CN112176089A - System and method for identifying single nucleotide polymorphism of panax plants - Google Patents

Abstract

The invention belongs to the technical field of plant identification, and particularly relates to a system and a method for identifying single nucleotide polymorphism of a ginseng plant. The detection system utilizes the basic principle of DNA amplification induced by damage and uses an immobilized capture probe to realize the detection of single nucleotide polymorphism of ginseng and American ginseng and the accurate and quick identification of the two medicinal materials. The scheme allows a small amount of samples to be detected, does not have the requirement on a thermal cycler or even a hot water bathtub, and solves the technical problems of expensive equipment and complicated operation process caused by the need of performing external amplification such as PCR (polymerase chain reaction) and the like in the SNP detection technology. The scheme can be applied to the practical operation of distinguishing and identifying the ginseng medicinal materials, and plays a beneficial role in protecting the rights and interests of consumers and ensuring the quality of the medicinal materials.

Description

System and method for identifying single nucleotide polymorphism of panax plants

Technical Field

The invention belongs to the technical field of plant identification, and particularly relates to a system and a method for identifying single nucleotide polymorphism of a ginseng plant.

Background

Ginseng (Asian Ginseng/Chinese Ginseng, Panax Ginseng) and American Ginseng (Panax Quinquefolius) are also used as Panax medicinal materials. Although the ginseng and American ginseng are extremely similar in appearance and morphology, the effects are different and the values are greatly different, so that the phenomenon that the fish eyes are mixed with beads and are not good enough often appears in the market, and the technology and means for quickly identifying ginseng and American ginseng are very important for protecting the rights and interests of consumers. The ginseng and the American ginseng can be effectively distinguished by using Single Nucleotide Polymorphism (SNP) of dammarenediol-II synthetase (DS) genes of panax.

At present, species identification often employs a method of detecting single nucleotide polymorphism, and among them, there are various methods of detecting SNP, including molecular hybridization identification, DNA sequencing, and the like. The method of using molecular hybridization is to hybridize a DNA probe to a PCR product containing a SNP site. The use of this method requires the use of various means to confirm whether probe hybridization contains a perfect complementary match or mismatch, such as adjusting the temperature, ionic strength, and choice of wash solution. Methods using molecular hybridization require extensive optimization for a particular system. DNA sequencing has become a popular alternative method of species identification. Although sequencing does not require extensive optimization, a high purity sample is still required for detection. Existing SNP detection techniques require external amplification (e.g., PCR), are expensive in equipment and cumbersome in operation, and have limited applicability in commercial settings.

Disclosure of Invention

The invention aims to provide a system for identifying single nucleotide polymorphism of Panax plants, which overcomes the technical problems of expensive equipment and complicated operation process caused by external amplification such as PCR (polymerase chain reaction) and the like required by the SNP detection technology.

In order to solve the technical problems, the technical scheme of the invention is as follows:

a system for identifying single nucleotide polymorphism of Panax plants comprises a detection unit for detecting DNA template, wherein the detection unit comprises a fixed fragment, and the DNA template and the fixed fragment are single-stranded DNA; the 5 'end of the immobilized fragment is immobilized on a solid support, the first base at the 3' end of the immobilized fragment is an antisense SNP base, and the immobilized fragment is complementarily paired with a DNA template.

By adopting the technical scheme, the technical principle and the beneficial effects are as follows: this protocol utilizes and improves LIDA ligation, a method that uses a ligase to perform the ligation reaction, instead of polymerase-based amplification. This method is called damage-induced DNA amplification (LIDA), and can simultaneously perform amplification and SNP discrimination at an isothermal temperature. T4 DNA ligase, unlike polymerase, is very sensitive to correct base pairing at the ligation site and therefore can be used as a tool for SNP discrimination. In the prior art, the LIDA ligation reaction is usually performed in a liquid phase environment, and all DNA fragments are in a free state. In order to realize the detection of the target SNP, fluorescent labeling needs to be carried out on a plurality of DNA strands, thereby increasing the complexity of the operation. In the scheme, only the fixed fragment is fixed on the solid phase support, and the detection of the target SNP can be realized only by the detection fragment marked by the reporter group, so that the operation steps and the complexity of the system are simplified.

Further, the sequence of the DNA template is a section of DNA sequence on the sense strand of the gene containing the SNP site, and the SNP site of the gene is positioned on the DNA template; the detection unit further comprises a LIDA mixed solution, wherein the LIDA mixed solution comprises T4 DNA ligase, a buffer solution, and a detection fragment, a universal fragment and a specific fragment which are single-stranded DNA; the 5 'end of the detection fragment is connected with a non-base functional group, the 3' end of the detection fragment is modified with a reporter group, and the detection fragment is in complementary pairing with the DNA template; the second base at the 5' end of the specific fragment is a sense SNP base, and the first base is an unpaired base.

Further, the DNA template comprises a class I template and a class II template; the type I template is from a sample to be detected; the 3 'end of the universal fragment is connected with the 5' end of the specific fragment to form a II-type template; the 5 'end of the detection fragment is connected with the 3' end of the fixed fragment and then is complementarily matched with the DNA template, and the unpaired base and the function group without the base are allelic.

By adopting the technical scheme, when the system is used for SNP detection, the I-type template is prepared from a sample to be detected, the I-type template is mixed with T4 DNA ligase, buffer solution, detection fragments, universal fragments and specific fragments, and is contacted with immobilized fixed fragments, so that LIDA ligation reaction occurs. After the reaction is finished, the report group signal is detected, so that whether the polymorphism of the mononucleotide exists in the sample to be detected can be known, and the species can be identified.

