Detection method of growth differentiation factor 15

Abstract

The invention relates to the field of visual auxiliary detection, in particular to a method for detecting a growth differentiation factor 15. The method comprises the following steps: acquiring a side image of a reagent to be detected by adopting an exogenous detection mode, performing optical path analysis without interference on the reagent to be detected, determining an optical irradiation band caused by exciting light of an optical read head, and determining image reflection characteristics; and calculating a pixel discrete characterization value to label an optical irradiation band, and controlling an amplification cycle process according to a subsequent adaptive labeling result of the optical irradiation band, including obtaining reflection excitation characteristics of the optical read head at different detection heights, determining a sampling position, and evaluating whether to maintain sampling at the current detection height of the optical read head. By evaluating the optical irradiation band, the influence of media in different areas of the reagent to be detected on sampling of the optical read head is considered from the image dimension, the sampling position is adjusted, more accurate data is obtained, and the detection accuracy of the growth differentiation factor 15 is improved.

Description

Technical Field

[0001] The present invention relates to the field of visually assisted detection, and in particular to a method for detecting growth differentiation factor 15. Background Art

[0002] Growth Differentiation Factor 15 (GDF15) is a secreted protein belonging to the transforming growth factor-β (TGF-β) superfamily that plays an important role in various physiological and pathological processes. In recent years, research on GDF15 as a biomarker has gradually attracted attention, particularly in areas such as cancer, cardiovascular disease, metabolic disorders, and neurodegenerative diseases. The development and application of its detection technology has become a research hotspot.

[0003] For example, Chinese Patent Publication No. CN117625755A discloses a method for detecting methylation of the GDF15 gene, comprising the following steps: step one, primer design; step two, sample collection; step three, nucleic acid extraction; step four, bisulfite conversion; and step five, methylation detection. The present invention uses a U-resistant specific Taq enzyme for PCR amplification, avoiding the conversion of a large number of unmethylated Cs to U during the bisulfite conversion process of ordinary Taq enzymes and high-fidelity Taq enzymes, which causes the inability to PCR amplify. Using the bisulfite conversion product as a template, the PCR amplification at different annealing temperatures was tested, with a temperature gradient of 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, and 62°C to ensure a suitable reaction temperature. The experimental process for methylation modification of the GDF15 gene is reasonable and efficient. Primers of different concentrations were tested, and it was found that the GDF15-F4 / R4 primer pair had the best amplification effect at a concentration of 0.4 μM, which is conducive to ensuring the reasonable selection of primer pairs.

[0004] However, the prior art still has the following problems:

[0005] The amplification cycle is an ongoing process. As the amplification cycle progresses, the properties of the medium within the test reagent may change, for example, precipitation, aggregation, and suspension. Under the influence of gravity, the conditions in different areas of the test reagent vary. Due to reflection, the feedback to the excitation light emitted by the optical reader varies, affecting detection accuracy. Summary of the Invention

[0006] To this end, the present invention provides a method for detecting growth differentiation factor 15 to overcome the problem in the prior art that the properties of the medium in the reagent to be tested may change as the amplification cycle proceeds, resulting in different medium properties in different areas of the reagent to be tested, and different feedback to the excitation light emitted by the optical reader during the reflection process, thereby reducing the detection accuracy.

[0007] To achieve the above object, the present invention provides a method for detecting growth differentiation factor 15, comprising:

[0008] Step S1, placing the reagent to be tested in a PCR instrument for amplification cycles;

[0009] Step S2, continuously collecting side images of the reagent to be tested to perform optical pathway analysis, including determining an optical illumination band corresponding to the excitation light of an optical reader in a PCR instrument based on the side images of the reagent to be tested, and determining image reflection characteristics of the optical illumination band;

[0010] Step S3, calculating a pixel discrete representation value for the optically illuminated band based on the image reflection feature to determine whether to mark the optically illuminated band;

[0011] Step S4, based on the annotation results of the optical image band, controlling the amplification cycle process, including:

