CN113699033A - Melting curve-based multiple digital nucleic acid analysis device and analysis method - Google Patents
Melting curve-based multiple digital nucleic acid analysis device and analysis method Download PDFInfo
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Abstract
The invention discloses a melting curve-based multiple digital nucleic acid analysis device and an analysis method, which relate to the field of nucleic acid analysis, and the device comprises a microfluidic chip, a flat PCR (polymerase chain reaction) instrument, a fluorescence detection system and a processor; the fluorescence detection system comprises an excitation light source, an optical filter and a camera; the multiplex digital nucleic acid analysis method comprises the steps of: dispersing a PCR reaction system into a large number of reaction micro units on a microfluidic chip, performing digital PCR amplification on the reaction micro units on a flat PCR instrument, performing temperature control and fluorescence detection on an amplification product, and obtaining and analyzing a melting curve. The invention realizes the discrimination and classification of the amplification products by performing the melting curve analysis on the nucleic acid amplification products in the micro-reaction unit for digital nucleic acid detection, thereby realizing the quantitative detection of the multiple digital nucleic acids. The melting curves of different amplification products are different through the design of nucleic acid amplification products, so that the amplification products are distinguished.
Description
Technical Field
The invention relates to the field of nucleic acid analysis, in particular to a melting curve-based multiple digital nucleic acid analysis device and method.
Background
Digital PCR is a method of accurate nucleic acid quantification. A standard PCR reaction is distributed into a large number of tiny reaction units, so that each reaction unit contains or does not contain one or more copies of target molecules, after amplification is finished, a fluorescence signal in the reaction unit with a template is remarkably enhanced, a threshold value is set, the fluorescence signal is classified as a positive point, and the copy number of the target molecules can be calculated by utilizing Poisson distribution through the proportion and the number of the positive reaction units and the negative reaction units.
Digital PCR requires quantitative detection by end-point fluorescence, typically an embedded fluorescent dye such as SybrGreen; or a hydrolysis probe. In the dye method experiment, free dye emits weak fluorescence, and after the free dye is combined with a DNA double strand, the free dye emits bright fluorescence, so that the micropores with the template emit fluorescence signals. The dye method has the advantages of convenient use, low cost and strong universality. However, since the dye can bind to any DNA double strand, it can also bind to non-specific double strands to produce false positive signals, and only one fluorescent signal can be emitted to distinguish different amplification products. The hydrolysis probe is represented by a TaqMan probe, the principle is that a probe specifically combined with a template is designed, the 5 ' end of the probe is marked with a donor fluorescent group, the 3 ' end of the probe is marked with an acceptor fluorescent group, when the probe is complete, the fluorescent group does not emit fluorescence because FRET is formed by the close distance between the fluorescent group and a quenching group, and when PCR reaction is carried out, the 5 ' end of the probe is cut because Taq enzyme has 5 ' -3 ' exonuclease activity, the fluorescent group and the quenching group are separated, the FRET between two fluorescent molecules is destroyed, and then fluorescence is emitted. The probe has high specificity, and can have different fluorescence channels, such as FAM and VIC, and detect different target fragments. However, probes are expensive, difficult to design, and limited fluorescence pathways are available.
The multiplex PCR is a PCR reaction in which more than two pairs of primers are added in the same PCR reaction system and a plurality of nucleic acid fragments are amplified simultaneously, and has the advantages of saving time, saving cost and saving samples. The quantitative detection of multiple digital nucleic acids is always difficult, and the current method for quantitative detection of multiple digital nucleic acids comprises the steps of using hydrolysis probes of multiple fluorescence channels, and changing the fluorescence intensity of an end point by adjusting the concentration of the probes so as to improve the detectable target of each channel. However, probes are expensive and require extensive optimization. Another method is based on fluorescent dyes that change the amplification efficiency by changing the primer concentration and thus the final fluorescence intensity. However, the specificity is poor since the dye can bind to any double stranded DNA molecule.
Melting curves are another dye-based multiplexing method. The rationale for this is that the thermal stability of a double-stranded nucleotide is affected by its length and base composition, and sequence changes can lead to changes in dsDNA melting behavior during warming. Since the fluorochromes used can only intercalate and bind to dsDNA, melting curves of different shapes can be generated by monitoring the change in fluorescence signal values during melting of dsDNA in real time. The melting temperature is the temperature required to cause half of the double helix structure of DNA to unwind. A plurality of amplification products in the same reaction unit can be distinguished by different melting curve shapes and melting temperatures. The method has low cost and solves the problem of poor specificity of the dye method. However, the implementation of a digital melting curve analysis requires a fixed, traceable physical location of the microreaction units. And the system can simultaneously realize temperature control and real-time fluorescence signal acquisition, so that no commercial instrument can realize the function of a digital melting curve at present.
