CN117305512B - Nucleic acid detection method for detecting soybean plant MON87705 - Google Patents
Nucleic acid detection method for detecting soybean plant MON87705 Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
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- C12Q2600/00—Oligonucleotides characterized by their use
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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Abstract
The invention relates to a nucleic acid detection method for detecting soybean plant MON87705, which constructs a lateral flow chromatography sensor by a double loop-mediated isothermal amplification technology, a strand displacement reaction and CRISPR/Cas12a and according to soybean internal reference genesLectinSD reaction sequence is designed according to LAMP product of specific sequence of transgenic soybean MON87705 transformant, trans-cleavage activity of reaction with CRISPR/Cas12a is utilized by base complementation principle, and detection results of LFB are respectively read in one detection flow, thus realizing soybean internal reference geneLectinLogical detection of specific sequences from transgenic soybean MON87705 transformants.
Description
Technical Field
The invention relates to a detection method for detecting soybean plant MON87705, in particular to a nucleic acid detection method for detecting soybean plant MON 87705.
Background
Currently, detection of transgenic foods often requires two assays of both internal standard genes and transgenic transformants, which greatly increases the cost and complexity of the assay and increases the assay time.
Loop-mediated isothermal amplification (LAMP-mediated isothermal amplification) technology was initiated by Notomi's team in 2000, and can isothermal amplify target nucleic acid templates to 109 copies or more in 1 hour or less, and has the advantages of a single enzyme system, high amplification efficiency and the like, and by combining with a lateral flow chromatography sensor (Lateral flow biosensor, LFB), high-sensitivity rapid visual detection can be realized under simple operation, but hapten labeling is often required for the combination of the two, and an antibody is often required to be used as an identification element for the constructed LFB, so that the cost is high.
In summary, how to provide a method for detecting transgenic food with simple operation, low cost, high speed, sensitivity and visualization is one of the problems in the fields of food safety and transgenic food.
Disclosure of Invention
The invention aims at overcoming the defects of the prior art and providing a nucleic acid sequence combination for detecting LAMP-SD-CRISPR of soybean plant MON87705 and a nucleic acid detection method.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in one aspect, the invention provides a nucleic acid sequence combination for detecting LAMP-SD-CRISPR of soybean plant MON87705, said nucleic acid sequence combination comprising a double LAMP primer set, SD long and short chain combinations, crRNA;
wherein the double LAMP primer group is selected from one or more of the following A-E groups:
primer set A, isLectinF3、LectinB3、LectinFIP、LectinBIP, F31, B31, FIP1 and BIP1 are respectively shown as SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4;
primer group B, isLectinF3、LectinB3、LectinFIP、LectinBIP, F32, B32+3, FIP2 and BIP2+3 are respectively shown as SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8;
primer set C, isLectinF3、LectinB3、LectinFIP、LectinBIP, F33, B32+3, FIP3 and BIP2+3 are respectively shown as SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 9, SEQ ID NO 6, SEQ ID NO 10 and SEQ ID NO 8;
primer set D, isLectinF3、LectinB3、LectinFIP、LectinBIP, F34, B34, FIP4 and BIP4 are respectively shown as SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14;
primer set E, isLectinF3、LectinB3、LectinFIP、LectinBIP, F35, B35, FIP5 and BIP5 are respectively shown as SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17 and SEQ ID NO. 18;
preferably, the double LAMP primer set is selected from primer sets A, C and D.
The crRNA is selected from one of the following sequences:
UAAUUUCUACUAAGUGUAGAUAGGACAACGGUGCCUUGGCC，SEQ ID NO:23；
UAAUUUCUACUAAGUGUAGAUCCGGACAUGAAGCCAUUUAC，SEQ ID NO:24；
UAAUUUCUACUAAGUGUAGAUCAAUUGAAGAGACUCAGGGU，SEQ ID NO:25。
in another aspect, the present invention provides a nucleic acid detection method for detecting soybean plant MON87705, comprising the steps of:
s1, performing LAMP reaction by two groups of primers;
s2, SD reaction with LAMP reaction products;
s3, CRISPR/Cas12a system reaction with an un-incubated SD reaction mixture;
s4, detecting SD reaction and CRISPR/Cas12a system reaction products by using LFB.
