Detection result interpretation method of portable constant-temperature nucleic acid amplification instrument

Abstract

The invention relates to a detection result interpretation method of a portable constant-temperature nucleic acid amplification instrument, and relates to the field of detection equipment, and the method comprises the following steps: placing a multi-tube PCR (Polymerase Chain Reaction) tube filled with an amplified chromogenic nucleic acid reagent and a colorimetric card in the same environment for photographing; performing image processing analysis on the picture, determining pixel positions of a plurality of test tubes and pixel positions of a standard color card, and performing image color extraction to obtain RGB values; correcting the color through an algorithm, and calculating a judgment value of the color of the reagent in the PCR tube; a color judgment threshold value is set to be 0.4, if the calculated judgment value is larger than the threshold value, it is judged that the PCR tube is positive, and if the calculated judgment value is smaller than the threshold value, it is judged that the PCR tube is negative. And the interpretation accuracy of the amplification detection result is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of detection equipment, and in particular to a method for interpreting detection results of a portable constant-temperature nucleic acid amplification instrument. Background Art

[0002] Traditional nucleic acid amplification analyzers are often bulky, inconvenient to carry and use, and the instruments are expensive, so only specialized medical institutions will purchase them. When nucleic acid testing is performed, it is necessary to go to these medical institutions for centralized sampling, and professionally trained medical staff will centrally process them in professional laboratories. The steps from sample collection to obtaining test results are cumbersome and time-consuming, and test results are often not obtained in a timely manner.

[0003] The nucleic acid detection part usually uses a complex optical path structure and expensive optical components (including lasers, lens groups, color filters, dichroic mirrors, photomultiplier tubes, etc.) to collect and process fluorescence signals. The specific process includes filtering the stray light of the laser excitation light with an excitation filter, focusing the filtered laser through the excitation lens group and irradiating it on the DNA fragment to excite fluorescence, and the fluorescence is shaped into parallel light by the emission lens group, and then the stray light is removed by the emission filter and focused by the lens into the photomultiplier tube or other receiving optical elements for light intensity signal collection; In addition, the excitation light and the emission light overlap in part of the optical path, so it is necessary to add a dichroic mirror to the optical path to convert the propagation direction of part of the optical path; The collected light intensity signal is then filtered, multi-stage amplified, AD sampled, data communicated, etc. through the circuit, and finally the fluorescence data to be collected is obtained. The whole process requires expensive optical and electronic components, and the optical-mechanical structure and circuit layout are very complex, which cannot achieve the portability and low cost of the instrument.

[0004] Therefore, in view of the above shortcomings, it is necessary to provide a method for interpreting the detection results of a portable constant temperature nucleic acid amplification instrument. Summary of the invention

[0005] 1. Technical issues to be resolved The technical problem to be solved by the present invention is to solve the problem that some colorimetric instrument detection only gives results through naked eye observation, which leads to misinterpretation, while the instrument of optical judgment method is expensive.

[0006] (II) Technical solution In order to solve the above technical problems, the present invention provides a method for interpreting the detection results of a portable constant temperature nucleic acid amplification instrument, comprising the following steps: Ⅰ. Place the multi-tube PCR tubes containing the amplified chromogenic nucleic acid reagent and the colorimetric card in the same environment and take photos; Ⅱ. Perform image processing and analysis on the photos, determine the pixel positions of multiple test tubes and the pixel positions of the standard color card, extract the image colors respectively, and obtain the RGB values; III. Correct the color through the algorithm and calculate the judgment value of the color of the reagent in the PCR tube; specifically: in, is the G value after calibration of the reagent in tube n; is the B value after calibration of the reagent in tube n; It is the maximum value of the parameters of the three RGB colors in tube n after correction; It is the minimum value of the parameters of the three RGB colors in tube n after correction; IV. Set the color judgment threshold to 0.4. If the calculated judgment value is greater than the threshold, the PCR tube is judged to be positive. If the calculated judgment value is less than the threshold, it is judged to be negative.

