CN117051084A

CN117051084A - Nucleic acid detection method using non-modified probe and application thereof

Info

Publication number: CN117051084A
Application number: CN202311053608.6A
Authority: CN
Inventors: 贾艳伟; 沈韧; 麦沛然; 马许愿
Original assignee: University of Macau
Current assignee: University of Macau
Priority date: 2023-08-21
Filing date: 2023-08-21
Publication date: 2023-11-14

Abstract

The invention discloses a nucleic acid detection method using a non-modified probe and application thereof, and relates to the technical field of nucleic acid detection. The invention utilizes the non-modified probe to match with the double-stranded DNA binding dye, and then judges the complementary degree of the target sequence and the probe through a melting curve, thereby realizing the nucleic acid detection. The method provided by the invention can reduce the detection cost, shorten the detection time and promote the large-scale application of gene mutation screening in resource-limited areas while ensuring the detection accuracy of nucleic acid.

Description

Nucleic acid detection method using non-modified probe and application thereof

Technical Field

The invention relates to the technical field of nucleic acid detection, in particular to a nucleic acid detection method using a non-modified probe and application thereof.

Background

Nucleic acids are used as genetic material of organisms, which when mutated have various effects on human health. On the one hand, mutation of genes occurring in humans may cause functional changes of related proteins, which in turn cause gene-related diseases. On the other hand, when DNA or RNA in the pathogen is mutated, the toxicity, transmissibility, drug resistance and other properties of the pathogen are possibly changed, so that the pathogens such as viruses, bacteria and the like can break through the human defense line and cause another infectious disease epidemic situation.

Based on this, researchers have developed various techniques for detecting gene mutations. The most common method among these is to detect the presence of a specific target sequence using a fluorescently labeled oligonucleotide probe. For example, in Taqman probes, a fluorescent group and a quenching group are modified at both ends of the oligonucleotide probe, respectively. When a specific target sequence exists, the Taqman probe is combined with the specific target sequence, under certain conditions, the probe is cut, and the distance between the fluorescent group and the quenching group is increased, so that a fluorescent signal is amplified. Other fluorescent probes, such as molecular beacon probes, employ different strategies to trigger conformational changes and fluorescent signal changes, but like Taqman probes, these probes are modified with a fluorescent group and a quenching group. Such modifications not only result in high cost of synthesis of the probes, but also make the probes difficult to preserve (light shielding, repeated freeze thawing should be avoided).

In addition to fluorescence-based detection methods, researchers have invented other signal-based nucleic acid mutation detection methods. For example, by using surface plasmon resonance techniques, the binding of an unmodified DNA probe to a specific target sequence on a metal surface can trigger a shift in resonance angle, and the DNA hybridization process can be monitored by measuring the change in resonance angle over time. In addition, electrochemical signals can be utilized to modify a DNA probe with ferrocene on the surface of a gold electrode, and the combination of the probe and a target sequence causes conformational change, so that the distance between the ferrocene and the gold electrode is changed, and the detected electric signal is enhanced or weakened accordingly, thereby achieving the purpose of detecting target nucleic acid. However, both surface plasmon resonance technology and electrochemical sensing technology require elaborate surface modifications and complex sensing modules, making these technologies very limited in practical applications.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide a nucleic acid detection method using a non-modified probe and application thereof. The nucleic acid detection method can reduce the detection cost, shorten the detection time and promote the large-scale application of gene mutation screening in resource-limited areas while ensuring the detection accuracy of nucleic acid through a non-modified probe, a stepwise reaction and a data standardization method.

The invention is realized in the following way:

in a first aspect, the present invention provides a method for detecting nucleic acid using a non-modified probe, comprising: carrying out asymmetric amplification on target nucleic acid, then adding a reaction solution containing an unmodified probe and a double-stranded DNA binding dye into the amplified system, gradually increasing the temperature, monitoring fluorescent signals of the double-stranded DNA binding dye to obtain a melting curve, and obtaining sequence information of the target nucleic acid by analyzing the melting curve;

the melting curve comprises a first melting peak generated at a lower temperature and a second melting peak generated at a higher temperature;

analyzing the melting curve comprises the steps of taking a second melting peak as a reference peak, normalizing the analysis result of the melting curve, and judging the result according to the normalized first melting peak to obtain the sequence information of the target nucleic acid;

the above-mentioned standardized method is a two-dimensional standardized method, which includes:

(1) Normalizing the change in fluorescence intensity: setting the temperature of thoroughly melting the double-chain amplification product as a base line, setting the corresponding-dF/dT value as 0, setting the peak value of a reference peak, namely the-dF/dT value thereof as 1, and expressing the height of a first melting peak as the concentration ratio of the single-chain product to the double-chain product in the amplification product;

(2) Normalizing the temperature: the starting temperature is set to a fixed temperature close to room temperature and the reference peaks in the different samples are set to the same melting temperature accordingly.

