CN103205496B - Method for rapidly detecting Bt gene in rice or rice processed goods - Google Patents
Method for rapidly detecting Bt gene in rice or rice processed goods Download PDFInfo
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Abstract
The invention belongs to the field of inspection of agricultural products, and discloses a method for rapidly detecting a Bt gene in rice or rice processed goods. The detecting method comprises the following steps of: (1) extracting a genome DNA (Deoxyribonucleic Acid) in the rice or rice processed goods as a template, and taking an upstream outer primer F3: SEQ, ID, NO.1, a downstream outer primer B3: SEQ, ID, NO.2, an upstream outer primer FIP: SEQ, ID, NO.3 and a downstream outer primer BIP: SEQ, ID, NO.4 to carry out LAMP amplification; and according to a reaction result, judging whether the rice or rice processed goods contains the Bt gene. By virtue of a series of LAMP reaction system, reaction condition optimization and primer specificity tests, the invention successfully establishes the LAMP method for rapidly detecting Bt gene in the rice processed goods, the detection limit achieves 0.005%, the method has the advantages that the operation is simple, a special instrument is not needed, the result is easy to observe; and the method is suitable for popularization on a basement layer.
Description
Technical field
The invention belongs to agricultural-food inspection field, relate to Bt Gene Detecting method in paddy rice or paddy rice processed goods.
Background technology
Along with population in the world 7,000,000,000 critical points of marching toward rapidly, the cultivated area of genetically modified crops surge continuously continue after 15 years soaring.According to (the International Service for the Acquisition of Agri-biotech Applications of International Agriculture biotechnology applications service centre, ISAAA) statistics, within 2011, global transgenic crop cultivated area reaches 1.6 hundred million hectares.2011, the country of plantation genetically modified crops had 29, and wherein, mainly plantation country is the U.S., Brazil, Argentina, India, Canada and Chinese.The cultivated area of China genetically modified crops occupies the 6th, the world, has reached 3,900,000 hectares in 2011
[1].In November, 2009, the Ministry of Agriculture of China has ratified magnificent extensive safety certificate of planting in Hunan Province with Bt63 insect-proof rice for No. 1, and this explanation transgenic paddy rice is within sight in the commercialization plantation of China.
Genetically modified crops receive much concern to the potential risk of HUMAN HEALTH and ecotope all the time
[2-3].Because genetically modified food lacks long-term safety testing data, thereby long-term edible safety cannot be determined, many countries such as European Union, the U.S., Japan and China have all formulated the laws and regulations on the management of genetically modified organism in succession, and genetically modified food is taked to tag control and the management system of tracing to the source.2012, Chinese exports, to the rice made products of European Union, contained transgene component owing to being detected, and again and again suffers to examine, circulates a notice of, removes frame.Therefore be, the trade exports of protection China, in the urgent need to setting up a kind of accurate, special, genetically modified crops detection method fast.Traditional detection of nucleic acids complex operation step, detection time, the detection and tracking in real time of the long scene of being therefore not suitable for detected; And need the expensive equipment such as pcr amplification instrument, testing cost is high, the knowledge to reviewer, operant level require high, can not meet existing detection of nucleic acids site supervision needs.
Compared with detecting with the transgenosis of raw material, processed goods is through some roads work program, raw material DNA inevitably suffers degraded to a certain degree in the course of processing, some physics, chemistry or the enzyme factor occurring in the course of processing also can affect the quality of DNA, and can produce retarding effect to the amplification of nucleic acid, these factors have all increased the difficulty of detection of GMOs in processed goods.
Summary of the invention
The object of the invention is the above-mentioned deficiency for prior art, a kind of Bt Gene Detecting method in paddy rice or paddy rice processed goods is provided.