[0007] As a further illustration of the present invention, preferably, a tube No. N is provided in the multi-tube PCR tube, and tube No. N is a negative quality control position for nucleic acid amplification; the remaining PCR tubes are reaction chambers for the test agent; the judgment value in tube No. N is first calculated and compared with the threshold value, if the judgment value of tube No. N is greater than the threshold value, then the amplification is judged to be invalid, and there is no need to judge the other PCR tubes; if the judgment value of tube No. N is less than the threshold value, then the amplification is judged to be valid, and then the other PCR tubes are calculated and judged.

[0008] As a further illustration of the present invention, preferably, the PCR tube and the colorimetric card need to be placed on the same white background plate at the same time before taking the photo.

[0009] As a further illustration of the present invention, preferably, a plurality of black triangles are provided on the color comparison card, and the color card position is calibrated by finding the triangle positions through software, thereby accurately obtaining the RGB value of the color card.

[0010] As a further illustration of the present invention, preferably, when performing calibration, the captured photo is enlarged and the triangle position is found again, and then the photo is reduced and the triangle position is found again, so as to further determine the position of the red, green and blue icons in the color card.

[0011] As a further illustration of the present invention, preferably, after collecting data, one third of the pixels with the highest values ​​are removed from the red, green and blue blocks respectively, and one third of the pixels with the lowest values ​​are removed, and the remaining one third of the pixel values ​​are retained.

[0012] As a further explanation of the present invention, preferably, the correlation coefficients of the remaining pixels are calculated respectively, specifically: in , and Corresponding to the correlation coefficients of red, green and blue respectively, , and The average of the pixel values ​​retained for red, green and blue respectively.

[0013] As a further illustration of the present invention, preferably, the color correction algorithm is: in , and are the RGB values ​​after calibration of tube n reagent, , and They are respectively the red, green and blue collection values ​​of tube n this time.

[0014] As a further illustration of the present invention, preferably, after taking photos, the RGB values ​​of a single PCR tube and a color card can be obtained and histograms can be established respectively, and the positive or negative result can be determined by comparing the similarity of the two histograms.

[0015] As a further illustration of the present invention, preferably, after the histogram is established, the Bhattacharyya distance of the two histograms is calculated. If the calculated distance is the smallest with the comprehensive distance of the red histogram of the color card, it is judged as positive; if the calculated distance is small with the green and blue distances of the color card and large with the red distance, it is judged as negative.

[0016] (III) Beneficial effects The above technical solution of the present invention has the following advantages: The present invention uses a common smart phone to synchronously take photos of a PCR tube and a customized standard RGB color card, uses software to perform image processing and analysis on the photos, determines the pixel positions of multiple test tubes and the pixel positions of the standard color card, and then respectively extracts the image color, uses a specific algorithm to calibrate the liquid in the PCR tube with the standard color card to calculate the color judgment value of the liquid in the PCR tube, and uses a determined threshold to perform positive and negative sample judgment, and can give a numerical detection result according to the calculated color judgment value, and then determine the concentration of the initial sample. Compared with the traditional naked eye interpretation method, it will not be disturbed by external environmental light, will not be affected by personal interpretation errors, and improves the accuracy of the interpretation of the amplification detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a schematic diagram of the arrangement of PCR tubes and color cards of the present invention; Figure 2 is a schematic diagram of PCR tube allocation of the present invention; Figure 3 It is a fitting curve diagram of the color judgment value and the corresponding nucleic acid concentration of the present invention. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution 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. 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.

[0019] A method for interpreting the detection results of a portable constant temperature nucleic acid amplification instrument, such as Figure 1 As shown, the following steps are included: Ⅰ. Install the portable constant temperature nucleic acid amplification instrument data analysis software on your mobile phone, open the software and use the camera function in the software to take pictures of the PCR tubes containing the amplified color-developing nucleic acid reagents and the standard color card placed on a white background.

