CN118067682B - Method for detecting growth differentiation factor 15 gene expression - Google Patents
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Abstract
The invention relates to the field of fluorescence detection, in particular to a method for detecting the expression of a growth differentiation factor 15 gene, which comprises the steps of setting an independent image acquisition unit on an optical read head of a PCR instrument to acquire liquid level images of all reagents, calculating scattering influence coefficients to divide the optical scattering influence types of single reagents, and then adjusting the sampling mode of an optical read head according to the optical scattering influence types of the reagents in a targeted manner.
Description
Technical Field
Relates to the field of fluorescence detection, in particular to a method for detecting the expression of a growth differentiation factor 15 gene.
Background
Growth differentiation factor 15 (Growth Differentiation Factor, GDF 15), a cytokine protein also known as bone morphogenic protein 1 (Bone Morphogenetic Protein, BMP-1) or cardiac arrest protein (Heart Inhibitor Protein), is an important factor that regulates cell growth, proliferation, differentiation and apoptosis, and thus, it is important to determine the gene expression concentration of growth differentiation factor 15 in many physiological and pathological processes, and in the prior art, the gene expression concentration of growth differentiation factor 15 is usually determined by means of real-time fluorescent quantitative PCR by related instruments;
for example, chinese patent publication No.: CN115216401a discloses a real-time fluorescence quantitative PCR appearance, and it includes frame, detection mechanism, heat lid, rack and incubation mechanism, the rack is in the frame, set up a plurality of test tube holes that are used for placing the test tube on the rack, the heat lid slides and sets up in the frame and towards the rack is close to, the heat is covered and has been seted up a plurality of shielding grooves that are parallel to each other, the detection hole that is used for the light source to pass has been seted up on shielding groove's the diapire, the detection hole with test tube in the test tube hole corresponds, incubation mechanism sets up the rack is kept away from one side of heat lid, incubation mechanism is right test tube on the rack heats. According to the application, the interference of the reflected light rays between the adjacent detection holes on the detected reagent can be reduced through the shielding groove, so that the accuracy of the detection result of the reagent is higher.
Such a device can be used for determining the gene expression concentration of the growth differentiation factor 15, but there are problems in the related art in that,
When the reagent in the test tube is detected by the optical reading head, the optical loop has certain difference due to the difference of the liquid level depth of the reagent, so that the detection result is influenced, and the solution may have certain precipitation or slight turbidity to influence the light path propagation and the accuracy of the data sampled by the optical reading head due to the higher concentration of the PCR reaction product in the stable stage in the amplification cycle process, so that the influence of the factors on the experimental result is not considered.
Disclosure of Invention
Therefore, the invention provides a method for detecting the expression of the growth differentiation factor 15 gene, which is used for solving the problems that in the prior art, the concentration of a PCR reaction product in a stable stage is higher in the amplification cycle process, a certain precipitation or slight turbidity possibly exists in a solution, the propagation of an optical path is influenced, the accuracy of data sampled by an optical reading head is influenced, and the experimental data is inaccurate.
To achieve the above object, the present invention provides a method for detecting the expression of a growth differentiation factor 15 gene, comprising:
step S1, preparing a reagent containing a gene primer, adding the reagent into a pore plate, and putting the pore plate into a precooled high-speed centrifuge for centrifugation, wherein the gene primer is GDF-15;
S2, carrying out amplification circulation on the centrifuged reagent through a PCR instrument, and arranging an independent image acquisition unit on an optical reading head of the PCR instrument to acquire liquid level images of the reagents;
S3, acquiring liquid level depth and turbidity according to the liquid level image, and calculating a scattering influence coefficient to divide the optical scattering influence category of the single reagent;
Step S4, selecting an optical sampling mode of the optical reading head for a single reagent according to the optical scattering influence category of the reagent, including,
Adjusting the sampling angle of the optical reading head to continuously change the position of a sampling datum point on the liquid level of the reagent, acquiring fluorescence intensities acquired by different sampling angles in real time, judging whether the optical sampling of a single reagent is abnormal or not based on a fluorescence intensity discrete difference value, and selecting an available sampling angle for the reagent, wherein the sampling datum point is a virtual intersection point of an irradiation vector of the optical reading head and the liquid level of the reagent;
Or, maintaining an initial sampling angle of the optical readhead;
and S5, constructing an amplification cycle curve according to the data obtained by optical sampling to calculate the relative expression quantity of the GDF-15.
