Method, device and electronic device for separating overlapping peaks of melting curves

Abstract

The present invention provides a method, device and electronic device for separating overlapping peaks of melting curves, and relates to the technical field of PCR detection, including: firstly obtaining the melting point peak position of each hole melting curve of a PCR reaction plate based on a conventional method, and determining the second-order derivative curve of each hole melting curve using a Savitzky‑Golay derivation method; then determining the initial overlapping peak position of the original melting curve according to the second-order derivative curve and the melting point peak position; then superimposing and fitting the initial overlapping peak position to generate two melting point peak curves; finally determining the target melting point peak that meets the error range according to the two melting point peak curves, and separating the target melting point peak. The method reduces background interference and improves the resolution of melting point peaks by calculating the second-order derivative curve of the melting curve, and better solves the problem that there is only one Tm value for the difficult melting curve melting point peak with overlapping peaks under the condition of large background interference.

Description

Technical Field

[0001] The invention relates to the technical field of PCR detection, and in particular to a method, a device and an electronic device for separating overlapping peaks of a melting curve. Background Art

[0002] After the PCR amplification reaction is completed, the negative derivative value of the fluorescence intensity, i.e., the melting curve, is often obtained by gradually increasing the temperature to degrade the amplified product, so as to examine the specificity of the amplified product or perform SNP typing detection. During the heating process, when the temperature reaches the half-melting temperature, a large amount of fluorescent dye will be released, and the fluorescence intensity will decrease rapidly, thus forming a high peak point on the melting curve. The temperature corresponding to the peak point is the Tm value, and the number and position of Tm are the focus of the investigation. When the amplitude characteristics of each peak point of the melting curve are obvious, it is not difficult to find the Tm value.

[0003] In some existing technologies, traditional methods such as cluster analysis and continuous wavelet transform are often used to analyze melting curves; some technologies provide a melting curve analysis method that can automatically analyze whether there is a peak in at least one of the two temperature ranges through a direct search algorithm; or by clustering the peak heights of the target melting peaks of multiple melting curves and combining them with standards, the category of the target gene is obtained. However, none of the above schemes involve more complex melting curves, that is, when the melting curve has large background interference and has hidden overlapping peaks, conventional methods are difficult to effectively solve. Summary of the invention

[0004] The purpose of the present invention is to provide a method, device and electronic device for separating overlapping peaks of melting curves, so as to alleviate the technical problem of the difficulty of complex melting curve analysis existing in the prior art.

[0005] In a first aspect, an embodiment of the present invention provides a method for separating overlapping peaks of melting curves, the method comprising: based on the pre-acquired melting point peak positions of the melting curves of each well of a PCR reaction plate, using a Savitzky-Golay derivation method to determine a second-order derivative curve of the melting curve of each well;

[0006] Determining the initial overlapping peak position of the original melting curve according to the second-order derivative curve and the melting point peak position;

[0007] The above initial overlapping peak positions are superimposed and fitted to generate two melting point peak curves;

[0008] A target melting point peak satisfying an error range is determined based on the two melting point peak curves, and the target melting point peak is separated.

[0009] In a possible embodiment, the melting point peak position of the melting curve of each well of the PCR reaction plate corresponds to the temperature (Tm) value of the melting point peak; based on the pre-acquired melting point peak position of the melting curve of each well of the PCR reaction plate, the step of determining the second-order derivative curve of the melting curve of each well using the Savitzky-Golay derivation method includes: according to the Savitzky-Golay derivation method, using a pre-set first derivation parameter to calculate the second-order derivative curve of the melting curve of each well; wherein the above-mentioned first derivation parameter includes: a window size with a highest determination coefficient and a polynomial order.

[0010] In a possible embodiment, the step of determining the initial overlapping peak position of the target melting curve based on the second-order derivative curve and the melting point peak position includes: determining the first region corresponding to each melting point peak based on a predetermined first range, and performing normalization processing; the first region includes the second-order derivative region of the melting curve selected within the first range; determining the minimum point of the second-order derivative curve within the first region; determining the minimum point that meets a predetermined first threshold as the initial overlapping peak position based on the relative amplitude of the minimum point; the condition of the predetermined first threshold includes: within the first region, the amplitude of the maximum point of the second-order derivative curve does not differ by more than two times.

