CN105969844A - Primer for detecting papaver somniferum l. based on LAMP technology, kit, and detecting method - Google Patents
Primer for detecting papaver somniferum l. based on LAMP technology, kit, and detecting method Download PDFInfo
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Abstract
The invention discloses a primer for detecting papaver somniferum l. based on an LAMP technology, a kit, and a detecting method. The primer for detecting papaver somniferum l. based on the LAMP technology is characterized by comprising a forward outer primer F3:5'-TCGATGAAAAATTCCCTGAAC-3', and a reverse outer primer B3:5'-CACGTTTAGGGGTACTGAT-3', a forward inner primer FIP and a reverse inner primer BIP. The invention aims to solve the shortcomings of the prior art to provide the primer for detecting papaver somniferum l. based on the LAMP technology, wherein the primer is rapid, simple and convenient, efficient, high in accuracy, high in specificity and high in sensitivity; the invention also aims to provide a kit including the primer for detecting papaver somniferum l. based on the LAMP technology; and in addition, the invention also aims to provide a method for detecting papaver somniferum l. by adopting the kit.
Description
Technical field
The present invention relates to a kind of for detecting the primer of Semen Papaveris, test kit and detection method, belong to biological technical field.
Background technology
Semen Papaveris formal name used at school: Papaver somniferum L., bloodroot, is the primary raw material producing Opium, immaturity
Fruit serosity Han milky, is Opium after system is dry, and its extract is also multiple ataractic source, as morphine, codeine and
The multiple alkaloid such as papaverine, wherein morphine is its main component, and processing is used as medicine, astringe the lung, astringing intestine to stop diarrhea, cough-relieving, pain relieving and hypnosis
Etc. effect, control chronic cough, chronic diarrhea, chronic dysentery, proctoptosis, all pains of trusted subordinate's muscles and bones；The meaning of formal name used at school " somniferum " is " hypnosis ", reflection
Go out it and there is narcoticness.Poppyseed Semen Papaveris is important food product, wherein contains healthy and helpful oils and fats, extensively
It is applied in salad all over the world, and poppy flower is splendid magnificent, is a kind of of great value ornamental plant.Semen Papaveris shell is micro-
Cold, sour-puckery flavor, slightly poisonous, containing alkaloids such as low amounts morphines.Another containing polysaccharide about 2.4%, hydrolysis can obtain lactose 10%, arabinose
6%, xylose 6%, rhamnose 4%, lactobionic acid Galacturonic acid 60%, 4-0-methyl glucose alduronic acid-0-
Methylglucuronic acid 4%, trace fucose Fucose, 2-0-methyl fucose 2-0-Methylfucose, 2-
0-methyl xylose 2-0-Methylxylose and glucuronic acid G-tucuronic acid, sedoheptose Sedoheptulose,
D-mannoheptulose D-Mannoheptulose, D-glyceryl-D-manna octulose D-Glycero-D-Mannooctulose,
Meso inositol Mesoinositol, erithritol Erythritol, have astringe the lung, astringing intestine to stop diarrhea, the effect of pain relieving, for chronic cough, chronic diarrhea,
Proctoptosis, epigastric pain, but the easy addiction of these product, unsuitable informal dress.At present some illegal retailers using Pericarpium Papaveris as food additive
Join in the food such as bottom material of chafing dish, halogen soup, barbecue flavoring, after making people edible, produce pleasant sensation, and gradually produce dependency with
Reach to solicit the purpose of frequent customer.If people is eaten for a long time the food containing Semen Papaveris composition, arise that excitement, dysphoria,
Perspire abnormal sweat, the chronic poisoning symptom such as malaise is felt sleepy, emaciation and sallow complexion, also can make that people is drowsiness, personality change, hypomnesis,
It is eaten for a long time and also results in Mental Subnormality, hallucination occurs；Be also easy to be vehicle driver feel sleepy, absent minded and cause friendship
Interpreter's event；With the addition of the food of Pericarpium Papaveris and be addicting owing to being very easy to, even go on road of drug abuse crime etc., this
The health of infringement is badly damaged consumer, had become the serious hidden danger of food safety already.China already forbids in plain text
Adding Semen Papaveris composition in food, Pericarpium Papaveris is listed in " administration of narcotic drug way " register by State Council, by Semen Papaveris
Shell is managed according to narcotic.Therefore, setting up one detection method quicker, sensitive, convenient is to ensure people's health
A healthy important means, it is that the technology backing of administrative law enforcement, new detection technique and method have become as one and compel to be essential
The political mission wanted is very urgent.