As a further aspect of the present invention, the LAMP reaction step used in the present method:
(1) Sample pretreatment: and extracting DNA from the biological sample to obtain a DNA template.
(2) All required reagents were diluted to the appropriate concentrations.
(3) Adding all reagents according to the system, reacting at 65 ℃ for 60 min, and keeping at 80 ℃ for 5 min to inactivate enzymes, and preserving the product at 4 ℃ or carrying out the next scheme.
As a further aspect of the invention, the SD reaction step used in the present method comprises:
long strand (shown as SEQ ID NO: 26) and short strand (shown as SEQ ID NO: 27) were dissolved to 10 μm at 10 x Cas12a-ssDNA Buffer II, long strand: short chain 2.3:2.2 volume ratio, and taking out 4.5 mu L and 0.5 mu L ddH 2 O is added into 25 mu L of LAMP products together and uniformly mixed, 2 mu L of LAMP products are taken out to carry out CRISPR/Cas12a reaction, and the mixture is stored at 4 ℃ for standby after incubation at the remaining 37 ℃ for 20 min.
As a further aspect of the invention, the CRISPR/Cas12a system reaction step used in the method:
the removed non-incubated 2 μl SD reaction mixture was added to the premixed CRISPR/Cas12a system reaction system, incubated at 37 ℃ for 20 min and stored at 4 ℃ for later use.
As a further aspect of the invention, the LFB detection reaction product used in the method comprises the steps of:
SD product detection: 99 [ mu ] L of running buffer (4 XSSC+10 mM Tris-HAc+0.002% TritionX-100+2% BSA+0.1% Tween 20) and 1 [ mu ] L of the sample well are addedSD product, spraying 1.5 mu L gold-labeled antibody on the upper edge of the LFB sample pad, inserting the sensor sample pad into the sample hole, waiting for several minutes, and observing the result (the color of CL is normal to indicate the chromatographic condition of the test paper, the color of TL is positive to indicate the result to be positive, namely the sample to be tested contains the soybean internal standard gene)LectinNucleic acids of (a).
CRISPR/Cas12a reaction product detection: adding 90 [ mu ] L of running buffer (4 XSSC+10 mM Tris-HAc+0.002% TritionX-100+2% BSA+0.1% Tween 20) and 10 [ mu ] L of CRISPR/Cas12a reaction product into a sample hole, spraying 1.5 [ mu ] L of gold-labeled antibody on the upper edge of a sample pad of the LFB, inserting the test paper sample pad into the sample hole, and waiting for a few minutes to observe a result (the appearance of red at CL indicates that the chromatographic condition of the test paper is normal, and the appearance of red at TL indicates that the result is negative, namely, the sample to be tested does not contain MON87705 transgenic soybean transformant components).
According to the two LFB detection results, a red band normally appears at TL and CL in the first detection result, and only the red band normally appears at CL in the second detection result, the transgenic soybean MON87705 transformant component can be judged to be detected in the sample; the presence of a red band at TL and CL in the first detection result and the presence of a red band at TL and CL in the second detection result can be determined that the transgenic soybean MON87705 transformant component is not detected in the sample; in the first detection result, only the CL normally appears in the red strip, and the soybean component is not detected in the sample; other situations occur, and the detection can be judged to be invalid.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the invention constructs the LFB by utilizing LAMP reaction, SD reaction and CRISPR/Cas12a system reaction;
2. the method can realize the soybean standard gene by only reading LFB results twice in one detection flow without limiting LFB to hapten marks and antibodiesLectinLogically detecting the specific sequence of the transformant of the transgenic soybean MON 87705;
3. the invention reduces the manufacturing cost of the LFB and has good detection specificity.
Drawings
The accompanying drawings, which are included to provide a further understanding of embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiments of the invention. In the drawings:
FIG. 1 shows the transgenic soybean MON87705 detection principle (A. LAMP principle; B. SD principle; C. CRISPR/Cas12a reaction principle; D. LFB principle).