Technical principle of the present solution referring to fig. 3, there are two cycles of the LIDA ligation reaction. In Cycle 1(Cycle 1), class I templates (DNA templates) are ligated with immobilized fragments (Gin-NH)₂) Hybridization (step 1.1) and ligation of the detection fragment (Det) is induced (step 2.1). At this time, the fragment (Gin-NH) was fixed₂) And the detection fragment (Det) is connected end to end under the action of ligase to form an immobilized long chain. Subsequently, the duplex (three formed of class i template, detection fragment and immobilized fragment) is dissociated (step 3), the class i template is separated from the immobilized long strand, and cycle 1 is repeated. Then, in Cycle 2(Cycle 2), the immobilized long strand is hybridized with the specific fragment (GSF) (step 1.2) and the specific fragment (GSF) is ligated with the universal fragment (Frag) (step 2.2) forming a duplex. Subsequently, the duplex is dissociated (step 3) to provide class II templates, which may be subjected to cycle 1. The number of detection fragments immobilized on the immobilized fragment is increased, and the detection fragments are further immobilized on a solid support, and the condition of SNP in the sample can be known by detecting a reporter group on the detection fragments. In this protocol, the inventors adapted the liquid phase LIDA to the solid phase LIDA and introduced the fluorescently labeled detection fragment in cycle 1, which eliminates the need for external amplification (e.g., PCR).

In the present embodiment, the antisense SNP base refers to a single base located on the antisense strand at the SNP site of the gene. A sense SNP base refers to a single base located on the sense strand at the SNP site of a gene. A Single Nucleotide Polymorphism (SNP) site has two allelic bases (deoxyribonucleotides), one on the sense strand of the gene and one on the antisense strand of the gene.

In conclusion, the method allows the detection of small amounts of sample, and the amplification of the gene of interest can be achieved by cycle 1 and cycle 2. In addition, the ligation product of the reaction will bind to the solid support, which overcomes the drawbacks of liquid phase LIDA. In liquid-phase LIDA, fluorescence labeling on both strands is required for energy resonance transfer (FRET) detection. The detection method can be carried out at room temperature, thereby eliminating the need of using a thermal cycler or even a hot water bathtub in the process, and having simpler and more convenient operation.

Further, the non-basic functional group is 1 ', 2' -dideoxyribose.

By adopting the technical scheme, the introduction of the non-base functional group can realize spontaneous proceeding of the ligation reaction and ensure the smooth implementation of the LIDA ligation reaction.

Further, the solid support is an aldehyde-based glass slide of the microfluidic biochip.

By adopting the technical scheme, the microfluidic detection equipment has the advantages of simple structure, convenience in preparation, high detection speed, capability of flexibly adjusting the sample loading mode according to actual requirements and the like.

Further, the detection unit is used for detecting SNP sites of dammarenediol-II synthetase genes of ginseng and American ginseng; when the SNP site of the ginseng is detected, the antisense SNP base of the fixed fragment is the base positioned on the antisense strand of the SNP site of the ginseng, and the sense SNP base of the specific fragment is the base positioned on the sense strand of the SNP site of the ginseng; when the SNP locus of American ginseng is detected, the antisense SNP base of the fixed fragment is the base positioned on an antisense strand on the SNP locus of American ginseng, and the sense SNP base on the specific fragment is the base positioned on a sense strand on the SNP locus of American ginseng.

By adopting the technical scheme, whether the sample is a pure ginseng, a pure American ginseng or a heterozygous species can be judged by detecting the SNP locus of the dammarenediol-II synthetase gene of the ginseng and the American ginseng, so that the accuracy of species identification is improved.

Further, the nucleotide sequence of the fixed fragment for detecting the SNP site of ginseng is: NH (NH)₂-5’-CAATTTAAG-3’；

The nucleotide sequence of the fixed fragment for detecting the SNP locus of the American ginseng is as follows: NH (NH)₂-5’-CAATTTAAA-3’；

The nucleotide sequence of the specific fragment for detecting the SNP locus of the ginseng is as follows: 5'-TCTTAAATTG-3', respectively;

the nucleotide sequence of the specific fragment for detecting the SNP locus of the American ginseng is as follows: 5'-TTTTAAATTG-3', respectively;

the nucleotide sequence of the detection fragment is as follows: 5 '-AbCACTTTC-3' -bio;

the nucleotide sequence of the universal fragment is as follows: 5 '-GAAAGTG-3';

wherein NH₂Represents a 5 'end amino modification, bio represents a 3' end biotin modification, and Ab represents a functional group without a base group.

Further, the LIDA mixed solution also comprises a free fragment, and the free fragment is consistent with the fixed fragment.

By adopting the technical scheme, the addition of the free fragment can increase the fluorescence intensity, thereby increasing the sensitivity of the detection.

Further, magnesium ions are also included in the LIDA mixed solution.

By adopting the technical scheme, the ionic strength of the LIDA mixed solution is improved by adding magnesium ions, and the association of chains on the solid surface is promoted.

Further, an identification method of a system for identifying single nucleotide polymorphisms of a ginseng plant comprises the following steps:

(1) probe immobilization and template acquisition: immobilizing the immobilized fragments on a solid support; obtaining a class i template from a sample;

(2) and (3) connecting:

comprises a connection process for detecting the SNP sites of the ginseng: contacting the LIDA mixed solution and the class I template with the fixed fragment and carrying out LIDA ligation reaction to obtain a first ligation product; the antisense SNP base of the fixed fragment is a base positioned on an antisense chain on the SNP site of the ginseng;

also comprises a connection process for detecting the SNP locus of the American ginseng; enabling the LIDA mixed solution and the class I template to contact the fixed fragment and carrying out LIDA ligation reaction to obtain a second ligation product; the antisense SNP base of the fixed fragment is a base positioned on an antisense chain on the SNP locus of the American ginseng;

(3) signals of the first ligation product and the second ligation product are detected separately.

Further, the duration of the LIDA ligation reaction was 4 h.