The defects of the prior art mainly lie in that:
1. the existing digital nucleic acid detection (such as digital PCR) is difficult to carry out multiple digital nucleic acid quantitative analysis and end point fluorescence quantitative detection due to the low specificity of a fluorescent dye lamp;
2. the multiplex digital PCR quantitative method using the hydrolysis fluorescent probe has high price and difficult probe design and is limited by the number of fluorescent channels;
3. the existing multiple digital PCR quantitative detection instrument with a plurality of fluorescence channels has high cost and complex system.
Therefore, those skilled in the art have made efforts to develop a multiplex nucleic acid analysis apparatus and analysis method with simple system, high flexibility, low cost, and strong specificity, which can not only break through the limitation of the number of fluorescence pathways, but also have strong specificity.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention is to develop a simple, flexible, low-cost, and highly specific multiplex nucleic acid amplification method and an apparatus for implementing the same.
To achieve the above object, the present invention provides a melting curve-based multiplex digital nucleic acid analyzer. A melting curve-based multiple digital nucleic acid analysis device comprises a microfluidic chip, a flat PCR instrument, a fluorescence detection system and a processor; the fluorescence detection system comprises an excitation light source, an optical filter and a camera.
Furthermore, the micro-fluidic chip comprises a sliding micro-fluidic chip, the sliding micro-fluidic chip comprises a lower chip and an upper chip, the lower chip and the upper chip are two glass flat plates which are in close contact, and a layer of lubricating oil is arranged between the two glass flat plates; the lower chip and the upper chip are respectively provided with discontinuous pipelines and holes, and after the lower chip and the upper chip are aligned, the discontinuous pipelines and holes of the lower chip and the discontinuous pipelines and holes of the upper chip form continuous flow channels.
Further, the sliding microfluidic chip realizes the loading of the sample. Then the positions of the upper chip and the lower chip are relatively changed through sliding, so that a large number of micro droplets can be generated, and the micro droplets can be used for various biochemical analysis applications including nano-scale PCR reaction.
Further, the sliding microfluidic chip can provide traceable physical positions of droplets for digital melt curve analysis.
Furthermore, the fluorescence detection system is controlled by the processor to monitor the change condition of the fluorescence signal of the micropores on the whole microfluidic chip along with the temperature.
Further, the flat PCR instrument and the fluorescence detection system are integrated into the same system.
The invention also provides a melting curve-based multiple digital nucleic acid analysis method, which comprises the following steps:
step 2, placing the micro-fluidic chip dispersed in the step 1 on a flat PCR instrument, performing digital PCR amplification on the micro-fluidic chip, and amplifying nucleic acid in each independent dispersed micro-reaction unit obtained in the step 1 to obtain an amplification product;
Further, the step 1 also comprises a method for dispersing the PCR reaction system, which comprises a micro-droplet, micropore or micro-chamber method, and is completed by fluid control equipment according to various driving forces of surface physical and chemical properties, centrifugal force, surface tension, gravity or shearing force; the PCR reaction body comprises nucleic acid embedded fluorescent dye, and the nucleic acid embedded fluorescent dye comprises ybrGreen, EvaGreen or PicoGreen.
Further, step 2 further comprises: the digital PCR amplification reaction may be PCR nucleic acid amplification requiring thermal cycling or isothermal nucleic acid amplification (isothermal amplification).
Further, the method for obtaining the digital melting curve in step 3 comprises the following steps:
setting a step-by-step temperature gradient, gradually raising the temperature from the temperature lower than the unwinding temperature of the double-stranded nucleic acid to the unwinding temperature of the double-stranded nucleic acid, and collecting fluorescence pictures at each temperature to obtain a series of fluorescence pictures changing along with the temperature; by monitoring the change of the fluorescence value of each micropore on the chip along with the change of time and fitting the change into a smooth curve, a melting curve of an amplification product of each micropore can be obtained; the first derivative is then taken to obtain a peak (Tm) plot of the melting curve.
Further, different amplification products can be distinguished according to the Tm value.
Further, the temperature gradient comprises steps of less than 0.25 degrees celsius; temperatures below the unwinding of double-stranded nucleic acids include 65 ℃; the temperature for unwinding the double-stranded nucleic acid included 95 ℃.