At present China goes back, for the detection of Semen Papaveris, the standard that neither one is unified, the method for predominantly detecting have chemical analysis,
Thin layer chromatography, immunoassay, gas chromatography mass spectrometry method and liquid chromatography etc..Chemical analysis is according to contained by Pericarpium Papaveris
Alkaloid and particular agent reaction solution make qualitative detection, simple rapidly but sensitivity is low；Thin layer chromatography is also according to small-mouthed jar
Alkaloid contained by foxtail millet shell detects, and can detect multiple alkaloid simultaneously, but detection sensitivity is the lowest, poor specificity, and
Easily pollute environment；Immunoassay is that the specific antibody utilizing the alkaloid of Semen Papaveris to prepare is to detect the existence of alkaloid antigen
Whether, detection sensitivity is up to 0.1-20ug/kg, and the method ratio is faster, flux is higher, sensitivity is the strongest, can conduct
Daily a large amount of sample sifter inspection survey is used, but the method can not accurate quantitative analysis, and antibody preparation more difficult again be difficult to preserve, repeat
Property the highest, false positive rate is high.Gas chromatography GC, GC-MS GC-MS, high performance liquid chromatography HPLC, liquid phase color
Spectrum-tandem mass spectrometry LC-MS: M&C etc. can be detected, although detection sensitivity is higher, but it is required for setting of costliness
Standby, environmental requirement is high, detection cycle length, testing cost is expensive, also need to the technical staff that professional training is crossed, be not suitable for basic unit and
Site Detection.Additionally said method is all indirectly to detect alkaloid, it is impossible to carry out Semen Papaveris source of species accurately
Qualitative.
The illegal phenomenon adding Pericarpium Papaveris in bottom material of chafing dish and snack products etc. has caused people to food at present
Paying much attention to and the extensive concern of the whole society of product safety problem, in order to provide law enforcement to depend on to the most accurately supervision law enforcement
According to, in the urgent need to can fast and accurate convenient again and applicable basic unit common lab Semen Papaveris source of species be carried out the most qualitative
Detection method.
Summary of the invention
The invention aims to overcome weak point of the prior art, it is provided that a kind of quick, easy, efficient, accurate
Really property height, high specificity, susceptiveness height, primer based on LAMP technology detection Semen Papaveris；
Another object of the present invention is to provide a kind of test kit for detecting Semen Papaveris comprising above-mentioned primer；
Another object of the present invention is to provide a kind of method using mentioned reagent box detection detection Semen Papaveris.
