CN110835645B - Digital PCR detection method - Google Patents
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- CN110835645B CN110835645B CN201810932950.6A CN201810932950A CN110835645B CN 110835645 B CN110835645 B CN 110835645B CN 201810932950 A CN201810932950 A CN 201810932950A CN 110835645 B CN110835645 B CN 110835645B
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The digital PCR detection method provided by the application can realize genotyping, mutation scanning, methylation research and the like by using a dye when preparing a nucleic acid amplification reaction solution to be detected, has high resolution and sensitivity, and reduces the detection cost. And the micro-droplet array can complete polymerase chain reaction on the same highly integrated digital PCR detector, and the melting curve analysis is carried out on the PCR product after the micro-droplet array is subjected to PCR amplification. Meanwhile, a fluorescence curve and a melting curve of the micro-droplet array can be obtained by a digital PCR detection method, and the traceless connection of the real-time monitoring of the whole PCR amplification process and the melting curve analysis of a PCR product can be completely realized. Therefore, the method realizes the copy number identification based on different fluorescence curves and the identification of nucleic acid amplification characteristics of different characteristic melting curves, realizes gene typing, mutation scanning and the like through a high-resolution melting curve, and completes the detection of the digital PCR more comprehensively, simply and efficiently.
Description
Technical Field
The application relates to the field of digital PCR detection and analysis, in particular to a digital PCR detection method.
Background
In recent years, digital PCR (dPCR) technology has rapidly developed. Polymerase Chain Reaction (PCR), is a molecular biology technique for amplifying specific DNA fragments. Digital PCR includes both droplet-type PCR detection methods and chip-type detection methods. In the chip-based detection method, the number of effective reaction cavities on a single chip is generally only thousands, which is far less than that of a micro-drop type, and the dynamic range of the chip-based digital PCR is narrower than that of the micro-drop type.
The micro-drop digital PCR system carries out micro-titration treatment on a sample before traditional PCR amplification, namely, a reaction system containing nucleic acid molecules is divided into thousands of nano-upgrade micro-drops, wherein each micro-drop contains no nucleic acid target molecules to be detected or contains one to a plurality of nucleic acid target molecules to be detected. After PCR amplification, each droplet was detected individually. However, in the process of digital PCR detection and analysis, for thousands of nano-scaled micro-droplet arrays, if a conventional digital PCR detection method wants to detect multiple target sequences simultaneously, multiple primers need to be designed for sequential detection, and repeated detection increases workload and consumes time and has low efficiency.
Disclosure of Invention
Therefore, it is necessary to provide a simple and efficient digital PCR detection method aiming at the problems of heavy workload, time consumption and low efficiency of the traditional digital PCR detection method.
The application provides a digital PCR detection method, which comprises the following steps:
s10, preparing a nucleic acid amplification reaction solution to be detected;
s20, micro-dripping the nucleic acid amplification reaction solution to be detected to form a micro-droplet array;
s30, performing polymerase chain reaction on the micro-droplet array, and acquiring a fluorescence curve of each micro-droplet in the micro-droplet array and a melting curve of each micro-droplet; and
s40, analyzing the micro-droplet array according to the fluorescence curve of each micro-droplet in the micro-droplet array and the melting curve of each micro-droplet to obtain the information of the nucleic acid to be detected.
In one embodiment, the step S30 includes:
s310, setting temperature parameters, time parameters and cycle times of the polymerase chain reaction;
s320, performing polymerase chain reaction on the micro-droplet array according to the temperature parameter and the time parameter, completing the cycle times in sequence, and acquiring a fluorescence curve of each micro-droplet in each cycle process; and
s330, cooling the micro-droplet array after the polymerase chain reaction amplification is completed, and heating at specific temperature intervals to obtain a melting curve of each micro-droplet.
In one embodiment, the step S320 includes:
s321, performing polymerase chain reaction on the micro-droplet array according to the temperature parameter and the time parameter to obtain a fluorescence image of the micro-droplet array;
s322, sequentially circulating according to the circulation times to obtain all fluorescence images of the micro-droplet array in the polymerase chain reaction process;
s323, acquiring fluorescence information of each micro-droplet in each circulation process according to all fluorescence images of the micro-droplet array; and
and S324, acquiring a fluorescence curve of each micro-droplet according to the fluorescence information of each micro-droplet in each circulation process, so as to obtain the fluorescence curve of the micro-droplet array.
In one embodiment, the step S330 includes:
s331, cooling the micro-droplet array subjected to polymerase chain reaction amplification to below 40 ℃;
s332, heating the micro-droplet array cooled to the temperature below 40 ℃ at a specific temperature interval to obtain a fluorescence image of the micro-droplet array corresponding to the temperature interval;
s333, acquiring fluorescence information of each micro-droplet corresponding to the temperature interval according to the fluorescence image of the micro-droplet array corresponding to the temperature interval; and
and S334, obtaining a melting curve of each micro-droplet according to the fluorescence information of each micro-droplet corresponding to the temperature interval, so as to obtain a melting curve of the micro-droplet array.
In one embodiment, the step S40 includes:
s410, acquiring the initial copy number of the nucleic acid of the micro-droplet array according to the fluorescence curve of the micro-droplet array; and
and S420, acquiring the nucleic acid information of the micro-droplet array according to the melting curve of the micro-droplet array.
In one embodiment, the step S410 includes:
s411, acquiring a Ct value corresponding to the fluorescence curve of each micro-droplet according to the fluorescence curve of the micro-droplet array;
s412, clustering is carried out according to the Ct value of the fluorescence curve of each micro-droplet, and sequencing is carried out from large to small in sequence to obtainGet x 1 ，x 2 ，....x n A category;
s413, according to the x 1 ，x 2 ，....x n Obtaining the number y of the micro-droplets corresponding to each category 1 、y 2 、....y n ；
S414, the number y of the micro-droplets corresponding to each category 1 、y 2 、....y n Obtaining said x 1 ，x 2 ，....x n The number y of the micro-droplets corresponding to each category 1 、y 2 、....y n Frequency distribution;
and S415, calculating the initial copy number of the nucleic acid of the micro-droplet array according to the frequency distribution.
In one embodiment, in the step S415, when x is greater than x 1 Number y of the micro-droplets of a class 1 And when the number of the initial copies of the nucleic acid of the micro-droplet array is larger than or equal to the characteristic value m, carrying out Poisson distribution fitting according to the frequency distribution to obtain a parameter lambda of Poisson distribution, thereby obtaining the initial copy number of the nucleic acid of the micro-droplet array.
In one embodiment, in the step S415, when x is greater than x 1 Number y of the micro-droplets of a class 1 When the characteristic value is less than the characteristic value m, the method comprises the following steps:
s4151 according to said x 2 ，....x n The number y of the micro-droplets corresponding to each category 2 、....y n The partial frequency distribution of (2), successively assuming the x 2 The initial copy number of the nucleic acid corresponding to the category is subjected to poisson distribution fitting to obtain a parameter lambda corresponding to each poisson distribution j (j＝0，1，2....)；
S4152, in a range [ lambda ] min ,λ max ]Inner search lambda j So as to minimize the sum of squared error err of frequency value and obtain the optimum lambda optimal ；
S4153, according to the optimal lambda optimal And calculating the initial copy number of the nucleic acid of the micro-droplet array.