In some embodiments, the asymmetric amplification described above comprises PCR amplification using non-limiting primers and limiting primers to obtain a mixture comprising single-stranded and double-stranded amplification products.

In some embodiments, the concentration of the unmodified probe is not less than one-half the concentration of the non-limiting primer after addition to the amplified system.

In some embodiments, the length of the unmodified probe is 12-36 nt.

In some embodiments, the length of the unmodified probe is 12-28 nt.

In some embodiments, the Tm value of the unmodified probe is 45 ℃ to 73 ℃.

In some embodiments, the Tm value of the unmodified probe is 50 ℃ to 70 ℃.

In some embodiments, the reaction solution further comprises an amplification inhibitor, wherein the amplification inhibitor comprises EDTA.

In some embodiments, the EDTA is added to the amplified system at a concentration of 0 to 10mM.

In some embodiments, the double-stranded DNA binding dye described above comprises LCGreen, evaGreen and SYBR Green I.

In some embodiments, the concentration of the double-stranded DNA binding dye is 1× to 8×.

In a second aspect, the present invention provides a non-modified probe for use in melt curve analysis, wherein the non-modified probe is a single stranded oligonucleotide, which does not have any modifying groups at both ends.

In some embodiments, the non-modified probes described above are added to the reaction system after amplification is complete.

In some embodiments, the length of the unmodified probe is 12-36 nt.

In some embodiments, the length of the unmodified probe is 12-28 nt.

In some embodiments, the Tm value of the unmodified probe is 45 ℃ to 73 ℃.

In some embodiments, the Tm value of the unmodified probe is 50 ℃ to 70 ℃.

In a third aspect, the present invention provides a nucleic acid detecting chip on which the above-described non-modified probe is loaded.

In a fourth aspect, the present invention provides a nucleic acid detection kit comprising the above-described unmodified probe.

In a fifth aspect, the present invention provides the use of the above-described nucleic acid detection method for detecting a mutation in a gene.

The invention has the following beneficial effects:

the invention utilizes the non-modified probe to match with the double-stranded DNA binding dye, and then judges the complementary degree of the target sequence and the probe through a melting curve, thereby realizing the nucleic acid detection. The method provided by the invention can reduce the detection cost, shorten the detection time, reduce the requirements on an optical detection system and a temperature control system and promote the large-scale application of gene mutation screening in resource-limited areas while ensuring the detection accuracy of nucleic acid.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of the detection of nucleic acid mutations by a non-modified probe according to the present invention;

FIG. 2 is a graph showing the results of the MCA result normalized to have a consistent melting temperature for the target peak at different temperature configurations in Experimental example 1;

FIG. 3 shows the presence of probe-directed amplification and the effect of EDTA elimination amplification during MCA of Experimental example 2;

FIG. 4 is a comparison of MCA results using non-modified probes of different lengths in Experimental example 3;

FIG. 5 is a comparison of the results of the non-modified probe MCA with different matching degree with the target sequence in experimental example 4;

FIG. 6 is a graph showing the effect of using different types of double-stranded DNA binding dyes at different concentrations on MCA results in Experimental example 5.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Among the numerous nucleic acid analysis methods, a fluorescent signal-based detection method is the mainstream, and an oligonucleotide probe having a fluorescent group and a quenching group modified therein is most often used for the detection of nucleic acid mutations. When the probe binds to a specific target sequence, the distance between the fluorescent group and the quenching group changes, thereby inducing a change in the fluorescent signal, so that information of the target sequence can be obtained from the change in the fluorescent signal. The method has high specificity, however, the modification group leads to high synthesis cost of the probe, which prevents the large-scale application of the probe in genetic variation screening.

Fusion curve analysis using double-stranded DNA binding dyes is a great cost advantage over the probe method described above. In melting curve analysis, the temperature rise is accompanied by denaturation and melting of double-stranded DNA, and the double-stranded DNA binding dye is detached, thereby causing a decrease in fluorescence signal. Since the thermal stability of double-stranded DNA is determined to the greatest extent by the degree of complementarity between the two DNA strands constituting it, sequence information of a specific nucleic acid can be extracted from the change in fluorescence signal with temperature. However, DNA dye-based melting curve analysis has lower specificity than the probe method described above, and is generally difficult to distinguish from single nucleotide mutations and the like. Wittwer et al combine the advantages of both methods and invent a so-called "unmodified" probe. However, the probes of this invention are not unmodified. In order to avoid interference of the probe to amplification, a blocking group is modified at the 3' end of the probe to avoid amplification by using the DNA polymerase as an anchor point.