In order to achieve the above object, the present invention uses below scheme:
A kind of primer based on LAMP technology detection Semen Papaveris, it is characterised in that including:
Forward outer primer F3:5'-TCGATGAAAAATTCCCTGAAC-3'；
Reversely outer primer B3:5'-CACGTTTAGGGGTACTGAT-3'；
Forward inner primer FIP and reverse inner primer BIP；
Wherein said forward inner primer FIP comprises F1c and F2 sequence:
5'-CCTGATAAGAAAGCCATGGATTCA-TGGGGTTAGTAGATAAAGAGTT-3'；
Wherein F1c:5'-CCTGATAAGAAAGCCATGGATTCA-3'；
F2:5'-TGGGGTTAGTAGATAAAGAGTT-3'；
Described reverse inner primer BIP includes containing B1c and B2 sequence:
5'-TACCATCTCTGAACTAAACAACAGG-TCAACTTTAGTCTTAAAAGCTCT-3'；
Wherein B1c:5'-TACCATCTCTGAACTAAACAACAGG-3'；
B2:5'-TCAACTTTAGTCTTAAAAGCTCT-3'。
A kind of test kit based on LAMP technology detection Semen Papaveris, it is characterised in that include:
Described primer includes:
Forward outer primer F3:5'-TCGATGAAAAATTCCCTGAAC-3'；
Reversely outer primer B3:5'-CACGTTTAGGGGTACTGAT-3'；
Forward inner primer FIP and reverse inner primer BIP；
Wherein said forward inner primer FIP comprises F1c and F2 sequence:
5'-CCTGATAAGAAAGCCATGGATTCA-TGGGGTTAGTAGATAAAGAGTT-3'；
Wherein F1c:5'-CCTGATAAGAAAGCCATGGATTCA-3'；
F2:5'-TGGGGTTAGTAGATAAAGAGTT-3'；
Described reverse inner primer BIP includes containing B1c and B2 sequence:
5'-TACCATCTCTGAACTAAACAACAGG-TCAACTTTAGTCTTAAAAGCTCT-3'；
Wherein B1c:5'-TACCATCTCTGAACTAAACAACAGG-3'；
B2:5'-TCAACTTTAGTCTTAAAAGCTCT-3'；
The wherein product specification of this test kit: 50 times.
Test kit as above, it is characterised in that described reactant liquor buffer 2 × composed of the following components:
Test kit as above, it is characterised in that described enzymatic solution is Bst archaeal dna polymerase and AMV reverse transcriptase mixes
Close liquid.
Any one test kit as above, it is characterised in that containing calcein in described 2 × reactant liquor buffer
Fluorescent dye.
A kind of method based on LAMP technology detection Semen Papaveris, it is characterised in that comprise the following steps:
A, employing nanometer magnetic bead extract the DNA of testing sample；
B, use test kit as claimed in claim 5, on ice, extract reaction solution buffer, primer, enzyme in proportion molten
Liquid, positive control, deionized water put into preparation premixed liquid in sterile tube；
C, the DNA of appropriate testing sample is added in the sterile tube in step B；
D, the test tube in step C is placed in AB7500 quantitative real time PCR Instrument, at 63 DEG C, keeps constant temperature 1 hour；
If E measuring samples occurs typical S amplification curve as positive control, be then judged to the positive, if with the moon
Property comparison equally there is no amplification curve, and be judged to feminine gender.
Method as above, it is characterised in that use nanometer magnetic bead to extract the concrete step of testing sample DNA in step A
Rapid:
A, sample preparation: the plant tissue addition 1mL extraction buffer taking 0.1g is ground；
B, cleaning nanometer magnetic bead: in taking-up immune nanometer magnetic bead to PCR pipe, use ddH2O cleans nanometer magnetic bead 3 times repeatedly,
DdH is sucked under the effect of Magnet2O；
C, combination: add the sample of 100 μ L in PCR pipe, with liquid-transfering gun compressing mixing sample and nanometer magnetic bead, under room temperature
Absorption；
D, cleaning: remove supernatant under the effect of Magnet, nanometer magnetic bead cleans 3 times；
E, cracking: add 50 μ L ddH2O is in PCR pipe, after liquid-transfering gun compressing mixing nanometer magnetic bead, and 95 DEG C, 10min；
F samples: use magnet adsorption nanometer magnetic bead, and after solution clarification in PCR pipe, supernatant is testing sample DNA.
