PCR (Polymerase Chain Reaction) fluorescence test method

Abstract

The invention provides a PCR (Polymerase Chain Reaction) fluorescence test method, which comprises the following steps of: 1, respectively injecting a plurality of groups of same fluorescent dyes with concentrations into test holes of a test chip, obliquely irradiating the test chip by using an excitation light source at a fixed angle, and acquiring a gray level image of excitation fluorescence of the test chip by using optical acquisition equipment; constructing a model for solving the minimum error between the variation coefficient of the calibration fluorescence values of the fluorescent dyes with multiple groups of concentrations and the expected variation coefficient, and solving the model to obtain a calibration coefficient; step 2, filling the obtained calibration coefficient into a calculation module of the optical acquisition equipment; and step 3, adding a reaction reagent into the test hole of the test chip, irradiating the test chip with an excitation light source, collecting a fluorescence grayscale image of the reaction reagent by the optical collection equipment, and calculating the fluorescence value of the reaction reagent through the internal calculation module. The method has the advantages of simplicity in operation and high accuracy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomolecule detection, and particularly relates to a PCR fluorescence testing method. BACKGROUND

[0002] Polymerase chain reaction (PCR) is a molecular biology technique used to amplify specific DNA fragments, which can be regarded as a special DNA replication in vitro. The biggest feature of PCR is that it can greatly increase the amount of DNA from a small amount. Therefore, whether it is a fossil of ancient organisms, the remains of historical figures, or a little hair, skin or blood, as long as a little DNA can be isolated, it can be amplified by PCR for comparison.

[0003] Because the conventional polypropylene PCR tube is used in the prior art, the heat conduction of polypropylene is slow, and the reaction time is usually more than 60 minutes. At the same time, a coaxial excitation light source and a fluorescence receiving system need to be matched. Because the excitation light source is perpendicular to the test chip, a large amount of excitation light will enter the fluorescence receiving system. A higher cost and larger size collimation system is needed to filter the reflected excitation light. And in the fluorescence experiment, only one kind of excitation light can be used for one collimation system. However, there are usually multiple fluorescence channels in the PCR instrument to use different fluorescent dyes for fluorescence experiments. Therefore, the existing fluorescence testing system has the problems of complex optical path setting, high cost, and inconvenient internal fluorescence channel switching, which leads to low detection efficiency.

[0004] Therefore, it is necessary to design a new type of PCR fluorescence detection method which can efficiently perform fluorescence experiments and fluorescence detection. SUMMARY

[0005] The present application provides a PCR fluorescence testing method, which can simplify the optical path of the PCR fluorescence testing device, and has the advantages of simple testing and high efficiency.

[0006] Other purposes and advantages of the present application can be further understood from the technical features disclosed in the present application.

[0007] To achieve one or part or all of the above purposes or other purposes, one of the technical solutions of the present application provides a PCR fluorescence test method, which comprises the following steps: step 1: selecting multiple groups of the same fluorescent dye with different concentrations and injecting them into test holes of a test chip, using an excitation light source to irradiate the test chip at a fixed angle, using an optical acquisition device to acquire a gray-scale image of the excited fluorescence of the test chip, calculating the average pixel value of the gray-scale image of the multiple groups of fluorescent dyes as multiple acquired fluorescence values, constructing a model for solving the minimum error between the variation coefficient of the calibration fluorescence value of the multiple groups of fluorescent dyes and the expected variation coefficient, and solving the model to obtain a calibration coefficient; step 2: filling the obtained calibration coefficient into a calculation module of the optical acquisition device; step 3: adding a reaction reagent into the test holes of the test chip, using the excitation light source to irradiate the test chip at the same irradiation angle as in step 1, using the optical acquisition device to acquire a fluorescence gray-scale image of the reaction reagent, and calculating the fluorescence value of the reaction reagent through the internal calculation module. The beneficial effects of this technical solution are that the excitation light source of the PCR test method of the present application adopts an inclined angle irradiation mode to irradiate the test chip, and then the optical acquisition device acquires the reflected excited fluorescence image. Since the inclined irradiation will cause uneven fluorescence irradiation, the calibration coefficient of a certain fluorescent dye at a certain concentration is calculated in advance, and the calibration coefficient is filled into the calculation module of the optical acquisition device. Therefore, during the subsequent detection of the reaction reagent, a more accurate fluorescence detection result can be obtained. The present application does not need to set a complex optical system, and the detection efficiency is high.

