The recombinase-mediated amplification Constant Temperature Detection method and kit of your detail herbal medicine American GinsengTechnical field
The invention belongs to your detail herbal medicine detection field and technical field of molecular biology, specifically, belong to and how to adopt(Recombinase-aid Amplification, RAA) Constant Temperature Detection technology is expanded with recombinase-mediated and combines special drawWhether the method for American Ginseng (Panax quinquefolius) ingredient is contained in object and fluorescence probe Identification chinese herbs medicine.
Background technique
Since 2017, China's traditional Chinese medicine new policies are intensively put into effect, the especially implementation of " traditional Chinese medicine method " and " Chinese medicine productionIndustry poverty alleviation action plan (2017-2020) year " publication, bring new opportunities for Traditional Chinese Medicine Industry Development.In this context, nationalChinese medicine supply scale continues to expand, and intermediate links resource optimization is promoted, and intensive Habitat producing mode highlights, " internet+"The novel methods of conducting trade are risen, and Chinese medicine circulation market accelerates transition and upgrade, and Import and export of Chinese traditional medicines trade increases.In last year China's import9.10 ten thousand tons of medicinal material, increase by 13.62% on year-on-year basis, 2.87 dollar/kilogram of average inlet price goes up 14.15% on year-on-year basis, Chinese medicine2.61 hundred million dollars of total import value, increase by 29.69% on a year-on-year basis, much higher than increasing by a year-on-year basis for China's import of goods volume 18.7%.ChinaThe principal item of import has longan, American Ginseng, pilose antler, west safflower, olibanum, myrrh and dragon's blood etc..
American Ginseng is that only one is on the list and imports and exports the kind that the value of imports in preceding ten kind is higher than export amount of money.It is many longThe people that phase manages American Ginseng both knows about, and import ginseng has many tricks that the good and bad jumbled together.The American ginseng of imported from America is veryIt is few, or even not as good as Canada.According in each Chinese herbal medicine industry analysis report, we have 1500 tons of American Ginseng or so of annual " import ", butAccording to statistics formally come in from customs less than 800 tons, it is or even less, and have and much turn to put into from Hong Kong, will not influenceImport volume.There is a large amount of mixed pseudo-, imitation behavior in China's American Ginseng at present, has seriously affected traditional Chinese medicine quality and medication peaceEntirely, how simply, fast and accurately to detect whether nominal American Ginseng Chinese medicine is that real American Ginseng is asked as urgently to be resolvedTopic.
Recombinase-mediated amplification (Recombinase-aid Amplification, RAA) technology be also it is a kind of at a constant temperatureIt can make the method for nucleic acid rapid amplifying.Unlike RPA, RAA amplification, which is used, to be obtained from bacterium or fungiRecombinase, under 37 DEG C of constant temperature, which can combine closely with primed DNA, the condensate of enzyme and primer be formed, when primer existsWhen searching the sequence of complete complementary therewith on template DNA, in single-stranded DNA binding protein (single-stranded DNABinding, SSB) with the help of, make template DNA unwinding, and under the action of archaeal dna polymerase, forms new DNA complementary strand, insteadAnswering product is also to be increased with exponential, can obtain that the amplification piece that agarose gel electrophoresis detects can be used usually in 1hSection.Fluorophor is added in RAA reaction system, monitors entire RAA amplification procedure in real time using the accumulation of fluorescence signal, 20 points, it can be achieved that quantitative and qualitative analysis to starting template in clock.Entirely react simple and quick, because not needing high temperature circulation, instituteYour to be particularly suitable for using in the non-laboratory testing place for there are a large amount of samples, quickly detected suitable for detail herbal medicine authenticityField.
Summary of the invention
It is an object of the present invention to provide the amplifications of the recombinase-mediated of your detail herbal medicine American Ginseng (Panax quinquefolius)Constant Temperature Detection method and kit.
In order to achieve the above object, the invention adopts the following technical scheme:
A kind of detection kit of your detail herbal medicine American ginseng-nucleic acid, comprising: American Ginseng forward primer, reverse primer andSpecificity fluorescent probe, wherein the American Ginseng forward primer nucleotide sequence is as shown in SEQ ID NO.1, the American Ginseng is anti-To primer nucleotide sequences as shown in SEQ ID NO.2, the nucleotide sequence of the specificity fluorescent probe such as SEQ IDNO.3,5 ' ends are marked with fluorescent reporter group, and 3 ' ends are marked with fluorescent quenching group.