In one embodiment, the step S420 includes:
s421, obtaining a melting temperature corresponding to the melting curve of each micro-droplet according to the melting curve of the micro-droplet array; and
s422, classifying the micro-droplet array according to the melting temperature to obtain the nucleic acid information of the micro-droplet array, and further obtaining the nucleic acid information of the nucleic acid to be detected.
In one embodiment, the digital PCR detection method further comprises:
s50, obtaining a high-resolution melting curve of the micro-droplet array, classifying the micro-droplet array, and obtaining the nucleic acid information of the micro-droplet array, such as genotyping, mutation detection and the like.
The digital PCR detection method provided by the application can realize genotyping, mutation scanning, methylation research and the like by using one dye when preparing the nucleic acid amplification reaction solution to be detected, has high resolution and sensitivity, and reduces the detection cost. And by the digital PCR detection method, the micro-droplet array can complete polymerase chain reaction on the same highly integrated digital PCR detector, and a PCR product is subjected to melting curve analysis after the micro-droplet array is subjected to PCR amplification. Meanwhile, by the digital PCR detection method, a fluorescence curve and a melting curve of the micro-droplet array can be obtained, and the real-time monitoring of the whole PCR amplification process and the traceless connection of the melting curve analysis of a PCR product can be completely realized. Therefore, genotyping or classification based on melting curves of different shapes is realized through the fluorescence curve and the melting curve of the micro-droplet array, so that qualitative and quantitative analysis of the micro-droplet array is realized, and detection of digital PCR is completed more comprehensively, simply and efficiently.
Drawings
FIG. 1 is a flowchart illustrating the overall steps of a digital PCR detection method provided herein;
FIG. 2 is a schematic diagram of a micro-droplet array obtained by the digital PCR detection method provided in the present application;
FIG. 3 is a schematic diagram of a fluorescence image of a micro-droplet array obtained by the digital PCR detection method provided in the present application;
FIG. 4 is a schematic diagram of a real-time fluorescence curve obtained by the digital PCR detection method provided in the present application;
FIG. 5 is a graph comparing the standard deviation of CPD with other methods for digital PCR quantitative detection of partial samples obtained by the digital PCR detection method provided in the present application;
fig. 6 is a schematic diagram of a melting curve obtained by the digital PCR detection method provided in the present application.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail below by way of embodiments and with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.
The application provides a digital PCR detection method, which comprises the following steps:
s10, preparing a nucleic acid amplification reaction solution to be detected;
s20, micro-dripping the nucleic acid amplification reaction solution to be detected to form a micro-droplet array;
s30, performing polymerase chain reaction on the micro-droplet array, and acquiring a fluorescence curve of each micro-droplet in the micro-droplet array and a melting curve of each micro-droplet; and
s40, analyzing the micro-droplet array according to the fluorescence curve of each micro-droplet in the micro-droplet array and the melting curve of each micro-droplet to obtain the information of the nucleic acid to be detected.
When the nucleic acid amplification reaction solution to be detected is prepared, classification of different types of variation can be realized by using one saturated fluorescent dye, high resolution and sensitivity are realized, and the detection cost of the digital PCR detector 1 is reduced. And by the digital PCR detection method, the micro-droplet array can complete Polymerase Chain Reaction (PCR) on the same highly integrated digital PCR detector, and the melting curve analysis is carried out on a PCR product after the micro-droplet array is subjected to PCR amplification. And acquiring a fluorescence curve of each micro-droplet in the process of performing polymerase chain reaction on the micro-droplet array. And after the micro-droplet array completes PCR amplification, performing high-low temperature circulation again to obtain a melting curve of each micro-droplet in the circulation process. By the digital PCR detection method, the fluorescence curve of the micro-droplet array and the melting curve of the micro-droplet array can be obtained, and the seamless connection of the real-time monitoring of the whole PCR amplification process and the melting curve analysis of a PCR product can be completely realized. Therefore, qualitative and quantitative analysis of the micro-droplet array is realized through the fluorescence curve and the melting curve of the micro-droplet array, and the detection of the digital PCR is completed more comprehensively, simply, conveniently and efficiently.
In one embodiment, the nucleic acid amplification reaction solution in step S10 includes a nucleic acid template to be detected, a reaction buffer solution, deoxyribonucleoside triphosphates, primers, a polymerase, a product labeling substance, and the like. The heat-resistant DNA polymerase can be FastStart Taq DNA polymerase, ex Taq, Z-Taq, accuPrime Taq DNA polymerase, HS Taq DNA polymerase and the like.
The nucleic acid amplification reaction solution may be a nucleic acid amplification reaction solution (may be referred to as a DNA amplification reaction solution) in which deoxyribonucleic acid (DNA) is used as a template, a reverse transcription nucleic acid amplification reaction solution (may be referred to as an RNA reverse transcription reaction solution) in which complementary deoxyribonucleic acid (cDNA), i.e., ribonucleic acid (RNA), is used as a template, or another nucleic acid amplification reaction solution, such as a loop-mediated isothermal amplification (LAMP) reaction solution. The DNA amplification reaction solution is characterized by containing dNTP required by DNA amplification, a buffer solution, inorganic salt ions, polymerase, a primer, a DNA template to be detected, a dye and the like. The dye in the reaction solution can indicate nucleic acid amplification, and may be a fluorescent dye such as SYBR Green combined with DNA.
In one embodiment, kits of reagents and solutions specific for use in digital PCR are prepared to reduce or avoid potential contamination of template DNA samples with exogenous DNA. All instruments and consumables used should be sterilized and dried at high temperature.
In one embodiment, when the reaction solution for nucleic acid amplification to be detected is prepared, the reaction solution for nucleic acid amplification to be detected is labeled with SYBR Green fluorescent dye. As SYBR Green fluorescent dye can be combined with all double-stranded DNA, the template has no selectivity, and the price is low, so that the SYBR Green fluorescent dye is suitable for detecting various target products. When a melting curve analysis is used, SYBR Green is generally used as a fluorescent dye. Since SYBR Green dyes are non-specific dyes, the dyes bind to double stranded DNA as long as there is amplification and fluorescence is greatly enhanced. The melting curve is formed by plotting the change of the fluorescence signal with respect to temperature in order to observe whether the amplified fluorescent fragment is the target gene to be detected in the digital PCR detection. If the melting curve is made to have only a single peak, the peak position is your annealing temperature and is a narrow peak, the product is a specific product of PCR amplification, if the peak position is not right or is a broad peak, the product may not be specific or a corresponding product is not designed, if a small peak exists before the peak of the melting curve, the product may be a primer dimer, and primer redesign needs to be considered. If the high resolution melting curve has slight temperature or curve shape changes, which indicate nucleotide changes, the nucleic acid sequence can be automatically typed by melting curve analysis of a large number of repeated micro-droplet arrays, by a dense curve analysis matching program, and the like.