[0012] By controlling the detection height of the optical reader, collecting side images of the reagent to be tested at different detection heights, determining the optical illumination band, calibrating the reflection excitation characteristics of each of the optical illumination bands, and determining the sampling position of the optical reader based on the reflection excitation characteristics in the different optical illumination bands;

[0013] Or, compare the side image of the reagent to be tested in the adjacent frame to determine whether to control the optical reader to maintain the current detection height sampling;

[0014] Step S5, obtaining the sampling output result of the PCR instrument and determining the relative expression level of GDF-15;

[0015] The image reflection characteristics include the pixel gradient difference along the vertical excitation light irradiation direction and the discrete value of the local chromaticity.

[0016] Furthermore, in step S2, the process of determining the image reflection characteristics of the optically illuminated band includes:

[0017] Clustering the side image of the test agent, determining the cluster edge, and determining the area corresponding to the cluster edge as the optical irradiation zone;

[0018] Dividing the optical illumination zone into a plurality of rectangular areas along the direction perpendicular to the excitation light illumination, calculating the pixel chromaticity difference between each rectangular area and the adjacent area, and determining the average pixel chromaticity difference as the pixel gradient difference;

[0019] A number of local observation areas are randomly selected in the optical illumination band, and the variance of the chromaticity of the pixels corresponding to the local observation areas is determined as the discrete value.

[0020] Furthermore, calculating the discrete representation value of the pixel for the optically illuminated band includes,

[0021] Determining a ratio of the pixel point gradient difference to a preset pixel point gradient difference threshold as a first pixel discrete factor;

[0022] Determining a ratio of the discrete value to a preset pixel discrete threshold as a second pixel discrete factor;

[0023] The pixel discrete representation value is obtained by weighted summing the first pixel discrete factor and the second pixel discrete factor.

[0024] Further, determining whether to mark the optical illumination band includes,

[0025] If the pixel discrete representation value is greater than or equal to a preset pixel discrete representation threshold, it is determined that the optical irradiation zone is marked;

[0026] If the pixel discrete representation value is less than a preset pixel discrete representation threshold, it is determined that the optical illumination zone is not marked;

[0027] Wherein, marking the optical irradiation zone includes marking the edge of the optical irradiation zone in the side image of the reagent to be tested.

[0028] Furthermore, based on the annotation results of the optical image bands, the amplification cycle process is controlled, wherein:

[0029] If the optical image band is marked, then by controlling the detection height of the optical reader, collecting the side image of the reagent to be tested at different detection heights, determining the optical illumination band, calibrating the reflection excitation characteristics of each optical illumination band, and determining the sampling position of the optical reader based on the reflection excitation characteristics in the different optical illumination bands;

[0030] If the optical image strip is not marked, it is determined whether to control the optical reading head to maintain the current detection height sampling.

[0031] Furthermore, by controlling the detection height of the optical reader, side images of the reagent to be detected at different detection heights are collected, wherein:

[0032] The optical reading head needs to be moved several times to adjust to different detection heights, and the single movement distance of the optical reading head is half of the width of the optical irradiation band.

[0033] Furthermore, the process of calibrating the reflection excitation characteristics of each of the optical illumination bands includes:

[0034] determining a number of profile features in an optically illuminated band;

[0035] Determine the chromaticity difference between each contour feature and the reference ring and the area of ​​the contour feature;

[0036] If the profile feature meets the reflection excitation condition, the profile feature is determined as a reflection excitation feature;

[0037] The reference ring is a strip-shaped area surrounding the contour feature, and the reflection excitation condition is that the chromaticity difference is greater than a predetermined chromaticity difference threshold and the area is greater than a predetermined area threshold.

[0038] Furthermore, the process of determining the sampling position of the optical read head based on the reflection excitation characteristics in different optical illumination bands includes:

[0039] Determine the optical reflection bands corresponding to different positions of the optical pickup head;

[0040] determining each reflection excitation feature in each optical reflection band;

[0041] Determine the absolute variance of the chromaticity difference and the absolute variance of the area corresponding to each optical reflection band and perform weighted summation to obtain a reflection excitation characteristic value;

[0042] An optical reflection band having the smallest reflection excitation characteristic value is determined, and a position of the optical reading head corresponding to the optical reflection band is determined as the sampling position.