Based on this, the inventors of the present invention devised a non-modified probe and used for nucleic acid detection by a method as shown in FIG. 1, which specifically comprises:

s1, carrying out asymmetric amplification on a target sequence.

Asymmetric amplification: with unequal amounts of a pair of primers, after several cycles, the low concentration of the primers is consumed and subsequent cycles only produce high concentrations of the primer extension products, resulting in large amounts of single stranded DNA (ssDNA), which are referred to as limiting and non-limiting primers, respectively. Because the two primers used in the PCR reaction are different in concentration, they are called asymmetric amplification.

In the present invention, the ratio of the restriction primer to the non-restriction primer is not limited as long as single-stranded and double-stranded products can be amplified. Other amplification conditions may be adjusted according to the specific sequence detected, and the invention is not limited thereto.

S2, adding an unmodified probe and a double-stranded DNA binding dye into the system after S1 amplification.

The non-modified probe in the present invention is a single-stranded oligonucleotide having no modification group at both ends.

The double-stranded DNA binding dye is capable of non-specifically binding to double-stranded DNA and, upon binding, fluoresces, and when double-stranded DNA dissociates, the signal decreases rapidly, indicating an increase in product by the fluorescent dye. The double-stranded DNA binding dye in the invention comprises, but is not limited to SYBR Green I, LC Green, eva Green and the like, and the type of the double-stranded DNA binding dye can be adjusted according to actual needs by a person skilled in the art; the concentration of the double-stranded DNA binding dye used may be 1X, 2X, 3X, 4X, 6X or 8X, or may be adjusted to other concentrations as required.

In the present invention, after adding the unmodified probe and the double-stranded DNA binding dye to a system containing the single-stranded and double-stranded amplification products, the unmodified probe is bound to the single-stranded amplification product, and the double-stranded DNA binding dye is bound to the double-stranded region after the double-stranded amplification product and the unmodified probe are bound to the single-stranded amplification product, respectively.

S3, gradually heating the mixed system in the S2 to obtain a melting curve, and then performing Melting Curve Analysis (MCA), wherein the MCA adopts a reference peak data standardization method.

Since the degree of complementarity between a probe and a particular target sequence affects the thermal stability of the probe-target sequence complex, the degree of complementarity of the target sequence to the probe can be determined by the melting temperature (Tm) of the complex in the results of a melting curve analysis.

As shown in FIG. 1 (a), as the temperature increases, the probe is detached from the target sequence, and the DNA dye originally bound to the double-stranded region is detached, so that fluorescence tends to decrease drastically, and a melting peak at a lower temperature is generated correspondingly. As the temperature continues to rise, the double-stranded amplification product is denatured, and a large amount of DNA dye falls off, resulting in a further dramatic drop in fluorescence, which corresponds to a melting peak at a higher temperature. As shown in fig. 1 (b), the second melting peak is used as a reference peak, the analysis result of the melting curve is normalized, and the result is interpreted according to the normalized first melting peak, so as to obtain the related information of the target sequence.

The reference peak data normalization method adopted in the invention is a novel two-dimensional normalization method, which normalizes the MCA result. As shown in fig. 1 (b), in the MCA results exhibited in the form of melting peaks, the second melting peak results from melting of the double-stranded amplification product at a higher temperature. The melting peak is used as a reference peak, and data are standardized so that the peak values of all the reference peaks are consistent and the corresponding melting temperatures are consistent, and on the basis, the first melting peak is analyzed to judge the complementation degree of the probe and the specific target sequence.

The two-dimensional normalization method comprises normalization of two dimensions of fluorescence intensity and temperature, and specifically comprises the following steps:

(1) Normalizing the change in fluorescence intensity: the temperature at which the double-stranded amplification product was completely melted was set as a baseline, the corresponding-dF/dT value was set to 0, and the peak value of the reference peak, i.e., its-dF/dT value was set to 1.

The change of the fluorescence intensity is normalized, and the result deviation caused by different exposure intensities can be eliminated. After fluorescence intensity normalization, the first melting peak is highly indicative of the concentration ratio of single-stranded to double-stranded products in the amplified product.

Due to the near room temperature, at the initial temperature, the non-uniformity of heating of the individual samples is negligible and the accuracy of the temperature sensor is also very high. By the above temperature normalization process, the non-uniformity of heating of different samples in the same test, the error of the temperature sensor, and the difference of heating conditions between different tests, the deviation of the results caused by these temperature-related factors is eliminated.