Referring to fig. 2, in one embodiment, the nucleic acid amplification reaction solution to be detected is micro-titrated in step S20 to form a micro-droplet array. The microdroplet array includes a plurality of microdroplets.
In one embodiment, a digital PCR detector, the digital PCR detector comprising: the device comprises a micro-droplet array generating device, a temperature control device, a signal collecting device, a quantitative analysis device and a controller. The micro-droplet array generating device is used for micro-dripping the nucleic acid amplification reaction solution to form the micro-droplet array, so that the micro-droplet array is formed in the micro-droplet container, and various target sequences can be detected simultaneously. The temperature control device is used for carrying out temperature circulation to realize nucleic acid amplification. The signal acquisition device is arranged opposite to the temperature control device and is used for acquiring signals of the micro-droplet array after nucleic acid amplification. The quantitative analysis device is connected with the signal acquisition device through a data line and is used for realizing the transmission of the fluorescence information of the micro-droplet array and carrying out quantitative analysis. The controller is respectively connected with the micro liquid drop array generating device, the temperature control device, the signal acquisition device and the quantitative analysis device and is used for controlling the micro liquid drop array generating device, the temperature control device, the signal acquisition device and the quantitative analysis device.
When the digital PCR detector works, the micro-droplet array generating device can micro-droplet the nucleic acid amplification reaction solution to be detected, so as to form the micro-droplet array. The microdroplet array includes a plurality of microdroplets. The temperature control device can perform nucleic acid amplification on the micro-droplet array. And the signal acquisition device acquires the fluorescence change image of the micro-droplet array in real time. The temperature control device carries out nucleic acid amplification reaction on the micro-droplet array, and the signal acquisition device acquires product signals of the micro-droplet array after the nucleic acid amplification reaction, such as signals of fluorescence, ultraviolet absorption, turbidity and the like. And analyzing the quantity of the droplets amplified by the obtained target sequence by utilizing the composition difference of the plurality of amplified and non-amplified micro droplets, and finally realizing qualitative and quantitative analysis of the nucleic acid molecules. By monitoring the change of the signal of the micro-droplet array in real time, the detection result has directness, and the problems of false positive and false negative in the micro-droplet array can be solved.
And generating the micro-droplet array in the micro-droplet container by the micro-droplet array generating device, and enabling the micro-droplet array to fall to the bottom of the micro-droplet container and irregularly pile up together. The micro-droplet arrays prepared by the micro-droplet array generating device are gathered at the middle part of the micro-droplet container in the downward sedimentation process, and are gathered together, and at the moment, a plurality of micro-droplet arrays are required to be paved at the bottom of the micro-droplet container before signal acquisition is carried out on the micro-droplet arrays.
In one embodiment, before signal acquisition of a plurality of the microdroplet arrays, high and low temperatures are performed by the temperature control device 20, and the plurality of the microdroplet arrays are tiled at the bottom of the microdroplet container. First, the micro-droplet array is warmed. Secondly, cooling the micro-droplet array. And thirdly, circulating the micro-droplet array at high and low temperatures until the micro-droplet array is paved on the bottom plate of the micro-droplet container. And finally, flatly paving the micro-droplet array in a micro-droplet container, and carrying out PCR amplification on the flatly paved micro-droplet array and carrying out photographing detection.
In one embodiment, a plurality of uniform-sized microdroplets may be generated by a microdroplet array generation device. Each of the micro-droplets is of a micron size. And quantitatively analyzing the plurality of micro-droplets based on fluorescence information of the plurality of micro-droplets when the plurality of micro-droplets have a uniform volume.
In one embodiment, the step S30 includes:
s310, setting temperature parameters, time parameters and cycle times of the polymerase chain reaction;
s320, performing polymerase chain reaction on the micro-droplet array according to the temperature parameter and the time parameter, completing the cycle times in sequence, and acquiring a fluorescence curve of each micro-droplet in each cycle process; and
s330, cooling the micro-droplet array after the polymerase chain reaction amplification is completed, and heating at specific temperature intervals to obtain a melting curve of each micro-droplet.
PCR consists of three basic reaction steps of denaturation-annealing (renaturation) -extension. The denaturation of the template DNA means that the template DNA is heated to 90-95 ℃ for a certain time, and then the double strand of the template DNA or the double strand DNA formed by PCR amplification is dissociated into a single strand so that it can be bound to the primer in preparation for the next reaction. Annealing (renaturation) of the template DNA and the primer means that the temperature of the template DNA is reduced to 50-60 ℃ after the template DNA is heated and denatured into single strands, and the primer is matched and combined with a complementary sequence of the single strand of the template DNA. The primer extension refers to that under the action of DNA polymerase, a DNA template-primer combination is synthesized into a new semi-preserved copy chain which is complementary with a template DNA chain by taking dNTP as a reaction raw material and a target sequence as a template according to the base pairing and semi-preserved copy principle under the action of 70-75 ℃, and the three processes of repeated cyclic denaturation, annealing and extension are carried out, so that more semi-preserved copy chains can be obtained, and the new chain can become a template of the next cycle. The amplification of the target gene to be amplified can be amplified by millions of times within 2-3 hours after each cycle is completed for 2-4 minutes.
Among them, annealing temperature is a relatively important factor affecting the specificity of PCR. After denaturation, the temperature is rapidly cooled to 40-60 ℃, so that the primer and the template can be combined. Since the template DNA is much more complex than the primer, the chance of collision binding between the primer and the template is much higher than between the complementary strands of the template. The annealing temperature and time depend on the length of the primer, the base composition and its concentration, and also the length of the target base sequence.
In the step S310, the micro droplet array performs PCR by repeating a cycle of the denaturation, annealing and extension steps about 30 to 50 times in the presence of primers, a DNA sample to be detected, and a thermostable DNA polymerase. The number of cycles is generally set to 30 to 50 cycles of the three-step process of denaturation, annealing and extension. The temperature parameter is typically set to 40-95 deg.c, and the time parameter is determined for each particular process.
In one embodiment, the step S320 includes:
s321, performing polymerase chain reaction on the micro-droplet array according to the temperature parameter and the time parameter to obtain a fluorescence image of the micro-droplet array;
s322, sequentially circulating according to the circulation times to obtain all fluorescence images of the micro-droplet array in the polymerase chain reaction process;
s323, acquiring the fluorescence information of each micro-droplet in each circulation process according to all fluorescence images of the micro-droplet array; and
and S324, acquiring a fluorescence curve of each micro-droplet according to the fluorescence information of each micro-droplet in each circulation process, so as to obtain the fluorescence curve of the micro-droplet array.
In the step S321, the step S321 includes:
firstly, heating the micro-droplet array to 95 ℃ for 4min to thermally start the enzyme in the micro-droplet array, and after the micro-droplet array finishes the thermal start of the enzyme, denaturing the micro-droplet array for 1min;
secondly, after the micro-droplet array is denatured, the temperature is reduced to 55 ℃, a primer is combined with a DNA template to form a local double chain, annealing (renaturation) is carried out for 1min, at the moment, the micro-droplet array is photographed through the signal acquisition device 30, and a fluorescence image of the micro-droplet array in the first circulation is obtained;
thirdly, heating the micro-droplet array to 70 ℃ and extending for 7min;
and finally, circulating for 45 times according to the three steps of denaturation, annealing (renaturation) and extension in sequence according to the circulation times, cooling to 4 ℃ after the circulation for 45 times, and storing the plurality of micro-liquids.