[0043] Furthermore, comparing the adjacent frame side image of the reagent to be tested to determine whether to control the optical reader to maintain the current detection height sampling includes:

[0044] Determine whether there is a marked optical irradiation band in the adjacent frame side image of the reagent to be tested;

[0045] If there is no marked optical irradiation zone in the adjacent frame side images of the reagent to be tested, it is determined that the optical reader maintains the current detection height sampling.

[0046] Furthermore, the amplification cycle process includes pre-denaturation, denaturation, annealing and extension.

[0047] Compared with the prior art, the beneficial effect of the present invention lies in that the present invention uses exogenous detection to collect side images of the reagent to be tested, without interfering with the reagent to be tested, performs optical pathway analysis, determines the optical irradiation band caused by the excitation light of the optical reader, determines the image reflection characteristics, and calculates the pixel discrete representation value to mark the optical irradiation band. Subsequently, the amplification cycle process is adaptively controlled based on the marking results of the optical irradiation band, including obtaining the reflection excitation characteristics of the optical reader at different detection heights, determining the sampling position, and evaluating whether to maintain the current detection height sampling of the optical reader. By evaluating the optical irradiation band, the influence of the medium in different areas of the reagent to be tested on the optical reader sampling is considered from the image dimension, the sampling position is adjusted, and more accurate data is obtained, thereby improving the accuracy of the detection of growth differentiation factor 15.

[0048] In particular, the present invention considers determining the optical irradiation band and determining the image reflection characteristics. In actual situations, the optical reader needs to emit excitation light to obtain the reflected fluorescence signal, and the excitation light is a relatively concentrated light beam, so an optical irradiation band is formed in the reagent to be tested, and in the image dimension, the reagent to be tested is easier to observe in the optical irradiation band. Therefore, the present invention detects the optical irradiation band from the image dimension and determines the image reflection characteristics of the optical irradiation band. In actual situations, the amplification cycle is a continuous process involving steps such as heating. The reagent to be tested may be precipitated, suspended, or aggregated, affecting the reflection of light, and under the influence of gravity, there may be differences in the gradient area, resulting in different reflection conditions, which in turn affects the sampling of the optical reader. Therefore, the present invention extracts image reflection characteristics from the image dimension, characterizes the impact of the reflection caused by the corresponding area of ​​the reagent to be tested on the sampling of the optical reader, marks the optical irradiation band, and subsequently adaptively controls the amplification cycle process, thereby improving the accuracy of the detection of growth differentiation factor 15.

[0049] In particular, the present invention controls the amplification cycle based on the annotation results of the optical image band. For the case of the marked optical image band, since the reflection conditions in the area are relatively discrete when observed from the image dimension, the detection height of the optical reader is continuously adjusted to take into account the reflection excitation characteristics in the formed optical irradiation band. In actual situations, larger precipitates or aggregates have a greater impact on reflection. Considering the reflection excitation characteristics in different areas, a better optical reader detection height is selected for sampling based on this. The present invention considers the impact of the medium in different areas of the reagent to be tested on the optical reader sampling from the image dimension, adjusts the sampling position, obtains more accurate data, and improves the accuracy of growth differentiation factor 15 detection.

[0050] In particular, for the case of unlabeled optical image bands, their image reflection characteristics perform better in preliminary image dimension considerations. By comparing the side images of the reagent to be tested in adjacent frames, it is determined whether it is accidental, and then the detection height of the optical reader is directly maintained, ensuring the efficiency of detection while ensuring reliability. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is a schematic diagram of the steps of the method for detecting growth differentiation factor 15 according to an embodiment of the invention;

[0052] Figure 2 A logic block diagram for determining whether to mark an optical illumination zone according to an embodiment of the invention;

[0053] Figure 3 A logic block diagram of controlling the amplification cycle process based on the annotation results of the optical image band according to an embodiment of the invention;

[0054] Figure 4 A logic block diagram for determining the sampling position of an optical pickup head according to an embodiment of the invention. DETAILED DESCRIPTION

[0055] In order to make the objects and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with embodiments; it should be understood that the specific embodiments described herein are merely used to explain the present invention and are not intended to limit the present invention.