By adopting the technical scheme, the SNP of the American ginseng can be detected only after the reaction time is more than 4 hours.

Further, in the step (3), a fluorescence signal of the first ligation product is obtained, wherein the intensity of the fluorescence signal is F1; obtaining a fluorescent signal of the second ligation product, the intensity of the fluorescent signal being F2;

when F1/F2 > 2, the sample contains pure ginseng;

when F1/F2 is less than 0.5, the sample contains pure American ginseng;

when the ratio of F1/F2 is more than or equal to 0.5 and less than or equal to 2, the sample contains ginseng and American ginseng.

By adopting the technical scheme, the species condition of the sample can be judged according to the condition of the fluorescence signal. When F1 is more than twice that of F2, the DNA of ginseng is much more than that of American ginseng, and the sample contains pure ginseng (at least the content of ginseng is the most). When F1 is less than one-half of F2, the DNA of American ginseng is much more than that of ginseng, and the sample contains pure American ginseng (at least the content of American ginseng is most). When the ratio of F1/F2 is more than 0.5 and less than 2, the sample is mixed with ginseng and American ginseng.

Drawings

Fig. 1 is a schematic view of the microfluidic detection apparatus of example 1 (showing the first PDMS slab and the functionalized substrate).

Fig. 2 is a schematic view of the microfluidic detection device of example 1 (showing a second PDMS slab and a functionalized substrate).

FIG. 3 is a schematic diagram of the ligation reaction scheme and the principle of example 1.

Fig. 4 is a graph of the effect of reaction time and match/mismatch type on LIDA for example 2.

FIG. 5 shows an embodiment3 free Gin-NH₂Or Quin-NH₂Influence on fluorescence intensity (2 h).

FIG. 6 shows free Gin-NH of example 3₂Or Quin-NH₂Influence on fluorescence intensity (4 h).

FIG. 7 is the results of the discrimination studies of example 4 using Gin gBlock and Quin gBlock as samples.

FIG. 8 is a graph of the effect of different concentrations of ligase on LIDA results for example 5 (100 CEU/. mu.L, NEB).

FIG. 9 is a graph of the effect of different concentrations of ligase on LIDA results for example 5 (75 CEU/. mu.L, NEB).

FIG. 10 is a graph of the effect of different concentrations of ligase on LIDA results for example 5(50 CEU/. mu.L, NEB).

FIG. 11 is a graph showing the effect of dilution of the ligase of example 5 on LIDA results (Gin gBlock,1 pg/. mu.L).

FIG. 12 is a graph of the effect of dilution of the ligase from example 5 on LIDA results (N2G, 1 pM).

FIG. 13 shows magnesium ions (Mg) in example 6²⁺) Effect of concentration on LIDA results.

Fig. 14 is a graph of the effect of QSF concentration on LIDA results for example 7.

FIG. 15 shows the results of the mixed sample assay in example 8.

FIG. 16 shows the results of the tests on the ginseng samples X1-X6 of example 9.

FIG. 17 shows the results of examining the ginseng root samples of example 9.

Detailed Description

The reference numbers are as follows: the device comprises a functionalized glass slide 1, a first PDMS flat plate 2, a first microfluidic channel 3, a first sample loading hole 4, a first sample sucking hole 5, a second PDMS flat plate 6, a fixed segment line 7 on the glass slide, a second microfluidic channel 8, a detection site 9, a second sample loading hole 10 and a second sample sucking hole 11.

Example 1

This example illustrates the efficacy of the present detection system and method by taking the detection of SNPs in Panax ginseng and Panax quinquefolium as an example. The inventor finds that ginseng and American ginseng are different at 5 SNP sites and can be used for identifying and distinguishing the two medicinal materials. Wherein, the NO.2SNP is the best SNP locus for distinguishing the ginseng and the American ginseng, and the best detection effect can be obtained. Therefore, this example was conducted to examine and explain the detection of NO.2SNP as an example. The DNA fragment information used in this protocol is detailed in Table 1, and all single-stranded and double-stranded oligonucleotides were purchased from IDT (Coralville, IA, USA). During the experiment, ginseng genomic DNA was extracted using a plant DNA extraction kit (Qiagen). Six ginseng powder samples (X1-X6, Sam Liu of Macan Biotechnology Ltd), eight ginseng plant root samples (2 in China, 3 in the United states, 3 in Korea). T4 DNA ligase was purchased from NEB and ThermoFisher.

Table 1: DNA fragment information

Table 1 notes: double underline indicates a single nucleotide polypeptide site; #: ab represents an abasic group and is 1 ', 2' -dideoxyribose, and a phosphate is bonded to the 5 '-end and a hydroxyl group is bonded to the 3' -end.