The melting temperature in the MCA results is indicative of the thermal stability of the probe-target sequence complex, and thus of the degree of complementarity of the probe to the target sequence. The effect of the temperature control system on MCA results is self-evident. Conventional MCA methods typically require precise, fine temperature control. The invention eliminates the result inconsistency caused by factors such as inaccurate temperature, uneven temperature distribution and the like through standardized treatment, and realizes high-resolution detection of the nucleic acid variant by using a very simple temperature control system.

The invention decomposes the complex reaction into a plurality of step reactions such as amplification, probe addition, fusion curve analysis and the like, realizes the non-modification of the probe and does not generate any interference to the amplification process. In contrast to the conventional method in which all reagents are previously added to the reaction tube to avoid contamination by later uncapping, the non-modified probe inevitably interferes with the amplification process, the present invention breaks down the complex multi-step reaction into multiple step reactions, and the reagents required for each step are added before the specific reaction process. In this way, the unmodified probe will not affect the earlier amplification process, and the efficiency of each reaction process can be optimized.

The invention can be performed on a Biorad CFX96 real-time fluorescence PCR instrument using an Off-chip reaction. However, to avoid contamination of the amplified products by the action of adding additional reagents after amplification, the present invention is preferably used with specific droplet manipulation techniques. Taking a Digital Microfluidic (DMF) technology as an example, on a digital microfluidic platform, liquid drops containing a sample are soaked in an oil environment, the liquid drops can freely and independently move through a programmed driving electrode, the fusion of the liquid drops can not cause pollution, and the step-by-step reaction can be realized.

Based on the foregoing, the inventors have further optimized the above detection method to obtain a better detection effect, where the optimized content is as follows:

(1) Avoiding the interference of the amplification reaction taking the probe as the anchor point on the analysis result of the melting curve.

Since the 3' end of the non-modified probe is not modified at all, the DNA polymerase can synthesize DNA by using the non-modified probe as an anchor point, and the non-modified probe is equivalent to an amplification primer. However, this phenomenon increases the complexity of the experiment. During MCA, even if DNA melting is completed within a few minutes, probe-anchored amplification is likely to occur.

To overcome this problem, the inventors propose two solutions, one of which is: an amplification inhibitor including EDTA is added simultaneously with the addition of the unmodified probe. Through verification, EDTA is used as an amplification inhibitor, can inhibit all amplification reactions in the MCA process, and ensures the consistency of MCA results. Preferably, the concentration of EDTA in the mixed system is 0 to 10mM, more preferably, the concentration of EDTA in the mixed system is 5mM.

The second step is: after each MCA was completed, the chip temperature was allowed to drop to room temperature, and then reheated, and a second round of melting curve analysis was performed on the same sample, and so on. It is verified that if multiple rounds of MCA are performed without EDTA, the same set of samples are repeatedly heated and cooled, probe-directed amplification proceeds more and more fully, and the evolution of multiple rounds of MCA results can also provide matching information between the probe and the target sequence. Therefore, the method can be used as a novel method for detecting nucleic acid by performing multiple rounds of melting curve analysis on a sample without inhibiting amplification.

During the multiple rounds of MCA experiments, the inventors found that the target melting peak in the second round of MCA results was significantly shifted to the right compared to the first round of results when no EDTA was added. When probe-directed amplification is present, the original probe extends to a certain length, and the Tm value of the probe-target sequence complex increases accordingly, which explains the phenomenon that the melting peak of the target shifts to the right. And after EDTA is introduced into the MCA reaction liquid, the MCA result of the second round is completely consistent with that of the first round, and the right shift phenomenon of the target melting peak disappears, so that the effect of EDTA on inhibiting amplification is fully verified.

(2) The unmodified probe is optimized.

In the field of nucleic acid detection, the length and Tm value of a probe are important factors that affect the detection effect. Based on this, the inventors have optimized the length and Tm value of the unmodified probe of the present invention. Experiments prove that when the length of the unmodified probe is 12 nt-36 nt and the Tm value is set to 45-73 ℃, all the probes can express obvious target melting peaks. As the probe length increases, the Tm value measured by MCA increases. As the probe becomes longer, the effect of increasing the Tm value increases with increasing length of 4nt, and the estimated Tm of the probes L32 and L36 is very close, and the measured Tm is the same. Meanwhile, when the probe length reaches 28nt and above, as the Tm of the first melting peak gradually approaches the Tm of the reference peak, the target melting peak starts to be partially fused with the reference peak, and the longer the probe, the more obvious the fusion trend.

In order to obtain a better detection effect, the better probe provided by the invention comprises the following components: the length is between 12nt and 28nt, and the corresponding Tm value is 50-70 ℃.

By the above-mentioned nucleic acid detection method, a non-modified probe which is a single-stranded oligonucleotide having no modification groups at both ends can be obtained. The length of the non-modified probe is 12nt to 36nt, the Tm value is 45 ℃ to 73 ℃, more preferably, the length of the non-modified probe is 12nt to 28nt, and the Tm value is 50 ℃ to 70 ℃.