In one embodiment, the temperature control device 20 heats up and cools down the micro-droplet array, the number of cycles is set to 45, and then each micro-droplet can acquire 45 fluorescence images in 45 cycles. Each of the microdroplets was cycled 45 times to obtain a total of 45 fluorescence images. And positioning each micro-droplet in 45 fluorescence images, and acquiring 45 fluorescence intensity values of each micro-droplet so as to acquire a fluorescence curve of each micro-droplet.
In one embodiment, in the step S323, first, a fluorescence intensity value of each of the microdroplets subjected to PCR amplification is obtained from each fluorescence image. Then, obtaining the fluorescence curve of each microdroplet subjected to PCR amplification according to the fluorescence intensity value of each microdroplet subjected to PCR amplification. And finally, obtaining the fluorescence curves of all the microdroplets subjected to PCR amplification, namely the fluorescence curves of the microdroplet array according to the fluorescence curve of each microdroplet subjected to PCR amplification.
Referring to fig. 3, in one embodiment, a fluorescence image of the microdroplet array is acquired and image tracking is performed. When acquiring the fluorescence curve of each micro-droplet array, each micro-droplet in each image needs to be separately positioned, and the fluorescence intensity of each micro-droplet is acquired. In the digital PCR detector, the actual ratio of each pixel of the fluorescence image is calibrated in an imaging system. According to the fluorescence image, the number of pixels corresponding to the diameter of the micro-droplet is extracted, so that the number of micrometers corresponding to the diameter is obtained, and the diameter of the micro-droplet can be obtained according to the number of the pixels.
In one embodiment, when tracking each of the micro-droplets, the NCAST image differentiation and clustering operations may be performed on the images acquired during each temperature cycle to identify the position of each of the micro-droplets, and further obtain the fluorescence intensity of the micro-droplet array.
In one embodiment, if the microdroplet moves no more than one microdroplet diameter during one temperature cycle, microdroplet tracking can be performed using the following method. When tracking each micro-droplet, the image tracking step for each micro-droplet is as follows:
firstly, identifying a picture shot in each temperature circulation process to obtain the circle center position of each micro-droplet;
then, comparing the circle center position of each micro-droplet identified currently with the circle center position of each micro-droplet in the previous circulation process;
and finally, if the circle center distance between the currently identified circle center position of each micro-droplet and the circle center position of each micro-droplet in the previous circulation process is smaller than the diameter of one micro-droplet, marking the micro-droplets as the same micro-droplet.
Referring to fig. 4, in one embodiment, a fluorescence curve of each of the microdroplets is obtained according to the fluorescence intensity value of each of the microdroplets during each temperature cycle. And summing the fluorescence intensity values of the parts of each micro-droplet in each temperature cycle process to obtain the fluorescence intensity value of each micro-droplet at a specific moment.
In one embodiment, to prevent the adjacent edge portions of each of the micro-droplets from interfering with each other, the fluorescence intensity value of each of the micro-droplets at a specific time is partially summed. By the fluorescence intensity value of the micro-droplet array in each temperature cycle process, the change condition of the micro-droplet array in the whole cycle process can be obtained, and the fluorescence curve of each micro-droplet can be obtained.
In one embodiment, the step S330 includes:
s331, cooling the micro-droplet array subjected to polymerase chain reaction amplification to below 40 ℃;
s332, heating the micro-droplet array cooled to the temperature below 40 ℃ at a specific temperature interval to obtain a fluorescence image of the micro-droplet array corresponding to the temperature interval;
s333, acquiring fluorescence information of each micro-droplet corresponding to the temperature interval according to the fluorescence image of the micro-droplet array corresponding to the temperature interval; and
and S334, acquiring a melting curve of each micro-droplet according to the fluorescence information of each micro-droplet corresponding to the temperature interval, so as to obtain the melting curve of the micro-droplet array.
After the PCR amplification reaction was completed, the melting curve was generated by gradually increasing the temperature while monitoring the fluorescence signal at each step. As different DNAs have different temperature melting lines, the fluorescent dye returns to a free state to reduce the fluorescent signal as the double-stranded DNA is denatured in the reaction, and the change in the fluorescent signal is plotted against the temperature. The melting curve is needed to be made in the amplification product dye method, and because the melting curve is due to the poor specificity of the dye method, whether the amplification product is the target product or not is examined through the melting curve. The melting temperature has a characteristic peak, and the specific product can be separated from other products such as a primer dimer region or non-specific products by using the characteristic peak.
In step S332, after PCR amplification, melting curve analysis is performed on the PCR product, so that the PCR amplification and the melting curve analysis of the PCR product are seamlessly connected. In the step S332, the temperature is decreased to below 40 ℃ by the temperature control device, and the temperature is sequentially raised to 95 ℃ at the temperature interval of 0.1 ℃, and the signal acquisition device 30 photographs the image once at each interval of 0.1 ℃ until the temperature is raised to 95 ℃. And then, after the photographing is finished, cooling the micro-droplet array to 4 ℃, and storing the micro-droplet array.
In one embodiment, the method of processing the fluorescence image when obtaining the fluorescence curve of each of the micro-droplets is the same as the method of processing the fluorescence image when obtaining the melting curve of each of the micro-droplets.
In the step S333, a fluorescence intensity corresponding to each of the micro-droplets is obtained from the fluorescence image acquired at intervals of 0.1 ℃, a curve relating to temperature and fluorescence intensity is plotted, and a peaked graph containing a peak is obtained by first differentiation.
Melting curve (Dissociation curve) refers to the curve of the degree of degradation of the double helix structure of DNA with increasing temperature. Melting curve analysis can be used to determine different reaction products, including non-specific products. The temperature at which the overall DNA duplex structure is degraded by half is called the melting temperature (Tm), and for DNA of different sequences, the Tm value is different. That is, the melting curve of a DNA is a fingerprint of a DNA corresponding to a specific DNA. According to the melting curve, the temperature at which the peak is located represents the Tm value (melting point temperature) of the double-stranded DNA molecule. The genotype can be judged according to the Tm value of the amplified product. The Tm of a DNA fragment depends on its length, G + C composition, sequence, strand complementarity, concentration, and buffer components, such as salts, dyes, and PCR enhancers.
Each melting curve represents a single instance of the product in each of the microdroplets, and is considered normal if one melting curve is unimodal and within a reasonable temperature range (typically 80-90 ℃), and may be non-specific amplified if the melting curve is bimodal. Thus, it is possible to determine whether the product is a single gene or not. The internal reference has a line of the internal reference, and the peak of two genes can not appear on the same melting curve.
For each high resolution melting curve analysis of the micro-droplet array, single nucleotide polymorphisms and scanning mutations can be detected.
In one embodiment, during the digital PCR detection process, the micro-droplet array is subjected to image acquisition by the signal acquisition device.