[0056] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are only used to explain the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0057] It should be noted that, in the description of the present invention, terms such as "up", "down", "left", "right", "inside", and "outside" indicating directions or positional relationships are based on the directions or positional relationships shown in the accompanying drawings. This is only for the convenience of description and does not indicate or imply that the device or element must have a specific orientation, be constructed and operated in a specific orientation. Therefore, it cannot be understood as a limitation on the present invention.

[0058] Furthermore, it should be noted that, in the description of the present invention, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood in a broad sense. For example, they may refer to fixed connections, detachable connections, or integral connections; mechanical connections or electrical connections; direct connections or indirect connections through an intermediate medium; and internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0059] See also Figure 1 , which is a schematic diagram of the steps of the method for detecting the growth differentiation factor 15 according to an embodiment of the present invention. The method for detecting the growth differentiation factor 15 according to an embodiment of the present invention comprises:

[0060] Step S1, placing the reagent to be tested in a PCR instrument for amplification cycles;

[0061] Step S2, continuously collecting side images of the reagent to be tested to perform optical pathway analysis, including determining an optical illumination band corresponding to the excitation light of an optical reader in a PCR instrument based on the side images of the reagent to be tested, and determining image reflection characteristics of the optical illumination band;

[0062] Step S3, calculating a pixel discrete representation value for the optically illuminated band based on the image reflection feature to determine whether to mark the optically illuminated band;

[0063] Step S4, based on the annotation results of the optical image band, controlling the amplification cycle process, including:

[0064] By controlling the detection height of the optical reader, collecting side images of the reagent to be tested at different detection heights, determining the optical illumination band, calibrating the reflection excitation characteristics of each of the optical illumination bands, and determining the sampling position of the optical reader based on the reflection excitation characteristics in the different optical illumination bands;

[0065] Or, compare the side image of the reagent to be tested in the adjacent frame to determine whether to control the optical reader to maintain the current detection height sampling;

[0066] Step S5, obtaining the sampling output result of the PCR instrument and determining the relative expression level of GDF-15;

[0067] The image reflection characteristics include the pixel gradient difference along the vertical excitation light irradiation direction and the discrete value of the local chromaticity.

[0068] Specifically, in implementation, there is no limitation on the form of PCR instrument used. The main component of PCR is the optical reader, which can irradiate the sample in the form of excitation light and detect the feedback fluorescence signal for analysis. The optical reader is divided into a vertical reader and a horizontal reader. In implementation, a horizontal reader is used. The horizontal reader can adaptively adjust the detection height, which will not be repeated here.

[0069] Specifically, a pre-arranged miniature industrial camera may be used to capture the side image of the test agent. There is no limitation on the miniature industrial camera, and those skilled in the art may select one based on the usage environment.

[0070] Specifically, the sample reagent is prepared using existing basic preparation steps, which, in some possible implementations, include:

[0071] Sample reagents included 9.2 μl of cDNA diluent and 10.8 μl of primer-mix solution;

[0072] The cDNA dilution buffer consists of:

[0073] cDNA template: 0.4 μL

[0074] ddH2O: 8.8 μL (enzyme-free water, to make up the volume)

[0075] Primer-Mix 10.8 μl contains:

[0076] qPCR Mix: 10 μL (containing SYBR Green, dNTPs, Taq enzyme, buffer, etc.).

[0077] Forward primer (F): 0.4 μL;

[0078] Reverse primer (R): 0.4 μL.