In some embodiments, the fluorescent reporter group of the specificity fluorescent probe be selected from FAM, VIC, JOE, TET,One of CY3, CY5, ROX, Texas Red or LC RED460, fluorescent quenching gene be selected from BHQ1, BHQ2, BHQ3,One of DABCY1 or TAMRA.
In some embodiments, the kit for detecting nucleic acid further includes primer mixed liquor, specificity fluorescent spyNeedle, A Buffer, B Buffer, RAA powdered reagent, American Ginseng standard items and ddH2At least one of O.
In some embodiments, the kit, wherein described ground A Buffer is 20%PEG；B Buffer is280mM MgAc。
In some embodiments, the kit, wherein the ingredient of the RAA powdered reagent is as follows: 1mmol/L dNTP, 90ng/ μ L SSB albumen, 120ng/ μ L recA recombination zymoprotein (SC-recA/BS-recA) or 30ng/ μ LRad51,30ng/ μ L Bsu archaeal dna polymerase, 100mmol/L Tricine, 20%PEG, 5mmol/L dithiothreitol (DTT),100ng/ μ L creatine kinase, Exo exonuclease.
In some embodiments, the kit for detecting nucleic acid, American Ginseng standard items are to contain American Ginseng full genomeThe positive plasmid of group partial sequence.
In some embodiments, the kit, the positive matter containing American Ginseng full-length genome partial sequenceThe sequence of grain is as shown in SEQ ID NO.4.
The present invention also provides a kind of RAA Constant Temperature Detection methods of American Ginseng, the DNA of sample to be tested are extracted, to test sampleThe DNA of product is template, in the forward primer of American Ginseng, reverse primer, specificity fluorescent probe and RAA powdered reagent, ABuffer, B Buffer and ddH2In the presence of O carry out real-time fluorescence RAA reaction, according to real-time fluorescence RAA amplification curve analysis toSample；Wherein the American Ginseng forward primer nucleotide sequence is as shown in SEQ ID NO.1, the American Ginseng reverse primer coreNucleotide sequence is as shown in SEQ ID NO.2, the nucleotide sequence of the specificity fluorescent probe such as SEQ ID NO.3,5 ' endsIt is marked with fluorescent reporter group, 3 ' ends are marked with fluorescent quenching group.
In some embodiments, the real-time fluorescence RAA response procedures are as follows: 39 DEG C, 40s, one circulations；39 DEG C, 30sOne circulation, 20min, 40 circulations.
Detection method of the present invention needs real-time fluorescence RAA after reaction, is analyzed using real-time fluorescence RAA instrument softPart analyzes sample to be tested according to the amplification curve of real-time fluorescence RAA.Preferably, the analysis sample to be tested is sample to be tested FAMChannel fluorescence curve is in " S " type and value≤35 CT, is judged as American Ginseng positive findings；When sample to be tested curve be not in " S " type orCT value > 35, is judged as American Ginseng negative findings.
The utility model has the advantages that
1, rapidly and efficiently: entire amplification only needs 20-30min can be completed, and amplification yield can achieve 109-1010It is a to copyShellfish；
2, easy to operate: not need special reagent, do not need to carry out the tedious steps such as the deformation of double-stranded DNA in advance, only needThe luminoscope of constant temperature is wanted, condition is milder；
3, high specific: the present invention is to American Ginseng others ingredient, ginseng (Panax ginseng C.A.Meyer), threeThe DNA of seven (Panax notoginseng (Burk.) F.H.Chen) etc. is not expanded.
4, highly sensitive: detectable limit of the invention can achieve 0.10fg/ μ L reaction.
5, identification is simple: according to real-time fluorescence data, directly judging amplification, is not necessarily to electrophoresis detection, is suitble to scene inspectionIt surveys.
Detailed description of the invention
Fig. 1 is 3 pairs of primer RAA amplification curve diagrams involved in the present invention.
Fig. 2 is RAA detection method to the sensitivity experiment figure of American Ginseng, is from left to right followed successively by 100fg/ μ L, 10.0fg/The amplification of the positive criteria product of μ L, 1.00fg/ μ L, 0.10fg/ μ L, 0.01fg/ μ L, 1.00ag/ μ L.