[0008] The calculation of the calibration coefficient in step 1 also includes calculating the calibration coefficient of different fluorescent dyes and filling the calibration coefficients of different fluorescent dyes into the calculation module of the optical acquisition device.

[0009] The excitation light source has multiple LED light sources, and in one PCR cycle, an excitation light control system controls the excitation light source to emit LED light of different wavelengths to irradiate the test chip in turn, and the optical acquisition device acquires images of the reflected fluorescence excited by LED light of different wavelengths in turn.

[0010] The wavelengths of the different LED light sources in the excitation light source are adapted to the excitation wavelengths of the different fluorescent dyes.

[0011] The test chip is a silicon-based test chip, and the silicon-based test chip is provided with multiple serpentine test holes, which are uniformly arranged on the test chip; a temperature control module is arranged below the test chip, and a glass surface is arranged above the test chip.

[0012] In step 1, at least three groups of the same fluorescent dye are selected, and the concentrations of the multiple groups of fluorescent dyes are diluted according to a gradient.

[0013] The step 1 further comprises dividing the fluorescent gray scale image into several regions, calculating the average pixel value of the fluorescent gray scale image in each region as the collected fluorescent value of each region; and constructing a model for solving the minimum error between the coefficient of variation of the calibration fluorescent value of each region and the expected coefficient of variation, and solving the model to obtain the calibration coefficient of each region.

[0014] The excitation light source forms an angle of 40-50° with the silicon-based test chip; the light source for irradiating the test chip with different concentrations of fluorescent dyes is the same light source, and the angle of the excitation light source irradiating the test chip is the same.

[0015] The constructed model for solving the minimum error between the coefficient of variation of the calibration fluorescent value of each region and the expected coefficient of variation is:

[0016]

[0017] K represents the number of different concentrations of fluorescent dyes, cv n is the coefficient of variation of the nth group of concentration fluorescent dyes, and e is the expected coefficient of variation, wherein the derivation formula of cv is:

[0018]

[0019] Data nj is the fluorescent value of the nth group of concentration fluorescent dyes after calibration; std is the standard deviation, mean is the average value, and Data nj The derivation formula of cv is:

[0020]

[0021] wherein x m represents the calibration coefficient of the mth test well on the test chip, a nm represents the collected fluorescent value of the nth group of concentration fluorescent dyes in the mth test well, and m represents the number of test wells of the test chip.

[0022] The expected coefficient of variation ranges from 2% to 3%.

[0023] The model for solving the minimum error between the coefficient of variation of the calibration fluorescent value of each region and the expected coefficient of variation is solved by the quasi-Newton method.

[0024] The optical acquisition device comprises an imaging lens, a fluorescent filter, a CMOS image sensor, and an FPGA image acquisition board, and the CMOS image sensor senses images;

[0025] The FPGA image acquisition template is provided with a calculation module, and the FPGA image acquisition template acquires images from the CMOS image sensor and processes the images.

[0026] Compared with the prior art, the beneficial effects of the present application mainly include: 1. The present application adopts a silicon-based test chip, and a plurality of serpentine test holes are formed on the chip by etching, so that the test speed can be effectively accelerated;

[0027] 2. The excitation light source of the present application is inclined to irradiate on the test chip at a certain angle, and the calibration coefficient of the corresponding fluorescent dye is calculated, which can reduce the complex optical path design, has the advantages of low cost, small structure and high test precision;

[0028] 3. The present application does not need to design a complex collimation system, and the excitation light entering the fluorescent receiving system is greatly reduced when the test chip is irradiated by the excitation light at an angle, at which time the low-cost CMOS camera with internally installed fluorescent filter can meet the signal-to-noise ratio requirement. The excitation light source of the present application can emit LED light of multiple different wavelengths, and in one PCR cycle, the control system can control the excitation light source to emit excitation light of different wavelengths in turn to detect the reaction reagent under different fluorescent channels, which has the advantage of more flexible test.

