CN105441561B - Detect primer, probe, standard items and the application of rutaceae allergen gene - Google Patents

Detect primer, probe, standard items and the application of rutaceae allergen gene Download PDF

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CN105441561B
CN105441561B CN201511019719.0A CN201511019719A CN105441561B CN 105441561 B CN105441561 B CN 105441561B CN 201511019719 A CN201511019719 A CN 201511019719A CN 105441561 B CN105441561 B CN 105441561B
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primer
allergen gene
citrus
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马兆成
邓秀新
吴金龙
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Huazhong Agricultural University
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Abstract

The invention discloses a kind of primer, Taqman probe, plasmid standard and their applications for detecting rutaceae allergen gene, primer therein is based on rutaceae allergen gene, including forward primer and reverse primer, the nucleotide sequence of the forward primer such as SEQ ID NO:(3 × n-2) shown in, the nucleotide sequence of the reverse primer such as SEQ ID NO:(3 × n-1) shown in, wherein the n is that any one is more than or equal to 1, the positive integer less than or equal to 3.The present invention is improved by detecting primer, Taqman probe, the nucleotide sequence of plasmid standard etc. that the absolute expression quantity of rutaceae allergen gene uses to Realtime Taqman PCR, the sensitizability that Rutaceae fruit eats part can be used to evaluate, it is significant to the genetic breeding improvement of rutaceae and food safety.

Description

Detect primer, probe, standard items and the application of rutaceae allergen gene
Technical field
The invention belongs to molecular biology of plants, human body allergology and food safety crossing domains, more specifically, relating to And a kind of primer, Taqman probe and plasmid standard for the expression of accurate quantification rutaceae allergen gene, and The application of their allergen gene expression quantitative detections in rutaceae.
Background technique
According to incompletely statistics, crowd about 4000~5,000 ten thousand people or so of the China to food hypersenstivity.The symptom of food hypersenstivity Usually local oral cavity pathological symptom, in addition to this, there are also some other symptoms, such as gastrointestinal symptoms (Nausea and vomiting, abdomen Rush down, stomach colic pain etc.), skin symptom (morbilli, dermatitis, eczema, angioneurotic edema, pruitus etc.), respiratory system disease Shape (rhinitis, asthma, swollen throat etc.) and cardiovascular system symptoms (anaphylactic shock etc.) [1].For patients, best doctor The scheme of controlling is the food allergen tried in elimination diet, or avoids the type of food hypersenstivity caused by meeting completely, but in reality Exist in the operation of border it is many difficult, so identification and to select some foods without anaphylactogen or low sensitization to autopath be one The relatively good solution of kind.
Citrus is a kind of fruit being favored by people, and is considered healthy food, nutrients rich in both at home and abroad Matter.For citrus other than pulp has important edible value, pericarp, blade, pollen all have edible value.Orange peel has Higher value added, micro- life can also be carried out using it by being not only rich in essential oil, pigment, pectin, hesperidine, dietary fiber Object fermentation, such as produces fruit vinegar, lactic acid drink, high protein feed and culturing edible fungus [2].Tangerine leaf is in Chinese medicine with the benefit that valence is high With value, have soothing the liver, promoting the circulation of qi, resolving sputum, desinsection and other effects [3].Orange Blossom pungent-warm, pleasant width diaphragm, stomach function regulating debulking is relieving cough and reducing sputum, With certain medical value, also there are health-care efficacy [4] with the citrus jasmine tea of citrus flower scenting.These Citrus procession product have There is special function, provides nutrition abundant for people.Although edible citrus has many benefits to human body, still there are many people to mandarin orange Tangerine group food allergy.Some researches show that in the eating property anaphylactogen of the DaLian, China area anaphylactia infant SPT positive, citrus Anaphylactogen positive rate 7.4% [5].In recruitment in hay fever pollen allergic patients, having 39% patient to citrous fruit Cross allergy symptom is shown, and the IgE mediated immunity reaction mechanism of all patients all tests (SPT) by skin prick Confirmation is positive [6].Existing research at present shows that Cit s 1, Cit s 2 and Cit s 3 are three kinds of main citrus anaphylactogens [7-11].Cit s 1 is the class rudiment element glycoprotein (GLPs) [11] of a kind of 24KD identified by patients serum IgE.Sweet orange suppression PROTEIN C it s 2 processed is another main generally existing anaphylactogen, is related to the process of plant various physiology and pathology [12].Cit s 3 is a kind of lipid transfer protein, is the member [9,10,13] of Panallergen family.In addition to this, Cit l 3 [14,15] are accredited as anaphylactogen in lemon and m andarin respectively with Cit r 3.Accordingly, having in detection citrus can The expression of these three allergen proteins in edible value part (such as fruit, flower, tender leaf), be evaluation its whether sensitization Index.