Object of the present invention can be achieved through the following technical solutions:
Bt Gene Detecting method in paddy rice or paddy rice processed goods, comprises following steps:
(1) extract paddy rice or paddy rice processed goods genomic dna;
(2) genomic dna extracting taking step (1), as template, carries out LAMP amplification with upstream outer primer F3:SEQ ID NO.1, downstream outer primer B3:SEQ ID NO.2, upstream inner primer FIP:SEQ ID NO.3, downstream inner primer BIP:SEQ ID NO.4;
(3) amplified production detects by two kinds of methods: A.2% agarose gel electrophoresis, and electrophoresis is complete, and the gel imaging system imaging of taking pictures, if electrophoresis result presents the typical scalariform band of LAMP, illustrates in paddy rice or paddy rice processed goods and contains Bt gene; B. get 15 μ L reaction product, add 2 μ L SYBR Green I dyestuffs, in a moment result is directly carried out to visual observation, if reaction solution shows green fluorescence, in sample, contain Bt gene, if show negative result of light orange.
Wherein, described paddy rice processed goods preferably rice, rice or rice wine.
Described LAMP reaction system is: 10 × Thermopol Reaction Buffer2.50 μ L, 25mM MgCl
23.00 μ L, 20mM dNTPs3.50 μ L, 8U/ μ L Bst archaeal dna polymerase 0.80 μ L, 20 μ M upstream inner primer FIP1.60 μ L, 20 μ M downstream inner primer primer BIP1.60 μ L, 10 μ M upstream outer primer F30.25 μ L, 10 μ M downstream outer primer B30.25 μ L, 5M trimethyl-glycine 1.00 μ L, 100ng/ μ L DNA profiling 1.00 μ L, nuclease free pure water 9.50 μ L, amount to 25 μ L.
Described LAMP response procedures is: 61 DEG C of isothermal reaction 60min, 80 DEG C of insulation 10min termination reactions.Beneficial effect:
The present invention is by a series of LAMP reaction system, reaction condition optimization experiment and primer specificity experiment, successfully set up the LAMP method for quick of Bt gene in rice made products, detection limit reaches 0.005%, that this method has advantages of is simple to operate, do not need specific apparatus, result to be easy to observe, and is suitable for basic unit and promotes.
And, consider the degraded of DNA in the paddy rice processed goods course of processing, the LAMP primer of the present invention's design can be controlled at suitable scope by the size of amplified fragments, avoids amplified fragments to cause greatly the shortcoming of false negative result and the little reduction atopic of amplified fragments.
Brief description of the drawings
Fig. 1 is for the part Bt gene order of LAMP design of primers.
Fig. 2 LAMP detects the specific test detected result of transgenic paddy rice, wherein M:DNA marker; 1; Transgenic paddy rice 2: non-transgenic paddy rice; 3: blank.
Fig. 3 different Mg
2+the impact of concentration on LAMP reaction expanding effect, wherein, A:LAMP reaction gel electrophoresis result; After B:LAMP reaction, add SYBR Green I coloration result; M:Marker DM2000; 1:Mg
2+concentration is 1mM; 2:Mg
2+concentration is 2mM; 3:Mg
2+concentration is 3mM; 4:Mg
2+concentration is 4mM; 5:Mg
2+concentration is 5mM; 6:Mg
2+concentration is 6mM.
The impact of Fig. 4 different B st archaeal dna polymerase addition on LAMP reaction expanding effect, wherein A:LAMP reaction gel electrophoresis result; After B:LAMP reaction, add SYBR Green I coloration result; M:Marker DM2000; 1:Bst archaeal dna polymerase addition is 1.6U; 2:Bst archaeal dna polymerase addition is 3.2U; 3:Bst archaeal dna polymerase addition is 4.8U; 4:Bst archaeal dna polymerase addition is 6.4U; 5:Bst archaeal dna polymerase addition is 8.0U.
The impact of Fig. 5 differential responses temperature on LAMP reaction expanding effect, wherein A:LAMP reaction gel electrophoresis result; After B:LAMP reaction, add SYBR Green I coloration result; M:Marker DM2000; 1: temperature of reaction is 56 DEG C; 2: temperature of reaction is 57 DEG C; 3: temperature of reaction is 58 DEG C; 4: temperature of reaction is 59 DEG C; 5: temperature of reaction is 60 DEG C; 6: temperature of reaction is 61 DEG C; 7: temperature of reaction is 62 DEG C; 8: temperature of reaction is 63 DEG C; 9: temperature of reaction is 64 DEG C; 10: temperature of reaction is 65 DEG C.