(1) Production of microfluidic biochip and probe fixation

The procedure for probe immobilization is shown in FIGS. 1 and 2. A PDMS slab with 16 microfluidic channels (referred to as the first microfluidic channel 3), here the first PDMS slab 2, was first sealed to an aldehyde-functionalized slide (75mm x 50mm, i.e. functionalized slide 1) to form a microfluidic biochip (fig. 1, only eight channels shown). Suction (through first loading well 4 and first suction well 5) in a mounting buffer (1.5M NaCl, 0.15M NaHCO)₃) In which the microfluidic channel is filled with a solution containing Gin-NH₂Or Quin-NH₂(25. mu.M) solution of (Gin-NH)₂And Quin-NH₂Not present in the same microfluidic channel) and the solution mixture was incubated for 1h at room temperature. After this time, the solution was aspirated and washed with a blank fixing buffer (1.5M NaCl, 0).15M NaHCO₃) The microfluidic channel is washed. Printed with Gin-NH₂Or Quin-NH₂In (1). As mentioned above, the first PDMS plate 2 is removed and Gin-NH is printed on the functionalized glass slide 1₂Line or/and Quin-NH₂A line (referred to as a stationary section line 7 on the slide). Will have a print of Gin-NH₂Line or/and Quin-NH₂The slide of the thread (functionalized slide 1) is immersed in NaBH₄In solution (0.75mL of 20 XPBS, 5mL of 95% ethanol, approximately 50mg of NaBH₄Diluted with deionized water to a final volume of 20mL), reduced for 15min, then washed with 1 x PBS and dried under nitrogen. The PDMS plate is then washed (to form a second PDMS plate 6), dried and placed on the slide again, and the microfluidic channel on the second PDMS plate 6 (called the second microfluidic channel 8) is to be connected with the Gin-NH printed in the previous step₂Line or/and Quin-NH₂The lines (stationary section lines 7 on the slide) are vertical (fig. 2). And a sample or other reagents are added into the second microfluidic channel 8 through the second sample loading hole 10 and the second sample sucking hole 11 (the second sample loading hole 10 and the second sample sucking hole 11 are positioned at two ends of the same second microfluidic channel 8). The intersection point of the second microfluidic channel 8 and the fixed segment line 7 on the glass slide is a detection site 9, and the fluorescence intensity on the detection site 9 is detected, so that the SNP condition of the sample to be detected can be obtained. In which fig. 1 and 2 only show eight channels on the first PDMS slab 2 and the second PDMS slab 6, and not all microfluidic channels are completely drawn.

The preparation method of the microfluidic biochip comprises the following steps: plates with microfluidic channels were prepared using Polydimethylsiloxane (PDMS) as the starting material, as described in "L.Wang et al, Flexible microarray construction and fast DNA hybridization connected on a microfluidic chip for a gridding house plant detection J.Agric.food Chem,2007,55, 10509-16". Channels (16 channels) are distributed on the first PDMS flat plate and the second PDMS flat plate in parallel, the depth of the channels is 35 μm, and the width of the channels is 150 μm. Through holes (one end is called a sample loading hole, and the other end is called a sample sucking hole) are arranged at two ends of each channel.

The slide (substrate format) was first loaded with 100mL (from 70mL 98% H)₂SO₄And 30mL of 30% H₂O₂Composition) was washed for 15min with Piranha solution (Piranha solution). After washing was complete and the slides were dried, the slides were washed with a solution of APTES (3-aminopropyltriethoxysilane) in ethanol (consisting of 2mLAPTES and 98mL absolute ethanol) in inert N₂The next treatment is carried out for 20 min. After completion of the above treatment, the slide was heated at 120 ℃. The slide was then reacted with 100ml of glutaraldehyde solution for 60 min. The preparation method of the glutaraldehyde solution comprises the following steps: glutaraldehyde was dissolved in 1 × PBS buffer (phosphate buffer), wherein the mass fraction of glutaraldehyde in the solution was 5% by weight. And (4) completing the aldehyde group functionalization of the glass slide through the operation to obtain the functionalized substrate.

(2) Joining process

Mixing 2 Xligase reaction buffer solution, detection fragment (Det), universal fragment (Frag) and Ginseng specific fragment or radix Panacis Quinquefolii specific fragment (GSF or QSF) to form solution I (wherein the concentration of each DNA fragment is 50 μ M). The DNA template (Gin gBlock or Quin gBlock or genomic DNA extracted from plant samples or N2G or N2Q or other samples to be tested) was heated to 95 ℃ and then cooled on ice for 3min before centrifugation. Then, the denatured DNA template (0.5. mu.L) was mixed with solution one (1.0. mu.L, 50. mu.M each of DNA fragments) and T4 DNA ligase (0.5. mu.L, wherein NEB ligase 400 CEU/. mu.L or TF ligase 5U/. mu.L) to obtain a LIDA mixed solution in a final volume of 2. mu.L. The LIDA mixed solution contained 1 Xligase reaction buffer (50mM Tris-HCl, 10mM MgCl)₂1mM ATP, 10mM DTT, pH7.5), 25. mu.M Det, 25. mu.M Frag, 25. mu.M GSF or QSF. Pipetting the LIDA mixed solution up and down several times for thorough mixing, and then pumping the LIDA mixed solution into the second microfluidic channel. The chip is soaked for 2 hours or 4 hours at room temperature, and then the liquid in the channel is sucked. The channels were washed sequentially with 1.0% SDS, 1 XPBS, then filled with streptavidin-Cy5 (50. mu.g/mL, 1 XPBS) for 15min, then washed with 1 XPBS/0.15% Tween-20. The PDMS plates were removed and the slides were washed with 1 x PBS and dried under nitrogen.

In the ligation process, damage-induced DNA amplification (LIDA) was performed, and the reaction scheme and principle thereof are illustrated with reference to fig. 3, taking the detection of SNP sites in ginseng as an example. There are two cycles in the ligation process, in Cycle 1(Cycle 1), a DNA template (from Gin or gQuin or genomic DNA extracted from a plant sample or other sample to be tested, which is denatured to obtain single-stranded DNA with the sense strand as the LIDA-reactive DNA template, here designated as class i template) is hybridized with an immobilized short-site specific probe (Gin-NH2, antisense, immobilized fragment) (step 1.1) and ligation of the detection fragment (Det, antisense) is induced (step 2.1). At this time, the immobilized fragment and the detection fragment are ligated end to end by the action of ligase to form an immobilized long chain. Subsequently, the duplex (formed by the three of the DNA template, the detection fragment and the immobilized fragment) is dissociated (step 3), and the DNA template is separated from the immobilized long chain to repeat cycle 1. Then, in Cycle 2(Cycle 2), the immobilized long strand is hybridized with the specific fragment (i.e. GSF, sense) (step 1.2) and ligated with the universal fragment (Frag, sense) (step 2.2). Subsequently, the duplexes are dissociated (step 3) to provide another DNA template (designated herein as class II template, which is shorter than the class I template) which can be entered into cycle 1. Thus, cycle 1 produced immobilized Det (fluorescence detection) that did not exceed the limit of free Det in solution. Cycle 2 increased the DNA template (for cycle 1, there were two class II and class I templates) to a lesser number that did not exceed GSF or Frag.