Method as above, it is characterised in that described immune nanometer magnetic bead is made by following technique:
1. the preparation of nanometer magnetic bead
Take 5.2g FeCl respectively3·6H2O、2.7g FeSO4·7H2The concentrated hydrochloric acid of O, 0.85mL 12.1moL/L is dissolved in
In 200mL water, ultrasonic deoxidation, after by above solution instill 250mL, 0.75moL/L NaOH solution；Stir at 80 DEG C, reaction
After end, utilize magnetic separator gained precipitation to be separated from reaction medium, then clean 3 times with deionized water, anhydrous second
Alcohol cleans 2 times, and is made into the Fe of 5g/mL3O4Magnetic nano-particle ethanol solution；Take 25mL Fe3O4Magnetic nano-particle
Ethanol solution, then it is diluted to 150mL, sonic oscillation 30min with dehydrated alcohol；Dropping 0.4mL APTES, stirs under room temperature
7h；React complete, Fe APTES modified with magnetic separator3O4Magnetic nano-particle separates from reaction medium, and with anhydrous
Ethanol solution cleans 3 times, is finally made into amination Fe of 10.5mg/mL3O4Nanometer magnetic bead ethanol solution；
2. the preparation of immunomagnetic beads
Take 0.5mL amination Fe3O4Nanometer magnetic bead ethanol solution, with 1mL PBS 3 times, supernatant abandoned by magnetic separator
Liquid；Add 5% (V/V) glutaraldehyde solution 2mL, shaken at room temperature crosslinking 2h, Magneto separate, abandon supernatant；Wash 3 times with PBS and abandon
After supernatant, it is respectively adopted 0.5mL antiviral antibody and is coated amination Fe3O4Nanoparticle, the fixing 2h of vibration under room temperature；Magneto separate,
Washing respectively 3 times with PBS and ultra-pure water, then use PBS constant volume, 4 DEG C of Refrigerator stores are standby, obtain immunomagnetic beads.
Some materials used in the present invention:
1.1 plants:
Flos Papaveris rhoeadis Papaver rhoeas and poppy seeds are purchased from pharmaceuticals.
1.2 main agents and consumptive material
(1) PCR reaction reagent: 10 × PCR buffer, dNTPs, MgCl2, Taq archaeal dna polymerase, precious biological engineering is (big
Even) company limited；
(2) DNA Marker DL1000,6 × loading buffer, precious biological engineering (Dalian) company limited；
(3) LAMP reaction reagent；
(4) Bst DNA polymerase large fragment, New England Biolabs company；
(5) glycine betaine (Betaine), the good biological company limited of Guangzhou prestige；
DNTPs, MgCl2, precious biological engineering (Dalian) company limited；
(6) 50 × TAE buffer: 242g Tris and 37.2g Na2EDTA 2H2O is dissolved in 800mL deionized water,
It is sufficiently stirred for dissolving, adds 57.lmL acetic acid, stir, finally with deionized water, solution is settled to 1L, dilute during use
Release 50 times；
(7) agarose, this biological company limited of prompt times of Guangzhou；
(8) 70% ethanol；
(9) LAMP primer (forward outer primer F3, forward inner primer FIP, reverse inner primer BIP, reverse outer primer B3) and
PCR primer (forward primer, downstream primer), the synthesis of precious biological engineering (Dalian) company limited；
(10) nanometer magnetic bead (magnetic nano particles, MNP) test kit is purchased from Beijing Jenner limited public affairs of science and technology
Department；
1.3 key instrument equipment
(1)-80 DEG C of deep freezer；
(2) the most freezing desk centrifuge of Labofuge400R；
(3) regular-PCR instrument and AB7500 quantitative real time PCR Instrument；
(4) France's UVP gel imaging system；
(5) DYY-7 type electrophresis apparatus, Beijing 61 bio tech ltd；
(6) micropipettor, 1000 μ L, 200 μ L, 100 μ L, 10 μ L, 2.5 μ L,；
(7) microwave oven；
(8) analytical balance, prunus mume (sieb.) sieb.et zucc. Teller-torr benefit Instrument Ltd.；
(9) autoclave sterilization pot；
(10) ultrapure water system, Milli-Q Academic, Millipore company；
(11) Shimadzu, Japan UV-120-02 type visible-ultraviolet spectrophotometer；
(12) superclean bench；
(13) eddy mixer；
(14) enzyme connection detector；
(15) AB7500 quantitative real time PCR Instrument；
In sum, beneficial effects of the present invention:
Utilize primer of the present invention, test kit and detection method detection Semen Papaveris, have quick, easy, efficient, accuracy is high,
High specificity, susceptiveness advantages of higher.