[0011] In a possible embodiment, the step of predetermining the first range includes: taking the temperature (Tm) value of the melting point peak corresponding to the melting point peak position as a base point, taking the standard melting point peak resolution as an interval, and determining the trough corresponding to the melting point peak at the left and right ends of the base point respectively; the standard melting point peak resolution is four times the minimum melting point peak resolution; wherein the minimum melting point peak resolution is 2°C;

[0012] If the above-mentioned trough does not exist at both ends of the above-mentioned base point, the above-mentioned standard melting point peak resolution is determined to be the first intermediate value; the minimum value of the above-mentioned first range is the temperature (Tm) value of the above-mentioned melting point peak minus the above-mentioned first intermediate value; the maximum value of the above-mentioned first range is the temperature (Tm) value of the above-mentioned melting point peak plus the first intermediate value;

[0013] If the above-mentioned troughs exist on both the left and right ends of the above-mentioned base point, then determine that the distances between the above-mentioned base point and the above-mentioned troughs on the left and right ends are T left and T right respectively; compare T left with the resolution of the above-mentioned standard melting point peak, and determine that the minimum value is the second intermediate value; compare T right with the above-mentioned resolution, and determine that the minimum value is the third intermediate value; then the minimum value of the above-mentioned first range is the temperature (Tm) value of the above-mentioned melting point peak minus the above-mentioned second intermediate value; the maximum value of the above-mentioned first range is the temperature (Tm) value of the above-mentioned melting point peak plus the third intermediate value.

[0014] In a possible embodiment, the step of superimposing and fitting the above-mentioned initial overlapping peak positions to generate two melting point peak curves includes: setting initial parameters of the initial function based on the initial overlapping peak positions; using the Levenberg-Marquardt algorithm to superimpose and fit the above-mentioned second-order derivative curves in the above-mentioned first region corresponding to each melting point peak based on the above-mentioned initial function to generate two functions to be optimized; optimizing the above-mentioned two functions to be optimized according to the above-mentioned initial parameters to generate two objective functions; wherein the above-mentioned objective function includes the objective parameters; and the two above-mentioned objective functions correspond to the two above-mentioned melting point peak curves respectively.

[0015] In a possible implementation, the objective function is a three-parameter Lorentz function: g(v)=a·c / [c+(vb) 2 ]; where: v is used to represent temperature, a is used to represent coefficient, b is used to represent the position of overlapping peaks after optimization, and c is used to represent variance.

[0016] In a possible embodiment, the step of determining a target melting point peak that satisfies an error range based on the two melting point peak curves, and separating the target melting point peaks comprises: determining a target melting point peak that satisfies an error range based on the melting point peak curves; the error range comprises: the range of the difference between the first sum of squares before superposition fitting and the second sum of squares after superposition fitting; separating the target melting point peaks, and determining the positions of the two melting point peaks after separation and the temperature values ​​corresponding to the melting point peaks.

[0017] In a second aspect, an embodiment of the present invention provides a device for separating overlapping peaks of melting curves, the device comprising: a first determination module, for determining a second-order derivative curve of the melting curve of each well of a PCR reaction plate based on the pre-acquired melting point peak position of the melting curve of each well by using a Savitzky-Golay derivation method;

[0018] A second determination module is used to determine the initial overlapping peak position of the original melting curve according to the second-order derivative curve and the melting point peak position;

[0019] A melting point peak curve generation module is used to perform superposition fitting on the initial overlapping peak positions to generate two melting point peak curves;

[0020] The separation module is used to determine a target melting point peak that meets the error range according to the two melting point peak curves, and separate the target melting point peak.

[0021] In a possible embodiment, the melting point peak position of the melting curve of each well of the PCR reaction plate corresponds to the temperature (Tm) value of the melting point peak; the above-mentioned first determination module is specifically used to: calculate the second-order derivative curve of the melting curve of each well according to the Savitzky-Golay derivation method using a pre-set first derivation parameter; wherein the above-mentioned first derivation parameter includes: a window size with a highest determination coefficient and a polynomial order.

[0022] In a third aspect, an embodiment of the present invention provides an electronic device, comprising a memory and a processor, wherein the memory stores a computer program executable on the processor, and when the processor executes the computer program, the steps of any one of the methods described in the first aspect are implemented.

[0023] In a fourth aspect, an embodiment of the present invention provides a computer-readable storage medium, wherein the computer-readable storage medium stores machine-executable instructions. When the computer-executable instructions are called and executed by a processor, the computer-executable instructions cause the processor to execute any method described in the first aspect above.