And acquiring an image of the micro-droplet array through the signal acquisition device. Wherein, the micro-droplet container is irradiated on the micro-droplet container by adopting an inclined angle from the upper part of the micro-droplet container. The signal acquisition device is adopted to realize periodic two-dimensional scanning of the micro-droplet array and real-time image acquisition. The fluorescence inside the micro-droplet array in the micro-droplet container is excited, collected by the objective lens of the signal acquisition device and enters the camera, and the camera acquires the fluorescence image of the micro-droplet array.
The signal acquisition device can be used for carrying out fluorescence imaging on the micro-droplet array, shooting a certain number of fluorescence images of the micro-droplets at one time, and then automatically identifying the droplet fluorescence in the images by using an image processing technology so as to obtain the fluorescence information of the droplets.
And the fluorescence detection assembly of the fluorescence signal detection device is used for collecting fluorescence information of the micro-droplet array containing the fluorescent substance, and transmitting the detected fluorescence information to a computer in the form of a fluorescence image for quantitative analysis. And shooting a certain number of fluorescence images of the micro-droplets at one time by adopting a fluorescence imaging detection method, and then automatically identifying the fluorescence of the droplets in the images by utilizing an image processing technology so as to obtain the fluorescence information of the droplets. The imaging range of the fluorescence imaging detection method is large, so that the requirement on the detection environment where the micro-droplet array is located during detection is low.
In one embodiment, the step S40 includes:
s410, acquiring the initial copy number of the nucleic acid of the micro-droplet array according to the fluorescence curve of the micro-droplet array; and
and S420, acquiring the nucleic acid information of the micro-droplet array according to the melting curve of the micro-droplet array.
In one embodiment, the step S410 includes:
s411, acquiring a Ct value corresponding to the fluorescence curve of each micro-droplet according to the fluorescence curve of the micro-droplet array;
s412, clustering is carried out according to the Ct value of the fluorescence curve of each micro-droplet, and sequencing is carried out from large to small in sequence to obtain x 1 ，x 2 ，…，x n A category;
s413, according to the x 1 ，x 2 ，....x n Obtaining the number y of the micro-droplets corresponding to each category 1 、y 2 、....y n ；
S414, the number y of the micro-droplets corresponding to each category 1 、y 2 、....y n Obtaining said x 1 ，x 2 ，…，x n The number y of the micro-droplets corresponding to each category 1 、y 2 、....y n Frequency distribution;
and S415, calculating the initial copy number of the nucleic acid of the micro-droplet array according to the frequency distribution.
In step S411, when the PCR cycle reaches the cycle number where the Ct value is reached, the PCR cycle just enters the true exponential amplification stage (logarithmic phase), and at this time, the small error is not amplified yet, so the reproducibility of the Ct value is excellent, that is, the Ct value obtained by amplifying the same DNA template at different times or amplifying the same DNA template in different microdroplet containers at the same time is constant. When the fluorescence curve corresponding to the micro-droplet is an amplification curve, the micro-droplet is indicated to contain the target gene component. When the fluorescence curve corresponding to the micro-droplet is a straight line, the micro-droplet is indicated to contain no target gene component.
And obtaining a Ct value from the obtained real-time fluorescence curve, deriving the fluorescence curve when the Ct value of each micro-droplet is obtained, and determining the initial cycle number of the fluorescence curve with fixed slope of the fluorescence curve as the required Ct value.
In one embodiment, in step S411, first, a derivative is taken from the fluorescence curve of each PCR-amplified microdroplet to obtain a slope of the fluorescence curve of each PCR-amplified microdroplet. And secondly, acquiring a numerical value with a constant slope in the slopes of the fluorescence curves of the microdroplets subjected to the PCR amplification according to the slopes of the fluorescence curves of the microdroplets subjected to the PCR amplification. And thirdly, acquiring the corresponding initial cycle number according to the fixed and unchangeable value of the slope, wherein the initial cycle number is the Ct value of each micro-droplet subjected to PCR amplification. And finally, obtaining the Ct values of all the microdroplets subjected to PCR amplification according to the Ct value of each microdroplet subjected to PCR amplification.
In one embodiment, in step S411, firstly, a defect value of the fluorescence threshold of each microdroplet subjected to PCR amplification is obtained according to the fluorescence curve of each microdroplet subjected to PCR amplification. And secondly, obtaining the corresponding cycle number according to the defect value of the fluorescence domain value of each microdroplet subjected to PCR amplification, wherein the cycle number is the Ct value of each microdroplet subjected to PCR amplification. And thirdly, obtaining the Ct values of all the microdroplets subjected to PCR amplification according to the Ct value of each microdroplet subjected to PCR amplification.
Wherein C in the Ct value represents Cycle, t represents threshold, and the Ct value means: the number of cycles that the fluorescence signal in each reaction tube has undergone to reach a set threshold. In real-time fluorescent PCR, the Ct value refers to the number of cycles that the fluorescent signal in each reaction tube undergoes when it reaches a set threshold. The fluorescence signal of the first 15 cycles of the PCR reaction is used as the fluorescence background signal, and the defect setting of the fluorescence threshold value is 10 times of the standard deviation of the fluorescence signal of 3-15 cycles, namely: threshold =10 × SDcycle 3-15.
In one embodiment, the fluorescence signal of the first 15 cycles of the PCR reaction is used as the fluorescence background signal, and the defect setting of the fluorescence threshold is 10 times the standard deviation of the fluorescence signal of 3-15 cycles, i.e.: threshold =10 × SDcycle 3-15. And acquiring the corresponding cycle number according to the defect value threshold of the fluorescence threshold value, wherein the cycle number is the Ct value. The Ct value is related to the starting DNA concentration by: the greater the initial copy number, the smaller the Ct value.
In the step S412, clustering is performed according to the Ct value of the fluorescence curve of each micro-droplet, and sorting is performed sequentially from large to small to obtain x 2 ，…，x n And (4) each category. Wherein the category corresponding to the dark droplets in the micro-droplet array is x 1 That is, the droplets in the micro-droplet array that do not contain the initial copy number of nucleic acid correspond to the category x 1 A category. Since the larger the initial copy number of nucleic acid, the smaller the Ct value, the Ct value corresponding to the dark droplet (negative droplet) is infinite at this time, that is, the x is 1 The Ct value for a class is infinity. By analogy, the x 2 The initial copy number of nucleic acids corresponding to the class is 1, x 3 The initial copy number of nucleic acid corresponding to the class is 2, x 4 The initial copy number of nucleic acids corresponding to class is 3, x 5 The initial copy number of nucleic acids for a class is 4, etc.
In one embodiment, in the step S415, when x is greater than x 1 Number y of the micro-droplets of the class 1 And when the frequency distribution is larger than or equal to the characteristic value m, carrying out Poisson distribution fitting according to the frequency distribution to obtain a parameter lambda of the Poisson distribution, thereby obtaining the initial copy number of the nucleic acid of the micro-droplet array.
Wherein the characteristic value m ranges from 0.5% to 10% of the total number of microdroplets in the microdroplet array.