Further, step 3 further comprises: by designing the amplification product or the amplification primer, the amplification products of different target nucleic acids have different characteristic melting curves or melting temperatures; predicting the melting temperature of the amplicon by adopting melting curve simulation software (uMELT); the melting temperature was verified on a real-time fluorescent quantitative PCR instrument.
Further, the method of quantitative analysis in step 3 includes a poisson distribution method.
Further, in order to realize the melting curve analysis, it is required that temperature control and fluorescence detection are simultaneously performed, and the physical location of the micro reaction unit can be tracked. The fluorescence detection module can perform ultra-sensitive detection on the fluorescence change of the micro reaction units, and detect hundreds to millions of micro reaction units.
Further, the invention designs the melting curve (including the melting temperature) of the amplification product as the specific fingerprint of the amplification; generating a large number of micro-reaction units with fixed physical positions and traceable physical positions on a micro-fluidic chip (particularly a sliding micro-fluidic chip) for amplification and melting curve analysis; the system is used for simultaneously realizing the temperature control and the real-time fluorescence detection of the microfluidic chip. Thereby realizing the analysis of the digital melting curve.
Furthermore, the results are classified through specific fingerprint amplification curves of different amplification products, so that the differentiation of different amplification products and the quantitative analysis of multiple digital nucleic acids are realized.
Further, by designing the amplification primers, different characteristic melting curves (including the shape, height and melting temperature of the melting curve) for different amplification products are realized. By analyzing the characteristic melting curve, information on the amplification product can be deduced, and further information on the target gene (e.g., concentration, purity, etc.) can be deduced.
In a preferred embodiment of the present invention, a melting curve-based multiplex digital nucleic acid analyzer and its operation principle will be described in detail.
In another preferred embodiment of the present invention, a method for performing multiplex PCR quantitative detection using the melting curve-based multiplex digital nucleic acid analyzer of the present invention will be described in detail.
The invention realizes the discrimination and classification of the amplification products by performing melting curve analysis on the nucleic acid amplification products in the micro-reaction unit for digital nucleic acid detection, thereby realizing the quantitative detection of multiple digital nucleic acids. By designing the nucleic acid amplification product, the melting curves of different amplification products can be different from each other, thereby realizing the differentiation of the amplification products.
The concrete technical effects are as follows:
1. the invention designs the melting curve (including melting temperature) of the amplification product as the specific fingerprint of the amplification by 1). 2) A large number of micro-reaction units with fixed physical positions and traceable physical positions are generated on the micro-fluidic chip (particularly a sliding micro-fluidic chip) for amplification and melting curve analysis. 3) The system is used for simultaneously realizing temperature control and real-time fluorescence detection of the microfluidic chip. Therefore, the analysis of the digital melting curve is realized, the results are classified through the specific fingerprint amplification curves of different amplification products, and the differentiation of the different amplification products and the quantitative analysis of the multiple digital nucleic acids are realized.
2. The amplification products (or amplification primers) are designed to enable the amplification products of different target nucleic acids to have different characteristic (fingerprint) melting curves (or melting temperatures);
3. by melting curve analysis, different amplification products are distinguished in micro-droplets or micro-pores, so that the detection and quantitative analysis of target nucleic acid are realized;
4. the invention adopts a single fluorescence channel, and the invention is not only suitable for digital PCR, but also can be used for other digital isothermal (isothermal) amplification reactions.
5. The sliding microfluidic chip can provide traceable physical positions of droplets for digital melt curve analysis.
6. The melting curve-based multiple digital nucleic acid analysis method provided by the invention only needs to use a nucleic acid embedded fluorescent dye and a single fluorescent channel; complex fluorescent hydrolysis probes and multi-channel fluorescent detector equipment are not required.
7. The invention can complete multiple digital PCR quantitative detection by using a single fluorescent channel, and realize multiple digital nucleic acid quantitative analysis. The system is simple and has high flexibility.
8. By designing the amplification products (and the amplification primers), the melting curves of the amplification products of different target nucleic acids are different, thereby realizing multiple detection. Theoretically, the limit of the number of fluorescence channels can be broken through. The conventional instrument only needs 3-6 fluorescence channels, and can not realize more quantitative detection.
The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.