[0079] Specifically, the process of reverse transcription to synthesize cDNA includes:

[0080] Take 1 μg of total RNA and place it in a 200 μl EP tube without enzyme, add 2 μl of DNA enzyme, add pure water to 16 μl, pipette gently 5-10 times, and incubate at 37°C for 15-30 minutes;

[0081] Add 4 μl of 5×RT mix to each tube, pipette gently 10 times to mix, react at 42°C for 50 min, and freeze at -80°C.

[0082] Specifically, in step S2, the process of determining the image reflection characteristics of the optically illuminated band includes:

[0083] Clustering the side image of the test agent, determining the cluster edge, and determining the area corresponding to the cluster edge as the optical irradiation zone;

[0084] The optical illumination band is divided into several rectangular areas along the direction perpendicular to the excitation light illumination, the pixel chromaticity difference between each rectangular area and the adjacent area is calculated, and the mean of the pixel chromaticity difference is determined as the pixel gradient difference; several local observation areas are randomly selected in the optical illumination band, and the variance of the pixel chromaticity corresponding to the local observation area is determined as the discrete value.

[0085] Specifically, it can be understood that since the optical irradiation band is formed by reflected light irradiation, it has obvious boundaries in the image. Therefore, the clustering edge can be identified by clustering the side images of the test agent, and then the optical irradiation band can be determined. The clustering algorithm can use the K-Means algorithm. Of course, other methods can also be used, which will not be repeated here.

[0086] It can be understood that when a horizontal reader is used, the vertical excitation light irradiation direction is the direction extending toward both ends of the test tube. When dividing the rectangular area, the width of the rectangular area can be determined based on the total length of the test tube, and is usually set to 1 / 10 of the total length.

[0087] In implementation, a reference line perpendicular to the bottom of the side image of the test agent can be constructed in the side image of the test agent, and several parallel rectangular areas can be constructed based on the reference line to cover the side image of the test agent, which will not be described in detail.

[0088] The area of ​​the local observation region is determined based on the total area of ​​the side image of the test agent and is set to 1 / 100 of the total area.

[0089] Specifically, calculating the discrete representation value of a pixel for an optically illuminated band includes,

[0090] Determining a ratio of the pixel point gradient difference to a preset pixel point gradient difference threshold as a first pixel discrete factor;

[0091] Determining a ratio of the discrete value to a preset pixel discrete threshold as a second pixel discrete factor;

[0092] The pixel discrete representation value is obtained by weighted summing the first pixel discrete factor and the second pixel discrete factor.

[0093] Specifically, the pixel gradient difference threshold and the pixel discrete threshold are determined based on the side images of the test agent recorded during the amplification cycle of several identical test agents, wherein:

[0094] Determine the average pixel gradient difference and the average discrete value in the side image of each test sample;

[0095] The pixel gradient difference threshold is set as the product of the average pixel gradient difference and the pixel offset coefficient;

[0096] The pixel discrete threshold is set to the product of the average discrete value and the pixel offset coefficient;

[0097] The pixel shift coefficient is set in the interval [1.15,1.3];

[0098] The weighting coefficient for the first pixel discrete factor is 0.45, and the weighting coefficient for the second pixel discrete factor is 0.55.

[0099] The present invention considers determining the optical irradiation band and determining the image reflection characteristics. In actual situations, the optical reader needs to emit excitation light to obtain the reflected fluorescence signal, and the excitation light is a relatively concentrated light beam, so an optical irradiation band is formed in the reagent to be tested, and in the image dimension, the reagent to be tested is easier to observe in the optical irradiation band. Therefore, the present invention detects the optical irradiation band from the image dimension and determines the image reflection characteristics of the optical irradiation band. In actual situations, the amplification cycle is a continuous process involving steps such as heating. The reagent to be tested may be precipitated, suspended, or aggregated, affecting the reflection of light. Under the influence of gravity, there may be differences in the gradient area, resulting in different reflection conditions, which in turn affects the sampling of the optical reader. Therefore, the present invention extracts image reflection characteristics from the image dimension, characterizes the impact of the reflection caused by the corresponding area of ​​the reagent to be tested on the sampling of the optical reader, marks the optical irradiation band, and subsequently adaptively controls the amplification cycle process, thereby improving the accuracy of the detection of growth differentiation factor 15.