Accompanying drawing explanation
Fig. 1 is the augmentation detection curve chart with primer of the present invention detection Semen Papaveris；
Wherein 1: Semen Papaveris positive control；2: containing the measuring samples of Semen Papaveris；3: negative control and Flos Papaveris rhoeadis sample.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention is described further:
Embodiment 1
Test kit of the present invention:
1. product specification: 50 times
2. feature:
Ring mediated isothermal amplification method loop-mediated isothermal amplification, LAMP are a kind of novel
Based on amplification method, only expand at a constant temperature with a kind of enzyme, be capable of identify that the 6 of 6 distinguished sequences of target DNA owing to using
Bar primer, so specificity is high, and amplification efficiency is the highest, it is possible to expand in a large number in the short time, and reaction result can be estimated and be sentenced
Fixed.
This test kit uses ring mediated isothermal amplification method, directly carries out reverse transcription loop with DNA (deoxyribonucleic acid) DNA for template
The simple test kit of mediated isothermality amplification, this test kit uses mixing reverse transcriptase and the enzymatic solution of archaeal dna polymerase, only need to will treat
Reagent in test sample product and this test kit mixes as requested and is placed at a temperature of certain 63 DEG C and reacts about 1 hour,
Amplification curve result of determination from AB7500 instrument.
3. test kit component content:
1) .LAMP reactant liquor buffer 0.5mL
2). primer 150 μ L
3). enzymatic solution 60 μ L
4). positive control (20 μ L)
5). comparison primer solution (20 μ L)
6). deionized water (1000 μ L)
4. operational approach:
1). the preparation of reagent:
A. the various reagent preserved at taking-20 DEG C at room temperature thaw, and be immediately placed on and preserve on ice after defrosting；
B. the preparation (carrying out on ice) of premixed liquid: take the response magnitude needed for detection, according to following table ratio (reaction)
It is divided in respectively in the sterile tube of premixed solution preparation of additionally preparation:
Wherein reaction buffer composition (2X):
Enzymatic solution:
Bst archaeal dna polymerase and AMV reverse transcriptase mixed liquor.
Containing calcein fluorescent dye in reaction buffer:
Calcein is initial is in fluorescent quenching state with being combined with manganese ion, the carrying out reacted along with LAMP, due to
Byproduct of reaction pyrophosphate ion capture the manganese ion being combined with calcein and make calcein recover free out thus
Send fluorescence, and the calcein once magnesium ion in reactant liquor is combined, and fluorescence also can be made to be strengthened.Primer:
Semen Papaveris universal primer sequence (5 '-3 ')
C. flicking after subpackage and hit test tube and make it mix, the brief centrifugation several seconds makes solution concentrate on bottom test tube；
2). sample-adding: be separately added into the DNA 2 μ L of the detected sample prepared in the test tube that subpackage is good, make total amount reach
To 20 μ L, negative control reaction uses deionized water 2 μ L to use with this opium poppy virus nucleic acid as sample, positive control reaction
Positive control, then cover test tube cap and touch mixing, brief centrifugation makes reaction solution concentrate on bottom test tube；
3). amplification: test tube is placed in AB7500 quantitative real time PCR Instrument, at 63 DEG C, keep constant temperature 1 hour, then
Directly observation amplified fluorescence curve；
4). result judges: if measuring samples occurs typical S amplification curve as positive control, be then judged to sun
Property, if there is no amplification curve as negative control, and it is judged to feminine gender.