[0029] In order to make the above and other objects, features and advantages of the present application more obvious and easy to understand, the following preferred embodiments are described in detail below, and the accompanying drawings are described as follows. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the specific embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiment description. Obviously, the drawings described below are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0031] Figure 1 is a schematic diagram of the fluorescent experiment detection system of the present application;

[0032] Figure 2 is a schematic diagram of the front surface of the silicon-based chip used in embodiment 1 of the present application;

[0033] Figure 3 is a schematic diagram of the side surface of the silicon-based chip used in embodiment 1 of the present application;

[0034] Figure 4 is a flow chart for calculating the fluorescent calibration coefficient in embodiment 1 of the present application;

[0035] Figure 5 is a fluorescent brightness diagram after injection of different concentrations of fluorescent dyes in embodiment 1.

[0036] 1, temperature control module; 2, test chip; 3, imaging lens; 4, fluorescence filter; 5, CMOS image sensor; 6, FPGA image acquisition board; 7, excitation light source; 8, collimating lens; 9, dichroic mirror; 10, LED light source. DETAILED DESCRIPTION

[0037] The foregoing and other technical contents, features and effects of the present application will be clearly presented in the following detailed description of a preferred embodiment in conjunction with the accompanying drawings. The directional terms mentioned in the following embodiment, such as up, down, left, right, front or back, etc., are only with reference to the direction of the accompanying drawings. Therefore, the directional terms used are for illustration and not for limitation of the present application.

[0038] Example 1

[0039] Reference Figure 1 The test system of the present application is composed of a test chip 2 to be tested, a fluorescence receiving system and an excitation light source 7, wherein the excitation light source 7 is arranged at an oblique angle at one end of the test chip 2 to be tested, and the fluorescence receiving system is perpendicular to the test chip 2 to be tested. The steps of the PCR fluorescence test method of the present application are as follows:

[0040] Step 1: Select multiple groups of different concentrations of the same fluorescent dye and inject them into the test wells of the test chip 2, and use the excitation light source 7 to irradiate the test chip at a fixed angle, the oblique irradiation angle is 40°-50°, because the irradiation angle will affect the reflection intensity and range of the excitation light, in order to minimize the reflection of the excitation light into the optical acquisition device to affect the collection of fluorescence, therefore the irradiation angle of 40°-50° is preferred in Example 1. The gray scale image of the excited fluorescence of the test chip is collected by the optical acquisition device, which is composed of an imaging lens 3, a fluorescence filter 4, a CMOS image sensor 5 and a FPGA image acquisition board 6. Since the excitation light source 7 is obliquely irradiated on the test chip 2, the excitation light entering the fluorescence receiving module will be greatly reduced, so the optical acquisition device with the fluorescence filter 4 installed inside can meet the needs of signal-to-noise ratio, without the need to design a complex collimation system to filter out excess fluorescence.

[0041] The test chip 2 is a silicon-based test chip, and a plurality of serpentine test holes are provided on the silicon-based test chip 2. The serpentine test holes are etched on the silicon-based chip, and the serpentine test holes are uniformly arranged on the test chip. There is a temperature control module 1 below the test chip, and the silicon-based chip 2 is in close contact with the temperature control module 1 for heat conduction, to ensure that the reagents in the test holes can effectively perform PCR reaction. There is a glass surface above the test chip 2, and the glass surface is excited by the PCR system light path and collects fluorescence. The specific structure of the silicon-based chip is shown in Figure 2 and Figure 3The silicon-based chip used in embodiment 1 includes 16 serpentine test holes, which are evenly arranged on the silicon-based chip. Since silicon cannot be excited, only the reaction reagent in the test hole can be excited, and the reflection of the silicon-based chip to the excitation light is much higher than that of the polypropylene PCR tube. Therefore, in embodiment 1, the high reflectivity of the silicon-based chip meets the use requirements of the obliquely arranged excitation light source by using the calibration coefficient calculated by the calculation module in the optical acquisition module (the calculation module is integrated in the FPGA image acquisition board 6), and a complex optical collimation system does not need to be designed, so that the structure of the device is greatly simplified, and the experiment is more convenient. The temperature control module 1 below the silicon-based chip heats the silicon-based chip to a suitable temperature, and the heating temperature in step 1 is 55°C.