FIG. 2 is a transmission electron micrograph of gold nanoparticles (A) and gold nano-nucleic acid signal probes (B) and an ultraviolet-visible absorption spectrum (C) of 400-600 nm.
FIG. 3 shows the LAMP product electrophoresis pattern of the MON87705 primer (A. Group 1 primer; B. Group 2 primer; C. Group 3 primer; D. Group 4 primer; E. Group 5 primer; lane M: D2000 DNA Marker; lane 1: negative control; lane 2: transgenic soybean MON87705 genomic template set).
FIG. 4 is a diagram ofLectinGene primer and MON87705 primer LAMP product electrophoretogram (L.LectinA gene primer; A. group 1 primers; B. group 3 primers; C. group 4 primers; lane M: d2000 A DNA Marker; lane 0: a negative control; lane 1: a non-transgenic soybean genome template set; lane 2: transgenic soybean MON87705 genome template group).
FIG. 5 shows the SD results of LAMP products (A. LFB band; B. Image J peak area; a:Lectinthe gene primer and the negative control; b:Lectina genetic primer and a non-transgenic soybean genome template; c:Lectina gene primer and a transgenic soybean MON87705 genome template; d: group 1 primers and non-transgenic soybean genome templates; e: group 1 primers and transgenic soybean MON87705 genomic templates; f: group 3 primers and non-transgenic soybean genome templates; g: group 3 primers and transgenic soybean MON87705 genomic templates; h: group 4 primers and non-transgenic soybean genome templates; i: group 4 primers and transgenic soybean MON87705 genomic template).
FIG. 6 shows the SD results of double LAMP products (A. LFB band; B. Image J peak area; a: negative control; b: group 1 primer and non-transgenic soybean genomic template; c: group 1 primer and transgenic soybean MON87705 genomic template; d: group 4 primer and non-transgenic soybean genomic template; e: group 4 primer and transgenic soybean MON87705 genomic template).
FIG. 7 shows primer addition optimization (A. LFB band; B. Image J peak area; a: 2. Mu.L) of double LAMP systemLectinA gene long primer and a negative control; b: 2. mu LLectinA long gene primer and a soybean genome template; c:2.5 Mu LLectinA gene long primer and a negative control; d:2.5 Mu LLectinGene long primers and soybean genome templates).
FIG. 8 shows SD long and short chain ratio optimization (A. LFB band; B. Image J peak area; a: negative control; b: soybean genome template).
FIG. 9 is a CRISPR/Cas12a response crRNA selection (A. LFB band; B. Image J peak area; a: negative control; b: non-transgenic soybean genomic template; c: transgenic soybean MON87705 genomic template).
FIG. 10 shows the specificity of SD response (A. LFB band; B. Image J peak area; a: soybean genomic template; b: maize genomic template; c: canola genomic template).
FIG. 11 shows the LAMP-LFB logic detection of the specificity of transgenic soybean MON87705 (A. LFB band; B. Image J peak area; a soybean genomic template SD results; b transgenic soybean MON87705 genomic template (10 wt%); c transgenic soybean MON87769 genomic template (100 wt%); d transgenic soybean GTS40-3-2 genomic template (100 wt%)).
FIG. 12 shows the sensitivity of LAMP-LFB logic detection of transgenic soybean MON87705 (A. LFB band; B. Image J peak area; a: soybean genome template SD results; b: non-transgenic soybean genome; c: transgenic soybean MON87705 genome template (10 wt%); d: transgenic soybean MON87705 genome template (1 wt%); e: transgenic soybean MON87705 genome template (0.1 wt%) f: transgenic soybean MON87705 genome template (0.01 wt%)).
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The present invention will now be described in more detail by way of examples with reference to the accompanying drawings, which are not intended to limit the invention thereto, but are illustrative only.