[0024] The present invention provides a method, device and electronic device for separating overlapping peaks of melting curves, the method comprising: firstly obtaining the melting point peak position of each well melting curve of a PCR reaction plate based on a conventional method, and determining the second-order derivative curve of each well melting curve using a Savitzky-Golay derivation method; then determining the initial overlapping peak position of the original melting curve according to the second-order derivative curve and the melting point peak position; then superimposing and fitting the initial overlapping peak position to generate two melting point peak curves; finally determining the target melting point peak that meets the error range according to the two melting point peak curves, and separating the target melting point peak. The method reduces background interference and improves the resolution of melting point peaks by calculating the second-order derivative curve of the melting curve, and better solves the problem that the melting point peak of the difficult melting curve with overlapping peaks has only one Tm value under the condition of large background interference, and achieves the technical effect of reducing difficulty and improving analysis efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 A schematic flow chart of a method for separating overlapping peaks of melting curves provided in an embodiment of the present invention;

[0027] Figure 2A melting curve of a reaction well and a corresponding second-order derivative curve graph provided by an embodiment of the present invention;

[0028] Figure 3 An original melting curve, a superimposed fitting melting curve and a comparison diagram of two separation curves provided in an embodiment of the present invention;

[0029] Figure 4 A schematic diagram of the structure of an electronic device provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings here can be arranged and designed in various different configurations.

[0031] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.

[0033] After the PCR amplification reaction is completed, the negative derivative value of the fluorescence intensity, i.e., the melting curve, is often obtained by gradually increasing the temperature to degrade the amplified product, so as to examine the specificity of the amplified product or perform SNP typing detection. During the heating process, when the temperature reaches the half-melting temperature, a large amount of fluorescent dye will be released, and the fluorescence intensity will decrease rapidly, thus forming a high peak point on the melting curve. The temperature corresponding to the peak point is the Tm value, and the number and position of Tm are the key points of the investigation.

[0034] When the amplitude characteristics of each peak point of the melting curve are obvious, it is not difficult to find the Tm value. For example, the patent "Melting Curve Analysis Method and Melting Curve Analysis Device" provides a melting curve analysis method that can automatically analyze whether there is a peak in at least one of two temperature ranges through a direct search algorithm. The patent "A Software Analysis Method for Multiple Melting Curves" obtains the category of the target gene by clustering the peak heights of multiple melting curves and combining them with standard products. We have also used cluster analysis, CWT (continuous wavelet transform), etc. to analyze the melting curve, but when the background interference of the melting curve is large and there are hidden overlapping peaks, conventional methods are difficult to effectively solve the problem.

[0035] To this end, the present application proposes a method, device and electronic device for separating overlapping peaks of a melting curve. The main theoretical basis is that the negative extreme point of the inverted peak shown in the second-order derivative curve is consistent with the peak point of the original curve, and the half-width of the inverted peak converges, which is conducive to revealing the hidden weak shoulder peak.

[0036] To facilitate understanding of this embodiment, a method for separating overlapping peaks of a melting curve disclosed in an embodiment of the present invention is first described in detail, see Figure 1 The flowchart of a method for separating overlapping peaks of a melting curve is shown. The method can be performed by an electronic device and mainly includes the following steps S110 to S140:

[0037] S110: based on the pre-acquired melting point peak position of each well of the PCR reaction plate melting curve, a second-order derivative curve of each well melting curve is determined using a Savitzky-Golay derivation method;

[0038] The melting point peak position of the melting curve of each well of the PCR reaction plate corresponds to the temperature (Tm) value of the melting point peak.

[0039] S120: determining the initial overlapping peak position of the original melting curve according to the second-order derivative curve and the melting point peak position;

[0040] S130: performing superposition fitting on the initial overlapping peak positions to generate two melting point peak curves;

[0041] S140: determining a target melting point peak that meets an error range according to the two melting point peak curves, and separating the target melting point peak.

[0042] In order to verify the effectiveness of the above method, the present invention uses a fluorescent quantitative PCR detection system to perform PCR amplification-melting experiments on multiple reagents, and analyzes the fluorescence intensity data collected in the melting section. Here, one of the data is used as an example, the Tm value resolution is 2°C, and the sampling interval is 0.3°C. The analysis is performed according to the above method. For specific operation results, see Figures 2 to 3 .

[0043] In one embodiment, the above step S110 includes: calculating the second-order derivative curve of each well melting curve according to the Savitzky-Golay derivation method using a preset first derivation parameter; wherein the first derivation parameter includes: a window size with a highest determination coefficient and a polynomial order.