Embodiment 2
The present invention detects the detection method of Semen Papaveris, comprises the following steps:
A, employing nanometer magnetic bead extract the DNA of testing sample；
By following technique making immune nanometer magnetic bead:
1. the preparation of nanometer magnetic bead
Take 5.2g FeCl respectively3·6H2O、2.7g FeSO4·7H2The concentrated hydrochloric acid of O, 0.85mL 12.1moL/L is dissolved in
In 200mL water, ultrasonic deoxidation, after by above solution instill 250mL, 0.75moL/L NaOH solution；Stir at 80 DEG C, reaction
After end, utilize magnetic separator gained precipitation to be separated from reaction medium, then clean 3 times with deionized water, anhydrous second
Alcohol cleans 2 times, and is made into the Fe of 5g/mL3O4Magnetic nano-particle ethanol solution；Take 25mL Fe3O4Magnetic nano-particle
Ethanol solution, then it is diluted to 150mL, sonic oscillation 30min with dehydrated alcohol；Dropping 0.4mL APTES, stirs under room temperature
7h；React complete, Fe APTES modified with magnetic separator3O4Magnetic nano-particle separates from reaction medium, and with anhydrous
Ethanol solution cleans 3 times, is finally made into amination Fe of 10.5mg/mL3O4Nanometer magnetic bead ethanol solution；
2. the preparation of immunomagnetic beads
Take 0.5mL amination Fe3O4Nanometer magnetic bead ethanol solution, with 1mL PBS 3 times, supernatant abandoned by magnetic separator
Liquid；Add 5% (V/V) glutaraldehyde solution 2mL, shaken at room temperature crosslinking 2h, Magneto separate, abandon supernatant；Wash 3 times with PBS and abandon
After supernatant, it is respectively adopted 0.5mL antiviral antibody and is coated amination Fe3O4Nanoparticle, the fixing 2h of vibration under room temperature；Magneto separate,
Washing respectively 3 times with PBS and ultra-pure water, then use PBS constant volume, 4 DEG C of Refrigerator stores are standby, obtain immunomagnetic beads.
Concrete extracting method:
A, sample preparation: the Semen Papaveris tissue addition 1mL extraction buffer taking 0.1g is ground；
B, cleaning nanometer magnetic bead: in taking-up immune nanometer magnetic bead to PCR pipe, use ddH2O cleans nanometer magnetic bead 3 times repeatedly,
DdH is sucked under the effect of Magnet2O；
C, combination: add the sample of 100 μ L in PCR pipe, with liquid-transfering gun compressing mixing sample and nanometer magnetic bead, under room temperature
Absorption；
D, cleaning: remove supernatant under the effect of Magnet, nanometer magnetic bead cleans 3 times；
E, cracking: add 50 μ L ddH2O is in PCR pipe, after liquid-transfering gun compressing mixing nanometer magnetic bead, and 95 DEG C, 10min；
F samples: use magnet adsorption nanometer magnetic bead, and after solution clarification in PCR pipe, supernatant is testing sample DNA.
B, on ice, extract reaction solution buffer in the following proportions, primer, enzymatic solution, positive control, deionized water are put into and gone out
Tube is prepared premixed liquid；Wherein premixed liquid primary first-order equation amount adds up to 20 μ L, including 10 μ L reaction buffers, 1.2 μ L enzymes
Solution, 3 μ L primers, 3.8 μ L deionized waters.3 μ L primers wherein include 0.12 μ L forward outer primer F3,0.12 μ L reversely outside
Primer B3,1.0 μ L forward inner primer FIP and 1.0 μ L reverse inner primer BIP；Ring primer LF0.38 μ L and LB0.38 μ L.