The non-modified probe can be used for preparing various nucleic acid detection products, such as a nucleic acid detection chip, a nucleic acid detection kit and the like. The products all comprise the non-modified probes, and target nucleic acid can be detected by the nucleic acid detection method. The design or selection of the structures of products such as nucleic acid detection chips, nucleic acid detection kits and the like or other reagents are conventional technical means in the art, and the structures or the selection can be correspondingly adjusted according to actual detection targets, so that the invention is not repeated.

By providing a nucleic acid detection chip or a nucleic acid detection kit for different disease symptoms in different scenes, the user only needs to provide a sample, and the time and effort required by screening the nucleic acid mutation can be greatly saved.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

The present example provides a nucleic acid detection method using a non-modified probe using an Off-chip reaction performed on a Biorad CFX96 real-time fluorescent PCR instrument.

Wherein the amplified target is a sequence with a length of 106bp in human amino hexosaminidase A (human hexosaminidase A) gene G269 isotopomer, and is shown as SEQ ID NO. 1. All the nucleic acid fragments used in this example were synthesized by the manufacturer (China), and the non-modified probes and target sequences involved were synthesized by online software [ (]http://sg.idtdna.com/calc/ analyzer) The melting temperature was estimated as shown in table 1:

TABLE 1 predicted melting temperatures for target sequences, primer sequences and probe sequences

The specific operation steps are as follows:

s1, before amplification, adding an unmodified probe and EDTA on a PCR tube cover;

s2, carrying out asymmetric amplification on a target sequence

The PCR reaction solution contained 1 XSSOFEst EvaGreen premix (Bio-Rad, USA), 1000nM upstream primer (XP) and 50nM downstream primer (LP). For positive samples, 3X 10 was added to the reaction solution ⁵ Template of each copy, and ultrapure water was added to the negative sample reaction solution in place of the template. The amplification procedure was: 2min at 95℃for a hot start, 95℃for 5s, 56℃for 10s for 50 cycles.

After the amplification is completed, a mixture of single-stranded and double-stranded amplification products is obtained.

S3, after amplification, mixing the probe, EDTA and the amplified product

After the amplification, the unmodified probe and EDTA previously added into the PCR tube cap were mixed with the amplified product by centrifugation, and the concentration of the unmodified probe after mixing was 1. Mu.M and the concentration of EDTA (Biyundian, china) was 5mM. The following MCA procedure was: 35 ℃ to 95 ℃, at intervals of 0.5 ℃, each temperature stay for 2s.

S4, collecting signals and processing data

Fluorescence signals were collected by a Biorad CFX96 real-time fluorescent PCR instrument. The obtained fluorescence signal adopts Savitzky-Golay smoothing algorithm to calculate the first derivative of temperature and take negative value to obtain-dF/dT value (F represents fluorescence intensity and T represents temperature), and MCA result can be displayed in the form of melting peak.

And then, carrying out 2-dimensional standardization treatment on the MCA result by a novel data standardization method. In the dimension of fluorescence, the melting peak (i.e., the second melting peak) caused by the double-stranded amplification product was set to 1. The temperature after complete denaturation of the double-stranded amplification product was set as a baseline, and the-dF/dT value corresponding to the baseline was set to 0. And the second melting peak is pulled to the same melting temperature in the dimension of the temperature, so that the temperature can be calibrated, and the inaccuracy of the temperature and the nonuniformity of the temperature distribution are eliminated.

The test results are shown in fig. 4 and 5.

Example 2

The present example provides a nucleic acid detection method using a non-modified probe, which employs an on-chip PCR reaction. Wherein the amplification targets are the same as in example 1, the specific operation steps are as follows:

s1, carrying out asymmetric amplification on a target sequence

The PCR reaction solution contained 1 XSSOFEst EvaGreen premix (Bio-Rad, USA), 1000nM upstream primer (XP) and 50nM downstream primer (LP). For positive samples, 3X 10 was added to the reaction solution ⁵ Template of each copy, and ultrapure water was added to the negative sample reaction solution in place of the template. The amplification procedure was: 2min at 95℃for a hot start, 95℃for 5s, 56℃for 10s for 50 cycles. The above reagents were added to a digital microfluidic chip, and droplets on the chip were surrounded by 3cSt of silicone oil after loading, and the temperature during PCR was controlled by a Bioer thermal cycler (Bioer, china).