[0042] The optical acquisition device processes the gray-scale images under multiple groups of concentration fluorescent dyes, calculates the average pixel value of the gray-scale images as multiple acquisition fluorescence values, constructs a minimum error model of the variation coefficient of the calibration fluorescence value of different concentrations and the expected variation coefficient, and solves the model to obtain the calibration coefficient;

[0043] The CMOS image sensor 5 in the optical acquisition device senses the image, and the FPGA image acquisition board 6 acquires the image from the CMOS image sensor 5 and performs subsequent processing. The subsequent processing here includes generating a fluorescent gray-scale image, segmenting the fluorescent gray-scale image, and segmenting based on the area of the serpentine test hole on the test chip 2. The serpentine test hole area is completely framed by a rectangular frame, and the rectangular frame on the test chip 2 is uniformly arranged. The FPGA image acquisition board 6 is provided with a calculation module, which includes an image processing algorithm for calculating the average pixels of the fluorescent gray-scale image and a scientific calculation and analysis algorithm for calculating the calibration coefficient. The solving method of the constructed model is to solve it by using the embedded function of the scientific calculation and analysis algorithm.

[0044] Step 2: Fill the obtained calibration coefficient into the calculation module of the FPGA image acquisition board 6. In this way, in the next fluorescent experiment of the reaction reagent, the calculation module calculates the acquisition fluorescence value, and the calibrated fluorescence value can be calculated by calling the internal calibration coefficient;

[0045] Because the excitation light source 7 is arranged obliquely, a filter system is not needed to filter the reflected excitation light, so that the calibration coefficient of different fluorescent dyes can be calculated. Common fluorescent dyes include FAM, ROX, HEX and CY5 dyes, which correspond to different fluorescent channels in a PCR system. The excitation fluorescent wavelengths of these dyes are different, and specific parameters are shown in Table 1 below. The fluorescent dyes are subjected to fluorescent experiments respectively to obtain the calibration coefficient corresponding to each fluorescent dye, and the calibration coefficients corresponding to different fluorescent dyes are filled into the calculation module of the optical acquisition device, so that the fluorescent calibration coefficients of different fluorescent channels under different illumination angles of the excitation light source 7 are obtained.

[0046] Table 1: Four fluorescent dyes and their characteristics

[0047]

[0048] Referring to the excitation light source 7 in Figure 1 The excitation light source 7 includes a collimating lens 8 at the light outlet of the light source, and LED light sources 10 arranged on the excitation light source 7. The LED light sources 10 include four types of LED light sources. In order to adapt to the four types of fluorescent dyes of the fluorescent channels mentioned above in Embodiment 1, a four-wavelength-in-one light source module is selected, and the LED light sources with wavelengths of 480 nm (FAM), 580 nm (ROX), 525 nm (HEX) and 638 nm (CY5) are integrated into one light outlet by a dichroic mirror 9.

[0049] Step 3: The reaction reagent is added to the test hole of the test chip 2, the excitation light source 7 is irradiated onto the test chip 2 using the same illumination angle as in Step 1, the optical acquisition device acquires the fluorescent gray scale image of the reaction reagent, and the fluorescent value of the reaction reagent is calculated by the internal calculation module. In one PCR cycle, the excitation light control system controls the excitation light source to emit LED light of different wavelengths to irradiate the test chip in turn, and the optical acquisition device acquires the image of the reflected fluorescent light excited by the LED light of different wavelengths in turn. The scheme of the present application can quickly perform multi-channel fluorescent experiments.

[0050] In order to better illustrate the calibration coefficient model component and solution in Step 1 of the present application, Embodiment 1 takes a group of specific parameters of fluorescent dyes as an example to illustrate the process of calibration coefficient calculation in detail.