(3) Detection process

Fluorescence imaging results were obtained using a fluorescence imager (BioRad) scanning chip, which had resolution and sensitivity consistent with the previously used Typhoon imager.

Example 2: LIDA reaction time study

This example investigated the reaction time of LIDA, which was performed for 2h and 4h after pumping the mixed solution into the second microfluidic channel ((2) connection procedure), see example 1 for the specific experimental procedure. And the DNA template used in this example was derived from a genomic american ginseng sample (the DNA template of this example was obtained after extraction of genomic DNA from american ginseng and subsequent denaturation treatment), using the american ginseng sample as the sole template, and using NEB ligase. Results of the experimentAs shown in fig. 4, the experimental groups (four groups in total) shown in the figure are, from left to right: reaction 2h with GSF and LIDA (group 1), 2h with QSF and LIDA (group 2), 4h with GSF and LIDA (group 3), 4h with QSF and LIDA (group 4). In each set, the left bar data refers to the use of a strip of Gin-NH on a slide₂The signals of the section lines are fixed, and the right column data indicate that the slide glass is used with Quin-NH₂Fixing the signal of segment line. In the figure, asterisks indicate significant differences in results (p)<0.01). From the experimental results, it can be seen that Quin-NH reacts with QSF and Quin template LIDA for 4h compared to 2h₂The signal of the interaction is significantly higher. On the other hand, in Gin-NH₂In the interaction, despite mismatch with the Quin template, there is still signal on the 2h LIDA when GSF is used. Thus, Quin-NH was immobilized₂The presence of the Quin sample can only be identified under the condition of 4h reaction.

LIDA can detect and distinguish genomic templates, PCR products and gBlock only if cycles 1 and 2 are performed simultaneously. If only cycle 1 is performed (i.e., no QSF and Frag are added), both the PCR product used as template (1 pg/. mu.L) and gBlocks (1 pg/. mu.L) are invalid. The only template that functioned only in cycle 1 was ss single stranded oligonucleotides (100nMN2G or N2Q, not 1nM of N2G or N2Q) (data not shown).

Example 3: study of enhanced fluorescence intensity (free fragment added to LIDA Mixed solution)

It is generally accepted that the relatively weak fluorescence intensity is caused by hybridization of the three substances involved in LIDA reaction cycle 1 (DNA template, Gin-NH2 or Quin-NH2, Det). The large size and low concentration of genomic templates would make it difficult to hybridize the triplexes together for assembly. Therefore, a small amount (10nM) of Quin-NH was added to the LIDA mixed solution₂The ligation reaction will be initiated in solution phase, increasing the abundance of ligation products and generating more template for cycle 1 by Frag and GSF for greater extension. Although this procedure will consume Det without producing immobilized Det for fluorescence detection, the benefit of the increased number of templates is to compensate for the weak loss of Det, which is usually excessive (25 μ M). The experimental results of this example are shown in the figure5(LIDA reaction 2h) and FIG. 6(LIDA reaction 2 h). In this example, free Gin-NH was also added to the LIDA mixed solution using quin sample as the sole template and NEB ligase₂Or Quin-NH₂(10 nM). In fig. 5, there are four experimental groups from left to right, respectively: using GSF without free Gin-NH₂Or Quin-NH₂(group 1) use of QSF without free Gin-NH₂Or Quin-NH₂(group 2) addition of free Gin-NH Using GSF₂(group 3) addition of free Quin-NH Using QSF₂(group 4). In fig. 6, there are four experimental groups from left to right, consistent with fig. 5, except for the LIDA reaction times. For FIGS. 5 and 6, in each set, the left histogram data refers to the use of a strip of Gin-NH on the slide₂The signals of the section lines are fixed, and the right column data indicate that the slide glass is used with Quin-NH₂Fixing the signal of segment line. As can be seen from the experimental data, when a small amount of Gin-NH was added₂/Quin-NH₂When the fluorescence intensity obtained was greatly increased. Thus, free Gin-NH₂Or Quin-NH₂The joining may initiate a connection. Thus, all subsequent experiments will use this aminated oligonucleotide (Gin-NH)₂/Quin-NH₂) Adding into LIDA mixed solution (adding free Gin-NH into GSF-containing LIDA mixed solution)₂And adding free Quin-NH into the LIDA mixed solution containing QSF₂)。

Example 4: distinguishing ratio Studies Using Gin gBlock and Quin gBlock as samples

Gin gBlock and Quin gBlock were used as templates in LIDA, for experimental procedures see example 3. The results of the observation are shown in fig. 7, from left to right, for four experimental groups: quin gBlock was used as DNA template and GSF was used (set 1), Quin gBlock was used as DNA template and QSF was used (set 2), Gin gBlock was used as DNA template and GSF was used (set 3), Gin gBlock was used as DNA template and QSF was used (set 4). In each set, the left bar data refers to the use of a strip of Gin-NH on a slide₂The signals of the section lines are fixed, and the right column data indicate that the slide glass is used with Quin-NH₂Fixing the signal of segment line.