Example 1 design and verification of the principle of the nucleic acid detection method for detecting soybean plant MON87705
(1) Principle design and verification
The invention combines LAMP, SD, CRISPR/Cas12a and LFB to detect transgenic soybean MON87705, the principle of which is shown in figure 1, and the soybean standard geneLectinThe loop-forming single-chain part in the product generated by LAMP reacts with double chains formed after long and short chains are incubated to release short-chain nucleic acid, the 3 '-end sequence of the nucleic acid template is upwards chromatographed along with a sensor after being complementarily combined by a gold nano-nucleic acid signal probe on LFB, the nucleic acid on a T line is complementarily combined with the 5' -end sequence of the short-chain nucleic acid, and a sandwich structure of a gold nano-nucleic acid signal probe-short chain-T line nucleic acid is formed at the T line, so that the T line is developed. And the C line is directly combined with the gold nano-nucleic acid signal probe for color development. In addition to the reactions that occur with SD, LAMP double-stranded products generated by the specific sequences of transgenic soybean MON87705 transformants can excite CRISPR/Cas12a reactions to activate the trans-single-strand cleavage activity of Cas12a, and cut short chains released by SD reactions, so that a sandwich structure of 'gold nano-nucleic acid signaling probes-short chains-T-line nucleic acids' cannot be formed at the T lines, and color cannot be developed. The C line is still directly combined with the gold nano-nucleic acid signal probe for color development.
The verification process of the invention relates to agarose gel electrophoresis, which comprises the following specific steps:
first, a 50 XTAE buffer (242.2 g tris was dissolved by heating with 500 mL water under stirring, then 500 mM disodium ethylenediamine tetraacetate solution (pH 8.0) was added, the pH was adjusted to 8.0 with glacial acetic acid, and water was added to a volume of 1000 mL) was used and diluted to 1 XTAE buffer. When agarose gel electrophoresis is carried out, firstly, distilled water is used for cleaning a gel preparation tool, and a proper gel preparation flat plate and a tooth comb are selected for supporting the gel preparation tool. Accurately weighing 2 g agarose dry powder, using 100 mL 1 xTAE buffer dissolved in 250 mL conical flask to prepare 2% agarose gel. Placing the sealing film with the conical bottle cap well ventilated into a microwave oven, heating and melting, cooling to be non-scalding, adding 5 mu L of EB solution (10 mg/mL), uniformly mixing, pouring into a gel preparation flat plate, taking off a tooth comb after the gel preparation flat plate is solidified, then placing the gel preparation flat plate and gel into an electrophoresis tank with a proper amount of 1 xTAE Buffer solution, discharging air in a comb hole, taking out, taking 5 mu L of product to be detected, adding 1 mu L of 6 xLoading Buffer, uniformly mixing, loading into the comb hole, adding a D2000 DNA Marker into a proper comb hole, placing the gel back into the electrophoresis tank after Loading, switching on a power supply, taking out the gel after electrophoresis for 25 min under 120V voltage, and observing the positions of the product to be detected and the Marker on an ultraviolet gel imaging system.
The verification process of the invention relates to the construction of the LFB, and comprises the following specific steps:
preparation of gold nanoparticles: the round-bottomed flask after the acid jar was immersed (overnight) was washed 3 times with tap water, distilled water and ultrapure water, respectively, and dried to be used as a reaction vessel. 100 mL of 1 mM chloroauric acid solution is boiled, 10 mL of 38.8 nM trisodium citrate is vertically added, (the gun head is changed once every time to avoid influencing the experimental result by overheat steam, a flask mouth is covered by tinfoil paper, a small mouth is penetrated out), the time is immediately counted and sealed to prevent volatilization, the color change is observed to be yellow-black-red, heating is stopped after 10 min, the water bath is cooled to room temperature (recommended to avoid light) (refrigerator refrigeration), and the mixture is stored at 4 ℃.
Preparation of gold nano-nucleic acid signaling probe: first, 1 μl of dATP at a concentration of 100 mM was added to 1 mL gold nanoparticles, after which the mixture was incubated at room temperature for 20 min. After that, 15. Mu.L of a 1% SDS solution was slowly added to the mixture and incubated for 10 min on a shaker. The NaCl solution (100. Mu.L, 0.2. 0.2M) was then added to the mixture at a rate of 20. Mu.L per ten minutes. A0.25 OD DNA fragment (shown in SEQ ID NO: 28)) The mixture was incubated in a water bath at 60℃for 3 h. After completion of incubation, the obtained solution was centrifuged at 12000 rpm for 15 min, the supernatant was discarded, and the precipitate was washed with PBS, and the obtained ruby precipitate was dispersed and stored in 20. Mu.L of an eluent (20 nM Na 3 PO 4 •12H 2 O+5% BSA+0.25% Tween-20+10% cross) is the gold nano-nucleic acid signal probe, and is stored at 4 ℃.