[0051] Referring to Figure 4The calculation process of the correction coefficient is as follows: three concentrations of fluorescent dyes are prepared, and the three concentrations of fluorescent dyes are diluted according to a gradient. In Example 1, the three concentrations of fluorescent dyes are high-concentration fluorescent dye (0.5 Lmol / L), medium-concentration fluorescent dye (0.25 Lmol / L), and low-concentration fluorescent dye (0.125 Lmol / L), and the fluorescent dyes in Example 1 are diluted according to a gradient of 2 times. In Example 1, only three concentrations of fluorescent dyes are used, and for the fluorescent calibration result, the more the number of groups of fluorescent dyes of different concentrations selected, the more accurate the calculated correction coefficient will be.

[0052] Three groups of fluorescent dyes are injected into the test chip, which is a silicon-based chip with 16 serpentine test holes. During the fluorescence experiment, the test chip needs to be heated to keep the temperature at 55℃. The test chip injected with fluorescent dyes is excited by an excitation light source, which is inclined to irradiate the test chip at a certain angle. During the fluorescence experiment, the excitation light source irradiates the test chip containing different concentrations of fluorescent dyes, and the angle of the excitation light irradiating the test chip is the same.

[0053] Above the test chip is an image acquisition device. The collected fluorescent gray-scale images can be seen in Figure 5 , Figure 5 The difference in fluorescent brightness under excitation of three concentrations is given. The higher the concentration of fluorescent dye, the higher the fluorescent brightness under excitation. The collected fluorescent gray-scale images are processed, and the average pixel value of the region of interest on the test chip is calculated using an image processing algorithm. The region of interest refers to a rectangular region framed on the silicon-based chip to select the test hole, and the framed rectangular regions are equal in size and each contains a test hole. The collected data is:

[0054]

[0055] Data1 in formula 1 is high-concentration data, Data2 is intermediate-concentration data, and Data3 is low-concentration data. Assuming that the calibration coefficient to be solved for this channel is X = {x1, x2,..., x 16}, then the corrected fluorescent values of each concentration can be obtained as shown in formula 2:

[0056]

[0057] The cv derivation formula for high, medium, and low concentrations is shown in formula 3:

[0058]

[0059] where std is the standard deviation, and mean is the average value.

[0060] Assuming the expected cv is e, then according to the coefficient of variation formula, the calibration coefficient model of the kth channel can be derived as

[0061] f(x) is the objective function, X is the constraint set and X=R m , that is, X is an m-dimensional real number space.

[0062] The expected coefficient of variation (cv) is required to be less than or equal to 5% in the standard of the polymerase chain reaction analyzer, and in the experiment, in order to improve the calibration effect, the requirement for the expected coefficient of variation is 2%≤e≤3%, and in this embodiment 1, 3% is selected and the model is solved.

[0063] Step 3: Obtain the optimization model for the kth channel, for the unconstrained optimization model of the channel, use quasi Newton method to solve the model, specifically use Matlab optimization function fminunc and set optimization parameter quasi-newton to solve the minimum value of the model to obtain the calibration coefficient of each well under the kth channel, thereby obtaining the well calibration coefficient matrix M of the channel. m×1 .

[0064] Step 4: Repeat steps 2 and 3 to calculate the calibration coefficient matrix of each well of other channels, and finally obtain the well calibration coefficient matrix of all channels as

[0065]

[0066] wherein, M k is an m-row 1-column matrix, and k∈[1,n].

[0067] In order to facilitate fast calculation and processing of image calculation model, the embedded function fminunc in scientific analysis software MATLAB R2014b can be used to calculate the calibration coefficient, and the software can be burned in the FPGA image acquisition board to improve the calculation efficiency.

[0068] The following is the data analysis of the fluorescence experiment of different concentrations of fluorescent dyes in embodiment 1, in order to evaluate the accuracy of the fluorescence calibration method, four fluorescence channels are used for fluorescence experiment, wherein the four fluorescence channels use FAM, ROX, HEX and CY5 fluorescent dyes for fluorescence experiment, and the 16 snake-shaped test holes of the silicon chip are numbered in order from bottom to top and left to right. The fluorescence value of each test hole under each fluorescence channel obtained by the image acquisition module is as shown in Tables 2-4.