[0020] The software will actively find the 4 triangle positions on the color card from the captured photos and read the marks to calibrate the color card position. It will rotate and zoom in and out according to the relative positions of the calibration positions found, further determine the positions of the red, green and blue icons in the color card, and collect RGB values ​​for all pixels in the red, green and blue areas respectively.

[0021] After collecting data, sort the RGB values ​​for each color block. All valid pixels in the red color block are sorted by R value, and the 1 / 3 pixels with the highest R value in the data are removed (these points may be caused by reflection problems during the shooting process), and the 1 / 3 pixels with the lowest R value in the data are removed (these points may be caused by the dark ambient light when shooting). The remaining 1 / 3 of valid data is retained, and the average value of their R values ​​is taken as the sampling value of the red color mark of this shooting. The same method is used to analyze the data of the green and blue areas to obtain the green color sampling value of the standard color card in this shooting. and blue color scale sampling value .

[0022] Ⅱ. The standard values ​​of the designed standard color card taken under the default optimal environment should be: red: 255, 0, 0; green: 0, 255, 0; blue 0, 0, 255. However, due to different ambient light each time, or the different response of each mobile phone to red, green and blue colors, there are certain differences in the data taken. The correlation coefficient is calculated according to the following algorithm formula: in , and Corresponding to the correlation coefficients of red, green and blue respectively.

[0023] like Figure 2 As shown, 1 to 7 are 7 test reagent reaction chambers, and N is the negative quality control position of nucleic acid amplification. The software will automatically analyze the position of the PCR eight-tube strip in the same photo, analyze the position of the reagent at the bottom of each reagent tube in the eight-tube strip, collect RGB values ​​of the reagent image area at the bottom of the reagent tube, and read the RGB values ​​of the liquid reagent parts of the eight reaction chambers 1 to N in the PCR tube 28 respectively. Because the negative is reddish and the positive is yellowish during reagent tube amplification, the valid sampling data of the 8 reagent tubes are sorted according to the size of the R value, and the highest value data of 1 / 3 and the lowest value data of 1 / 3 in the valid data are removed respectively. The average value of the remaining 1 / 3 valid value data is taken as the RGB sampling value of the reaction tube, which is recorded as , and . Where n is 1 to 7 and N represents the serial number of each reagent tube.

[0024] Ⅲ. Because the ambient light of each shot is different, and different models of mobile phones have different response efficiencies to red, green and blue when shooting, color correction is performed using the following algorithm formula: in , and They are the RGB values ​​after calibration of reagent tube n.

[0025] The color judgment value is calculated according to the following algorithm formula: in, is the G value after calibration of the reagent in tube n; is the B value after calibration of the reagent in tube n; It is the maximum value of the parameters of the three RGB colors in tube n after correction; It is the minimum value of the parameters of the three RGB colors in tube n after correction; IV. The color judgment threshold is determined to be 0.4 based on the test results of strong positive, positive, weak positive and negative reagents. Among them, tube N is a negative quality control position. If the color judgment value of tube N is greater than 0.4 during the judgment process, the amplification is judged to be invalid. If the value is less than 0.4, the amplification is judged to be valid. Then the color judgment values ​​of tubes 1 to 7 are judged respectively. If it is greater than 0.4, it is judged to be positive, and if it is less than 0.4, it is judged to be negative. The corresponding judgment values ​​are given to facilitate the operator to quantitatively determine the initial nucleic acid concentration.

[0026] In order to verify the feasibility of the above method, the present invention also provides an embodiment: Prepare nucleic acid at concentrations of 1, 10, and 10 2 , 10 3 , 10 4 , 10 5 , 10 6 The Mycoplasma pneumoniae virus sample with 10 copies / μL was placed in tubes 1 to 7 of the 8-tube PCR concatenation to perform nucleic acid amplification experiments and obtain its amplification products.