When both the free oligonucleotide and the immobilized oligonucleotide were ginseng (group 3), Gin gBlock was at Gin-NH₂The fluorescence intensity on the lanes was strongest with a Differentiation Ratio (DR) of 9.7 (in group 3, Gin-NH was used)₂Fluorescence intensity of the column divided by Quin-NH₂The DR value was obtained from the fluorescence intensity of the column). Similarly, Quin gBlock was performed in Quin-NH when both free and immobilized oligonucleotides were in Panax quinquefolium (group 2)₂The intensity of fluorescence on the lanes was high with a discrimination ratio of 3.5 (in group 2, Quin-NH was used)₂Fluorescence intensity of the column divided by Gin-NH₂The DR value was obtained from the fluorescence intensity of the column). In the case where the two SNP fragments are not complementary (i.e., QSF for immobilized Gin-NH)₂And application of GSF to immobilized Quin-NH₂) Whichever template is used, the fluorescence intensity is low to background. Because each iteration of the LIDA cycles 1 and 2 must experience a mismatch when the SNP fragments do not match, resulting in low fluorescence intensity.

Example 5: effect of ligase concentration

To determine whether ligase (NEC) below 100 CEU/. mu.L could be used to save costs, we tested three lower ligase concentrations (25, 50 and 75 CEU/. mu.L), the assay method being referred to in example 4, where only the enzyme type and concentration were adjusted. Although the liquid phase LIDA was used at lower concentrations, the solid phase LIDA showed a sharp decrease in fluorescence intensity compared to 100 CEU/. mu.L (FIGS. 8-10), and in 25 CEU/. mu.L ligase, the signal was not detectable, so we continued to use the working concentration of 100 CEU/. mu.L.

Since both ligase stocks (400 CEU/. mu.L for the first or 2000 CEU/. mu.L for the second) contained 50% glycerol (v/v), we analyzed the effect of viscosity reduction upon dilution. 2000 CEU/. mu.L and 400 CEU/. mu.L ligase stocks were purchased from New England Biolabs (NEB). The stock solution of 2000 CEU/. mu.L (group 1 diluted with water, group 2 diluted with glycerol) was first diluted with water or 50% glycerol (v/v), and the stock solution of 400 CEU/. mu.L was used directly (group 3), the final enzyme concentration was 100 CEU/. mu.L, and the glycerol content became 12.5%. Observations indicate that none of the ligase dilutions prepared from the 2000 CEU/. mu.L stock solutions showed any evidenceFluorescence signal, signal only observed when 400 CEU/. mu.L stock was used (FIG. 11). Therefore, we believe that the first is a 400 CEU/. mu.L stock solution to be preferred. In FIG. 11, Gin-NH was immobilized₂Fluorescence intensities of Gin solution phase oligonucleotide and Gin template (Gin gBlock,1pg/μ L): the 2000CEU/L ligase stock (NEB) was diluted with water, the 2000CEU/L ligase stock (NEB) was diluted with 50% glycerol, or the 400CEU/L ligase stock (NEB). The final concentration of NEB ligase was 100 CEU/L. In FIG. 12, Gin-NH was immobilized₂Fluorescence intensities of solution-phase Gin oligonucleotide and Gin template (N2G, 1 pM): directly using 5U/. mu.L ligase stock (TF) (final concentration 1.25U/. mu.L), water 1: 1, diluted with 50% glycerol 1: 1, and then used for reaction (final concentration 0.625U/L or 125 CEU/. mu.L), or NEB ligase (final concentration 100CEU/L) was used directly. As a result, it was found that TF ligase at a final concentration of 1.25U/. mu.L was better than NEB ligase at a final concentration of 100 CEU/. mu.L.

Example 6: mg (magnesium)²⁺Influence of concentration

In this example, Mg was detected²⁺The effect of concentration on assay efficacy and the results are shown in figure 13. The QSF, Quin gBlock group strength is significantly weaker than the GSF, Gin gBlock group strength. The QSF and Quin gBlock group refers to the use of QSF and Quin gBlock in the connection process and the use of a glass slide with Quin-NH₂Fixing the fragment line signal, containing free Quin-NH in LIDA mixed solution₂(10 nM); the GSF, Gin gBlock group refers to the use of GSF, Gin gBlock, with Gin-NH on the slide during ligation₂Fixing the signals of the segment lines, containing free Gin-NH in LIDA mixed solution₂(10 nM). The difference between these two LIDA reactions is that QSF, Quin gBlock has a pair of AT base pairs AT the SNP site, while GSF, Gin gBlock has a pair of GC base pairs AT the SNP site. This indicates that association of chains on the solid surface is the rate-limiting step of the reaction. Part of this problem may be the negative charge on the slide surface, which has not been considered before. To solve this problem, more MgCl is added₂To increase the ionic strength (Na + is a ligase inhibitor, so NaCl cannot be added). In FIG. 13, the ligation buffer already contained 10mM Mg²⁺Due to the factThis will add additional Mg²⁺Addition to buffer gave 15, 20, 25 and 30 mM. Each Mg²⁺The concentration gradient contained GSF, Gin gBlock group (left) and QSF, Quin gBlock group (right). The GSF, Gin gBlock groups are measured with the left Y-axis, and the QSF, Quin gBlock groups are measured with the right Y-axis. We observed that the strength of the QSF, Quin gBlock groups varies with Mg²⁺The concentration increased until 20mM, and then the intensity decreased. On the other hand, the GSF, Gin gBlock group is not limited by Mg²⁺Effect, although the intensity drops to near background noise when the concentration reaches 25 mM. Thus, 20mM Mg²⁺For all subsequent LIDA experiments.