Anaphylactogen studies shorter mention to citrus level of allergen in the world at present, and is only directed to citrus anaphylactogen Cit S 2 develops enzyme-linked immunosorbent assay kit (enzyme linked immunosorbent assay, ELISA), can be directly For detecting navel orange (Navel orange), blood orange (Blood orange), bright Buddhist nun tangerine shaddock (Minneola orange), lemon (Lemon), the anaphylactogen Cit s of the Citrus Cultivars such as grape fruit (Grapefruit), satsuma orange (Satsuma mandarin) 2 contents, while finding that 2 content difference of Cit s is larger [16] in the citrus fruit of different cultivars.But still without detect remaining The method of two 3 protein levels of anaphylactogen Cit s1 and Cit s is unable to the sensitizability of integral level evaluation citrus fruit. There is research based on proteomics detection of platform allergen protein, is confined to mass spectrographic sensitivity, is only able to detect part lemon The presence [17] of middle allergen protein.In addition to this, there are also researchs to pass through sxemiquantitative inverse transcription polymerase chain reaction and Real- Time PCR detects allergen gene expression, and discovery Ke Liman fourth tangerine anaphylactogen Cit s 1 and Cit s 3 represents 2 kinds and leads to The anaphylactogen often expressed in fruit and pollen, and the anaphylactogen that Cit s 2 is represented only expresses [6] in citrus pulp.However, The method of relative quantification used by this is studied can not accurate detection go out the expression of citrus allergen gene.Therefore, It is capable of the method for entirety, quantification evaluation citrus sensitization at present.
Bibliography:
[1] the anaphylaxis problem Chinese food of the certain foods of Bai Qingyun and nutrition, 2005,7:54-55.
[2] the comprehensive utilization Zhejiang Agriculture science of Qiao Haiou, Ding Xiaowen, Zhang Qingzhu orange peel, 2003,2 (03): 31- 35.
[3] Cui Guojing, Liu Fang, He Qiang tangerine leaf, tangerine pith, the character of tangerine seed and the capital medical value medicine, 2013,05.
[4] Li Yun citrus scented tea baking technical research modern agriculture science and technology, 2013,24,278-278.
[5] Dalian anaphylactia infant skin prick test interpretation of result [D] the medical courses in general of the Dalian Area Qin Peng 121 are big Learn .2012
[6]Rosa Anna Iorio,Stefano Del Duca,et al.Citrus Allergy from Pollen to Clinical Symptoms.PLOS ONE,2013,8(1):e53680.
[7]M.D.J.Sastre,M.M.San Ireneo,M.T.Laso,D.Barber, M.Lombardero.Different patterns of allergen recognition in children allergic to orange.Journal of Allergy and Clinical Immunology,2004(113):175-177.
[8]O.Ahrazem,M.D.G.López-Torrejón,R.Sánchez-Monge,J.Sastre, Lombardero,D.Barber,G.Salcedo.Orange germin-like glycoprotein Cit s1:an equivocal allergen.International archives of allergy and immunology,2006 (139):96-103.