In one embodiment, the characteristic value m may be 5% of the total number of microdroplets.
When said x is 1 Number y of the micro-droplets of a class 1 When the number of the dark droplets in the micro-droplet array is larger than or equal to the characteristic value m 1 Greater than or equal to the characteristic value m. At this time, the number of dark droplets in the micro-droplet array plays a role in calculating the initial copy number of nucleic acid of the micro-droplet array as a whole, thereby the number y of micro-droplets 1 、y 2 、....y n And performing Poisson distribution fitting on the frequency distribution to obtain a corresponding parameter lambda of the Poisson distribution.
Suppose that: the microdroplets in digital PCR contain a starting DNA copy number of x, and according to mathematical statistical theory, the probability distribution function P of x = k (k =0,1,2,3.. Once.) fits the poisson probability model, where λ is the average molecular copy number contained in the microdroplet.
Therefore, the expectation value mu and the variance sigma are obtained through the Poisson distribution model 2 It can be seen that the expected value μ is λ and the variance σ is 2 Is lambda. Therefore, it is known that the copy number of the target DNA molecule contained in each microdroplet in the digital PCR is λ, and thus the determined λ value enables quantitative detection of nucleic acid.
Wherein, for the droplet-based PCR, the initial copy number contained in a single droplet satisfies the Poisson distribution.
Wherein λ is the starting DNA copy number contained on average in the microdroplet. The average starting copy number contained in each droplet is represented by CPD (copies per copy).
At this time, the initial number of copies of nucleic acids of the microdroplet array is λ multiplied by the number of microdroplets of the microdroplet array.
Assuming that the total volume of the nucleic acid amplification reaction solution to be detected is V (the volume of each micro-droplet is V), the concentration c (copy/. Mu.L) of the nucleic acid amplification reaction solution to be detected is:
therefore, the quantitative detection of DNA can be realized by determining the value of lambda.
In one embodiment, in the step S415, when x is greater than x 1 Number y of the micro-droplets of the class 1 When the value is less than the characteristic value m, the method comprises the following steps:
s4151 according to said x 2 ，…，x n The number y of the micro-droplets corresponding to each category 2 、…，y n The partial frequency distribution of (2), successively assuming the x 2 Class-corresponding initial copies of said nucleic acidsCounting, and carrying out Poisson distribution fitting to obtain a parameter lambda corresponding to each Poisson distribution j (j＝0，1，2....)；
S4152, in a range [ lambda ] min ,λ max ]Inner search lambda j So that the sum of squared frequency error err is minimized to obtain the optimal lambda optimal ；
S4153 according to said optimum lambda optimal And calculating the initial copy number of the nucleic acid of the micro-droplet array.
When said x is 1 Number y of the micro-droplets of a class 1 When the number of the dark droplets in the micro-droplet array is less than the characteristic value m, the number of the dark droplets in the micro-droplet array has no effect on the overall calculation of the initial copy number of the nucleic acid of the micro-droplet array, and can be ignored.
Biorad System in the case of 20000 droplet systems, it is generally recommended that the sample DNA concentration is not greater than 6CPD. In the course of practical experiments, k>4, the difference in Ct value becomes small, and it is difficult to distinguish the initial copy number of one droplet as 4 or 5 from the Ct value, so that only the x value can be used 2 ，…，x n Incomplete sampling of each class was performed to fit the Poisson distribution.
In one embodiment, an interval λ is given min ,λ max ]At [ lambda ] min ,λ max ]Searching in interval, calculating the minimum sum of squares err of errors, and selecting the optimal lambda optimal So that the sum of squared errors is minimized.
In one embodiment, when the parameter λ of the poisson distribution is obtained, the parameter λ is estimated by using a maximum likelihood estimation method.
In one embodiment, the method of estimating the parameter λ may also be a moment estimator, a sequential statistics method, or a maximum likelihood method.
By adopting the method, the number of negative dark liquid drops is not required to be ensured, and the accuracy and the stability are much higher than those of estimation by singly adopting one frequency point.
In the step S4153, the initial copy number of nucleic acids of the micro-droplet array is the optimal lambda optimal Multiplied by the number of microdroplets of the microdroplet array.
In one embodiment, the parameter λ of the poisson distribution is point estimated using a least squares method based on the incomplete samples.
In a practical process, the digital PCR quantitative detection method can determine the initial copy number of the nucleic acid of the micro-droplet array with high precision without depending on a standard curve.
Meanwhile, the problem of false positive in the micro-droplet array can be solved through a real-time fluorescence curve. True absolute quantification is achieved by processing the fluorescence curve of the microdroplet array and performing statistical corrections independent of the homogeneity assumption.
The digital PCR quantitative detection method not only gets rid of the dependence on the standard fluorescence curve and eliminates the problem of uncertain quantitative results caused by the standard fluorescence curve, but also solves the limitation of a droplet-type digital PCR endpoint detection mode, breaks the limitation of parameter estimation on the whole sample to be detected by only adopting data of one p (x = 0), and improves the accuracy of digital PCR quantitative detection.
The digital PCR quantitative detection method is adopted without ensuring the number of negative empty liquid drops. Meanwhile, the accuracy of the overall optimal parameter estimation by adopting the multidimensional frequency distribution data is much higher than that of the estimation by singly adopting the data of p (x = 0).
Each fluorescence curve represents the change process of a curve with useful information, and the change process participates in the droplet sample information to realize real-time monitoring so as to set an algorithm to eliminate the mutual influence between adjacent droplets.
The digital PCR detection method depends on an abstract mathematical model, realizes repeatability and high sensitivity, enlarges the dynamic range, and can realize monitoring by using a small amount of liquid drops. More information is covered with a small amount of data. Meanwhile, the digital PCR quantitative detection method avoids the error of the prior Poisson distribution probability model, realizes absolute quantification and is more visual. And, all data can be combined by the digital PCR quantitative detection method, so that the generation of random errors can be avoided. And acquiring a fluorescence curve of the liquid drop sample, and monitoring the change of the fluorescence brightness of the liquid drop sample in real time to remove false positives, eliminate the mutual influence between adjacent liquid drops and provide a more accurate data source for a subsequent quantitative analysis model.
Referring to FIG. 5, the Poisson distribution fit obtained for the cases where the initial copy numbers of the partial nucleic acids were 0,1,2,3, respectively. Wherein the abscissa is the average starting copy number (CPD) contained in each of the microdroplets. The ordinate is the standard deviation (Std Dev, std) of the mean initial copy number contained in each of the microdroplets. The average starting copy number contained in each of the microdroplets is represented by CPD (copies per copy). It can be seen that the standard deviation of the mean initial copy number in CPD contained in each of the microdroplets obtained using a portion of the initial copy number of the nucleic acid is smaller than the standard deviation of the mean initial copy number in CPD obtained by other algorithms. Therefore, the average starting copy number contained in each of the microdroplets obtained by the digital PCR detection method is more accurate with the value of CPD. The results of 1000 simulations on 20000 droplets indicate. The estimation method using only a single point can cover only a limited concentration range, and the estimation accuracy deteriorates sharply as the concentration of the sample increases. And by adopting an incomplete poisson distribution fitting algorithm, along with the increase of the concentration of the sample, the estimation precision is not obviously deteriorated, and the concentration of the nucleic acid amplification reaction solution to be detected can be doubled. For the case of a small number of droplets, the incomplete poisson distribution fitting algorithm (partial sampling poisson distribution fitting algorithm) still has good reliability.