Fig. 3 is specificity experiments figure of the RAA detection method to American Ginseng.
Specific implementation method
Below by way of specific embodiment, the present invention is further described, and however, it is not limited to this.
Embodiment 1: primer and probe design screening
The present invention searches for American Ginseng complete genome sequence to American Ginseng in Genebank database, soft using DNAMAN 6.0Multisequencing is compared in part, finds out conservative section.3 groups of primer and probes are devised in conservative region, and in NCBI dataBLAST comparison is carried out in library, the sequence of primer and probe is as shown in table 1.
To the positive plasmid of American Ginseng conserved region gene sequence, the base sequence of the plasmid is remembered as shown in SEQ ID NO.4Property amplification,
ATGGCCCATCACGCCCAACAGGTAAAAGGGGTGCATGGCATGCATGGAAATCTAGAAAGGTACCATCGCGTGAGCTAAGCCCCATCGTGTTGAGCAACCAAAATCCAACGCAAGGAGATAAATCTTGAGTCGAGGTTAACACGACCTTTTTTGTTTCATGTGTGAGGCGAAGAGGTCCACGGCCCGTAGCACGCCGTGGCAGGGCCCAACACGCCCAGAGGCAGCACCACGCACACGCAAACAGGCAACGCCAACCAGGACCATCGAATGAAATGGCGGCAAACATGAAAAAAGCTTGCACTACACCATTTCCTCGAGCTA(SEQ ID NO.4)
The reagent of amplification is as follows:
Including primer mixed liquor, specificity fluorescent probe, A Buffer, B Buffer, RAA powdered reagent, American Ginseng markQuasi- product and ddH2O.Described ground A Buffer is 20%PEG；B Buffer is 280mM MgAc.
The ingredient of the RAA powdered reagent is as follows: 1mmol/L dNTP, 90ng/ μ L SSB albumen, 120ng/ μ LRecA recombinates zymoprotein (SC-recA/BS-recA) or 30ng/ μ L Rad51,30ng/ μ L Bsu archaeal dna polymerase, 100mmol/L Tricine, 20%PEG, 5mmol/L dithiothreitol (DTT), 100ng/ μ L creatine kinase, Exo exonuclease.
The real-time fluorescence RAA response procedures are as follows: 39 DEG C, 40s, one circulations；39 DEG C, 30s mono- circulation, 20min, 40A circulation.
Detection method of the present invention needs real-time fluorescence RAA after reaction, is analyzed using real-time fluorescence RAA instrument softPart analyzes sample to be tested according to the amplification curve of real-time fluorescence RAA.
Positive sample amplification curve is as shown in Figure 1.
1 primer and probe sequence of table:
By Fig. 1 result as it can be seen that the amplification curve of first group of primer and probe is the most typical, there are apparent exponential phase and platformPhase has compared with high fluorescent (ordinate value), and CT value smaller (abscissa corresponding to the crosspoint of curve and threshold line) is tiedFruit analysis is shown in Table 2.Other primed probe curve lifting heights are lower, and CT value is larger, and plateau is unobvious；Or do not expandIncrease, missing inspection occurs.Illustrate the reproduction speed of first group of primer and probe purpose product faster, more, amplified reaction efficiencyIt is higher.
The analysis of 2 primed probe the selection result of table
| Group/result | CT value | Fluorescence intensity |
| First group | 16.97 | 690000 |
| Second group | 35.21 | 500000 |
| Third group | 40.21 | 450000 |
Embodiment 2: kit assemble method
The kit American Ginseng
Kit for detecting nucleic acid of the present invention further includes primer mixed liquor, specificity fluorescent probe, A Buffer, BBuffer, RAA powdered reagent, American Ginseng standard items and ddH2O.Kit of the present invention, wherein described ground A BufferFor 20%PEG；B Buffer is 280mM MgAc.Kit of the present invention, wherein the RAA powdered reagent atDivide as follows: 1mmol/L dNTP, 90ng/ μ L SSB albumen, 120ng/ μ L recA recombination zymoprotein (SC-recA/BS-recA)Or 30ng/ μ L Rad51,30ng/ μ L Bsu archaeal dna polymerase, 100mmol/L Tricine, bis- sulphur of 20%PEG, 5mmol/L Soviet UnionSugar alcohol, 100ng/ μ L creatine kinase, Exo exonuclease.