[0100] Specifically, determining whether to label the optical irradiation zone includes:

[0101] If the pixel discrete representation value is greater than or equal to a preset pixel discrete representation threshold, it is determined that the optical irradiation zone is marked;

[0102] If the pixel discrete representation value is less than a preset pixel discrete representation threshold, it is determined that the optical illumination zone is not marked;

[0103] Wherein, marking the optical irradiation zone includes marking the edge of the optical irradiation zone in the side image of the reagent to be tested.

[0104] Specifically, the purpose of setting the pixel threshold is to characterize the situation where the pixel gradient difference is close to the pixel gradient difference threshold and the discrete value is close to the pixel discrete threshold. In implementation, the pixel discrete characterization threshold is selected in the interval [1.08, 1.25].

[0105] Specifically, based on the annotation results of the optical image band, the amplification cycle process is controlled, wherein:

[0106] If the optical image band is marked, then by controlling the detection height of the optical reader, collecting the side image of the reagent to be tested at different detection heights, determining the optical illumination band, calibrating the reflection excitation characteristics of each optical illumination band, and determining the sampling position of the optical reader based on the reflection excitation characteristics in the different optical illumination bands;

[0107] If the optical image strip is not marked, it is determined whether to control the optical reading head to maintain the current detection height sampling.

[0108] Specifically, by controlling the detection height of the optical reader, side images of the reagent to be detected at different detection heights are collected, wherein:

[0109] The optical reading head needs to be moved several times to adjust to different detection heights, and the single movement distance of the optical reading head is half of the width of the optical irradiation band.

[0110] It is understandable that based on this, the reagents to be tested can be relatively completely covered to avoid missing areas, which will not be elaborated here.

[0111] Specifically, the process of calibrating the reflection excitation characteristics of each of the optical illumination bands includes:

[0112] determining a number of profile features in an optically illuminated band;

[0113] The chromaticity difference between each contour feature and the reference ring and the area of ​​the contour feature are determined. It can be understood that the chromaticity difference between the contour feature and the reference ring is the difference between the chromaticity mean within the contour feature and the chromaticity mean within the reference ring.

[0114] If the profile feature meets the reflection excitation condition, the profile feature is determined as a reflection excitation feature;

[0115] The reference ring is a strip-shaped area surrounding the contour feature, and the reflection excitation condition is that the chromaticity difference is greater than a predetermined chromaticity difference threshold and the area is greater than a predetermined area threshold.

[0116] Specifically, there is no limitation on the method of identifying contour features. For example, an image segmentation algorithm may be used, or other methods may be used, which will not be described in detail.

[0117] When constructing the reference ring, the original contour feature can be magnified, and the strip area formed by the contour feature after being magnified 1.2 times and the original contour feature is determined as the reference ring.

[0118] The purpose of setting the chromaticity difference threshold is to characterize the situation where the chromaticity of the reflection feature is significantly different from the surrounding chromaticity. The chromaticity difference threshold is determined based on the chromaticity mean inside the contour feature and is determined between 0.25 and 0.5 times the chromaticity mean.

[0119] The area threshold is pre-set, wherein side images of several identical test agents during amplification cycles are obtained in advance, and the optical irradiation band is determined, the average area of ​​the contour features therein is determined, and the area threshold is set to 1.25 times the average area.

[0120] Specifically, the process of determining the sampling position of the optical read head based on the reflection excitation characteristics in different optical illumination bands includes:

[0121] Determine the optical reflection bands corresponding to different positions of the optical pickup head;

[0122] determining each reflection excitation feature in each optical reflection band;

[0123] Determine the absolute variance of the chromaticity difference and the absolute variance of the area corresponding to each optical reflection band and perform weighted summation to obtain a reflection excitation characteristic value;

[0124] In implementation, the absolute variance of the chromaticity difference corresponds to a weight of 0.5, and the absolute variance of the area corresponds to a weight of 0.5.