The impact of Fig. 6 differential responses time LAMP reaction expanding effect, wherein A:LAMP reaction gel electrophoresis result;
After B:LAMP reaction, add SYBR Green I coloration result; M:DM2000DNA Marker; 1. reaction 15min; 2. reaction 30min; 3. reaction 45min; 4. reaction 60min; 5. reaction 75min.
The experiment of Fig. 7 LAMP primer specificity, wherein A:LAMP reaction gel electrophoresis result; After B:LAMP reaction, add SYBR Green I coloration result; M:Marker DM2000; 1. non-transgenic paddy rice; 2. blank; 3. transgenic paddy rice.
Fig. 8 LAMP reaction sample sensitivity test, wherein A:LAMP reaction gel electrophoresis result; After B:LAMP reaction, add SYBR Green I coloration result; M:Marker DM2000; 1-8: standard transgenic paddy rice sample sneaks into 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005% sample; 9: non-transgenic paddy rice; 10: blank.
The LAMP of Fig. 9 rice and rice wine detects, wherein A:LAMP reaction gel electrophoresis result; After B:LAMP reaction, add SYBR Green I coloration result; M:DM2000DNA Marker; A: transgenosis rice; B: non-transgenic rice; C: blank; 1: cooked sample; 2: rice wine fermenting 1d; 3: rice wine fermenting 2d; 4: rice wine fermenting 3d.
Embodiment
Embodiment 1
The extraction of 1.1DNA and the detection of extract
DNA extraction method is with reference to the CTAB method in GB/T19495.3-2004.The mensuration of DNA mass concentration and purity: measure respectively the ultraviolet absorption value of nucleic acid at 260nm and 280nm place with ultraviolet spectrophotometer, with the ratio calculation purity of the ultraviolet absorption value at 260nm and 280nm place.Get DNA concentration and be greater than 20ng/ μ L, the extract of A260/280 between 1.7～2.0 is as template.Blank uses sterilized water as template.1.2 design of primers
That chooses that GenBank issues turns the bacillus thuringiensis toxoprotein gene Cry1Ac(GeneBank accession number Y09787 containing in Bt paddy rice) gene is as amplification template, utilize online primer-design software Primer Exploer Version5.0(https: //primerexplorer.jp/lamp5.0.0/index.html) carry out design of primers (Fig. 1), after design of primers completes, carry out online BLAST detection, finally in 407 to 590 regions, 183bp place has designed a set of LAMP reaction primer (F3 altogether, B3, FIP, BIP), synthesized by Shanghai Jierui Biology Engineering Co., Ltd, primer sequence is in table 1.
Table 1LAMP reaction primer sequence
The pcr amplification of 1.3 primer specificity inspections
Adopt the reaction system of 25 μ L, Qi Zhonghan: pcr amplification premixture 12.5 μ L, outer primer F3 and B3 mixture (final concentration is 0.4 μ M) 1 μ L, DNA profiling 100ng.Pcr amplification reaction program is: a.94 DEG C denaturation 5min; B.94 DEG C sex change 30s, 58 DEG C of annealing 30s, 72 DEG C are extended 30s, 40 circulations; C. last 72 DEG C are extended 10min.PCR product after 2% agarose gel electrophoresis, the gel imaging system imaging of taking pictures.
Carry out regular-PCR amplification with outer primer F3 and the B3 of LAMP reaction, through agarose gel electrophoresis, discovery only has transgenic paddy rice to amplify the band that a length is 183bp, non-transgenic paddy rice and the blank product that amplifies useless, as Fig. 2, cut this band and reclaim purifying order-checking, the sequencing result of Ying Jun biotech firm shows that this fragment is to be positioned at Bt gene (CryIA (c) gene) 407-590(5 '-3 ') a fragment.