In primer mixed liquor of the present invention, the forward primer base sequence is described as shown in SEQ ID NO.1Shown in the base sequence SEQ ID NO.2 of reverse primer, the mol ratio of forward primer and reverse primer is SEQ ID NO.1:SEQ ID NO.2 is 1:1.The specific probe base sequence of American Ginseng provided by the invention is as shown in SEQ ID NO.3, probe5 ' ends be marked with FAM fluorescent reporter group, 3 ' ends are marked with BHQ1 fluorescent quenching group.American Ginseng standard provided by the inventionProduct include the positive plasmid of American Ginseng conserved region gene sequence, and the base sequence of the plasmid is as shown in SEQ ID NO.4.
Embodiment 3: kit application method
1, the extraction of positive sample nucleic acid
1.1, plant genome DNA nucleic acid nucleic acid extraction: is extracted using CTAB improved method.
1. taking identical tissue powder that 2 × CTAB extraction buffer of 700 μ L preheated in 65 DEG C of water-baths is added, gently stirIt is dynamic, in the sterile centrifugation tube of 1.5ml, 40min is kept the temperature in 65 DEG C of water bath or insulating box, is gently shaken every 10min；
2. after cooling 2min, isometric chloroform is added: isoamyl alcohol (24:1) is uniformly mixed, and l0000r/min is centrifuged 10min,Supernatant is taken to be transferred in the isopropanol centrifuge tube of 600 μ L；
3. jog 30s is to the DNA floccule that can be seen；
4. after l0000r/min is centrifuged 1min, outwelling liquid immediately and retaining white DNA pellet, stand upside down after standing 60s, be addedThe sodium acetate of 75% ethyl alcohol of 720 μ L and 80 μ L 5M, is placed at room temperature for 30min；
5. l0000r/min is centrifuged 1min, liquid is outwelled, adds 800 μ L, 75% ethyl alcohol, after standing 30min,After l0000r/min is centrifuged 30s, liquid is outwelled；
6. after several minutes, 50 μ L 0.5 × TE buffers are added in the DNA after drying, it is placed in 37 DEG C of insulating box about 15h；
7. being placed in -20 DEG C of preservations, spare.
2, the configuration of RAA reaction system: the corresponding RAA reaction dry powder pipe of each test sample is (as described in examples of implementation 2Reagent), each reactive component and the volume being added are as shown in table 3 in each RAA reaction dry powder pipe.
Table 3:
A Buffer is 20%PEG；B Buffer is 280mM MgAc.
3, the RAA reaction tube for having configured reaction system is placed in ABI7500 amplification instrument, is carried out according to following procedureRAA amplification: 39 DEG C, 40s, one circulations；39 DEG C, 30s mono- circulation, 20min, 40 circulations.The channel each circulating collection FAMFluorescence.
4, American Ginseng positive or negative result is determined according to fluorescence curve judgement and CT value after expanding.
Determine result: FAM channel fluorescence curve is in " S " type and value≤35 CT, is judged as American Ginseng positive findings；When to be measuredSample curve is not in " S " type or CT value > 35, be judged as American Ginseng negative findings (this criterion: according to embodiment 1,2,4,5, it 6 obtains).
Embodiment 4: the assessment of RAA detection kit of the present invention in practical applications
Experiment (2 reagent of examples of implementation and the detection side of examples of implementation 3 of blind sample are carried out using kit of the present inventionMethod), 34 parts of your detail herbal medicine detected；The experimental results showed that the first primer of the invention be to that can distinguish American Ginseng, with nidoPCR positive coincidence rate is very high.In 34 parts, (primer is the same, and reality is matched according to traditional PCR amplification is practical for nest-type PRCThan), there are 18 parts for positive findings, 16 parts are negative findings (No. 34 are negative), and being 18 parts by the result that RAA method detects isThe positive has 15 parts also for negative findings, and it is different (No. 34 samples are the positive) that there are a positive findings, to this sample DNA intoRow PCR amplification is simultaneously sequenced, and sequencing result shows the sample as the positive, and it is higher accurate to illustrate that RAA detection reagent of the invention hasRate, the results are shown in Table 4.That is RAA detection has 19 parts of positives, 15 parts of feminine genders, and it is positive that traditional nest-type PRC, which only has 18 parts,Property as a result, 16 parts be negative findings.