LFB assembly: after mixing and incubating 0.5. 0.5 OD of T-line nucleic acid (shown as SEQ ID NO: 29) with C-line nucleic acid (shown as SEQ ID NO: 30) with an equal volume of 1.1 mg/mL streptavidin overnight, the T-line and C-line solutions were fed into the three-dimensional spray point platform spray head 1, fixed at a position 1.1 cm from the lower edge of the NC membrane, and the C-line solutions were fed into the three-dimensional spray point platform spray head 2, fixed at a position 1.6 cm from the lower edge of the NC membrane, so that the distance between the T-line spray head and the C-line spray head was 5.0 mm. And sticking the NC film on the PVC bottom plate, setting the scribing speed to be 1.0 [ mu ] L/cm, and uniformly spraying the scribing solution on the NC film. Drying the sprayed NC film at 37 ℃ for 3 h, then adhering the water absorbing pad to the backboard by tightly adhering the upper edge of the backboard, and carefully trowelling; bonding the bonding pad to a proper position of the back plate, and carefully trowelling; the sample pad was adhered to the back plate against the lower edge of the back plate, and carefully smoothed. Cutting the stuck test paper strip into test paper with the width of 3.5 and mm by a programmable strip cutting machine, and placing the cut test paper into a packaging bag with a drying agent for storage at normal temperature for standby.
Characterization of gold nanoparticles and gold nanoparticle-nucleic acid signaling probes is shown in fig. 2.
(2) Primer selection
The DNA genome (50 ng/. Mu.L) extracted from transgenic soybean MON87705 with 5 sets of LAMP primers designed in Table 1 was reacted according to the single LAMP system in Table 2, and the detection results of LAMP products by 2% agarose gel electrophoresis are shown in FIG. 3. Wherein the primers of the 1 st group, the 3 rd group and the 4 th group can specifically amplify the DNA genome extracted from the transgenic soybean MON87705, and the three groups of primers are selected for subsequent experiments. By soybean internal standard geneLectinGene primer, group 1, group 3 and group 4 transgenic soybean MON87705 primers were used for non-transgenic soybean genome (50 ng/μl) And transgenic soybean MON87705 genome (50 ng/μl) was reacted according to the LAMP system in table 2, LAMP products were detected by 2% agarose gel electrophoresis (containing 0.1 μg/mL EB) (fig. 4), group 3 primers produced unexpected products when amplifying non-transgenic soybean genome, so the following experiments were performed using group 1 and group 4 primers. Meanwhile, the LAMP products of the primers were subjected to SD reaction, and the results are shown in FIG. 5, and it is clear that the LAMP products of the primers do not interfere with the SD reaction results, and the subsequent experiments can be performed. Respectively using group 1 and group 4 primers and soybean internal standard genesLectinThe gene primers were reacted according to the double LAMP system in Table 3, and SD reaction was performed with LAMP products, and the reaction products were detected with LFB, and the results are shown in FIG. 6.
TABLE 1 LAMP primer group sequences
TABLE 2 Single LAMP reaction System
TABLE 3 Dual LAMP initial reaction System
Example 2 detection condition optimization results
(1) Primer addition amount of double LAMP System
Standard genes in soybeanLectinThe gene long primer is added with 2.5 mu L instead, the other conditions are the same as the double LAMP system in Table 3, and the SD reaction result LFB is detected, and the result is shown in FIG. 7. Compared with the original condition, 2.5 mu L soybean internal standard genes are usedLectinThe gene long primer has obvious color improvement, and 2.5 mu L is added in the subsequent testLectinAnd (5) gene long primers.