[0044] In an embodiment of the present invention, firstly, the melting point peak position of the melting curve of each well of the PCR reaction plate can be obtained based on conventional methods, and then based on the Savitzky-Golay derivation method, the appropriate window size and polynomial order are set to obtain the second-order derivative curve of the melting curve of each well.

[0045] Among them, the conventional method can be a melting curve extreme value or a clustering method. For example, in this embodiment, a clustering method is used to obtain the initial melting curve of each well position in the melting section of a PCR experiment.

[0046] The method for setting the appropriate window size and polynomial order can be: calculate the second-order derivative curve of the melting curve of each well under different window sizes and polynomial orders, and take the window size and polynomial order with the highest determination coefficient. Usually, based on the Savitzky-Golay derivation method, the melting curve is processed with the determination coefficient as an indicator, and the determination coefficient under different window sizes (generally an odd number, 3, 5, 7, 9, etc.) and polynomial orders (generally 1, 2, 3) is calculated. The corresponding window size and polynomial order with the largest determination coefficient are taken, and on this basis, the second-order derivative polynomial coefficient is obtained, and the corresponding second-order derivative value in the window is calculated. The center value in the window is taken as the value in the sliding window, and then the sliding window is translated to obtain the second-order derivative value of each point. Note that for the front and rear half window positions of the data, since there is generally no melting point peak, it can be omitted or replaced by the second-order derivative value calculated by the first and last windows.

[0047] For example: the determination coefficient of the melting curve of a reaction well A under different window sizes and polynomial orders is obtained. It is found that when the window size is 5 and the order is 2, it has the largest determination coefficient. On this basis, its second-order derivative curve is obtained.

[0048] In one implementation, the above step S120 includes:

[0049] (1) firstly, according to a predetermined first range, determining a first region corresponding to each melting point peak and performing a normalization process;

[0050] The step of presetting the first range includes:

[0051] First, take the temperature (Tm) value of the melting point peak corresponding to the melting point peak position as the base point, and use the standard melting point peak resolution as the interval to determine the trough corresponding to the melting point peak at the left and right ends of the base point respectively; wherein the standard melting point peak resolution is four times the minimum melting point peak resolution; wherein the minimum melting point peak resolution is 2°C.

[0052] If there are no troughs on both sides of the base point, the standard melting point peak resolution is determined to be the first intermediate value; the minimum value of the first range is the temperature (Tm) value of the melting point peak minus the first intermediate value; the maximum value of the first range is the temperature (Tm) value of the melting point peak plus the first intermediate value.

[0053] If there are troughs on both the left and right ends of the base point, the distances between the base point and the troughs on the left and right ends are determined to be T 左 and T 右 ; Compare T 左 and the standard melting point peak resolution, determine the minimum value to be the second intermediate value; compare T 右 and the size of the resolution, determine the minimum value to be the third intermediate value;

[0054] The minimum value of the first range is the temperature (Tm) value of the melting point peak minus the second intermediate value; the maximum value of the first range is the temperature (Tm) value of the melting point peak plus the third intermediate value.

[0055] For example, in the embodiment of the present invention, the first range given in advance can be obtained according to the following principle: Statistical data show that different target fragments can be distinguished when the Tm values ​​differ by more than 2°C, and 2°C is used as the melting point peak resolution, and the melting point peak position is set as Tm. Based on this, the trough corresponding to the peak point is searched at both ends of the range of 8°C, which is 4 times the resolution (standard melting point peak resolution). If there is a trough, its distance from Tm is set to Tm. 左 、T 右 If there is no trough, then the temperature is taken as 8°C, that is, the range is: [Tm-min(8, T 左 ),Tm+min(8,T 右 )].

[0056] To reduce rounding errors, the corresponding sampling point range can be used to represent the second-order derivative region of the melting curve within the range, which is set as region A (i.e., the first region). The normalization method can be the maximum and minimum normalization method. Assume that the maximum value of the melting curve in the region is X max , the minimum value is X min , then its normalized melting curve value X(i) is:

[0057]

[0058] In the embodiment, Tm = 66.6 ° C, region A is: [58.2 ° C, 74.4 ° C], the corresponding sampling interval is [1, 55], the initial temperature T0 = 58.2 ° C, the sampling interval △ T is 0.3 ° C, the melting point peak is located at the 29th sampling point, and the melting curve in the reaction well A region A is as follows Figure 2 As shown (Note: the horizontal axis of the graph is marked with sampling points).