S2, after the reaction amplification is finished and the chip is cooled to normal temperature, moving a liquid drop containing an amplification product through a driving electrode, mixing the liquid drop with a liquid drop containing an unmodified probe, EDTA and EvaGreen dye, wherein the concentration of the unmodified probe after mixing is 1 mu M, the concentration of EDTA (Biyun, china) is 5mM, and the concentration of EvaGreen dye (Biotechnology, U.S.) is 4X. In the subsequent MCA process, a heating plate with direct current power supply is adhered to the bottom of the chip to heat the sample, and a K-type film temperature sensor is adhered to the surface of the chip to measure the temperature.

The digital microfluidic chip is adopted, and the chip is loaded with a non-modified probe. Which has a chromium electrode of size 2mm x 2mm arranged on a glass substrate and a layer of SU-8 3010 of thickness about 10 μm is applied as a dielectric layer by photolithographic techniques over the electrode. Another SU-8 3050 having a thickness of 50-60 μm is also applied over the dielectric layer by photolithographic techniques as a "barrier" against droplet drift. The top plate is also glass, and one surface of the top plate is plated with ITO as a grounding electrode. After the bottom plate and the top plate are subjected to the hydrophobic treatment of Teflon, the upper part and the lower part are assembled together by using double faced adhesive tape with the thickness of 250 mu m, wherein the surface of the top plate with ITO faces downwards. The electrical signal used to drive the electrodes was a sine wave of 90-100vrms,1khz when loading and moving the droplets.

S3, fluorescence signals are collected through a Nikon SMZ1270 fluorescence microscope (Nikon, japan). The obtained fluorescence signal adopts Savitzky-Golay smoothing algorithm to calculate the first derivative of temperature and take negative value to obtain-dF/dT value (F represents fluorescence intensity and T represents temperature), and MCA result can be displayed in the form of melting peak.

After such processing, as shown in FIG. 1, the second melting peaks of all samples will overlap, and the first melting peaks caused by denaturation of the probe-target sequence complex can be aligned using the overlapping second melting peaks as reference peaks, and thus target sequence-related information can be obtained.

In this embodiment, with the chip described above, the addition of reagents by droplet fusion techniques does not introduce contamination, as the droplets are surrounded by oil. Meanwhile, by utilizing a digital microfluidic technology, the liquid drops can be flexibly and independently controlled, the stepwise reaction is realized, and the reaction efficiency of each reaction stage is maximized.

Experimental example 1

To verify the data normalization method of the present invention, this experimental example tested 3 sets of samples: in the group A, a temperature sensor is arranged at the bottom of a chip, and a heating plate is powered by a 15V direct current power supply; in the group B, a temperature sensor is arranged on the surface of a chip, and a heating plate is powered by a 15V direct current power supply; in group C, the temperature sensor is placed on the chip surface, and the heating plate is powered by a 12V DC power supply, as shown in FIGS. 2 (a) and 2 (b). The sequence and detection method in this experimental example are the same as in example 2.

These three groups of samples represent different temperature configurations. The effect of inaccuracy of the temperature sensor on the results can be tested by groups a and B. The effect of different heating efficiencies on the results can be tested by groups B and C, with the sample heating rate being lower when the heater chip is powered by 12V than by 15V. As shown in fig. 2 (c), after normalizing the MCA results, the first melting peak induced by the probe-target complex in the 3 groups of samples had a uniform melting temperature even though the temperature configuration was different.

Under the condition that the heating plate is supplied with constant voltage by a direct current power supply, the temperature sensor is only responsible for recording temperature, and no control algorithm exists between the temperature sensor and the heating plate, the experimental result proves that the data processing method can enable a simple and even fuzzy temperature control system to be applied to distinguishing fine gene differences. Therefore, by using the data normalization method of the present invention, a high-precision temperature control system is no longer a necessary condition for detecting gene mutation. For a personalized temperature control system, the workload brought by debugging the temperature control algorithm is also greatly reduced.

Experimental example 2

To verify the effect of amplification inhibitors and rounds of MCA on the detection effect, the experimental example designed a series of unmodified probes as shown in fig. 3 (a) and tested the results of MCA in the absence of EDTA and in the presence of 5mM EDTA, respectively. The sequence and detection method in this experimental example are the same as in example 2.

As shown in FIG. 3 (b), when EDTA was not added, the Tm value of the probe measured by MCA was greater than the Tm value estimated by software. In particular, for probes P1 and P2, the predicted Tm for P1 is approximately 49℃below the predicted Tm for P2, 53.4 ℃. However, in the MCA results, the Tm of P1 is significantly higher than that of P2. After introducing 5mM EDTA, the MCA reaction was performed, and then the detected Tm values of several probes were found to have the same tendency as the estimated Tm values, i.e., in descending to ascending order of Tm: p1< P2< P3.