Example 7: effect of QSF concentration

Even if the template is perfectly complementary to the oligonucleotide, QSF is in Quin-NH₂The signal on the trace is always weak. The intensity increased significantly when the concentration of QSF increased from 25 μ M to 30 μ M (as shown in figure 14). In this embodiment, the four QSF groups in FIG. 14 refer to the use of QSF, Quin gBlock, with Quin-NH on the slide during the attachment process₂Fixing the fragment line signal, containing free Quin-NH in LIDA mixed solution₂(10 nM); a GSF panel is prepared by using GSF, Gin gBlock, and Quin-NH on glass slide during connection₂Fixing the signals of the segment lines, containing free Gin-NH in LIDA mixed solution₂(10 nM). As can be seen from the results of the experiments, the concentration of QSF was in response to Quin-NH₂The Quin gBlock signal on the lane may have an effect. Quin gBlock (1.0 pg/. mu.L) was used as template, to which 10nM free Quin-NH was added₂100CEU ligase, soaking and incubating for 4 h. The last data bar is associated with 25 μ M GSF, which is in Gin-NH₂Line 1.0 pg/. mu.L Gin gBlock was used. Thus, in subsequent LIDA experiments, the GSF and QSF fragments increased in concentration from 25. mu.M to 30. mu.M, while the other fragments remained at 25. mu.M (both referring to the final concentration in the LIDA ligation mix solution).

Example 8: detection for mixed samples

Pure (100%) and mixed (50% each) template solutions were prepared and tested using the LIDA method. As shown in FIG. 15, in 100% of the templates, onlyOne signal is very strong, namely a high GSF signal of p.gin (ginseng) and a high QSF signal of p.quin (american ginseng). On the other hand, in the 50% hybrid template, both the GSF and QSF signals are strong. In fig. 15, from left to right, there are: a100% Gin gBlock panel, a 100% Quin gBlock panel, and a 50% Gin gBlock + 50% Quin gBlock panel were used (the concentration of the genome was 1.0 pg/. mu.L). In FIG. 15, GSF, free Gin-NH was used on the left side of each group₂Fixed with Gin-NH₂The micro flow channel of (2); right side use QSF, free Quin-NH₂On which Quin-NH is immobilized₂The micro flow channel of (1). The experimental general process is as follows: the immobilized fragments were immobilized on the chip for 1h, ligated for 4h (LIDA reaction). Finally, LIDA mixed solution (2. mu.L total) was composed of 1. mu.L oligonucleotide-containing solution-0.5 uL template and 0.5. mu.L ligase (5U/. mu.L TF). The first solution containing oligonucleotides had the following composition: 50 μ M Det, 50 μ M Frag, 60 μ M GSF or QSF, 20nM Gin-NH₂Or Quin-NH₂、40mM MgCl₂(wherein 20mM is from 2 Xligase reaction buffer and the other 20mM is from additional additions).

Example 9: detection of plant samples

Unknown genomic ginseng samples were analyzed for X1-X6 (see FIG. 16 for results), and their intensity ratios (Gin/Quin, Difference ratio) were calculated. The experimental general process is as follows: extracting genome of ginseng sample X1-X6, and obtaining DNA template (single strand) after denaturation treatment; adding 10nM Gin-NH to the LIDA mixed solution₂Or Quin-NH₂30 μ M GSF or QSF, 25 μ M Det, 25 μ M Frag, 10mM Mg²⁺(additional addition, 10mM Mg in 2 Xbuffer by itself²⁺) 100 CEU/. mu.L ligase (NEB), soaking (LIDA reaction) for 4 h. The calculation method of the distinguishing ratio comprises the following steps: gin detected fluorescence intensity/Quin detected fluorescence intensity. Gin detected LIDA mixed solution containing free Gin-NH₂GSF, and using a catalyst with Gin-NH₂Fixing a segment line; the LIDA mixed solution for detecting the Quin contains free Quin-NH₂QSF, and use of a carrier with Quin-NH₂Fixing the segment line. The experimental results are shown in fig. 16, and the discrimination ratio calculation results are shown in table 2.

Table 2: identifying Gin/Quin discrimination ratios

X1	X2	X3	X4	X5	X6
						0.40	2.20	173.49	0.19	8.36	0.15

In table 2, ratios greater than 2 are for pure ginseng and ratios less than 0.5 are for pure ginseng. The ratio is very large for pure ginseng and very small for American ginseng. A high ratio (e.g., X3) indicates that the sample is likely to be pure p.ginseng, and a low ratio (e.g., X4 and X6) indicates that the sample is likely to be pure p.quinquefolius. The remaining three samples (X1, X2, X5) appeared to be hybrids of the two species.

In FIG. 16, Gin detection and Quin detection were performed for each sample for the fluorescence intensities detected for X1-X6 in order from left to right. The detection can be performed on the same microfluidic chip. Gin detected as: the extracted sample genome is a DNA template source; in the LIDA mixed solution, use Det, Frag, GSF, free Gin-NH₂(ii) a Using Gin-NH immobilised on microfluidic chips₂A micro flow channel. Quin testThe test result is as follows: the extracted sample genome is a DNA template source; in the LIDA mixed solution, Det, Frag, QSF, free Quin-NH were used₂(ii) a Quin-NH2 microchannels were used, immobilized on microfluidic chips.

We also obtained several samples of ginseng roots. The root samples were powdered, plant DNA was extracted, and then analyzed using LIDA. Samples of American ginseng (American ginseng) were labeled AmG (section from wingowski), AmG2 (section from wingowski) and AmG3 (whole root from canlux), respectively. Korean ginseng (Korean ginseng) sample was labeled KorG (slice from Vancouver), KorG2 (ginseng tea); furthermore, RedG is a slice of red ginseng from China. The ginseng (Chinese ginseng) samples were labeled as AuChG (whole root from china) and AuChG2 (whole root from china). The experimental results are shown in fig. 17, although AmG, AmG2 and AmG3 are considered as American ginseng (American ginseng), only AmG2 and AmG3 are true. AuChG, AuChG2, KorG, KorG2 and RedG, which are considered to be ginseng (Chinese ginseng), are also only in accordance with AuChG2 and RedG.