Wherein said primer includes:
Forward outer primer F3:5'-CCTTTGATTTCTACGAGATGAC-3'；
Reversely outer primer B3:5'-ACCACTACACTCCCCTC-3'；
Forward inner primer FIP and reverse inner primer BIP；
Wherein said forward inner primer FIP comprises F1c and F2 sequence:
5'-TCCAAGCCAAATAAACGATTTTGAAGTGAGGCACATATTCAAATGAAGG-3'；
Wherein F1c:5'-TCCAAGCCAAATAAACGATTTTGAA-3'；
F2:5'-GTGAGGCACATATTCAAATGAAGG-3'；
Described reverse inner primer BIP includes containing B1c and B2 sequence:
5'-GGAAACGTCGAAACACAAGAAGAGAGGAGGTTGTGCATGTTG-3'；
Wherein B1c:5'-GGAAACGTCGAAACACAAGAAG-3'；
B2:5'-AGAGGAGGTTGTGCATGTTG-3'。
C, the DNA of appropriate testing sample is added in the sterile tube in step B；Concrete steps: in the test tube that subpackage is good
Be separately added into the DNA 2 μ L of the detected sample prepared, make total amount reach 20 μ L, negative control reaction use deionized water and
The nucleic acid 2 μ L of Flos Papaveris rhoeadis uses the DNA with poppy seeds as sample, positive control, then covers test tube cap and touch mixing,
Brief centrifugation makes reaction solution concentrate on bottom test tube；
D, the test tube in step C is placed in AB7500 quantitative real time PCR Instrument, at 63 DEG C, keeps constant temperature 1 hour；
E, result judge: if measuring samples occurs typical S amplification curve as positive control, be then judged to sun
Property, if there is no amplification curve as negative control, and it is judged to feminine gender.
Claims (8)
1. a primer based on LAMP technology detection Semen Papaveris, it is characterised in that including:
Forward outer primer F3:5'-TCGATGAAAAATTCCCTGAAC-3'；
Reversely outer primer B3:5'-CACGTTTAGGGGTACTGAT-3'；
Forward inner primer FIP and reverse inner primer BIP；
Wherein said forward inner primer FIP comprises F1c and F2 sequence:
5'-CCTGATAAGAAAGCCATGGATTCA-TGGGGTTAGTAGATAAAGAGTT-3'；
Wherein F1c:5'-CCTGATAAGAAAGCCATGGATTCA-3'；
F2:5'-TGGGGTTAGTAGATAAAGAGTT-3'；
Described reverse inner primer BIP includes containing B1c and B2 sequence:
5'-TACCATCTCTGAACTAAACAACAGG-TCAACTTTAGTCTTAAAAGCTCT-3'；
Wherein B1c:5'-TACCATCTCTGAACTAAACAACAGG-3'；
B2:5'-TCAACTTTAGTCTTAAAAGCTCT-3'。
2. a test kit based on LAMP technology detection Semen Papaveris, it is characterised in that include:
Described primer includes:
Forward outer primer F3:5'-TCGATGAAAAATTCCCTGAAC-3'；
Reversely outer primer B3:5'-CACGTTTAGGGGTACTGAT-3'；
Forward inner primer FIP and reverse inner primer BIP；
Wherein said forward inner primer FIP comprises F1c and F2 sequence:
5'-CCTGATAAGAAAGCCATGGATTCA-TGGGGTTAGTAGATAAAGAGTT-3'；
Wherein F1c:5'-CCTGATAAGAAAGCCATGGATTCA-3'；
F2:5'-TGGGGTTAGTAGATAAAGAGTT-3'；
Described reverse inner primer BIP includes containing B1c and B2 sequence:
5'-TACCATCTCTGAACTAAACAACAGG-TCAACTTTAGTCTTAAAAGCTCT-3'；
Wherein B1c:5'-TACCATCTCTGAACTAAACAACAGG-3'；
B2:5'-TCAACTTTAGTCTTAAAAGCTCT-3'；
The wherein product specification of this test kit: 50 times.
Test kit the most according to claim 2, it is characterised in that described reactant liquor buffer 2 × by following components group
Become:
Test kit the most according to claim 3, it is characterised in that described enzymatic solution be Bst archaeal dna polymerase and AMV inverse
Transcriptase mixed liquor.