The method solves the problem of false positive and false negative of the result by the digital PCR quantitative detection method. The sequencing platform has high sample throughput, and can detect hundreds of samples at the same time. Meanwhile, the detection of a plurality of sites can be carried out by utilizing different types of fluorescence, the detection speed is accelerated, and the experiment cost is reduced. The method has the advantages that the rare detection fragments are separated from a large number of complex backgrounds by adopting the digital PCR detector through micro-titration treatment, the operation steps are greatly simplified, the preparation time and the detection time are effectively saved, the result interpretation is visual and reliable, the method has the characteristic of stable implementation, the detection sensitivity and the detection accuracy meet the requirements of accurate quantification, and the detection sensitivity and the detection accuracy are improved.
In one embodiment, the step S420 includes:
s421, obtaining a melting temperature corresponding to the melting curve of each micro-droplet according to the melting curve of the micro-droplet array; and
s422, classifying the micro-droplet array according to the melting temperature to obtain the nucleic acid information of the micro-droplet array, and further obtaining the nucleic acid information of the nucleic acid to be detected.
The Tm values of DNAs having different sequences are different from each other. That is, the melting curve of a DNA is a fingerprint of a DNA corresponding to a specific DNA. According to the melting curve, the temperature at which the peak is located represents the Tm value (melting point temperature) of the double-stranded DNA molecule. The genotype can be judged according to the Tm value of the amplified product. By classifying and dividing the same melting curve, genotyping or classifying the melting curves in different shapes, and comparing the melting curves with the melting curve of the target gene, non-specific false positives can be removed, and sequences different from the target gene are excluded.
In one embodiment, when the micro-droplet array is classified by a melting curve, algorithms such as decision trees, bayesian, artificial neural networks, K-nearest neighbor, support vector machines and association rule based classification, bagging and Boosting can be used.
In one embodiment, the digital PCR detection method further comprises:
s50, obtaining a high-resolution melting curve of the micro-droplet array, classifying the micro-droplet array, and obtaining the nucleic acid information of the micro-droplet array, such as genotyping, mutation detection and the like.
According to the melting curve, the specificity of nucleic acid amplification of the micro-droplet array, whether a primer dimer phenomenon exists in a nucleic acid amplification layer process or not can be obtained.
And acquiring a high-resolution melting curve of the micro-droplet array according to the change of the fluorescence signal value of the micro-droplet array in the dsDNA melting process. The high-resolution melting curve is a new gene analysis technology for forming melting curves of different forms based on different melting temperatures of mononucleotides, has extremely high sensitivity, can detect the difference of single basic groups, has low cost, high flux, high speed and accurate result, is not limited by detection sites, and realizes real closed-tube operation. The HRM analysis technology plays an important role in mutation scanning, single nucleotide polymorphism analysis, methylation research, genotyping, sequence matching and the like. The thermal stability of a double-stranded nucleotide is affected by its length and base composition, and sequence changes can result in altered melting behavior of dsDNA during warming. Since the fluorescent dye used can only be intercalated and bound to dsDNA, differences in PCR products can be visualized by real-time PCR techniques by detecting changes in fluorescence signal values during melting of dsDNA in real-time to generate melting curves of different shapes. Meanwhile, the gene classification or classification based on the melting curves of different shapes can be realized for the test population by means of professional analysis software.
In one embodiment, in forming the micro-droplet array in step S20, a transient acceleration micro-droplet generation method or a variable speed period micro-droplet generation method may be used.
In one embodiment, a saturated dye is used to analyze the pcr product in the amplification reaction solution in step S10.
In one embodiment, when performing qualitative classification analysis on a plurality of microdroplets, high resolution melting analysis (HRM) can be used to analyze PCR reaction products using a saturation dye without using sequence specific probes. The thermal stability of double-stranded nucleotides (dsDNA) is affected by their length and base composition, and sequence changes can lead to changes in melting behavior of dsDNA during warming. Since the fluorescent dye used can only be intercalated and bound to dsDNA, differences in PCR products can be visualized by real-time PCR techniques by detecting changes in fluorescence signal values during melting of dsDNA in real-time to generate melting curves of different shapes. Meanwhile, the gene classification or classification based on the melting curves of different shapes can be realized for the test population by means of professional analysis software.
In one embodiment, the primer design has three basic principles. First, the primer is closely complementary to the sequence of the template. Second, the formation of stable dimers or hairpins between primers is avoided. Again, the primer is unable to initiate DNA polymerization (i.e., mismatches) at sites of the template that are not of interest. The three basic principles are realized by considering a plurality of factors, such as the length of the primer, the length of the product, the Tm value of the sequence, the internal stability of the double strand formed by the primer and the template, the energy value of the primer dimer and the hairpin structure, the priming efficiency at the mismatch site, the GC content of the primer and the product, and the like. Meanwhile, the primer can be modified aiming at special detection, such as increasing restriction enzyme sites, introducing mutation and the like.
The Tm value of the sequence corresponding to the position of the template to the primer is about 72 ℃ to optimize the renaturation conditions. There are several methods for calculating the Tm value, such as the nearest neighbor method used in Oligo software according to the formula Tm =4 (G + C) +2 (a + T).
Referring to fig. 6, in one embodiment, the melting curve of the microdroplet array obtained by the digital PCR detection method is plotted by the negative first derivative of the change of fluorescence signal and temperature, and the temperature at which the peak is located represents the Tm value (melting point temperature) of the double-stranded DNA molecule. The genotype can be judged according to the Tm value of the amplified product. From this, it can be seen that there are two melting temperatures Tm1 and Tm2. The melting temperature Tm1 and the melting temperature Tm2 correspond to two different types of DNA, respectively, so that the target DNA in the micro-droplet array can be distinguished.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present application. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (7)
1. A digital PCR detection method, comprising:
s10, preparing a nucleic acid amplification reaction solution to be detected;
s20, micro-dripping the nucleic acid amplification reaction solution to be detected to form a micro-droplet array;
s30, performing polymerase chain reaction on the micro-droplet array, and acquiring a fluorescence curve of each micro-droplet in the micro-droplet array and a melting curve of each micro-droplet; and
s411, acquiring a Ct value corresponding to the fluorescence curve of each micro-droplet according to the fluorescence curve of the micro-droplet array;
s412, clustering is carried out according to the Ct value of the fluorescence curve of each micro-droplet, and sequencing is carried out from large to small in sequence to obtain x 1 ，x 2 ，....x n A category;
s413 according to the x 1 ，x 2 ，....x n Obtaining the number y of the micro-droplets corresponding to each category 1 、y 2 、....y n ；
S414, the number y of the micro-droplets corresponding to each category 1 、y 2 、....y n Obtaining said x 1 ，x 2 ，....x n The number y of the micro-droplets corresponding to each category 1 、y 2 、....y n Frequency distribution;
s415, (1) when x is 1 Number y of the micro-droplets of a class 1 When the number of the initial copies of the nucleic acid of the micro-droplet array is larger than or equal to the characteristic value m, carrying out Poisson distribution fitting according to the frequency distribution to obtain a parameter lambda of Poisson distribution, thereby obtaining the initial copy number of the nucleic acid of the micro-droplet array;
(2) When said x is 1 Number y of the micro-droplets of the class 1 When the characteristic value is less than the characteristic value m, the method comprises the following steps:
s4151 according to said x 2 ，....x n The number y of the micro-droplets corresponding to each category 2 、....y n The partial frequency distribution of (2), successively assuming the x 2 The initial copy number of the nucleic acid corresponding to the category is subjected to poisson distribution fitting to obtain a parameter lambda corresponding to each poisson distribution j (j＝0，1，2....)；
S4152, in a range [ lambda ] min ,λ max ]Inner search lambda j So as to minimize the sum of squared error err of frequency value and obtain the optimum lambda optimal ；
S4153, according to the optimal lambda optimal Calculating the initial copy number of the nucleic acid of the micro-droplet array; and
and S420, acquiring the nucleic acid information of the micro-droplet array according to the melting curve of the micro-droplet array.
