embodiment, fruits and vegetables reference gene and raspberry real-time fluorescent PCR amplification condition are 95 DEG C, 10min；
95 DEG C of 15s, 60 DEG C of 1min, 40 circulations, for preferably, real-time fluorescence PCR method used in the present invention is Taqman
Fluorescence probe method.
In the present invention, raspberry contains 4 raspberry, black raspberry, yellow raspberry, tupelo gum certain kind of berries Breeds containing raspberry.
In another aspect of the present invention, there is provided glimmering in real time for fruits and vegetables composition universal testing kit, raspberry composition
Light PCR specific detection agents box, the oligonucleotide sequence or the composition are included in the kit.
Kit provided by the invention is used for real-time PCR detection fruits and vegetables composition and raspberry into dtex including the present invention
Specific primer and operation instructions.
In one embodiment, raspberry specific primer of the invention is set according to chloroplast gene matk sequences respectively
Meter.In one embodiment, raspberry is included in the kit（By taking raspberry as an example）Specific amplification target sequence is:
GTTATTCGTTGGAACCGTAGGAATTCCTTTCTTCAATCAGGAAGGAATAGATTTAGATATATTATCCAAATTGTTAA
CCCCGTCCATAAACCTTTTACATCAAAATCGAACCCACCCTGTCGATTGGTATGAATTTATCACAAATGCAGTTTTT
TCAGTC (SEQ No.7), in a preferred embodiment, the operation instructions of the kit are included to then
The description of real-time PCR detection raspberry specific primer and amplification condition.It is of the invention in a specific embodiment
Kit for detecting raspberry composition also includes reference substance.Preferably, reference substance includes positive reference substance and negative controls.
In one embodiment, negative control is aseptic double-distilled water.
In one embodiment, the real-time fluorescence PCR detection method detects that the absolute sensitivity of raspberry composition is：
Raspberry and black raspberry composition minimum 0.001ng/ μ L, the yellow minimum 0.0001ng/ μ L of raspberry composition.
In one embodiment, the minimum detectability of the real-time fluorescence PCR detection method is the mangrove of volume ratio 1%
Certain kind of berries composition, the black raspberry of volume ratio 0.1% and yellow raspberry composition.
Again on the one hand, the invention provides the composition or the kit to differentiate fruit juice（Except juice）, fruit
Application in the raspberry product such as sauce in raspberry composition.
Real-time fluorescence quantitative PCR adds the probe or fluorescence dye of fluorescence labeling i.e. on the basis of conventional PCR method
Material, with the accumulation of PCR primer, the fluorescence signal that probe or dyestuff are sent strengthens, and fluorescence monitoring system can receive fluorescence
Signal, i.e., a DNA is often produced, just has a fluorescence molecule to be formed, whole PCR courses of reaction are with the increasing of cycle-index
Add, exponentially rule increases the target gene fragment being amplified, and corresponding changes fluorescence with amplification by detecting in real time
Signal intensity, try to achieve Ct (cycle threshold, Ct) value.The fluorescence letter of amplified production in Ct values, i.e. PCR amplification procedures
Number reach the amplification cycles number passed through during the threshold value of setting, the logarithm of it and the starting copy number of template has linear close
System, template DNA amount is more, and fluorescence is fewer up to the period of threshold value, i.e. Ct values are smaller, quantitative to starting template and fixed so as to realize
Property analysis, so as to can detect composition to be measured.
The present invention's differentiates that the real-time fluorescence PCR of template detects by primer or probe and the specific hybrid of template
Method specificity is high, and false positive is low；Complete stopped pipe can be used to detect, be post-processed without PCR, avoid cross pollution and vacation sun
Property, shorten the reaction time.The fluorescence after PCR amplifications is detected, is exaggerated reaction signal, sensitivity greatly improves.The present invention's
Method dexterously used the DNA efficient amplifications of PCR technologies, the specificity of nucleic acid hybridization and detection technique of fluorescence quick and
Sensitiveness, there are simple to operate, time saving and energy saving, reliable results and accurate sensitive.Use the real-time fluorescence PCR of the present invention
Detection method, the characteristics of its is simple, quick, special and sensitive, are suitable for fruit juice on domestic and international market（Except juice）, jam
Deng the qualitative detection of raspberry composition in product.
Brief description of the drawings
Fig. 1 is that the typical amplification that fruits and vegetables occur is bent when utilizing real-time PCR detection fruits and vegetables internal reference primed probe versatility
Line.Berry and common fruit dredge 1-40：Raspberry（Northern land, brilliance, blue gold, Biloxi）, yellow raspberry, raspberry（Victory
Profit）, black raspberry, Cranberry, linberry, Binchuan summer black grape, blackcurrant, cape gooseberry, pomegranate, mock-strawberry, strawberry, hawthorn, Mi
Monkey peach, mulberries, cherry, red bayberry, carambola, apple, pears, peento, flowering peach, orange, tangerine, apricot, watermelon, pawpaw, cucumber, loquat, pineapple,
Lichee, longan, banana, tomato, matrimony vine, mango, olive；Negative control 41-48 is respectively：It is pine tree, metasequoia, agaric, mushroom, green
Algae, Escherichia coli, yeast, blank.