[9]G.López‐Torrejón,M.Ibanez,O.Ahrazem,R.Sánchez‐Monge,J.Sastre, M.Lombardero,D.Barber,G.Salcedo.Isolation,cloning and allergenic reactivity of natural profilin Cit s 2,a major orange allergen.Allergy,2005(60):1424- 1429.
[10]J.F.Crespo,M.Retzek,K.Foetisch,E.Sierra-Maestro,A.B.Cid-Sanchez, C.Y.Pascual,A.Conti,A.Feliu,J.Rodriguez,S.Vieths,S.Scheurer.Germin-like protein Cit s 1and profilin Cit s 2are major allergens in orange(Citrus sinensis)fruits.Molecular Nutrition&Food Research,2006(50):282-290.
[11]G.Poltl,O.Ahrazem,K.Paschinger,M.D.Ibanez,G.Salcedo, I.B.H.Wilson.Molecular and immunological characterization of the glycosylated orange allergen Cit s 1.Glycobiology,2006(17):220-230.
[12]B.Haarer,S.S.Brown.Structure and function of profilin.Cell motility and the cytoskeleton,1990(17):71-74.
[13]R.Sánchez-Monge,M.Lombardero,F.J.García-Sellés,D.Barber, G.Salcedo.Lipid-transfer proteins are relevant allergens in fruit allergy.Journal of Allergy and Clinical Immunology,1999(103):514-519.
[14]D.Ebo,O.Ahrazem,G.Lopez-Torrejon,C.Bridts,G.Salcedo, W.Stevens.Anaphylaxis from mandarin(Citrus reticulata):identification of potential responsible allergens.International archives of allergy and immunology,2007(144):39-43.
[15]O.Ahrazem,M.D.G.López-Torrejón,R.Sánchez-Monge,J.Sastre, M.Lombardero,D.Barber,G.Salcedo.Lipid transfer proteins and allergy to oranges.International archives of allergy and immunology,2005(137):201-210.
[16]Kiyota K,Kawatsu K,Sakata J,et al.Development of sandwich ELISA for quantification of the orange allergen profilin(Cit s 2).Food and Agricultural Immunology,2016,27(1):128-137.
[17]I.A.Serra,L.Bernardo,A.Spadafora,P.Faccioli,C.Canton, S.Mazzuca.The Citrus clementina Putative Allergens:From Proteomic Analysis to Structural Features.Journal of Agricultural and Food Chemistry,2013(61):8949- 8958.
Summary of the invention
Aiming at the above defects or improvement requirements of the prior art, the purpose of the present invention is to provide a kind of plants of detection Rutaceae Primer, Taqman probe, plasmid standard and their application of object allergen gene, wherein by Realtime Primer, the Taqman probe, plasmid standard that the Taqman PCR detection absolute expression quantity of rutaceae allergen gene uses Nucleotide sequence etc. improve, compared with prior art being capable of sensitive, Absolute quantification all citrus anaphylactogens of detection The expression of gene can be used to evaluate rutaceae fruit sensitizability, to the improvement of the genetic breeding of rutaceae and Food safety is significant.
To achieve the above object, according to one aspect of the present invention, a kind of detection rutaceae allergy former base is provided The primer of cause, which is characterized in that the primer is based on rutaceae allergen gene, including forward primer and reverse primer, institute The nucleotide sequence for stating forward primer preferably has such as SEQ ID NO:(3 × n-2) shown in nucleotide sequence, it is described reversely to draw The nucleotide sequence of object preferably has such as SEQ ID NO:(3 × n-1) shown in nucleotide sequence, wherein the n be it is any one A positive integer for being more than or equal to 1 and being less than or equal to 3.
It is another aspect of this invention to provide that a kind of Taqman probe for detecting rutaceae allergen gene is provided, It is characterized in that, the nucleotide sequence of the Taqman probe have such as SEQ ID NO:(3 × n) shown in nucleotide sequence, Described in n be any one be more than or equal to 1 and be less than or equal to 3 positive integer.