The optimization Test of embodiment 2LAMP reaction conditions
Primitive reaction system and response procedures
The reaction system of 25 μ L comprises: the each 1.6 μ L of the inner primer FIP of 20 μ M and BIP, the each 0.25 μ L of the outer primer F3 of 10 μ L and B3,10 × Bst DNA polymerase reaction damping fluid (20mM Tris-HCl (pH 8.8), 10mM KCl, 10mM (NH
4)
2sO
4, 4mM MgSO
4, 0.1%Triton X-100) and 2.5 μ L, the MgCl of 25mM
23 μ L, the dNTPs3.5 μ L of 10mM, the Bst archaeal dna polymerase 0.8 μ L of 8U/ μ L, trimethyl-glycine (Betaine) the 1 μ L of 5M, DNA profiling 100ng, adds aqua sterilisa polishing.Response procedures is: 61 DEG C of isothermal reaction 60min, 80 DEG C of 10min are with termination reaction.
(1) impact of different magnesium ion concentrations on LAMP reaction expanding effect:
Mg in reaction system
2+concentration is made as 1.0,2.0,3.0,4.0,5.0 successively, 6.0mmol/L, carries out respectively LAMP reaction, thereby determines suitable Mg
2+concentration, the results are shown in Figure 3.
The stem circular DNA of the final synthetic all size of LAMP reaction, what amplified production presented on 2% sepharose an is not strand but typical scalariform band.Fluorescence dye SYBR Green I can be sent very strong fluorescence in the time that double-stranded DNA is combined, and not with double-stranded combination only send faint fluorescence.Therefore, the quantity of the double-stranded DNA that reaction generates is directly proportional to the fluorescence signal intensity sending, thereby can detect the double-stranded DNA quantity that LAMP system generates according to fluorescence signal intensity, thereby the result of LAMP reaction is detected.If reaction solution shows green fluorescence, in sample, contain Bt gene, if show negative result of light orange.
React each concentration from Fig. 3 A: LAMP and all can amplify band, but amplification is best when concentration 3mM; React each concentration from Fig. 3 B: LAMP and all send green fluorescence, when concentration is 3mM, fluorescence is the strongest, and this is consistent with the result of agarose gel electrophoresis, therefore chooses Mg
2+concentration is that 3mM is optimal concentration.(2) impact of different B st archaeal dna polymerase addition on LAMP reaction expanding effect:
In this experiment, Bst archaeal dna polymerase enzyme concn is 8U/ μ L, and the Bst archaeal dna polymerase addition that design addition is followed successively by test reaction system is made as 0.2,0.4 successively, 0.6,0.8,1.0 μ L, be that addition is respectively 1.6,3.2,4.8,6.4,8.0U, carry out respectively LAMP reaction, thereby determine suitable BstDNA polysaccharase addition, result is shown as Fig. 4.Can be found out by Fig. 4 A: in the time that addition is 1.6U, do not have notch cuttype band to occur, addition starts to occur notch cuttype band while being 3.2U, and along with the increase band brightness of addition strengthens gradually, but the band difference in brightness of 6.4U and 8.0U is little, consider the factor such as amplification efficiency and experimentation cost, choose 6.4U for the suitableeest addition, concentration is that the Bst polysaccharase of 8 μ L adds 0.8 μ L.From Fig. 4 B: when addition is 1.6U, reaction tubes hue preserving is orange constant, illustrates amplified reaction does not occur.In the time that addition increases to 3.2U, from the orange green that becomes, there is amplified reaction in color, and this is consistent with electrophoresis result.
(3) impact of differential responses temperature on LAMP reaction expanding effect:
Temperature of reaction directly affects the activity of enzyme, and the optimal reactive temperature of Bst archaeal dna polymerase is approximately 60～65 DEG C.This test chooses respectively 56,57,58,59, and 60,61,62,63,64,65 DEG C of 10 temperature are carried out LAMP reaction, and result as shown in Figure 5.