[0069] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070] Table 2: High-concentration fluorescent dye fluorescence data

[0070]

[0071]

[0072]

[0073]

[0074] Table 3: Fluorescence data collected at medium concentration of fluorescent dye

[0075]

[0076]

[0077]

[0078]

[0079] Table 4: Fluorescence data collected at low concentration of fluorescent dye

[0080]

[0081]

[0082]

[0083]

[0084] The 10 data measured in each test well above were obtained by repeatedly collecting 10 gray scale images of excited fluorescence at the same fluorescence channel and concentration, and calculating the average pixel value of the gray scale images in the test well.

[0085] The collected fluorescence values at each concentration in each fluorescence channel were subjected to scientific calculation and analysis algorithm to calculate the calibration coefficient, and the obtained calibration coefficient of each well in each fluorescence channel is shown in Table 5 below.

[0086] Table 5: Calibration coefficient of each well in each fluorescence channel

[0087]

[0088]

[0089] The calibration coefficient of each well in Table 5 was multiplied by the collected fluorescence value of each well in Table 2-Table 4 to obtain the calibrated fluorescence value, and the coefficient of variation of the calibrated fluorescence value was calculated, and the coefficient of variation of the collected fluorescence value in Table 2-Table 4 without calibration was calculated to obtain the data in Table 6 below.

[0090] The calibration coefficient of each well in Table 5 was multiplied by the collected fluorescence value of each well in Table 2-Table 4 to obtain the calibrated fluorescence value, and the coefficient of variation of the calibrated fluorescence value was calculated, and the coefficient of variation of the collected fluorescence value in Table 2-Table 4 without calibration was calculated to obtain the data in Table 6 below.

[0091] Table 6 calibration coefficient of each well of each fluorescence channel

[0092]

[0093]

[0094] Table 6 after calibration, the coefficient of variation meets the requirement of less than 5%, at the same time, the discrete degree of the coefficient of variation of each test well is obviously reduced, which weakens the influence of uneven distribution of light source on fluorescence acquisition.

[0095] In order to evaluate whether the calibration coefficient will affect the fluorescence linearity, it is also necessary to calculate the fluorescence linearity. The greater the fluorescence linearity, the better the acquired fluorescence data. Generally, the requirement for fluorescence linearity is not less than 99%. The calculation of fluorescence linearity is to calibrate the fluorescence values of different concentrations by using the calibration coefficient, and then to verify whether it affects the linearity by using the linear regression coefficient r = ∑(Xi-X)(Yi-Y / sqrt[∑(Xi-X) 2 ×∑(Yi-Y) 2 ], wherein Xi is the calibrated fluorescence value of each concentration (taking the average of three sample data of each concentration), X is the average value of the calibrated fluorescence of each concentration, Yi is the concentration of the measured fluorescent dye, and Y is the average value of each concentration of the measured fluorescent dye. The experimental results are as shown in Table 7.

[0096] Table 7 fluorescence linearity before and after calibration

[0097] Fluorescence channel Pre-calibration fluorescence linearity (%) Post-calibration fluorescence linearity (%) FAM 99.70 99.70 ROX 99.63 99.62 HEX 99.95 99.95 CY5 99.10 99.10

[0098] From the calculation results of Table 7, the fluorescence calibration method of the present application does not affect the original fluorescence linearity.

[0099] The above describes in detail the PCR fluorescence test method provided by the present application. The structure and working principle of the present application are described by using specific examples. The above description of the examples is only used to help understand the method and core idea of the present application. It should be pointed out that, for ordinary skilled persons in the technical field, some improvements and modifications can be made to the present application without departing from the principle of the present application, and these improvements and modifications also fall within the scope of protection of the claims of the present application.