Another aspect according to the invention provides a kind of plasmid standard for detecting rutaceae allergen gene, It is characterized in that, the standard items are used for quantitative PCR detection rutaceae allergen gene expression quantity absolute quantitation, the standard items Nucleotide sequence have the nucleotide sequence as shown in SEQ ID NO:10~SEQ ID NO:12 any one.
Another aspect according to the invention provides above-mentioned primer, Taqman probe or plasmid standard in detection rue Application in section's plant hypersensitive protogene.
Contemplated above technical scheme through the invention provides compared with prior art and can be used for absolute quantitation rue The method of savoury herb allergen gene, filled up detection citrus allergen gene expression blank, designed primer and Taqman probe has the characteristics that high specificity, high sensitivity, quantification, efficient, stable, energy accurate quantitative analysis sweet orange allergy former base Because of expression.People can use this method and carry out allergen gene detection of expression to the Citrus Cultivars of rutaceae, to comment The sensitization of valence difference Citrus Cultivars, to breeding low irritability Citrus Cultivars, special consumer selects edible low irritability citrus Kind and raising food safety have great importance.
Taqman probe has sequence-specific, is bonded only to complementary region, and fluorescence signal and the copy number of amplification have There is one-to-one relationship.The present invention is based on citrus genome, all genes related to citrus allergy are excavated, and using special Property strong, high sensitivity, quantification, efficient, stable Taqman probe technique absolute quantitation detection is carried out to these genes, not only Be conducive to explore expression rule of the citrus anaphylactogen on citrus different tissues, and then breeding low irritability Citrus Cultivars, it can also With for evaluate different citruses whether sensitization, be conducive to special consumer and select edible low irritability Citrus Cultivars, eaten to improving Product have great importance safely.
Primer, Taqman probe, the plasmid standard that rutaceae allergen gene is detected in the present invention provide use In the detection method of rutaceae anaphylactogen absolute quantitation, it can be used for evaluating the sensitization of rutaceae (such as citrus).Base It, can in the method for detecting citrus allergen gene expression quantity of the exploitations such as above-mentioned primer, Taqman probe, plasmid standard With system, comprehensive, quantization detection citrus allergen gene expression quantity, and then evaluate citrus sensitization or potential sensitization Property.
Detailed description of the invention
Fig. 1 is the primer for developing allergen gene absolute quantitation and Taqman probe and its techniqueflow of application of the invention Figure；
Fig. 2 is agarose gel electrophoresis figure；Primer is used such as general primer in table 3；
Fig. 3 is that Realtime Taqman PCR standard curve is established；The slope of standard curve is -3.10~-3.58, is expanded Increasing Efficiency 90%~110% has good correlation (r between Ct value and the logarithm of plasmid copy number2>0.9)；
Fig. 4 is that sweet orange respectively organizes allergen gene expression quantity to detect；It is detected in sweet orange blade, pollen, pericarp, pulp There is higher expression to Cit s 1.01, Cit s 2.01,3.01 gene of Cit s.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and It is not used in the restriction present invention.As long as in addition, technical characteristic involved in the various embodiments of the present invention described below Not constituting a conflict with each other can be combined with each other.
Embodiment 1: the information retrieval of sweet orange allergen gene and its homologous gene excavate
The retrieval of 1.1 allergen proteins
International Union of Immunological Societies subordinate anaphylactogen name special commission website (http: // Www.allergen.org/ known allergens in Citrus) be can get, as shown in table 1.
1 citrus allergens of table
1.2 excavate allergen gene based on sweet orange genome
Pass through the middle allergen protein Cit s 1.01 (GenBank Protein P84159), Cit published an article The amino acid sequence of s2.01 (GenBank Protein CAI23765) and Cit s 3.01 (GenBank Protein P84161) Column search for corresponding mistake in sweet orange genome (http://citrus.hzau.edu.cn/orange/) by sequence homology Quick protogene simultaneously obtains gene order, as shown in table 2.