[0059] (2) then determining the minimum point of the second-order derivative curve in the first region;

[0060] The minimum point of the second-order derivative of the melting curve corresponds to the melting point peak of the melting curve, and all minimum points in area A are determined (Note: the minimum point is the one with an amplitude lower than the amplitude of the adjacent points on the left and right).

[0061] (3) According to the relative amplitude of the minimum point, the minimum point that meets a predetermined first threshold is determined as the initial overlapping peak position.

[0062] According to the analysis results of multiple data, if there are overlapping peaks of a melting point peak, the amplitudes of the overlapping peaks of its second-order derivative curve should not differ much, and the predetermined first threshold can be set to 2, that is, the amplitudes of the maximum points in the region should not differ by more than twice.

[0063] Assume that the absolute value of the relative amplitude of the second-order derivative corresponding to the melting point peak in region A (note: the difference between the minimum point and the adjacent higher peak) is Y0, and the corresponding temperature b10 is an initial position where overlapping peaks may exist. In region A, take the maximum minimum point with an absolute value of amplitude exceeding Y0 / 2, and its corresponding position b20 is another initial position where overlapping peaks may exist. If there is no minimum point with an absolute value of amplitude exceeding Y0 / 2 in region A, then there is no overlapping peak.

[0064] For example: The second derivative curve corresponding to the melting curve of region A is Figure 2 It can be seen that the melting point peak position, i.e. the 29th sampling point, corresponds to Y0=7.79, and the position of the maximum minimum point where the absolute value of the amplitude exceeds Y0 / 2 in region A is the position corresponding to the 45th sampling point, and its relative amplitude is 4.52, i.e. the two initial positions of the overlapping peaks are: b10=29, b20=45.

[0065] In one implementation, the above step S130 includes:

[0066] First, the initial parameters of the initial function are set based on the initial overlapping peak positions;

[0067] Then, the Levenberg-Marquardt algorithm is used to superimpose and fit the second-order derivative curves in the first region corresponding to each melting point peak based on the initial function to generate two functions to be optimized; and the two functions to be optimized are optimized according to the initial parameters to generate two objective functions; wherein the objective function includes the target parameters; and the two objective functions correspond to the two melting point peak curves respectively.

[0068] Referring to the spectrum overlapping peak separation theory and the melting curve formation mechanism, when there are two peaks superimposed on the melting point peak, the specific function can be assumed to be a three-parameter Lorentz function g(T) = a·c / [c+(Tb) 2 ], where T is the temperature, a, b, c are the parameters to be optimized, and c represents the variance, which must be greater than 0. The overlapping peak function is the superposition of two three-parameter Lorentz functions, which is:

[0069] G(T)=g1(T)+g2(T)=a1·c1 / [c1+(T-b1) 2 ]+a2·c2 / [c2+(T-b2) 2 ];(1)

[0070] Among them, g1(T) and g2(T) are the objective functions respectively; T is used to represent temperature, a1 and a2 are used to represent coefficients, b1 and b2 are used to represent the positions of overlapping peaks after optimization, and c1 and c2 are used to represent variances.

[0071] An initial setting of the parameter values ​​is as follows: b1 = b10, b2 = b20, a1 = c1 = a2 = c2 = 1. In the embodiment, the fitting optimization separation is performed under the three-parameter Lorentz function.

[0072] In an embodiment of the present invention, the Levenberg-Marquardt nonlinear optimization algorithm is used to perform curve fitting on the melting curve in region A corresponding to each melting point peak based on formula (1) to obtain the final fitting result, i.e., the parameter combination {a1, b1, c1, a2, b2, c2}.

[0073] In the embodiment, the parameter combination obtained by superposition of the three-parameter Lorentzian function is: {0.97, 28.9, 98, 0.31, 43.2, 25.7}. It can be seen that the positions of the two overlapping peak sampling points are b1=28.9 and b2=43.2, which are slightly deviated from the initial values ​​(b10=29, b20=45).

[0074] In one implementation, the above step S140 includes:

[0075] (1) determining a target melting point peak that meets an error range based on the melting point peak curve; the error range includes: a range of a difference between a first square sum before superposition fitting and a second square sum after superposition fitting;

[0076] (2) Separating the target melting point peaks, determining the positions of the two melting point peaks after separation and the temperature values ​​corresponding to the melting point peaks.