As shown in fig. 3 (c), after each MCA was completed, the chip temperature was reduced to room temperature, and the chip was reheated, and a second round of melting curve analysis was performed on the same sample. By comparing the MCA results of the first and second rounds of samples of each group, it was found that the target melting peak in the MCA results of the second round was significantly shifted to the right compared to the results of the first round when no EDTA was added. When the probe-directed amplification exists, the original probe extends to a certain length, and the Tm value of the probe-target sequence complex is correspondingly increased, thereby explaining the phenomenon that the melting peak of the target is shifted to the right. And after 5mM EDTA is introduced into the MCA reaction liquid, the MCA result of the second round is completely consistent with that of the first round, the right shift phenomenon of the target melting peak disappears, and the effect of EDTA on inhibiting amplification is fully verified.

Experimental example 3

In order to verify the influence of the length of the unmodified probe on the detection effect, a series of probes are designed for the target sequence in the embodiment 1, and the lengths are from the shortest 12nt to the longest 36nt, and are specifically divided into: 12nt, 16nt, 20nt, 24nt, 28nt, 32nt and 36nt, the GC content of these probes was designed to be approximately 50%, and the predicted Tm of the probes falls in the range of 45℃to 73℃as shown in Table 1. The detection method in this experimental example is the same as that in example 1 and example 2.

As shown in fig. 4 (a), in the on-chip test, all lengths of probes successfully expressed significant target melting peaks.

For the above samples, the present experimental example also performed an off-chip test on a conventional PCR apparatus.

As shown in FIG. 4 (b), the off-chip results are substantially identical to the on-chip test results. However, in the test tube, the tendency of the target melting peak of the long probe to fuse with the reference peak is more remarkable. When the probe length reaches 32nt or more, the target melting peak is even completely fused with the reference peak, so that a very broad fusion peak appears in the corresponding MCA result. The possibility of causing such a difference in Off-chip versus on-chip test results is the different data processing methods under the two settings.

For on-chip results, a 15-point smoothing algorithm was used when deriving the fluorescence values for temperature. The off-chip result is processed by the software built in the PCR instrument, and although details of the processing algorithm cannot be obtained, the off-chip result can be inferred according to the MCA result, and the smoothing algorithm used by the off-chip result can adopt a larger data window.

Experimental example 4

In order to verify the resolution of the invention for detecting gene mutation, a series of non-modified probes are designed in the experimental example. The sequence and detection method in this experimental example are the same as in example 2.

Wherein, L20 is perfectly matched with the target sequence, and the other three probes respectively have a single nucleotide mutation compared with L20. In L20 (A), one G in L20 is substituted by A; l20 (C) represents a G.fwdarw.C mutation; l20 (T) represents a G.fwdarw.T mutation. As shown in Table 1, the predicted Tm values of L20 and the target sequence are 1-2℃higher than those of the other three probes. And in the three probes which are not completely complementary with the target sequence, the Tm value of the probe is close to that of the target sequence compound, and the difference of the Tm value can be as low as 0.1 ℃.

As shown in FIG. 5 (a), the MCA test result on the chip shows that the high specificity of the invention for detecting nucleic acid mutation can easily distinguish whether the probe is completely matched and complementary with the target sequence, and can also distinguish different single nucleotide mutations. Even for different mutations, the difference in predicted Tm was as low as 0.1 ℃, and the melting peaks corresponding to MCA did not coincide.

While the off-chip MCA results do not exhibit as high a resolution as on-chip. As shown in FIG. 5 (b), the MCA performed in the PCR apparatus can only distinguish whether the probe is perfectly complementary to the target sequence, but cannot distinguish between different mutations with relatively close Tm. From the instructions of the PCR instrument CFX96 (Bio-Rad, usa) used in this experimental example, which had an inter-well temperature difference of ±0.4 ℃ within 10 seconds of heating to 90 ℃, while the MCA procedure used in the present invention had an increase of 35 ℃ to 95 ℃ with an interval of 0.5 ℃, each temperature remaining for 2 seconds, it was concluded that the inter-well temperature difference was greater during the actual MCA. This also explains why off-chip MCA cannot resolve differences between samples when Tm is closer.

As can be seen from the above results, the present invention has higher specificity in detecting nucleic acid mutations than conventional PCR apparatus. The resolution which can be achieved by a special high-resolution melting curve analysis instrument can be achieved by means of a simple temperature element only. In addition, in the present invention, a single melting curve analysis can be completed within 3 minutes, and the time is greatly shortened compared to 30 minutes required on a PCR apparatus. The method for detecting the mutation of the nucleic acid by using the non-modified probe, the stepwise reaction and the data standardization can reduce the detection cost, shorten the detection time and promote the large-scale application of the gene mutation screening in the resource-limited region while ensuring the detection accuracy of the nucleic acid.