Drawings
Fig. 1 is a schematic diagram of the principle of performing multiple digital nucleic acid detection and quantitative analysis based on the digital melting curve analysis of the sliding microfluidic chip according to a preferred embodiment of the present invention;
FIG. 2 is a graph of the melting curves and peaks of five plasmids of another preferred embodiment of the present invention A-E) melting curves F-J) of S.aureus, A.baumannii, S.pneumoniae, H.influenzae, and K.pneumoniae;
FIG. 3 is a melting curve and peak plot of another preferred embodiment of the present invention;
FIG. 4 is a graph showing the quantitative results of the mixing of target sequence molecules of S.aureus, A.baumannii, S.pneumoconiae, H.influenzae, and K.pneumoconiae at the ratio of 1:3:9:27:81,3:9:27:81:1,9:27:81:1:3,27:81:1:3:9, 81:1:3:9:27, respectively, according to another preferred embodiment of the present invention.
Detailed Description
The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.
Example 1 melting curve-based multiplex digital nucleic acid analysis device and its operating principle.
The melting curve-based multiplex digital nucleic acid analyzer of the present invention performs multiplex PCR quantitative detection. The device comprises a microfluidic chip containing 2240 micropores, a flat PCR instrument and a fluorescence detection system, wherein the fluorescence module consists of a bracket, two excitation light sources, an optical filter and a camera, and the change condition of fluorescence signals of the micropores on the whole microfluidic chip along with temperature can be monitored in real time through the control of a processor. The working principle is shown in figure 1, after the microfluidic chip is added into a PCR reaction system in a sliding mode, the microfluidic chip is placed into a flat PCR instrument for nucleic acid amplification, then a fluorescence detection system is used for simultaneously controlling temperature and collecting real-time fluorescence signals through a controller, finally a melting curve is obtained, the melting curve is analyzed, and multiple digital nucleic acid detection and quantitative analysis are achieved.
Example 2 quantitative detection of multiplex PCR was performed using the melting curve-based multiplex digital nucleic acid analysis device of the present invention.
The method comprises the following specific steps:
primers are respectively designed aiming at the genes of five bacteria to be detected, namely aureus, Acinetobacter baumannii, Streptococcus pneumniae, Haemophilus influenza and Klebsiella pneumniae, and the primer sequences are as follows:
Staphylococcus aureus F:AGTCACGTCTCGATCGAACA
Staphylococcus aureus R:GAAACTTGACCACGATCCGG
Acinetobacter baumannii F:GGCTGGACATCATCAACTGC
Acinetobacter baumannii R:GTCGGCCTGATCTCGTATGA
Streptococcus pneumoniae F:GCACACTCAACTGGGAATCC
Streptococcus pneumoniae R:ATGCAACCGTTCCCAACAAT
Haemophilus influenza F:CTGGTGTTGCGGCTAAAAGT
Haemophilus influenza R:TCATTAACTGGGGCTTCGGT
Klebsiella pneumoniae F:ACACAATCGCCCGTTGAAC
Klebsiella pneumoniae R:CCCGGTTAGATCCATGGTGA
melting temperature of the amplicons was predicted using melting curve simulation software (uMELT) at 77.75 deg.C, 79.75 deg.C, 81.75 deg.C, 84.5 deg.C, 90 deg.C, respectively, such that the melting temperature of each fragment differs by at least 1.5 deg.C.
2. The melting temperature was verified on a real-time fluorescent quantitative PCR instrument, and a 10. mu.L reaction system included rtaq 5. mu.L, 20 XEvaGreen 0.5. mu.L, upstream primer (10. mu.M) 0.2. mu.L, downstream primer (10. mu.M) 0.2. mu.L, BSA (2mg/mL) 0.5. mu.L, plasmid (100 fg/. mu.L) 2. mu.L, and ddH2O 8. mu.L. The reaction conditions are as follows: pre-denaturation at 95 ℃ for 180s, denaturation at 95 ℃ for 10s, annealing at 62 ℃ for 15s, and extension at 72 ℃ for 30s for 35 cycles. Melting curve analysis was then performed, the melting curve program was as follows: the temperature was gradually increased from 70 ℃ to 94 ℃ with a temperature gradient of 2.2 ℃/s at 95 ℃ for 60s, 40 ℃ for 60 s. The melting temperatures were 78.24 + -0.04 deg.C, 80.21 + -0.02 deg.C, 82.23 + -0.04 deg.C, 84.77 + -0.10 deg.C, 89.57 + -0.04 deg.C, respectively. And the software predicted temperature is substantially the same.
3. In order to characterize the system, five genes were subjected to digital amplification and digital melting curve analysis, respectively.