2 citrus allergen gene information of table
2 citrus allergen gene of embodiment quantifies primer and the design of Taqman probe
It is compared according to homologous sequence, searches the region of sequence specific, primer and probe designs in this region, to guarantee it Absolute quantitation can be carried out to these three genes.Primer and probe in the present invention uses 5.0 software design of Primer, primer and The nucleotide sequence of probe is as shown in table 3 or sequence table SEQ ID NO:1~SEQ ID NO:9.
3 primer of table and Taqman probe
Note: the F in table is forward primer, and R is reverse primer, and P is probe.
Embodiment 3Realtime Taqman PCR standard items template prepares and the foundation of standard curve
3.1Realtime Taqman PCR standard items template prepares
3.1.1 it is subcloned to PMD18-T carrier
It usesAfter PCR System9700 carries out standard PCR amplification, by PCR product with 1.5% TAE (Fig. 2) is detected in ultraviolet imager after agarose gel electrophoresis.PCR system such as table 4, program such as table 5, PCR Master Mix (2X) is purchased from Thermo Fisher Scientific (article No.: K0171).Template cDNA comes from embodiment 4.1.Primer is using such as Shown in table 3.
4 PCR system of table
5 PCR reaction condition of table
By PCR product to cut corresponding adhesive tape on ultraviolet imager after 1.5% TAE agarose gel electrophoresis.It usesThe E.Z.N.A.TMGel Extraction kit kit recycles PCR product.Operation specifically turn one's head such as Under: (1) gel that cuts is put into 1.5ml centrifuge tube in, 65 DEG C of water-baths are to gel after 600 μ l Binding Buffer are added It is completely dissolved.When dissolution, centrifuge tube is often rocked；(2) by HiBind DNA column sleeve on 2ml centrifuge tube；(3) by DNA and solidifying Glue mixed liquor is transferred in HiBind DNA column, and 1000 × g is centrifuged 1min, abandons filtrate；(4) HiBind DNA column is put back into 2ml Centrifuge tube is added 500 μ l SPW Wash Buffer, 1000 × g and is centrifuged 1min, abandons filtrate；In the same way again with 500 μ l SPW Wash Buffer washed once, and abandon filtrate；(5) HiBind DNA column is put back into 2ml centrifuge tube, 13000 × g centrifugation It uncaps after 2min and stands 5min；(6) HiBind DNA column is placed in new 1.5ml centrifuge tube, 20 μ l ddH20,37 DEG C of trainings are added It supports and stands 5min in case；(7) 13000 × g obtain product after being centrifuged 2min, and Nanodrop1000 is used to measure concentration.
Recovery product is connect with pMD18-T carrier, such as 6,16 DEG C of table of specific linked system overnight.pMD18-T Vector Kit is purchased from precious bioengineering (Dalian) Co., Ltd (article No.: 6011).
6 linked system of table
Connection product is converted into Escherichia coli.Concrete operations are as follows: (1) by competence (TransGenDH5 It α) is placed on ice, stands 20-30min；(2) 5-10 μ l connection product is added in super-clean bench into 50 μ l competence, gently inhales and beats It mixes, stands 20-30min；(3) 2min is stood on ice after centrifuge tube being placed in 42 DEG C of water-bath 90s；(4) to centrifugation in super-clean bench 600 μ l liquid LB are added in pipe and are placed on 37 DEG C of shaking table 45-80min；(5) after centrifuge tube being removed from shaking table, 5000rpm centrifugation 2min abandons supernatant, retains 200 μ l liquid LB；(6) after mixing is played in bacterium and liquid LB suction in centrifuge tube, it is applied to the Amp's containing antibiotic On LB plate；(7) next day observes clonal growth situation, and by bacterium colony PCR, (the raw work bioengineering in Shanghai is limited with sequence verification Company).PCR system and program such as table 4 and table 5, the primer such as table 3.