(2) SD long and short chain ratio optimization
More long chains may saturate the short chains as much as possible, but may also affect the efficiency of subsequent transgene specific transformation assays, so long chains (as shown in SEQ ID NO: 26) are mixed with short chains (as shown in SEQ ID NO: 27) in ratios of 2.1:2.0,2.2:2.1,2.3:2.2,2.4:2.3,2.5:2.4 and made up to 5 parts by volume with water for SD reactions, and the results are detected by LFB as shown in FIG. 8. The best detection effect can be obtained when the addition ratio of the long and short chains is 2.3:2.2, so that the ratio of the SD long and short chains in the subsequent test is 2.3:2.2.
(3) CRISPR/Cas12a responsive crRNA selection
Group 1 primers and soybean internal standard genesLectinThe double LAMP products of the gene primers perform CRISPR/Cas12a reaction, and the reaction products are detected by LFB, and the result is shown in figure 9. Cas12a cleavage efficiency was highest when using GMO crRNA2 (as shown in SEQ ID NO: 24), and subsequent experiments selected GMO crRNA2 for use.
Example 3 detection Performance of the detection method
(1) Specificity (specificity)
Under optimal experimental conditions, the specificity of LAMP and SD reaction on soybean genome is tested, and SD reaction results after LAMP are carried out by using soybean genome (50 ng/MuL), corn genome (100 ng/MuL) and rape genome (100 ng/MuL) are detected by LFB, as shown in FIG. 10. LAMP and SD reactions have good specificity, only soybean samples will trigger the reaction.
Under optimal experimental conditions, the specificity of the method for detecting transgenic soybean MON87705 constructed by combining LAMP, SD and CRISPR/Cas12a with LFB was tested. The genome was extracted using transgenic soybean MON87705 (10 wt%), transgenic soybean MON87769 (100 wt%), transgenic soybean GTS40-3-2 (100 wt%), and then diluted to a concentration of 50 ng/μl, and the detection was performed by the method of detecting transgenic soybean MON87705 constructed by LAMP, SD, and CRISPR/Cas12a in combination with LFB, and the reaction results were detected by LFB, and the results are shown in fig. 11. The LAMP, SD and CRISPR/Cas12a combined LFB constructed method for detecting transgenic soybean MON87705 can specifically identify the genome of the transgenic soybean MON87705, and has good specificity.
(2) Sensitivity of
To obtain qualitative detection limits of the methods for detecting transgenic soybean MON87705 constructed by LAMP, SD and CRISPR/Cas12a combined with LFB, transgenic soybean MON87705 was milled and added to 100 g non-transgenic soybean powder at 10,1,0.1,0.01 g, and mixed uniformly to obtain soybean samples containing 10,1,0.1,0.01 wt% of transgenic soybean MON87705, the genome was extracted, and the test was performed with 50 ng/μl genome, and the detection results were detected by LFB, as shown in fig. 12.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (7)
1. An LFB detection combination for detecting LAMP-SD-CRISPR of soybean plant MON87705, characterized in that said LFB detection combination comprises a double LAMP primer set, an SD long and short chain combination, a crRNA and LFB sensor nucleic acid combination;
the double LAMP primer group is a first primer group or a second primer group:
the first primer group isLectinF3、LectinB3、LectinFIP、LectinBIP, F31, B31, FIP1 and BIP1 are respectively shown as SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4;
the second primer group isLectinF3、LectinB3、LectinFIP、LectinBIP, F34, B34, FIP4 and BIP4 are respectively shown as SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14;
in the SD long chain and short chain combination, a long chain sequence is CGACTTATTGAGTTGTAACTTTCCCGA, and is shown as SEQ ID NO. 26; the short chain sequence is ACAACTCAATAAGTCG, and is shown as SEQ ID NO. 27;
the crRNA is selected from one of the following sequences: UAAUUUCUACUAAGUGUAGAUAGGACAACGGUGCCUUGGCC, as shown in SEQ ID NO. 23;
UAAUUUCUACUAAGUGUAGAUCCGGACAUGAAGCCAUUUAC, as shown in SEQ ID NO. 24;
the LFB sensor nucleic acid combination comprises a T-line nucleic acid, a C-line nucleic acid, and a gold nano-nucleic acid signaling probe; the sequence of the T line nucleic acid is TGAGTTGTTTTTTT, and is shown as SEQ ID NO. 29; the sequence of the nucleic acid of the C line is TTTTTTATAAGTCG, and is shown as SEQ ID NO. 30; the sequence of the gold nano-nucleic acid signal probe is CCCCCCCGACTTAT, and is shown as SEQ ID NO. 28.