Can be found out by Fig. 5 A: in the time that temperature of reaction is 56 DEG C, amplified band is not obvious, in the time that temperature of reaction is 57～65 DEG C, all can amplify notch cuttype band, and band brightness flop is not obvious, but can find out that when temperature of reaction is 61.0 DEG C, band is the brightest the most clear, be optimal reactive temperature so select 61 DEG C.From Fig. 5 B, add fluorescence dye after all reaction tubess all become green, consistent with electrophoresis result.
(4) impact of differential responses time on LAMP reaction expanding effect:
Choose respectively 15,30,45,60, a 75min5 time carries out LAMP reaction, thereby determines the shortest time that can complete LAMP reaction.
For the accuracy that ensures reaction result must ensure certain reaction times, for the minimum time of determining that reaction is required, this test chooses 30,45,60,75min4 time is carried out respectively LAMP reaction, thereby determine the shortest time that can complete LAMP reaction, result as shown in Figure 6.
From Fig. 6 A: when reaction 15～45min, do not occur specific band, during to 60min, band is clear gradually, and 60min and 75min band difference little, so, consider amplification efficiency, choosing 60min is optimal reactive temperature.Be orange from the reaction tubes of Fig. 6 B: 15min, 30min and 45min, negative.It is green that the reaction tubes of 60min and 75min is, and illustrates amplification has occurred, and result is consistent with electrophoresis result.
The reaction system of final optimization pass is: LAMP amplified reaction cumulative volume is 25 μ L, wherein comprises: 10 × Thermopol reaction buffer, 2.5 μ L, 5mol/L trimethyl-glycine 1 μ L, the MgCl of 25mmol/L
23 μ L, 20 μ M upstream inner primer FIP1.60 μ L, 20 μ M downstream inner primer primer BIP1.60 μ L, 10 μ M upstream outer primer F30.25 μ L, 10 μ M downstream outer primer B30.25 μ L, the dNTPs3.5 μ L of 20mmol/L, the Bst archaeal dna polymerase 0.8 μ L of 8U/ μ L, 100ng/ μ L template DNA 1 μ L, uses sterilizing deionized water to mend to 25 μ L.
The LAMP response procedures of final optimization pass is: 61 DEG C of isothermal reaction 60min, 80 DEG C of constant temperature 10min are with termination reaction.
Product detects by two kinds of methods: A.2% agarose gel electrophoresis, and electrophoresis is complete, the gel imaging system imaging of taking pictures; B. get 15 μ L reaction product, add 2 μ L SYBR Green I dyestuffs, in a moment result is directly carried out to visual observation.
The test of embodiment 3LAMP detection specificity
The LAMP method that adopts above-mentioned optimal conditions to set up, taking transgenic paddy rice R76-4 and non-transgenic paddy rice as material, extract genomic dna as reaction template according to method 2.2.1, taking distilled water as template is as blank, carry out LAMP reaction, with checking LAMP atopic, result as shown in Figure 7.
From Fig. 7 A: only go out specific band taking transgenic paddy rice (Cry 1Ac gene) as the sample amplification of template, and all do not amplify specific band taking non-transgenic paddy rice and sterilized water as the sample of template, illustrate that reaction does not exist false positive, and result is accurate, there is good specificity.Same from Fig. 7 B: the coloration result of non-transgenic and blank is negative, and transgenosis sample coloration result is positive, consistent with electrophoresis result.
The test of embodiment 4LAMP detection sensitivity
Non-transgenic paddy rice and transgenic paddy rice are spent the night-80 DEG C of freeze thawing, then pulverize with height pulverizer, in proportion non-transgenic and transgenic paddy rice are mixed again, after mixed powder freeze-drying, extract DNA according to embodiment 1 method, the LAMP reaction conditions of having optimized according to embodiment 2 reacts.Mixed transgenic paddy rice proportion is respectively: 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, and 0%, each sample do three parallel, result as shown in Figure 8:
From Fig. 8 A: transgenic paddy rice content all can amplify specific band 10%～0.005% time but not transgenic paddy rice blank does not all have specific band, illustrative experiment reliable results, the detection of the transgenic paddy rice LAMP detection method of foundation is limited to 0.005%.Known from Fig. 8 B: the reaction tubes color of 10%～0.005% sample becomes green, and feminine gender and blank sample reaction tubes color are orange, result is consistent with electrophoresis result, the result and gel electrophoresis result the indifference that add fluorescence dye sensitivity for analysis is described, accurately and reliably.