[0077] Calculate the sum of squared errors before and after fitting. If the error L2 norm is ≤0.1, the requirement is met. The Tm position after separation can be calculated based on the initial temperature T0 of region A, the sampling interval △T and the position of the overlapping peak sampling points: Tm1=T0+(b1-1)*△T, Tm2=T0+(b2-1)*△T.

[0078] In the embodiment, when the three-parameter Lorentz function is superimposed, the error L2 norm = 0.08 ≤ 0.1, and c1 = 98, c2 = 25.7, both > 0, meet the requirements. Overlap peak Tm1 = 58.2 + (28.9-1) * 0.3 = 66.57 ° C, Tm2 = 58.2 + (43.2-1) * 0.3 = 70.86 ° C, before and after separation and fitting graphs are as follows Figure 3 shown.

[0079] The invention provides a method for separating overlapping peaks of a melting curve. The Savitzky-Golay derivation method is adopted to reduce background interference and improve the resolution of melting point peaks, and the initial position of overlapping peaks in a difficult melting curve is determined. Then, a Levenberg-Marquardt algorithm is used to perform superposition curve fitting on a single melting point peak. The problem that the difficult melting point peak with large background interference and overlapping peaks has only one Tm value is preferably solved. The method is easy to understand and easy to implement.

[0080] An embodiment of the present invention provides a device for separating overlapping peaks of melting curves, the device comprising:

[0081] A first determination module is used to determine the second-order derivative curve of the melting curve of each well of the PCR reaction plate based on the melting point peak position of the melting curve of each well of the PCR reaction plate obtained in advance by using the Savitzky-Golay derivation method;

[0082] A second determination module is used to determine the initial overlapping peak position of the original melting curve according to the second-order derivative curve and the melting point peak position;

[0083] A melting point peak curve generation module is used to perform superposition fitting on the initial overlapping peak positions to generate two melting point peak curves;

[0084] The separation module is used to determine the target melting point peak that meets the error range according to the two melting point peak curves, and separate the target melting point peak.

[0085] In one embodiment, the melting point peak position of the melting curve of each well of the PCR reaction plate corresponds to the temperature (Tm) value of the melting point peak; the above-mentioned first determination module is specifically used to: calculate the second-order derivative curve of the melting curve of each well according to the Savitzky-Golay derivation method using a pre-set first derivation parameter; wherein the first derivation parameter includes: the window size with the highest determination coefficient and the polynomial order.

[0086] The separation device for overlapping peaks of melting curves provided in the embodiments of the present application can be specific hardware on the device or software or firmware installed on the device, etc. The device provided in the embodiments of the present application, its implementation principle and the technical effect produced are the same as those in the aforementioned method embodiments. For brief description, the parts not mentioned in the device embodiment can refer to the corresponding contents in the aforementioned method embodiments. Those skilled in the art can clearly understand that for the convenience and simplicity of description, the specific working processes of the systems, devices and units described above can all refer to the corresponding processes in the aforementioned method embodiments, and will not be repeated here. The separation device for overlapping peaks of melting curves provided in the embodiments of the present application has the same technical features as the separation method for overlapping peaks of melting curves provided in the aforementioned embodiments, so it can also solve the same technical problems and achieve the same technical effects.

[0087] An embodiment of the present application further provides an electronic device. Specifically, the electronic device includes a processor and a storage device. The storage device stores a computer program, and when the computer program is executed by the processor, it executes the method described in any one of the above-mentioned embodiments.

[0088] Figure 4 A structural schematic diagram of an electronic device provided in an embodiment of the present application, the electronic device 400 includes: a processor 40, a memory 41, a bus 42 and a communication interface 43, wherein the processor 40, the communication interface 43 and the memory 41 are connected via the bus 42; the processor 40 is used to execute an executable module stored in the memory 41, such as a computer program.

[0089] The memory 41 may include a high-speed random access memory (RAM), and may also include a non-volatile memory, such as at least one disk memory. The communication connection between the system network element and at least one other network element is realized through at least one communication interface 43 (which may be wired or wireless), and the Internet, wide area network, local area network, metropolitan area network, etc. may be used.

[0090] The bus 42 may be an ISA bus, a PCI bus, or an EISA bus, etc. The bus may be divided into an address bus, a data bus, a control bus, etc. For ease of representation, Figure 4Only one bidirectional arrow is used in the diagram, but this does not mean that there is only one bus or only one type of bus.