2. A nucleic acid detection method for detecting soybean plant MON87705 using the LFB detection combination of claim 1, said method comprising the steps of:
s1, performing LAMP reaction by using DNA extracted from a biological sample as a template and adopting the double LAMP primer group to obtain an LAMP reaction product;
s2, carrying out SD reaction on the LAMP reaction product and the SD long-chain and short-chain combination to obtain an SD reaction mixture, and incubating the SD reaction mixture for later use;
s3, performing CRISPR/Cas12a system reaction on the non-incubated SD reaction mixture and the crRNA;
s4, constructing an LFB sensor by adopting the LFB sensor nucleic acid combination, performing first LFB detection on the incubated SD reaction mixture by adopting the LFB sensor, and performing second LFB detection on a CRISPR/Cas12a system reaction product by adopting the LFB sensor;
s5, judging the LFB detection result: according to the two LFB detection results, a red stripe normally appears at the T line and the C line in the first LFB detection result, and a red stripe normally appears at the C line only in the second LFB detection result, judging that the transgenic soybean MON87705 transformant component is detected in the sample; the T line and the C line in the first LFB detection result normally show red stripes, and the T line and the C line in the second LFB detection result also normally show red stripes, and the transgenic soybean MON87705 transformant component is not detected in the sample; in the first LFB detection result, only a red stripe normally appears at the C line, and the soybean component is not detected in the sample; and if other conditions occur, judging that the detection is invalid.
3. The detection method according to claim 2, characterized in that the reaction system of the LAMP reaction is:
the double LAMP primer group, whereinLectinFIP (FIP)LectinThe final concentration of BIP was 1. Mu.M and the addition amount was 2.5. Mu.L, respectively, the final concentrations of FIP1, BIP1, FIP4 and BIP4 were 0.8. Mu.M and the addition amount was 2. Mu.L, respectively,LectinF3、Lectinthe final concentrations of B3, F31, B31, F34 and B34 were 0.1. Mu.M and the addition amounts were 0.25. Mu.L, respectively; deoxynucleotide solution mixture with a final concentration of 1.4 mM and an addition amount of 3.5. Mu.L; 10X Isothermal Amplification Buffer, 1X final concentration and 2.5. Mu.L of additive amount; betaine, final concentration of 0.8M, addition of 4 μl; magnesium sulfate with a final concentration of 5 mM and an addition of 1.25. Mu.L; and Bst enzyme, the final concentration is 320U/mL, and the addition amount is 1 mu L; and reacting the DNA extracted from the biological sample in a buffer system with the total volume of 25 mu L at 65 ℃ for 60 min.
4. The method according to claim 2, wherein the SD reaction system is:
SD long chain and short chain with the concentration of 10 mu M are added into LAMP reaction products of 25 mu L, and the volume ratio is 2.3:2.2 SD long chain and short chain combination 4.5 mu L, 0.5 mu L ddH is added 2 After O is uniformly mixed, taking out 2 mu L, and incubating for 20 min at 37 ℃ in a buffer system.
5. The detection method according to claim 2, wherein the CRISPR/Cas12a system reaction system is:
2. al of the non-incubated SD reaction mixture; crRNA with final concentration of 0.5 mu M; lbaCas12a nucleic at a final concentration of 0.5. Mu.M; and incubating for 20 min at 37 ℃ in a buffer system with a total volume of 20 [ mu ] L.
6. Use of the LFB assay combination of claim 1 or the assay method of claim 2 in the assay of transgenic soybean MON87705 components in a soybean preparation.
7. Use of the LFB assay combination of claim 1 in the preparation of a kit for the detection of transgenic soybean MON87705 components in a soybean preparation.
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