The foregoing is merely an example of the present invention and common general knowledge in the art of designing and/or characterizing particular aspects and/or features is not described in any greater detail herein. It should be noted that, for those skilled in the art, without departing from the technical solution of the present invention, several variations and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.

SEQUENCE LISTING

<110> Aoga Zhuhai Biotech Co., Ltd

<120> a system and method for identifying single nucleotide polymorphism of Panax plant

<130> 2020-10-11

<160> 10

<170> PatentIn version 3.5

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Claims (10)

1. A system for identifying single nucleotide polymorphisms of a Panax plant, comprising: the kit comprises a detection unit for detecting a DNA template, wherein the detection unit comprises a fixed fragment, and the DNA template and the fixed fragment are single-stranded DNA; the 5 'end of the immobilized fragment is immobilized on a solid support, the first base at the 3' end of the immobilized fragment is an antisense SNP base, and the immobilized fragment is complementarily paired with a DNA template.

2. The system according to claim 1, wherein the single nucleotide polymorphism of Panax species is identified by: the sequence of the DNA template is a section of DNA sequence on a sense strand of a gene containing SNP sites, and the SNP sites of the gene are positioned on the DNA template; the detection unit further comprises a LIDA mixed solution, wherein the LIDA mixed solution comprises T4 DNA ligase, a buffer solution, and a detection fragment, a universal fragment and a specific fragment which are single-stranded DNA; the 5 'end of the detection fragment is connected with a non-base functional group, the 3' end of the detection fragment is modified with a reporter group, and the detection fragment is in complementary pairing with the DNA template; the second base at the 5' end of the specific fragment is a sense SNP base, and the first base is an unpaired base.

3. The system according to claim 2, wherein the polynucleotide polymorphism of Panax species is selected from the group consisting of: the DNA template comprises a class I template and a class II template; the type I template is from a sample to be detected; the 3 'end of the universal fragment is connected with the 5' end of the specific fragment to form a II-type template; the 5 'end of the detection fragment is connected with the 3' end of the fixed fragment and then is complementarily matched with the DNA template, and the unpaired base and the function group without the base are allelic.

4. The system according to claim 3, wherein the polymorphism of the Panax plant is one of the single nucleotide polymorphisms: the non-basic functional group is 1 ', 2' -dideoxyribose; the solid support is an aldehyde-based glass slide of the microfluidic biochip.

5. The system for identifying single nucleotide polymorphisms of a Panax plant according to any one of claims 1-4, wherein: the detection unit is used for detecting SNP sites of dammarenediol-II synthetase genes of ginseng and American ginseng; when the SNP site of the ginseng is detected, the antisense SNP base of the fixed fragment is the base positioned on the antisense strand of the SNP site of the ginseng, and the sense SNP base of the specific fragment is the base positioned on the sense strand of the SNP site of the ginseng; when the SNP locus of American ginseng is detected, the antisense SNP base of the fixed fragment is the base positioned on an antisense strand on the SNP locus of American ginseng, and the sense SNP base on the specific fragment is the base positioned on a sense strand on the SNP locus of American ginseng.

6. The system according to claim 5, wherein the single nucleotide polymorphism of Panax species is identified by:

core of fixed fragment for detecting SNP site of ginsengThe nucleotide sequence is: NH (NH)₂-5’-CAATTTAAG-3’；

The nucleotide sequence of the fixed fragment for detecting the SNP locus of the American ginseng is as follows: NH (NH)₂-5’-CAATTTAAA-3’；

The nucleotide sequence of the specific fragment for detecting the SNP locus of the ginseng is as follows: 5'-TCTTAAATTG-3', respectively;

the nucleotide sequence of the specific fragment for detecting the SNP locus of the American ginseng is as follows: 5'-TTTTAAATTG-3', respectively;

the nucleotide sequence of the detection fragment is as follows: 5 '-AbCACTTTC-3' -bio;

the nucleotide sequence of the universal fragment is as follows: 5 '-GAAAGTG-3';

wherein NH₂Represents a 5 'end amino modification, bio represents a 3' end biotin modification, and Ab represents a functional group without a base group.

7. The system according to claim 6, wherein the single nucleotide polymorphism of Panax species is identified by: the LIDA mixed solution also comprises a free fragment, and the free fragment is consistent with the fixed fragment; the LIDA mixed solution also comprises magnesium ions.

8. The method according to claim 5, wherein the method comprises the steps of: the method comprises the following steps:

(1) probe immobilization and template acquisition: immobilizing the immobilized fragments on a solid support; obtaining a class i template from a sample;

(2) and (3) connecting:

comprises a connection process for detecting the SNP sites of the ginseng: contacting the LIDA mixed solution and the class I template with the fixed fragment and carrying out LIDA ligation reaction to obtain a first ligation product; the antisense SNP base of the fixed fragment is a base positioned on an antisense chain on the SNP site of the ginseng;

also comprises a connection process for detecting the SNP locus of the American ginseng; enabling the LIDA mixed solution and the class I template to contact the fixed fragment and carrying out LIDA ligation reaction to obtain a second ligation product; the antisense SNP base of the fixed fragment is a base positioned on an antisense chain on the SNP locus of the American ginseng;

(3) signals of the first ligation product and the second ligation product are detected separately.

9. The authentication method according to claim 8, wherein: the duration of the LIDA ligation reaction was 4 h.

10. The authentication method according to claim 9, wherein: in the step (3), obtaining a fluorescence signal of the first ligation product, wherein the intensity of the fluorescence signal is F1; obtaining a fluorescent signal of the second ligation product, the intensity of the fluorescent signal being F2;

when F1/F2 > 2, the sample contains pure ginseng;

when F1/F2 is less than 0.5, the sample contains pure American ginseng;

when the ratio of F1/F2 is more than or equal to 0.5 and less than or equal to 2, the sample contains ginseng and American ginseng.

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