5. according to any one test kit described in claim 2-4, it is characterised in that contain in described 2 × reactant liquor buffer
Calcein fluorescent dye.
6. a method based on LAMP technology detection Semen Papaveris, it is characterised in that comprise the following steps:
A, employing nanometer magnetic bead extract the DNA of testing sample；
B, use test kit as claimed in claim 5, on ice, extract reaction solution buffer, primer, enzymatic solution, sun in proportion
Property comparison, deionized water put in sterile tube preparation premixed liquid；
C, the DNA of appropriate testing sample is added in the sterile tube in step B；
D, the test tube in step C is placed in AB7500 quantitative real time PCR Instrument, at 63 DEG C, keeps constant temperature 1 hour；
If E measuring samples occurs typical S amplification curve as positive control, then it is judged to the positive, if right with feminine gender
According to equally there is no amplification curve, and it is judged to feminine gender.
Method the most according to claim 6, it is characterised in that use nanometer magnetic bead to extract testing sample DNA's in step A
Concrete steps:
A, sample preparation: the plant tissue addition 1mL extraction buffer taking 0.1g is ground；
B, cleaning nanometer magnetic bead: in taking-up immune nanometer magnetic bead to PCR pipe, use ddH2O cleans nanometer magnetic bead 3 times repeatedly, at Magnet
Effect under suck ddH2O；
C, combination: add the sample of 100 μ L in PCR pipe, with liquid-transfering gun compressing mixing sample and nanometer magnetic bead, inhale under room temperature
Attached；
D, cleaning: remove supernatant under the effect of Magnet, nanometer magnetic bead cleans 3 times；
E, cracking: add 50 μ L ddH2O is in PCR pipe, after liquid-transfering gun compressing mixing nanometer magnetic bead, and 95 DEG C, 10min；
F samples: use magnet adsorption nanometer magnetic bead, and after solution clarification in PCR pipe, supernatant is testing sample DNA.
Method the most according to claim 6, it is characterised in that described immune nanometer magnetic bead is made by following technique:
1. the preparation of nanometer magnetic bead
Take 5.2g FeCl respectively3·6H2O、2.7g FeSO4·7H2The concentrated hydrochloric acid of O, 0.85mL 12.1moL/L is dissolved in 200mL
In water, ultrasonic deoxidation, after by above solution instill 250mL, 0.75moL/L NaOH solution；Stirring at 80 DEG C, reaction terminates
After, utilizing magnetic separator gained precipitation to be separated from reaction medium, then clean 3 times with deionized water, dehydrated alcohol is clear
Wash 2 times, and be made into the Fe of 5g/mL3O4Magnetic nano-particle ethanol solution；Take 25mL Fe3O4Magnetic nano-particle is anhydrous
Ethanol solution, then it is diluted to 150mL, sonic oscillation 30min with dehydrated alcohol；Dropping 0.4mL APTES, stirs 7h under room temperature；
React complete, Fe APTES modified with magnetic separator3O4Magnetic nano-particle separates from reaction medium, and uses dehydrated alcohol
Solution cleans 3 times, is finally made into amination Fe of 10.5mg/mL3O4Nanometer magnetic bead ethanol solution；
2. the preparation of immunomagnetic beads
Take 0.5mL amination Fe3O4Nanometer magnetic bead ethanol solution, with 1mL PBS 3 times, supernatant abandoned by magnetic separator；
Add 5% (V/V) glutaraldehyde solution 2mL, shaken at room temperature crosslinking 2h, Magneto separate, abandon supernatant；Wash 3 times with PBS and abandon supernatant
After liquid, it is respectively adopted 0.5mL antiviral antibody and is coated amination Fe3O4Nanoparticle, the fixing 2h of vibration under room temperature；Magneto separate, uses
PBS and ultra-pure water wash 3 times respectively, then use PBS constant volume, and 4 DEG C of Refrigerator stores are standby, obtain immunomagnetic beads.
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