2. The digital PCR detection method of claim 1, wherein the S30 comprises:
s310, setting temperature parameters, time parameters and cycle times of the polymerase chain reaction;
s320, performing polymerase chain reaction on the micro-droplet array according to the temperature parameter and the time parameter, completing the cycle times in sequence, and acquiring a fluorescence curve of each micro-droplet in each cycle process; and
s330, cooling the micro-droplet array subjected to polymerase chain reaction amplification, and heating at specific temperature intervals to obtain a melting curve of each micro-droplet.
3. The digital PCR detection method of claim 2, wherein the S320 comprises:
s321, performing polymerase chain reaction on the micro-droplet array according to the temperature parameter and the time parameter to obtain a fluorescence image of the micro-droplet array;
s322, sequentially circulating according to the circulation times to obtain all fluorescence images of the micro-droplet array in the polymerase chain reaction process;
s323, acquiring fluorescence information of each micro-droplet in each circulation process according to all fluorescence images of the micro-droplet array; and
and S324, acquiring a fluorescence curve of each micro-droplet according to the fluorescence information of each micro-droplet in each circulation process, so as to obtain the fluorescence curve of the micro-droplet array.
4. The digital PCR detection method of claim 2, wherein the S330 comprises:
s331, cooling the micro-droplet array subjected to polymerase chain reaction amplification to below 40 ℃;
s332, heating the micro-droplet array cooled to the temperature below 40 ℃ at a specific temperature interval to obtain a fluorescence image of the micro-droplet array corresponding to the temperature interval;
s333, acquiring fluorescence information of each micro-droplet corresponding to the temperature interval according to the fluorescence image of the micro-droplet array corresponding to the temperature interval; and
and S334, acquiring a melting curve of each micro-droplet according to the fluorescence information of each micro-droplet corresponding to the temperature interval, so as to obtain the melting curve of the micro-droplet array.
5. The digital PCR detection method of claim 1, wherein the characteristic value m ranges from 0.5% to 10% of the total number of microdroplets in the microdroplet array.
6. The digital PCR detection method of claim 1, wherein the S420 comprises:
s421, obtaining a melting temperature corresponding to the melting curve of each micro-droplet according to the melting curve of the micro-droplet array; and
s422, classifying the micro-droplet array according to the melting temperature to obtain the nucleic acid information of the micro-droplet array, and further obtaining the nucleic acid information of the nucleic acid to be detected.
7. The digital PCR detection method of claim 1, further comprising:
s50, obtaining a high-resolution melting curve of the micro-droplet array, classifying the micro-droplet array, and obtaining the nucleic acid information of the micro-droplet array, such as genotyping, mutation detection and the like.
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| CN201810932950.6A CN110835645B (en) | 2018-08-16 | 2018-08-16 | Digital PCR detection method |
| JP2020560539A JP7094524B2 (en) | 2018-01-24 | 2019-01-24 | Digital PCR detector, digital PCR quantitative detection method, quantitative analysis method of digital PCR of different volumes, digital PCR detection method, nucleic acid detection microsphere, nucleic acid detection microsphere production method, nucleic acid detection microsphere reagent kit and high throughput nucleic acid detection method |
| CA3089411A CA3089411C (en) | 2018-01-24 | 2019-01-24 | Digital pcr detection apparatus, digital pcr quantitative detection method, multi-volume digital pcr quantitative analysis method, digital pcr detection method, nucleic acid detection microsphere, preparation method of nucleic acid detection microsphere, nucleic acid detection microsphere kit and high-throughput nucleic acid detection method |
| US16/964,183 US20210032680A1 (en) | 2018-01-24 | 2019-01-24 | Digital pcr detection apparatus, digital pcr quantitative detection method, multi-volume digital pcr quantitative analysis method, digital pcr detection method, nucleic acid detection microsphere, preparation method of nucleic acid detection microsphere, nucleic acid detection microsphere kit and high-throughput nucleic acid detection method |
| EP19743502.7A EP3739059B1 (en) | 2018-01-24 | 2019-01-24 | Detection instrument for digital pcr and quantitative detection method for digital pcr |
| CA3188153A CA3188153A1 (en) | 2018-01-24 | 2019-01-24 | Digital pcr detection apparatus, digital pcr quantitative detection method, multi-volume digital pcr quantitative analysis method, digital pcr detection method, nucleic acid detection microsphere, preparation method of nucleic acid detection microsphere, nucleic acid detection microsphere kit and high-throughput nucleic acid detection method |
| EP23206494.9A EP4293673A3 (en) | 2018-01-24 | 2019-01-24 | Detection instrument for digital pcr and quantitative detection method for digital pcr |
| PCT/CN2019/072974 WO2019144907A1 (en) | 2018-01-24 | 2019-01-24 | Detection instrument for digital pcr, quantitative detection method for digital pcr, quantitative analysis method for digital pcr having different volume, detection method for digital pcr, microsphere for nucleic acid test, preparation method for microsphere for nucleic acid test, kit for microsphere for nucleic acid test, and detection method for high-throughput nucleic acid |
| JP2022096041A JP2022120133A (en) | 2018-01-24 | 2022-06-14 | Digital pcr detection apparatus, digital pcr quantitative detection method, multi-volume digital pcr quantitative analysis method, digital pcr detection method, nucleic acid detection microsphere, preparation method of nucleic acid detection microsphere, nucleic acid detection microsphere kit, and high-throughput nucleic acid detection method |
| JP2023042563A JP2023075307A (en) | 2018-01-24 | 2023-03-17 | Digital pcr detection apparatus, digital pcr quantitative detection method, multi-volume digital pcr quantitative analysis method, digital pcr detection method, nucleic acid detection microsphere, preparation method of nucleic acid detection microsphere, nucleic acid detection microsphere kit, and high-throughput nucleic acid detection method |
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