Embodiment 5 rice processed goods LAMP detect
(1) rice LAMP detects: transgenosis rice and non-transgenic rice are made to rice through boiling, sample and use the LAMP detection method of having set up to detect, the results are shown in Figure 9.
(2) rice wine LAMP detects: by after 100% transgenosis rice and the elutriation of non-transgenic rice, boiling is dried in the air cool to room temperature, and mounted box after trickle, mixed song is fermented 3 days.Every 24h samples and is stored in 4 DEG C, with the LAMP detection method detection of having set up, the results are shown in Figure 9.
From Fig. 9 A: utilize LAMP method all can amplify obvious specific band with the rice of transgenosis rice processing and the rice wine of fermentation 1-3d, and the detected result of non-transgenic rice processed goods and blank is all negative.And can find out that by Fig. 9 B transgenosis rice processed goods coloration result is positive, non-transgenic rice processed goods and blank sample coloration result are negative, consistent with electrophoresis result.The LAMP detection method of visible foundation can effectively detect the transgenic rice goods through boiling, fermentation complete processing, can detect for the transgenosis of rice and rice wine, and directly add method that fluorescence dye carries out visual detection consistent with the result of traditional agarose gel electrophoresis, and there is easy, advantage fast.
Claims (2)
1. in paddy rice or paddy rice processed goods
btgene Detecting method, is characterized in that comprising following steps:
(1) extract paddy rice or paddy rice processed goods genomic dna;
(2) genomic dna extracting taking step (1) is as template, with upstream outer primer F3:SEQ ID NO.1, downstream outer primer B3:SEQ ID NO.2, upstream inner primer FIP: SEQ ID NO.3, downstream inner primer BIP:SEQ ID NO.4 carry out LAMP amplification; Described LAMP reaction system is: 10 × Thermopol reaction buffer, 2.50 μ L, 25 mM MgCl
23.00 μ L, 20 mM dNTPs 3.50 μ L, 8 U/ μ L
bstdNA polysaccharase 0.80 μ L, 20 μ M upstream inner primer FIP 1.60 μ L, 20 μ M downstream inner primer primer BIP 1.60 μ L, 10 μ M upstream outer primer F3 0.25 μ L, 10 μ M downstream outer primer B3 0.25 μ L, 5 M trimethyl-glycine 1.00 μ L, 100 ng/ μ L DNA template 1.00 μ L, nuclease free pure water 9.50 μ L, amount to 25 μ L; Described LAMP response procedures is: 61 DEG C of isothermal reaction 60min, 80 DEG C of insulation 10min termination reactions;
(3) amplified production detects by two kinds of methods: A. 2% agarose gel electrophoresis, and electrophoresis is complete, and the gel imaging system imaging of taking pictures, if electrophoresis result presents the typical scalariform band of LAMP, illustrates in paddy rice or paddy rice processed goods and contains
btgene; B. get 15 μ L reaction product, add 2 μ L SYBR Green I dyestuffs, in a moment result is directly carried out to visual observation, if reaction solution shows green fluorescence, in sample, contain
btgene, if show negative result of light orange.
2. in paddy rice according to claim 1 or paddy rice processed goods
btgene Detecting method, is characterized in that described paddy rice processed goods is rice, rice or rice wine.
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| CN101343665A (en) * | 2008-08-26 | 2009-01-14 | 天津市农业科学院中心实验室 | Method for quick detection of transgenic pest-resistant rice |
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| CN101343665A (en) * | 2008-08-26 | 2009-01-14 | 天津市农业科学院中心实验室 | Method for quick detection of transgenic pest-resistant rice |
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