Further, in the step S3, a scattering influence coefficient is calculated according to the formula (1),
In the formula (1), G represents a scattering influence coefficient, H represents a liquid level depth, H0 represents a liquid level depth threshold, L represents a turbidity, L0 represents a preset turbidity standard threshold, α represents a liquid level depth weight coefficient, and β represents a turbidity weight coefficient.
Further, in the step S3, the process of classifying the optical scattering influence type of the single agent includes,
Comparing the scattering influence coefficient of the single reagent with a preset scattering influence coefficient comparison threshold value,
If the scattering influence coefficient is greater than or equal to the scattering influence coefficient contrast threshold, judging that the reagent is in a strong optical scattering influence type;
And if the scattering influence coefficient is smaller than the scattering influence coefficient contrast threshold value, judging that the reagent is in a weak optical scattering influence type.
Further, the selecting the optical sampling method in the step S4 includes,
If the reagent is in a category of strong optical scattering influence, selecting whether the optical sampling aiming at a single reagent is abnormal or not, and selecting an available sampling angle aiming at the reagent;
If the reagent is of the weak optical scattering influence type, then the initial sampling angle of the optical read head is selected to be maintained.
Further, in the step S4, the process of adjusting the sampling angle of the optical pickup includes,
Adjusting the optical reading head to be vertical to the reagent liquid level, wherein the sampling datum point is positioned at the center of the reagent liquid level;
adjusting the sampling angle of the optical reading head so that the sampling reference points are respectively positioned on the preset offset points;
The offset points are positioned on a virtual circle with the liquid level center and the liquid level center as a reference and a preset radius, and the distances between adjacent offset points on the virtual circle are equal.
Further, in the step S4, the process of determining whether the optical sampling is abnormal includes,
Comparing the fluorescence intensity discrete difference value with a preset standard fluorescence intensity discrete difference threshold value,
And if the fluorescence intensity discrete difference value is larger than the standard fluorescence intensity discrete difference threshold value, judging that the optical sampling is abnormal.
Further, in the step S4, the process of calculating the discrete difference value of the fluorescence intensity includes,
In the process of adjusting the sampling angle of the optical reading head, recording the fluorescence intensity acquired by the optical reading head when the sampling reference point is at the offset point, calculating the fluorescence intensity discrete difference value according to the formula (2),
In the formula (2), C represents a fluorescence intensity discrete difference value, pi represents a fluorescence intensity sampled by the optical pickup when the sampling reference point is at the i-th offset point, Δp represents an average value of fluorescence intensities sampled by the optical pickup when the sampling reference point is at each offset point, n represents the number of offset points, and i is an integer greater than 0.
Further, in said step S4, selecting an available sampling angle for said reagent comprises,
In the step S4, selecting an available sampling angle for the reagent includes,
Comparing the fluorescence intensities acquired by the optical reading head when the sampling datum points are at each offset point so as to solve the fluorescence intensity difference corresponding to each offset point;
if the fluorescence intensity difference corresponding to the offset point is smaller than the preset fluorescence intensity difference threshold, judging that the sampling angle corresponding to the offset point is available;
the fluorescence intensity difference is the average difference value of the fluorescence intensity corresponding to the single offset point and the fluorescence intensity corresponding to the rest offset points.
Further, in the step S1, the centrifugation temperature is 4 ℃, the centrifugation rotation speed is 3000rpm, and the centrifugation is maintained for 5min.
Further, in the step S2, the amplification cycle includes,
Pre-denaturation for 2min15s at 95 ℃;
Denaturation for 15s at 95 ℃;
annealing for 30s at 60 ℃;
extending for 30s at 72 ℃.
Compared with the prior art, the invention sets an independent image acquisition unit on the optical read head of the PCR instrument to acquire liquid level images of all reagents, calculates scattering influence coefficients to divide optical scattering influence categories of single reagents, and then adjusts the sampling mode of the optical read head according to the optical scattering influence categories of the reagents in a targeted way.
In particular, the invention obtains the liquid level depth and turbidity through the liquid level image of the reagent, calculates the scattering influence coefficient, and then divides the optical scattering influence category of the reagent, in the actual situation, the test tube containing the sample has a certain depth, the optical path length from the optical reading head to the liquid level of the sample is short, the optical path difference exists, and along with the increase of the cycle times of the amplification cycle, the situation that partial precipitation or slight turbidity of the reagent exists after the increase of the product possibly exists, and certain scattering exists, and when the optical path length is longer, the phenomenon is aggravated, and the reality of the fluorescence intensity sampled by the optical reading head is influenced, therefore, the invention takes the phenomenon into consideration for characterization, calculates the scattering influence coefficient through the liquid level depth, provides data support for the subsequent adaptive adjustment of the optical sampling mode, further verifies the reliability of the optical sampling, provides support for the data choice, and further improves the accuracy and reliability of the experiment.