Through analyzing sequencing result, 3 recombinant plasmids, respectively pMD18-T-Cit s1.01, pMD18-T- are successfully constructed Cit s2.01、pMD18-T-Cit s3.01。
WithAxyPrep Plasmid DNA small volume of reagent box extracts recombinant plasmid.Concrete operations are as follows: (1) taking The bacterium solution of 4ml overnight incubation in LB culture medium, 12000 × g are centrifuged 1min, abandon supernatant to the greatest extent；(2) add 250 μ l Buffer S1 Suspended bacterial precipitating, suspending needs uniformly, should not there are small fungus blocks；(3) plus 250 μ l Buffer S2, mild and fully on It is lower overturning 6 times be uniformly mixed crack thallus sufficiently, this step no more than 5min；(4) add 350 μ l Buffer S3, mildly simultaneously Mixing 8 times is fully spun upside down, 12000 × g is centrifuged 10min；(5) the centrifugation supernatant in aspiration step 4 and it is transferred to preparation It manages (being placed in 2ml centrifuge tube), 12000 × g is centrifuged 1min, abandons filtrate；(6) pipe will be prepared and puts back into centrifuge tube, add 500 μ l Buffer W1,12000 × g are centrifuged 1min, abandon filtrate；(7) pipe will be prepared and puts back into centrifuge tube, add 700 μ l Buffer W2, 12000 × g is centrifuged 1min, abandons filtrate；It washed once again with 700 μ l Buffer W2 in the same way, abandon filtrate；(8) will It prepares pipe to put back into 2ml centrifuge tube, uncaps after 12000 × g centrifugation 2min and stand 5min；(9) pipe will be prepared and moves into new 1.5ml In centrifuge tube, adds 60 μ l, 65 DEG C of deionized waters preparing periosteum center, be stored at room temperature 1min.12000 × g is centrifuged 1min.It uses Nanodrop 1000 measures recombinant plasmid concentration (table 7), is stored in -20 DEG C of refrigerators.
7 standard concentration of table
The foundation of 3.2Realtime Taqman PCR standard curve
Realtime Taqman is carried out using constructed 10 times of serial dilutions of plasmid standard as standard items template PCR。Premix Ex TaqTM(Probe qPCR) is purchased from precious bioengineering (Dalian) Co., Ltd (article No.: RR390A).According to The system of table 8 and the program of table 9 carry out Realtime Taqman PCR reaction.It, can be 0.1~1.0 when reactivity worth is poor Primer concentration, optimization reaction are adjusted within the scope of μM；In 20 μ l reaction systems, the additive amount of standard items template is usually in 100ng Hereinafter, gradient dilution can be carried out when necessary, optimal standard items template additive amount is determined.
8 Realtime Taqman PCR system of table
9 Realtime Taqman PCR program of table
Using the logarithm of standard items template various concentration as abscissa, recurring number is ordinate mapping, obtains standard curve (Fig. 3).The results show that the slope of standard curve is -3.10~-3.58, and amplification efficiency 90%~110%, CtValue and plasmid are copied (r is had good correlation between the logarithm of shellfish number2>0.9).Therefore according to the C of unknown sampletValue and regression equation To the copy number accurate quantification of starting template.Plasmid pMD18-T Vector length is 2692bp.Use origin9.0 software point It analyses data and draws Fig. 3.
Specific calculation formula is:
Amplification efficiency calculation formula: E=10(-1/slope)-1
Its validity and application are verified in the absolute expression quantity detection of 4 sweet orange pulp allergen gene of embodiment
4.1 preparation of samples
4.1.1 sample acquires
Picking in 330 days is in Central China agricultural after red summer orange (Citrus sinensis [L.] Osbeck) mellow fruit is about spent The national citrus breeding center of university, sample are divided into pericarp, pulp, after liquid nitrogen frozen, are placed in -80 DEG C of refrigerators storages.Blade takes From young leaves on the spring tip.Flower is derived from florescence, same liquid nitrogen frozen processing.