[0125] An optical reflection band having the smallest reflection excitation characteristic value is determined, and a position of the optical reading head corresponding to the optical reflection band is determined as the sampling position.

[0126] The present invention controls the amplification cycle based on the annotated results of the optical image band. For the annotated optical image band, since the reflection conditions in the area are relatively discrete when observed from the image dimension, the detection height of the optical reader is continuously adjusted to take into account the reflection excitation characteristics in the formed optical irradiation band. In actual situations, larger precipitates or aggregates have a greater impact on reflection. Considering the reflection excitation characteristics in different areas, a more optimal optical reader detection height is selected for sampling. The present invention considers the impact of the medium in different areas of the reagent to be tested on the optical reader sampling from the image dimension, adjusts the sampling position, obtains more accurate data, and improves the accuracy of growth differentiation factor 15 detection.

[0127] Specifically, comparing the side images of the reagent to be tested in adjacent frames to determine whether to control the optical reader to maintain the current detection height sampling includes:

[0128] Determine whether there is a marked optical irradiation band in the adjacent frame side image of the reagent to be tested;

[0129] If there is no marked optical irradiation zone in the adjacent frame side images of the reagent to be tested, it is determined that the optical reader maintains the current detection height sampling.

[0130] Specifically, the amplification cycle process includes pre-denaturation, denaturation, annealing and extension:

[0131] In the implementation, the initial denaturation was 2 min 15 s at 95 °C;

[0132] Denaturation for 15 s at 95°C;

[0133] Annealing for 30 s at 60 °C;

[0134] Extension 30s, temperature 72℃.

[0135] For the case of unlabeled optical image bands, its image reflection characteristics perform better in preliminary image dimension considerations. By comparing the side images of the reagent to be tested in adjacent frames, it is determined whether it is accidental, and then the detection height of the optical reader is directly maintained, ensuring the efficiency of detection while ensuring reliability.

[0136] Specifically, the relative expression level of GDF-15 can be determined based on the sampling output results using the relative quantification method. The relative quantification method is a simple method for analyzing relative changes in gene expression and relative expression levels in real-time quantitative PCR experiments, and will not be described in detail here.

[0137] Thus far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art may make equivalent changes or substitutions to the relevant technical features, and the technical solutions after such changes or substitutions will fall within the scope of protection of the present invention.

Claims

1. A method for detecting growth differentiation factor 15, characterized in that: include: Step S1, placing the reagent to be tested in a PCR instrument for amplification cycles; Step S2, continuously collecting side images of the reagent to be tested to perform optical pathway analysis, including determining an optical illumination band corresponding to the excitation light of an optical reader in a PCR instrument based on the side images of the reagent to be tested, and determining image reflection characteristics of the optical illumination band; Step S3, calculating a pixel discrete representation value for the optically illuminated band based on the image reflection feature to determine whether to mark the optically illuminated band; Step S4, based on the annotation results of the optical image band, controlling the amplification cycle process, including: By controlling the detection height of the optical reader, collecting side images of the reagent to be tested at different detection heights, determining the optical illumination band, calibrating the reflection excitation characteristics of each of the optical illumination bands, and determining the sampling position of the optical reader based on the reflection excitation characteristics in the different optical illumination bands; Or, compare the side image of the reagent to be tested in the adjacent frame to determine whether to control the optical reader to maintain the current detection height sampling; Step S5, obtaining the sampling output result of the PCR instrument and determining the relative expression level of GDF-15; The image reflection characteristics include the pixel gradient difference along the vertical excitation light irradiation direction and the discrete value of the local chromaticity.

2. The method for detecting growth differentiation factor 15 according to claim 1, characterized in that: In step S2, the process of determining the image reflection characteristics of the optically illuminated band includes: Clustering the side image of the test agent, determining the cluster edge, and determining the area corresponding to the cluster edge as the optical irradiation zone; Dividing the optical illumination zone into a plurality of rectangular areas along the direction perpendicular to the excitation light illumination, calculating the pixel chromaticity difference between each rectangular area and the adjacent area, and determining the average pixel chromaticity difference as the pixel gradient difference; A number of local observation areas are randomly selected in the optical illumination band, and the variance of the chromaticity of the pixels corresponding to the local observation areas is determined as the discrete value.