Claims

1. A PCR fluorescence assay method, characterized in that, Includes the following steps: Step 1: Select multiple concentrations of the same fluorescent dye and inject them into the test holes of the test chip. Use an excitation light source to illuminate the test chip at a fixed angle. Use an optical acquisition device to acquire grayscale images of the excitation fluorescence of the test chip. Calculate the average pixel value of the grayscale images of the multiple concentrations of fluorescent dye as multiple acquired fluorescence values. A model is constructed to minimize the error between the coefficient of variation and the expected coefficient of variation of the calibration fluorescence values ​​of multiple concentrations of fluorescent dyes. The calibration coefficient is obtained by solving this model. Step 2: Fill the obtained calibration coefficients into the calculation module of the optical acquisition device; Step 3: Add the reaction reagent into the test hole of the test chip, and use the same irradiation angle as in Step 1 to irradiate the test chip. The optical acquisition device acquires the fluorescence grayscale image of the reaction reagent and calculates the fluorescence value of the reaction reagent through the internal calculation module.

2. The PCR fluorescence detection method according to claim 1, characterized in that, The calculation of the calibration coefficient in step 1 also includes calculating the calibration coefficient for different fluorescent dyes and filling the calibration coefficients corresponding to different fluorescent dyes into the calculation module of the optical acquisition device.

3. The PCR fluorescence detection method according to claim 2, characterized in that, The excitation light source contains multiple LED light sources. In one PCR cycle, the excitation light control system controls the excitation light source to emit LED light of different wavelengths in sequence to irradiate the test chip, and the optical acquisition device sequentially acquires images of reflected fluorescence excited by LED light of different wavelengths.

4. The PCR fluorescence detection method according to claim 3, characterized in that, The different LED light source wavelengths within the excitation light source are adapted to the excitation wavelengths of the different fluorescent dyes.

5. The PCR fluorescence detection method according to claim 1, characterized in that, The test chip is a silicon-based test chip, and the silicon-based test chip is provided with a plurality of serpentine test holes, which are evenly arranged on the test chip; The test chip has a temperature control module below it and a glass surface above it.

6. The PCR fluorescence detection method according to claim 5, characterized in that, In step 1, at least three groups of the same fluorescent dye are selected, and the concentrations of multiple groups of fluorescent dyes are diluted in a gradient manner.

7. The PCR fluorescence detection method according to claim 5, characterized in that, Step 1 further includes dividing the fluorescence grayscale image into several regions, calculating the average pixel value of the fluorescence grayscale image in each region, and using it as the collected fluorescence value for each region. A model is constructed to minimize the error between the coefficient of variation and the expected coefficient of variation of the calibration fluorescence values ​​at different concentrations in each region. The calibration coefficient for each region is obtained by solving this model.

8. The PCR fluorescence detection method according to claim 7, characterized in that, The excitation light source is at a 40°-50° angle to the silicon-based test chip; The test chips with different concentrations of fluorescent dyes were irradiated by the same light source, and the excitation light source irradiated the test chips at the same angle.

9. A PCR fluorescence detection method according to claim 8, characterized in that, The constructed model for minimizing the error between the coefficient of variation and the expected coefficient of variation when solving for calibration fluorescence values ​​at different concentrations is as follows: K represents the amount of fluorescent dye at different concentrations, cv n Let be the coefficient of variation for the nth concentration of fluorescent dye, and e be the expected coefficient of variation. The derivation formula for cv is: Data nj Here are the fluorescence values ​​after calibration for the nth concentration of fluorescent dye; std represents the standard deviation, mean represents the mean, and Data... nj The derivation formula is as follows: Where x m a represents the calibration coefficient of the m-th test hole on the test chip. nm This represents the fluorescence value collected in the m-th test well of the n-th concentration fluorescent dye, where m represents the number of test wells on the test chip.

10. A PCR fluorescence detection method according to claim 9, characterized in that: The expected coefficient of variation range is: 2% ≤ e ≤ 3%.

11. A PCR fluorescence detection method according to claim 10, characterized in that: The model for minimizing the error between the coefficient of variation and the expected coefficient of variation of calibrated fluorescence values ​​at different concentrations is solved using the quasi-Newton method.

12. The PCR fluorescence detection method according to claim 11, characterized in that, The optical acquisition device includes an imaging lens, a fluorescent filter, a CMOS image sensor, and an FPGA image acquisition board, wherein the CMOS image sensor senses images; The FPGA image acquisition template is equipped with a computing module, which acquires images from the CMOS image sensor and performs image processing.

Images