4.1.2 RNA is extracted using TRIZOL method
Reagent is purchased from Invitrogen, and article No. is 15596-026.RNA extraction is grasped to specifications Make.Specific steps are as follows: (1) takes appropriate amount of sample to be directly placed into mortar liquid feeding nitrogen, grind rapidly, by 50-100mg sample/ Trizol is added in ml Trizol, is placed at room temperature for 5min, cracks it sufficiently；(2) .12,000rpm are centrifuged 5min, abandon precipitating；⑶. Chloroform is added by 200ul chloroform/ml Trizol, oscillation is placed at room temperature for 15min after mixing；(4) .4 DEG C of 12,000g is centrifuged 15min； (5) draws upper strata aqueous phase, until in another centrifuge tube；(6) is added isopropanol by 0.5ml isopropanol/ml Trizol and mixes, room temperature Place 5-10min；(7) .4 DEG C 12,000g are centrifuged 10min, abandon supernatant, and RNA is sunken to tube bottom；(8) presses 75% ethyl alcohol of 1ml/ml 75% ethyl alcohol is added in Trizol, mildly vibrates centrifuge tube, and suspend precipitating；(9) .4 DEG C 8,000g are centrifuged 5min, as far as possible abandoning supernatant； (10) 5-10min is dried or be dried in vacuo to room temperature；Can use 30ul sterilizing DEPC water dissolve RNA sample, 55-60 DEG C, 5- 10min；(12) .NanoDrop1000 measures total rna concentration, and RNA is saved in -80 DEG C.
4.1.3 reverse transcription synthesizes cDNA
Above-mentioned total serum IgE is handled 30 minutes with DNaseI (Invitrogen, USA) to remove contaminating genomic DNA.Then By RNA sample according to RevertAidTM First Strand cDNA Synthesis Kit kit (Fermentas, Lithuania operating method reverse transcription) synthesizes the first chain cDNA: taking 1-1.5 μ g total serum IgE as reverse transcription template (20 μ l bodies System), the dilution of 50 μ l distilled waters is added after the completion of reverse transcription, NanoDrop1000 (Thermo Scientific, USA) measurement is total It is spare to be stored in -20 DEG C of refrigerators for cDNA concentration.Sample cDNA concentration is shown in Table 10.
The cDNA concentration of 10 sweet orange pericarp of table, pulp, blade, flower
The absolute expression quantity verifying Realtime Taqman PCR of allergen gene quantifies allergy in 4.2 detection sweet orange pulp The validity of original method
Using sweet orange pulp cDNA as template, according to 8 configuration scheme of table, program is run according to table 9, carries out Realtime Taqman PCR.After collecting data, according to the Ct value and regression equation for establishing standard curve, to each allergy in sweet orange pulp cDNA Protogene copy number carries out accurate quantification.
As a result such as Fig. 4, method proposed by the present invention quantification can detect Cit s 1.01, Cit s in sweet orange pulp 2.01, the absolute expression quantity of 3.01 gene of Cit s finds Cit s 1.01 and 3.01 gene of Cit s in pericarp than in pulp Height, 2.01 gene of Cit s are higher than in pericarp in pulp.According to the SCI article delivered it is found that Cit s in sweet orange pulp 1.01, Cit s 2.01, Cit s 3.01 these three allergen proteins all have been found and identify.Therefore, the present invention can be special Anisotropic ground, delicately, quantification, steadily detect Cit s 1.01, Cit s 2.01,3.01 base of Cit s in sweet orange fruit Because of expression.Accordingly, method provided by the invention is a kind of reliably method for detecting rutaceae allergen gene, tool There is important application value.
Above-described embodiment is based on identified allergen gene Information Authentication sweet orange pulp allergen gene and absolutely expresses Validity is measured, and sweet orange pericarp, blade, the flower absolute expression quantity of allergen gene are detected, evaluates these three edible portions The sensitization divided.