Firstly, after the chip is subjected to hydrophobic treatment, the upper chip and the lower chip are immersed in oil, and the upper chip and the lower chip are assembled at the initial positions. Configuring a PCR reaction system, wherein the 25ul reaction system comprises: 12.5 μ L rtaq, 1.25 μ L20 × Eva Green, 0.5 μ L upstream primer (10 μ M concentration), 0.5 μ L downstream primer (10 μ M concentration), 1.25 μ L BSA (2mg/mL), 5 μ L plasmid (100 fg/μ L concentration), and 4 μ L ddH2O 4. The sample solution is injected into the chip from the inlet, 2240 micropore arrays are formed by sliding, and the sample solution is placed on a flat plate PCR instrument for digital amplification, wherein the PCR program is as follows: pre-denaturation at 95 ℃ for 240s, denaturation at 95 ℃ for 50s, annealing at 62 ℃ for 30s, extension at 72 ℃ for 1min, and performing 35 cycles, wherein when the Evagreen fluorescent dye is combined with the double-stranded DNA, the micropores with the templates present fluorescent signals.
4. Finally, the melting curve is analyzed, and the program of the melting curve is set as follows: the temperature is gradually increased from 70 ℃ to 95 ℃ at intervals of 0.2 ℃ for 60s at 95 ℃ and 60s at 40 ℃, 5 pictures are taken at each degree, and a series of fluorescence pictures changing along with the temperature are obtained.
5. After the system is characterized, the system is used for carrying out multiple digital amplification and melting curve analysis to realize the quantification of the genes of the five bacteria. The melting curves of S.aureus, A.baumannii, S.pneumoniae, H.influenzae and K.pneumoniae were combined in the ratio of 1:3:9:27:81,3:9:27:81:1,9:27:81:1:3,27:81:1:3:9, 81:1:3:9:27, respectively, and the ratios of the primers to the five bacteria were adjusted on a real-time quantitative fluorescence PCR apparatus so that the peak heights of the melting curves of the target sequences were approximately the same, and it was confirmed that the peak heights of the melting curves of the five genes were approximately the same when the primer ratios of the S.aureus, A.baumannii, S.pneumoniae, H.influenzae and K.pneumoniae were 7:4:6:7:6 in this order. The reaction system of the multiplex PCR comprises: rtaq 12.5. mu.L, 20 × EvaaGreen 1.25. mu.L, five genes 'upstream primers (concentration 10. mu.M) 0.35. mu.L, 0.2. mu.L, 0.3. mu.L, 0.35. mu.L, 0.3. mu.L, five genes' downstream primers (concentration 10. mu.M) 0.35. mu.L, 0.2. mu.L, 0.3. mu.L, 0.35. mu.L, 0.3. mu.L, BSA (2mg/mL) 1.25. mu.L, five plasmid template mixtures, the remaining volume being filled up with ddH 2O. The PCR program and the melting curve program are the same as the single-digit PCR and melting curve analysis program.
6. And (3) data analysis: using the first picture (70 ℃) as a mask, using a Python program to circle all the positive points by threshold, automatically aligning all the pictures with the mask and obtaining the average value of the fluorescence intensity of each positive hole of each picture, i.e. the average value of the fluorescence intensity of each microwell varying with temperature, and filtering the raw data with a Savitzky-Golay filter to obtain the melting curve of the molecules in each microwell, as shown in the left A, B, C, D and part E of fig. 2; taking the negative first derivative, a peak map of the melting curve can be obtained, as shown in the right F, G, H, I and J parts of FIG. 2, different amplification products have different Tm values, and the Tm values of the five genes are 77.77 + -0.17 deg.C, 80.20 + -0.19 deg.C, 81.87 + -0.19 deg.C, 84.76 + -0.19 deg.C, and 89.52 + -0.21 deg.C, respectively, which are substantially consistent with the result of qPCR. When performing multiplex PCR and melting curve analysis, different amplification products can be distinguished according to Tm values, and still can be distinguished by melting curves when two or more amplification products are contained in one microwell, as shown in FIG. 3, a microwell contains melting curves and peak maps A-B) of two or more molecules, and melting curves and peak maps C-D) of amplification products of S.aureue and H.influezae in one microwell, and melting curves and peak maps E-F) of amplification products of S.aureue, S.pnesoniae, and K.pnesoniae in one microwell, and peak maps G-H) S.aureue, S.pnesoniae, H.influezae, and K.pnesoniae amplification products in one microwell, and melting curves and peak maps G-H) of amplification products of S.auxsoniae, and K.pnesoniae, and the melting curves and peak maps of amplification products of Poissoniae are quantified, as shown in Poisson a Poisson graph, and the result is finally quantified by Poisson graph, as shown in FIG. 4, quantitative results are shown for the mixture of target sequence molecules of s.aureus, a.baumannii, s.pneumoniae, h.influenzae, and k.pneumoniae in the ratios of 1:3:9:27:81,3:9:27:81:1,9:27:81:1:3,27:81:1:3:9, 81:1:3:9:27, respectively.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (10)
1. A melting curve-based multiple digital nucleic acid analysis device is characterized by comprising a microfluidic chip, a flat PCR instrument, a fluorescence detection system and a processor; the fluorescence detection system comprises an excitation light source, an optical filter and a camera.