Fig. 2 is to use raspberry primed probe specific detection result.1 is raspberry in figure（Triumph）, 2 be yellow raspberry, 3 be
Black raspberry, 4-36 be followed successively by blueberry, Cranberry, mock-strawberry, mulberries, Kiwi berry, strawberry, blackcurrant, Binchuan summer black grape, cherry,
Red bayberry, carambola, apple, pears, peento, flowering peach, jujube, orange, tangerine, apricot, watermelon, pawpaw, mango, cucumber, loquat, pineapple, lichee, dragon
Eye, tomato, matrimony vine, olive, ginkgo, banana and blank control.
Fig. 3 is to use raspberry primed probe coverage testing result.Cover raspberry, three black raspberry, yellow raspberry product
Population, totally 10 kinds, wherein 1 is raspberry（Australia is red, the U.S. 22, Nova, Mick, early red, triumph, unknown）, 2 be Huang
Raspberry, 3 be black raspberry（2 kinds）.
Fig. 4 is to use raspberry primed probe absolute sensitivity testing result.A, B represents raspberry/muscat grape respectively
Blended fruit juice expands obtained typical amplification curve and standard curve, and C, D represent that black raspberry/summer black grape blended fruit juice expands respectively
Increase obtained typical amplification curve and standard curve, E, F represent that yellow raspberry/watermelon blended fruit juice expands obtained typical case's expansion respectively
Increase curve and standard curve.1-7 represents that raspberry DNA concentration is 100 ng/ μ L, 10 ng/ μ L, 1 ng/ μ L, 0.1 ng/ μ respectively
L, 0.01ng/ μ L, 0.001 ng/ μ L, 0.0001 ng/ μ L, each dilution factor 3 are parallel.
Fig. 5 is to use raspberry primed probe relative sensitivity testing result.A is raspberry/muscat grape blended fruit juice
Raspberry composition expand to obtain typical amplification curve, B be black raspberry/summer black grape blended fruit juice raspberry composition expand to obtain
Typical amplification curve, the raspberry composition that C is yellow raspberry/watermelon blended fruit juice expand obtained typical amplification curve, 1-5 difference
It is 0.1%, 1%, 10%, 50% to represent volume ratio, the samples of juice of 100% ratio mixing.
Fig. 6 is using raspberry primed probe amplification working process analog sample lower bound Detection of Stability result.A is 1% mangrove
Typical amplification curve that the raspberry composition of the certain kind of berries/muscat grape blended fruit juice expands to obtain, B are 0.1% black raspberry/summer black grape
The raspberry composition of blended fruit juice expands obtained typical amplification curve, the raspberry composition that C is 0.1% yellow raspberry/watermelon blended fruit juice
Expand obtained typical amplification curve, P1, P2, P3 represent fresh raspberry, black raspberry, yellow raspberry DNA respectively.1-3 distinguishes
Represent fresh, pasteurization, the blended fruit juice sample of autoclaving processing.
Embodiment
The present invention is further illustrated by way of embodiment, but the present invention is not limited only to following implementation
Example.
Embodiment 1
Although specific embodiments of the present invention are described, those skilled in the art will appreciate that not
The present invention can be variously changed and be modified on the premise of deviateing the scope or spirit of the invention.Thus, this invention is intended to
Cover to fall all these changes and modification in claims and its range of equivalency.
Claims (8)
1. the composition for being used as fruits and vegetables internal reference by the detection of real-time fluorescence PCR method includes fruits and vegetables internal reference gene antisense oligonucleotide primer
Thing is to SEQ ID No.1 ~ SEQ ID No.2 and probe SEQ ID No.3 sequences.
2. detecting the composition of raspberry composition by real-time fluorescence PCR method, the composition includes raspberry specific oligonucleotide
Primer pair SEQ ID No.4 and SEQ ID No.5 and probe SEQ ID No.6 sequences.
3. according to the composition described in claim 1 and 2,3 ' ends of wherein specific probe are connected with a fluorescent quenching group
TAMRA, 5 ' ends are connected with a fluorescent reporter group FAM.
4. the real-time fluorescence PCR detection method of raspberry composition, methods described is including the use of any one of claim 1-3
Specific oligonucleotide primer pair and probe.
5. differentiating the kit of raspberry composition for real-time fluorescence PCR detection method, the kit includes claim 1-3 institutes
The composition stated.
6. the specific oligonucleotide primer pair and probe any one of claim 1-3 in small berries product raspberry into
Application in point.
7. the method according to claim 11, including step：
a）Obtain from the STb gene sample for treating raspberry product；
b）Real time PCR amplification is carried out using specific primer probe, amplification system such as table 1, internal reference, raspberry real-time fluorescence PCR are anti-
The amplification program is answered to be：50 DEG C of 2 min, 95 DEG C of pre-degenerations 10 min, 95 DEG C of 15 s, 60 DEG C of 1 min of 40 circulations.
8. the method described in claim 7, including the system that real-time fluorescent PCR amplification is carried out to the STb gene sample and
Condition.
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Cited By (5)
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| CN110734998A (en) * | 2019-11-04 | 2020-01-31 | 中国检验检疫科学研究院 | Primers, method and kit for identifying NFC orange juice and FC orange juice |
| CN110734998B (en) * | 2019-11-04 | 2022-04-12 | 中国检验检疫科学研究院 | Primers, method and kit for identifying NFC orange juice and FC orange juice |
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