[0027] In the present invention, three different mobile phone models are selected to take photos of the group of test tubes under three different ambient lights, namely mobile phone model 1 in a weak light environment, mobile phone model 2 in a normal light environment, and mobile phone model 3 in a strong light environment. Then, RGB data is collected using software, and the color judgment values ​​corresponding to the positive samples of each nucleic acid concentration of each mobile phone model are calculated, as shown in the following table: The mean of the color judgment values ​​of the three mobile phone models and the corresponding nucleic acid concentrations were nonlinearly fitted, and the fitting curve was as follows: Figure 3 As shown, the relationship between nucleic acid concentration and color determination value is obtained: in, is the color judgment value, is the nucleic acid concentration.

[0028] when ≥1, the corresponding ≥0.4, it is considered positive. When <1, the corresponding <0.4, judged as negative.

[0029] Next, the three different mobile phone models were used to take photos of the same group of test tubes under three different ambient lights. The color judgment values ​​of test tube No. N were calculated to be less than 0.4, and the nucleic acid amplification experiment was judged to be valid. The specific test data of tubes No. 1 to 7 were as follows: The corresponding nucleic acid concentration can be calculated through the relationship between nucleic acid concentration and color judgment value, as shown in the following table: Through experimental data and judgment results, it was found that even if different models of mobile phones were used to take photos under different ambient lights, after the algorithm was calibrated through the color card mark in the present invention, the color judgment values ​​of each reagent reaction tube were very close, and it was possible to judge that the color judgment value of tube N was less than the threshold, and the amplification was determined to be valid. The amplification results of tubes 1 to 7 were further judged to be positive, positive, positive, positive, negative, negative, and negative, respectively, and the corresponding color judgment values ​​and corresponding nucleic acid concentrations could be given to facilitate the operator to perform data analysis and give correct judgment results.

[0030] In addition, the present invention also provides another interpretation method, specifically: Ⅰ. Read the image area containing reagents in a single PCR tube, divide it into three channels: red, green, and blue, and construct a histogram for each channel. Taking the red channel as an example, construct a histogram containing 256 intervals, ranging from 0 to 256, to represent the distribution of pixel values ​​in the red channel. Similarly, construct histograms for the green and blue channels. Perform the same operation on the red, green, and blue areas in the standard color card.

[0031] Ⅱ. Use the Bhattacharyya distance formula to calculate the distance between the reagent color histogram and the red, green and blue histograms in the standard color card. 1. Compare with the red histogram: For the red channel, the Bhattacharyya distance is: in, is the reagent red histogram, Red histogram for the color card is the index value of the histogram. Similarly, the distance between the green channel histogram of the reagent color and the green channel histogram of the red area of ​​the standard color card is calculated. The distance between the blue channel histogram of the reagent color and the blue channel histogram of the red area of ​​the standard color card .

[0032] 2. Compare with the green histogram: For the red color channel, the Bhattacharyya distance is: in, is the reagent red histogram, Green histogram of the color card, and calculate the corresponding distance between the green channel and the blue channel and .

[0033] 3. Compare with the blue histogram: For the red channel, the Bhattacharyya distance is: in, is the reagent red histogram, The blue histogram of the color card and the corresponding distance between the green channel and the blue channel are calculated and .

[0034] Ⅲ. After the calculation is completed, compare these distances. If the comprehensive distance between the reagent color and the red histogram of the standard color card (which can be the average or weighted average of the three channel distances) is the smallest, then the reagent color is more inclined to red and is judged as positive; if the comprehensive distance between the reagent color and the green and blue histograms of the standard color card is smaller than red and closer to yellow (yellow can be regarded as a mixture of green and blue), then it is judged as negative. For example: Calculate the integrated distance between the reagent color and the red histogram: Calculate the integrated distance to the green histogram: Calculate the integrated distance to the blue histogram: like and , it is judged as positive, if and Relatively small, If it is relatively large, it can be judged as negative.