3. The method for detecting growth differentiation factor 15 according to claim 2, characterized in that: In step S3, calculating the discrete pixel representation value for the optically illuminated band includes: Determining a ratio of the pixel point gradient difference to a preset pixel point gradient difference threshold as a first pixel discrete factor; Determining a ratio of the discrete value to a preset pixel discrete threshold as a second pixel discrete factor; The pixel discrete representation value is obtained by weighted summing the first pixel discrete factor and the second pixel discrete factor.

4. The method for detecting growth differentiation factor 15 according to claim 3, characterized in that: Determine whether to mark the optical irradiation zone including, If the pixel discrete representation value is greater than or equal to a preset pixel discrete representation threshold, it is determined that the optical irradiation zone is marked; If the pixel discrete representation value is less than a preset pixel discrete representation threshold, it is determined that the optical illumination zone is not marked; Wherein, marking the optical irradiation zone includes marking the edge of the optical irradiation zone in the side image of the reagent to be tested.

5. The method for detecting growth differentiation factor 15 according to claim 1, characterized in that: Based on the annotation results of the optical image band, the amplification cycle process is controlled, wherein, If the optical image band is marked, then by controlling the detection height of the optical reader, collecting the side image of the reagent to be tested at different detection heights, determining the optical illumination band, calibrating the reflection excitation characteristics of each optical illumination band, and determining the sampling position of the optical reader based on the reflection excitation characteristics in the different optical illumination bands; If the optical image strip is not marked, it is determined whether to control the optical reading head to maintain the current detection height sampling.

6. The method for detecting growth differentiation factor 15 according to claim 1, characterized in that: By controlling the detection height of the optical reader, side images of the reagent to be detected at different detection heights are collected, wherein: The optical reading head is adjusted to different detection heights by moving several times, and a single moving distance of the optical reading head is half of the width of the optical irradiation band.

7. The method for detecting growth differentiation factor 15 according to claim 1, characterized in that: The process of calibrating the reflective excitation characteristics of each of the optical illumination bands includes: determining a number of profile features in an optically illuminated band; Determine the chromaticity difference between each contour feature and the reference ring and the area of ​​the contour feature; If the profile feature meets the reflection excitation condition, the profile feature is determined as a reflection excitation feature; The reference ring is a strip-shaped area surrounding the contour feature, and the reflection excitation condition is that the chromaticity difference is greater than a predetermined chromaticity difference threshold and the area is greater than a predetermined area threshold.

8. The method for detecting growth differentiation factor 15 according to claim 1, characterized in that: The process of determining the sampling position of the optical read head based on the reflection excitation characteristics in different optical illumination bands includes: Determine the optical reflection bands corresponding to different positions of the optical pickup head; determining each reflection excitation feature in each optical reflection band; Determine the absolute variance of the chromaticity difference and the absolute variance of the area corresponding to each optical reflection band and perform weighted summation to obtain a reflection excitation characteristic value; An optical reflection band having the smallest reflection excitation characteristic value is determined, and a position of the optical reading head corresponding to the optical reflection band is determined as the sampling position.

9. The method for detecting growth differentiation factor 15 according to claim 1, characterized in that: Compare the side images of the reagent to be tested in adjacent frames to determine whether to control the optical reader to maintain the current detection height sampling, including: Determining whether there is a marked optical irradiation band in the adjacent frame side image of the reagent to be tested; If there is no marked optical irradiation zone in the adjacent frame side images of the reagent to be tested, it is determined that the optical reader maintains the current detection height sampling.

10. The method for detecting growth differentiation factor 15 according to claim 1, characterized in that: The amplification cycle process includes pre-denaturation, denaturation, annealing and extension.

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