2. The multiplexed digital nucleic acid analysis device according to claim 1, wherein the microfluidic chip comprises a sliding microfluidic chip, the sliding microfluidic chip comprises a lower chip and an upper chip, the lower chip and the upper chip are two plates in close contact, and a layer of lubricating oil is arranged between the two plates; and the lower chip and the upper chip are respectively provided with a pipeline and a hole.
3. The multiplexed digital nucleic acid analysis device according to claim 1, wherein the flat PCR instrument and the fluorescence detection system are integrated into the same system.
4. A melting curve based multiplexed digital nucleic acid analysis method, comprising the steps of:
step 1, dispersing a PCR reaction system into a large number of reaction micro units on a microfluidic chip to obtain dispersed reaction micro units, wherein the volumes of the micro reaction units can be the same or different;
step 2, placing the micro-fluidic chip dispersed in the step 1 on a flat PCR instrument, performing digital PCR amplification on the micro-fluidic chip, and amplifying nucleic acid in each independent dispersed micro-reaction unit obtained in the step 1 to obtain an amplification product;
step 3, analyzing a digital melting curve, performing temperature control and fluorescence detection on the micro-reaction unit amplified in the step 2 to obtain a melting curve of the micro-reaction unit, and analyzing the melting curve; and finally, carrying out accurate quantitative analysis on the target nucleic acid according to the proportion of the positive micro-reaction unit in the total micro-reaction unit, the volume of the micro-reaction unit and the amplification reaction efficiency factor.
5. The multiplex digital nucleic acid analysis method according to claim 3, wherein the step 1 further comprises a method of dispersing the PCR reaction system comprising a microdroplet, microwell or microchamber method, by a fluid control device, according to a plurality of driving forces of surface physical and chemical properties, centrifugal force, surface tension, gravity or shear force; the PCR reaction body comprises nucleic acid embedded fluorescent dye, and the nucleic acid embedded fluorescent dye comprises SybrGreen, EvaGreen or PicoGreen.
6. The multiplexed digital nucleic acid analysis method according to claim 3, wherein step 2 further comprises: the digital PCR amplification reaction may be PCR nucleic acid amplification requiring thermal cycling, or isothermal nucleic acid amplification (isotermal amplification).
7. The multiplexed digital nucleic acid analysis method according to claim 3, wherein the method for obtaining the digital melting curve in step 3 comprises:
setting a step-by-step temperature gradient, gradually raising the temperature from the temperature lower than the unwinding temperature of the double-stranded nucleic acid to the unwinding temperature of the double-stranded nucleic acid, and collecting fluorescence pictures at each temperature to obtain a series of fluorescence pictures changing along with the temperature; by monitoring the change of the fluorescence value of each micropore on the chip along with the change of time and fitting the change into a smooth curve, the melting curve of the amplification product of each micropore can be obtained; the first derivative is then taken to obtain a peak (Tm) plot of the melting curve.
8. The multiplexed digital nucleic acid analysis method according to claim 6, wherein the temperature gradient comprises steps of less than 0.25 degrees Celsius; the temperature below unwinding of the double-stranded nucleic acid comprises 65 ℃; the temperature at which the double-stranded nucleic acid unwinds comprises 95 ℃.
9. The multiplexed digital nucleic acid analysis method according to claim 3, wherein step 3 further comprises: by designing the amplification product or the amplification primer, the amplification products of different target nucleic acids have different characteristic melting curves or melting temperatures; and predicting said melting temperature of the amplicon using melting curve simulation software (uMELT); the melting temperature was verified on a real-time fluorescent quantitative PCR instrument.
10. The multiplexed digital nucleic acid analysis method of claim 3 wherein the means for quantitative analysis in step 3 comprises a Poisson distribution method.
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