Equally, RAA detection being carried out using of the invention second group and third group primer, only 12 parts of discovery are the positive,22 parts are negative findings (second group)；The detection of third group, only 10 parts are the positive, and 24 parts are feminine gender.(specific experiment mistakeJourney is omited).
4:34 parts of table unknown blind sample testing results (RAA testing result)
Embodiment 5: the sensitivity test of kit of the present invention
The American Ginseng standard items plasmid that kit described in the embodiment of the present invention 2 provides extracts positive plasmid, is used in combinationNanoDrop measures the concentration of positive plasmid, and it is diluted to respectively 100fg/ μ L, 10.0fg/ μ L, 1.00fg/ μ L,Six 0.10fg/ μ L, 0.01fg/ μ L, 1.00ag/ μ L concentration gradients carry out sensitivity test.
Testing result is as shown in Fig. 2, be from left to right followed successively by 100fg/ μ L, 10.0fg/ μ L, 1.00fg/ μ L, 0.10fg/ μL, the amplification of the positive criteria product of 0.01fg/ μ L, 1.00ag/ μ L, it can be seen that RAA amplified fluorescence of the invention triesThe sensitivity of agent and detection is better than regular-PCR detection method up to 0.10fg/ μ L, accuracy, shows RAA constant temperature inspection of the inventionTest agent box and detection method have the sensitivity of height to the diagnosis of American Ginseng.And identical primer is used, using traditionalPCR carries out expansion sign, and sensitivity can only detect the amount (summary of specific experiment process) of 1.00ag/ μ L.RAA of the invention has than passingHigh 10 times of the sensitivity of system method.And other second and third group sensitivity it is very low, second group of lowest detection threshold values is10.0fg/ μ L, the lowest detection threshold values of third group are 100fg/ μ L.
Test example 6: the specific test of kit of the present invention
In order to detect the specificity of kit of the present invention, using the detection method in example 3, respectively to ginseng (PanaxGinseng C.A.Mey), Radix Notoginseng (Panax notoginseng (Burk.) F.H.Chen) etc. 20 be not American Ginseng sampleIt is detected, analyzes this kit to American Ginseng (Panax quinquefolius) and your detail herbal medicine other common compositionsDetection case.
Testing result shows: only there is normal amplification, negative control (ddH in American Ginseng sample2) and ginseng (Panax OGinseng C.A.Mey), other 20 samples such as Radix Notoginseng (Panax notoginseng (Burk.) F.H.Chen) do not occurIt expands (being the amplification curve of American Ginseng and ginseng as shown in Figure 3).The above results explanation, RAA Constant Temperature Detection kit of the present inventionEnergy specific amplification goes out the target sequence in American Ginseng, without cross reaction occurs with other your detail herbal medicine nucleic acid.Illustrate this hairBright method and kit specificity are good, do not occur false negative.
Meanwhile 2-3 designed by the invention carries out same specificity experiments to primer, it is found that these primers cannot be veryGood specifically distinguishes different samples, and specificity is not fine (summary of specific experiment data).
In the case where lacking any element specifically disclosed herein, limitation, may be implemented illustrated and described hereinInvention.Used terms and expressions method is used as the term of explanation rather than limits, and is not intended in these terms and tableUp to any equivalent for excluding shown and described feature or part thereof in the use of method, and it should be realized that various remodeling existIt is all feasible in the scope of the present invention.It is therefore to be understood that although specifically being disclosed by various embodiments and optional featureThe present invention, but the modifications and variations of concept as described herein can be used by those of ordinary skill in the art, and recognizeIt is fallen into for these modifications and variations within the scope of the present invention of the appended claims restriction.
It is described herein or record article, patent, patent application and every other document and can electronically obtainThe content of information to a certain extent in full include herein by reference, just as each individual publication by specific and singleSolely point out by reference.Applicant retains from any of any this article, patent, patent application or other documentsAnd all material and information are incorporated into the right in the application.
Sequence table
<110>food of Zhejiang Province drug inspection research institute Zhejiang China, Shan Cehe knight Biotechnology Co., Ltd department of traditional Chinese medicineInstitute of Chinese materia medica, institute
<120>the recombinase-mediated amplification Constant Temperature Detection method and kit of your detail herbal medicine American Ginseng
<130> 18-100070-00006636
<141> 2018-09-11
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