To sum up, the method provided by the present invention for absolute quantitation sweet orange allergen gene, has filled up detection citrus mistake The blank of quick antigen gene expressed.The present invention not only retrieved allergen gene in sweet orange genome, and clone the piece of these genes Primer and Taqman probe, energy precise Identification sweet orange allergen gene, while the present invention has also been devised for constructing standard items in section Establish the standard curve for Absolute quantification, and based on this standard curve to four sweet oranges eat part allergen gene into Row absolute quantitation.The method for sweet orange absolute quantitation that the present invention develops, using this method to the citrus of rutaceae Kind carries out the detection of allergen gene expression, so that the sensitization of different Citrus Cultivars and its different edible parts is evaluated, To breeding low irritability Citrus Cultivars, special consumer, which selects edible low irritability Citrus Cultivars and improves food safety, to be had Important meaning.
Plasmid standard in the present invention can be used for establishing standard curve, be based on the standard curve as amplification template, can Absolute quantitation is carried out to the allergen gene of a variety of edible tissues of rutaceae.The sequence of plasmid standard such as SEQ ID It is to be led by the way that the coding domain segment that quantitative primer is expanded will be used for by TA clone shown in No.10~SEQ ID No.12 Enter in PMD18-T carrier；The code area (coding sequence, CDS) expanded has such as SEQ ID No.10~SEQ ID Nucleotide sequence shown in No.12, respectively corresponds Cit s 1.01, Cit s 2.01, Cit s 3.01 totally 3 genes.
Rutaceae in the present invention both includes citrus plant (such as tangerine, mandarin orange, shaddock, orange, lemon), also including it His common citrus (such as trifoliate orange, kumquat).The primer of rutaceae allergen gene is detected in the present invention, Taqman is visited Needle, plasmid standard are particularly suitable for all sweet orange kinds, and other Citrus Cultivars of part.Those skilled in the art is easy Understand, the foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within principle.

Claims (4)

1. a kind of primer for Realtime Taqman PCR detection rutaceae allergen gene, which is characterized in that should Primer is based on rutaceae allergen gene, including forward primer and reverse primer, the nucleotide sequence of the forward primer With such as SEQ ID NO:[(3 × n) -2] shown in nucleotide sequence, the nucleotide sequence of the reverse primer has such as SEQ ID NO:[(3 × n) -1] shown in nucleotide sequence, wherein the n is that any one is more than or equal to 1 and is less than or equal to 3 just Integer.
2. a kind of Taqman probe for Realtime Taqman PCR detection rutaceae allergen gene, feature Be, the nucleotide sequence of the Taqman probe has such as SEQ ID NO:(3 × n) shown in nucleotide sequence, wherein the n It is more than or equal to 1 and the positive integer less than or equal to 3 for any one.
3. a kind of plasmid standard for Realtime Taqman PCR detection rutaceae allergen gene, feature It is, which is used for quantitative PCR detection rutaceae allergen gene expression quantity absolute quantitation, the nucleosides of the standard items Sequence set just like SEQ ID NO:10~SEQ ID NO:12 any one shown in nucleotide sequence.
4. plasmid standard described in Taqman probe or claim 3 described in primer as described in claim 1, claim 2 exists Realtime Taqman PCR detects the application in rutaceae allergen gene.
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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Citrus Allergy from Pollen to Clinical Symptoms;Rosa Anna Iorio et al;《PLOS ONE》;20130130;第8卷(第1期);e53680
germin-like protein cits 1 and profilin cits 2 are major allergens in orange(Citrus sinensis) fruits;JF Crespo et al;《Molecular Nutrition and food research》;20061231;第117卷(第2期);第282-290页
The Citrus clementina Putative Allergens: From Proteomic Analysis to Structural Features;Ilia Anna Serra et al;《Journal of agricultural and food chemistry》;20131231;第8949-4958页

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