[0091] Among them, the memory 41 is used to store programs, and the processor 40 executes the program after receiving the execution instruction. The method executed by the device for flow process definition disclosed in any embodiment of the above-mentioned embodiment of the present invention can be applied to the processor 40 or implemented by the processor 40.

[0092] The processor 40 may be an integrated circuit chip with signal processing capabilities. In the implementation process, each step of the above method can be completed by the hardware integrated logic circuit or software instructions in the processor 40. The above processor 40 can be a general-purpose processor, including a central processing unit (CPU), a network processor (NP), etc.; it can also be a digital signal processor (DSP), an application-specific integrated circuit (ASIC), a field-programmable gate array (FPGA), or other programmable logic devices, discrete gates or transistor logic devices, discrete hardware components. The methods, steps and logic block diagrams disclosed in the embodiments of the present invention can be implemented or executed. The general-purpose processor can be a microprocessor or the processor can also be any conventional processor. The steps of the method disclosed in the embodiments of the present invention can be directly embodied as a hardware decoding processor to execute, or can be executed by a combination of hardware and software modules in the decoding processor. The software module can be located in a mature storage medium in the field such as a random access memory, a flash memory, a read-only memory, a programmable read-only memory, or an electrically erasable programmable memory, a register, etc. The storage medium is located in the memory 41, and the processor 40 reads the information in the memory 41 and completes the steps of the above method in combination with its hardware.

[0093] Corresponding to the above method, an embodiment of the present application also provides a computer-readable storage medium, which stores machine-executable instructions. When the computer-executable instructions are called and executed by a processor, the computer-executable instructions prompt the processor to execute the steps of the above method.

[0094] In the embodiments provided in the present application, it should be understood that the disclosed devices and methods can be implemented in other ways. The device embodiments described above are merely schematic. For example, the division of the units is only a logical function division. There may be other division methods in actual implementation. For example, multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or direct coupling or communication connection shown or discussed can be through some communication interfaces, and the indirect coupling or communication connection of the devices or units can be electrical, mechanical or other forms.

[0095] The units described as separate components may or may not be physically separated, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed on multiple network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0096] In addition, each functional unit in the embodiments provided in the present application may be integrated into one processing unit, or each unit may exist physically separately, or two or more units may be integrated into one unit.

[0097] If the functions are implemented in the form of software functional units and sold or used as independent products, they can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of the present invention, in essence, or the part that contributes to the prior art, or the part of the technical solution, can be embodied in the form of a software product, which is stored in a storage medium and includes several instructions for enabling a computer device (which can be a personal computer, electronic device, or network device, etc.) to perform all or part of the steps of the methods described in each embodiment of the present invention. The aforementioned storage medium includes: various media that can store program codes, such as a USB flash drive, a mobile hard disk, a read-only memory (ROM), a random access memory (RAM), a magnetic disk or an optical disk.

[0098] It should be noted that similar numbers and letters represent similar items in the accompanying drawings. Therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings. In addition, the terms "first", "second", "third", etc. are only used to distinguish the description and cannot be understood as indicating or implying relative importance.

[0099] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for separating overlapping peaks of melting curves, characterized in that: include: Based on the pre-obtained melting point peak position of each well of the PCR reaction plate melting curve, the second-order derivative curve of each well melting curve is determined using the Savitzky-Golay derivation method; Determining the initial overlapping peak position of the original melting curve according to the second-order derivative curve and the melting point peak position; Performing superposition fitting on the initial overlapping peak positions to generate two melting point peak curves; Determine a target melting point peak that meets an error range according to the two melting point peak curves, and separate the target melting point peak; Wherein, determining the initial overlapping peak position of the original melting curve according to the second-order derivative curve and the melting point peak position comprises: determining a first region corresponding to each melting point peak according to a predetermined first range, and performing normalization processing; the first region includes a second-order derivative region of the melting curve selected within the first range; determining a minimum point of the second-order derivative curve within the first region; According to the relative amplitude of the minimum point, the minimum point that meets the predetermined first threshold is determined as the initial overlapping peak position; the condition of the predetermined first threshold includes: within the first region, the amplitude difference of the maximum point of the second-order derivative curve does not exceed twice.

2. The method for separating overlapping peaks of melting curves according to claim 1, characterized in that: The melting point peak position of the melting curve of each well of the PCR reaction plate corresponds to the temperature (Tm) value of the melting point peak; The steps of determining the second derivative curve of each well melting curve using the Savitzky-Golay derivation method include: According to the Savitzky-Golay derivation method, the second-order derivative curve of the melting curve of each well is calculated using the preset first derivative parameter; The first derivative parameters include: a window size with a highest determination coefficient and a polynomial order.