[0035] 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 make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for interpreting the detection results of a portable constant temperature nucleic acid amplification instrument, characterized in that: The following steps are involved: Ⅰ. Place the multi-tube PCR tubes containing the amplified chromogenic nucleic acid reagent and the colorimetric card in the same environment and take photos; Ⅱ. Perform image processing and analysis on the photos, determine the pixel positions of multiple test tubes and the pixel positions of the standard color card, extract the image colors respectively, and obtain the RGB values; III. Correct the color through the algorithm and calculate the judgment value of the color of the reagent in the PCR tube; specifically: in, is the G value after calibration of the reagent in tube n; is the B value after calibration of the reagent in tube n; It is the maximum value of the parameters of the three RGB colors in tube n after correction; It is the minimum value of the parameters of the three RGB colors in tube n after correction; IV. Set the color judgment threshold to 0.

4. If the calculated judgment value is greater than the threshold, the PCR tube is judged to be positive. If the calculated judgment value is less than the threshold, it is judged to be negative.

2. The method for interpreting the detection results of a portable constant temperature nucleic acid amplification instrument according to claim 1, characterized in that: A tube No. N is provided in the multi-tube PCR tube, and tube No. N is a negative quality control position for nucleic acid amplification; the other PCR tubes are reaction chambers for the test agent; the judgment value in tube No. N is first calculated and compared with the threshold value. If the judgment value of tube No. N is greater than the threshold value, it is determined that this amplification is invalid, and there is no need to judge other PCR tubes; if the judgment value of tube No. N is less than the threshold value, it is determined that this amplification is valid, and then the other PCR tubes are calculated and judged.

3. The method for interpreting the detection results of a portable constant temperature nucleic acid amplification instrument according to claim 2, characterized in that: Before taking pictures, the PCR tube and the colorimetric card must be placed on the same white background.

4. The method for interpreting the detection results of a portable constant temperature nucleic acid amplification instrument according to claim 3, characterized in that: There are several black triangles on the color card. The software is used to find the triangle positions to calibrate the color card position, and then the RGB value of the color card is accurately obtained.

5. The method for interpreting the detection results of a portable constant temperature nucleic acid amplification instrument according to claim 4, characterized in that: When calibrating, zoom in on the photo and find the triangle position again, then zoom out on the photo and find the triangle position again to further determine the position of the red, green and blue icons in the color card.

6. The method for interpreting the detection results of a portable constant temperature nucleic acid amplification instrument according to claim 5, characterized in that: After collecting the data, remove one-third of the pixels with the highest values ​​in the red, green and blue blocks respectively, and then remove one-third of the pixels with the lowest values, and retain the remaining one-third of the pixel values.

7. The method for interpreting the detection results of a portable constant temperature nucleic acid amplification instrument according to claim 6, characterized in that: The correlation coefficients of the remaining pixels are calculated respectively, as follows: in , and Corresponding to the correlation coefficients of red, green and blue respectively, , and The average of the pixel values ​​retained for red, green and blue respectively.

8. The method for interpreting the detection results of a portable constant temperature nucleic acid amplification instrument according to claim 7, characterized in that: The algorithm for color correction is: in, , and They are respectively the red, green and blue collection values ​​of tube n this time.

9. The method for interpreting the detection results of a portable constant temperature nucleic acid amplification instrument according to claim 1, characterized in that: After taking photos, the RGB values ​​of a single PCR tube and a color card can be obtained and histograms can be established respectively. Positive or negative can be determined by comparing the similarity of the two histograms.

10. The method for interpreting the detection results of a portable constant temperature nucleic acid amplification instrument according to claim 9, characterized in that: After the histogram is established, the Bhattacharyya distance between the two histograms is calculated. If the calculated distance is the smallest with the comprehensive distance of the red histogram of the color card, it is judged as positive; if the calculated distance is small with the green and blue distances of the color card and large with the red distance, it is judged as negative.

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