Experimental example 5

Double-stranded DNA binding dyes are an important part of the method of the invention, and variations in type and concentration may have an effect on the MCA results. To test this effect, the present experimental example tested the results of melting curve analysis using LCGreen (1 x to 3 x), evaGreen (1 x to 4 x) and SYBR Green I (2 x to 8 x), respectively. The sequence and detection method in this experimental example are the same as in example 2.

As shown in fig. 6, after two-dimensional normalization of experimental data, a target melting peak of the same Tm can be obtained even when different dyes are used. This represents the superiority of the present invention over the witter invention, namely: the dye type is not limited to LCGreen and other saturated probes, so that the application range of the invention is greatly expanded. Since the type and concentration of double-stranded DNA binding dye does not affect the MCA results, it is recommended that the user select the appropriate dye based on different experimental conditions and fluorescence collection systems when using the present invention for nucleic acid analysis. For example, in the above experimental examples, when the off-chip reaction is performed, a significant fluorescent signal can be obtained using EvaGreen at a concentration of about 1×; when the on-chip reaction is performed, the whole fluorescence intensity is reduced due to the small sample volume, and the experimental example uses EvaGreen (4 x) with higher concentration to obtain a fluorescence signal in the acquirable range of the corresponding optical instrument.

Notably, when different concentrations of SYBR Green I were used, the Tm of the target melting peak was the same but the peak was different. What causes this peak difference is that SYBR Green I has different binding preference for nucleic acid sequences of different lengths, and when the SYBR Green I concentration is low, it preferentially binds longer double-stranded DNA amplification products, so that insufficient dye can bind the probe-target complex, resulting in a lower target melting peak. However, the key of the analysis results of the melting curve is that the difference in peak value does not affect the Tm value corresponding to the melting peak, and the difference does not have a fundamental influence on the experimental results. This result further shows the versatility of the present invention.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting a nucleic acid using a non-modified probe, comprising: carrying out asymmetric amplification on target nucleic acid, then adding a reaction solution containing an unmodified probe and a double-stranded DNA binding dye into the amplified system, gradually increasing the temperature, monitoring fluorescent signals of the double-stranded DNA binding dye to obtain a melting curve, and obtaining sequence information of the target nucleic acid by analyzing the melting curve;

the normalization method is a two-dimensional normalization method, comprising:

(1) Normalizing the change in fluorescence intensity: setting the temperature of thoroughly melting the double-chain amplification product as a base line, setting the corresponding-dF/dT value as 0, setting the peak value of a reference peak, namely the-dF/dT value thereof as 1, and expressing the height of a first melting peak as the relative concentration ratio of the single-chain product to the double-chain product in the amplification product;

2. The method of claim 1, wherein the asymmetric amplification comprises PCR amplification using non-limiting primers and limiting primers to obtain a mixture comprising single-stranded and double-stranded amplification products.

3. The method according to claim 2, wherein the concentration of the unmodified probe after addition to the amplified system is not less than one-half of the concentration of the non-limiting primer.

4. The method for detecting a nucleic acid according to claim 3, wherein the length of the non-modified probe is 12 to 36nt;

preferably, the length of the non-modified probe is 12-28 nt;

preferably, the Tm value of the unmodified probe is 45 ℃ to 73 ℃;

preferably, the Tm value of the modified probe is 50℃to 70 ℃.

5. The method of detecting nucleic acid according to claim 4, wherein the double-stranded DNA binding dye comprises LCGreen, evaGreen and SYBR Green I;

preferably, the concentration of the double-stranded DNA binding dye is 1× to 8×.

6. The method for detecting nucleic acid according to claim 5, wherein the reaction solution further contains an amplification inhibitor, the amplification inhibitor comprising EDTA;

preferably, the EDTA is added to the amplified system at a concentration of 0 to 10mM.

7. A non-modified probe for melting curve analysis, characterized in that the non-modified probe is a single-stranded oligonucleotide, which does not have any modification groups at both ends;

preferably, the non-modified probe is added to the reaction system after amplification is completed;

preferably, the length of the non-modified probe is 12-36 nt;

preferably, the length of the non-modified probe is 12-28 nt;

preferably, the Tm value of the unmodified probe is 45 ℃ to 73 ℃;

preferably, the Tm value of the unmodified probe is 50 ℃ to 70 ℃.

8. A nucleic acid detecting chip, wherein the non-modified probe according to claim 7 is supported on the nucleic acid detecting chip.

9. A nucleic acid detection kit comprising the unmodified probe of claim 7.

10. Use of the nucleic acid detection method according to any one of claims 1 to 6 for detecting a mutation in a gene.