3. The method for separating overlapping peaks of melting curves according to claim 1, characterized in that: The step of predetermining the first range includes: Taking the temperature (Tm) value of the melting point peak corresponding to the melting point peak position as the base point, taking the standard melting point peak resolution as the interval, the trough corresponding to the melting point peak is determined at the left and right ends of the base point respectively; the standard melting point peak resolution is four times the minimum melting point peak resolution; wherein the minimum melting point peak resolution is 2°C; If the trough does not exist at both ends of the base point, the standard melting point peak resolution is determined to be the first intermediate value; the minimum value of the first range is the temperature (Tm) value of the melting point peak minus the first intermediate value; the maximum value of the first range is the temperature (Tm) value of the melting point peak plus the first intermediate value; If the troughs exist at both ends of the base point, the distances between the base point and the troughs at the left and right ends are determined to be T 左 and T 右 ; Compare T 左 and the size of the standard melting point peak resolution, determine the minimum value to be the second intermediate value; compare T 右 and the size of the resolution, determining the minimum value as the third intermediate value; The minimum value of the first range is the temperature (Tm) value of the melting point peak minus the second intermediate value; the maximum value of the first range is the temperature (Tm) value of the melting point peak plus the third intermediate value.

4. The method for separating overlapping peaks of melting curves according to claim 3, characterized in that: The step of performing superposition fitting on the initial overlapping peak positions to generate two melting point peak curves comprises: Setting initial parameters of the initial function based on the initial overlapping peak positions; Using the Levenberg-Marquardt algorithm, based on the initial function, the second-order derivative curves in the first region corresponding to each melting point peak are superimposed and fitted to generate two functions to be optimized; The two functions to be optimized are optimized according to the initial parameters to generate two objective functions; wherein the objective function includes the objective parameters; and the two objective functions correspond to the two melting point peak curves respectively.

5. The method for separating overlapping peaks of melting curves according to claim 4, characterized in that: The objective function is a three-parameter Lorentz function: g(v)=a·c / [c+(vb) 2 ]; Where: v is used to represent temperature, a is used to represent coefficient, b is used to represent the position of overlapping peaks after optimization, and c is used to represent variance.

6. The method for separating overlapping peaks of melting curves according to claim 5, characterized in that: The step of determining a target melting point peak that meets an error range according to the two melting point peak curves and separating the target melting point peak comprises: Based on the melting point peak curve, determining a target melting point peak that meets an error range; the error range includes: a range of a difference between a first square sum before superposition fitting and a second square sum after superposition fitting; The target melting point peaks are separated, and the positions of the two melting point peaks after separation and the temperature values ​​corresponding to the melting point peaks are determined.

7. A device for separating overlapping peaks of melting curves, characterized in that: include: A first determination module is used to determine the second-order derivative curve of the melting curve of each well of the PCR reaction plate based on the melting point peak position of the melting curve of each well of the PCR reaction plate obtained in advance by using the Savitzky-Golay derivation method; The second determination module is used to determine the initial overlapping peak position of the original melting curve according to the second-order derivative curve and the melting point peak position; wherein, determining the initial overlapping peak position of the original melting curve according to the second-order derivative curve and the melting point peak position includes: determining the first region corresponding to each melting point peak according to a predetermined first range, and performing normalization processing; the first region includes the second-order derivative region of the melting curve selected within the first range; determining the minimum point of the second-order derivative curve in the first region; determining the minimum point that meets a predetermined first threshold as the initial overlapping peak position according to the relative amplitude of the minimum point; the condition of the predetermined first threshold includes: in the first region, the amplitude difference of the maximum point of the second-order derivative curve does not exceed two times; A melting point peak curve generating module, used for performing superposition fitting on the initial overlapping peak positions to generate two melting point peak curves; A separation module is used to determine a target melting point peak that meets an error range based on the two melting point peak curves, and separate the target melting point peak.

8. An electronic device comprising a memory and a processor, wherein the memory stores a computer program that can be run on the processor, characterized in that: When the processor executes the computer program, the steps of the method according to any one of claims 1 to 6 are implemented.

9. A computer-readable storage medium, characterized in that: The computer-readable storage medium stores machine executable instructions. When the computer-executable instructions are called and executed by a processor, the computer-executable instructions prompt the processor to execute the method according to any one of claims 1 to 6.

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