In particular, the invention considers the adjustment of the sampling angle of the optical reading head, verifies the discrete difference value of the fluorescence intensity of different offset points in the adjustment process, judges whether the optical sampling is abnormal or not, in the actual situation, if the reagent is in the category of strong optical scattering influence, the reliability of the fluorescence intensity sampled by the optical reading head is easy to influence, especially, under the influence of the condition that the scattering phenomenon is strong and the superimposed optical path is long, in the sampling area above the test tube, the data sampled by different sampling angles may have differences in different sampling areas, and the sampling reliability of the optical reading head is doubtful, therefore, the offset points surrounding the center of the reagent liquid level are considered to be arranged, the sampling reference point is positioned on the offset points by adjusting the angle of the optical reading head, so that the optical reading head verifies the fluorescence intensity sampled by the optical reading head after the offset of the original sampling angle, thereby providing support for data choosing and further improving the accuracy and reliability of the experiment.
In particular, under the condition that the reagent is in the weak optical scattering influence category, the influence is small, the initial sampling angle is maintained, and the experimental efficiency is ensured.
Drawings
FIG. 1 is a method for detecting expression of growth differentiation factor 15 gene according to an embodiment of the invention;
FIG. 2 is a logical block diagram of the classification of optical scattering effects of individual agents according to an embodiment of the invention;
FIG. 3 is a logical block diagram of a selected optical read head of an embodiment of the invention for a single reagent optical sampling mode;
FIG. 4 is a logic diagram of an embodiment of the invention to determine whether an anomaly exists for a single optical sample of a reagent.
Detailed Description
In order that the objects and advantages of the invention will become more apparent, the invention will be further described with reference to the following examples; it should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
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 merely for explaining the technical principles of the present invention, and are not intended to limit the scope of the present invention.
It should be noted that, in the description of the present invention, terms such as "upper," "lower," "left," "right," "inner," "outer," and the like indicate directions or positional relationships based on the directions or positional relationships shown in the drawings, which are merely for convenience of description, and do not indicate or imply that the apparatus or elements must have a specific orientation, be constructed and operated in a specific orientation, and thus should not be construed as limiting the present invention.
Referring to fig. 1 to 4, fig. 1 is a logic diagram of a method for detecting the expression of the growth differentiation factor 15 gene according to an embodiment of the invention, fig. 2 is a logic diagram of a method for classifying the optical scattering effect of a single reagent according to an embodiment of the invention, fig. 3 is a logic diagram of a mode of optical sampling of a selected optical read head according to an embodiment of the invention for a single reagent, fig. 4 is a logic diagram of an embodiment of the invention for determining whether an abnormality exists for optical sampling of a single reagent, a method for detecting the expression of the growth differentiation factor 15 gene according to the invention includes:
step S1, preparing a reagent containing a gene primer, adding the reagent into a pore plate, and putting the pore plate into a precooled high-speed centrifuge for centrifugation, wherein the gene primer is GDF-15;
S2, carrying out amplification circulation on the centrifuged reagent through a PCR instrument, and arranging an independent image acquisition unit on an optical reading head of the PCR instrument to acquire liquid level images of the reagents;
S3, acquiring liquid level depth and turbidity according to the liquid level image, and calculating a scattering influence coefficient to divide the optical scattering influence category of the single reagent;
Step S4, selecting an optical sampling mode of the optical reading head for a single reagent according to the optical scattering influence category of the reagent, including,
Adjusting the sampling angle of the optical reading head to continuously change the position of a sampling datum point on the liquid level of the reagent, acquiring fluorescence intensities acquired by different sampling angles in real time, judging whether the optical sampling of a single reagent is abnormal or not based on a fluorescence intensity discrete difference value, and selecting an available sampling angle for the reagent, wherein the sampling datum point is a virtual intersection point of an irradiation vector of the optical reading head and the liquid level of the reagent;
Or, maintaining an initial sampling angle of the optical readhead;
and S5, constructing an amplification cycle curve according to the data obtained by optical sampling to calculate the relative expression quantity of the GDF-15.
Specifically, the invention does not limit the specific structure of the PCR instrument, the optical reading head of the PCR instrument is a core component, the detection position and the sampling angle can be adjusted to detect the intensity of the fluorescent signal, and the invention is the prior art and is not repeated.
Specifically, the specific structure of the image acquisition unit is not limited, and the image acquisition unit can be an industrial camera capable of shooting a depth image and a two-dimensional image, so that the depth of a liquid level can be obtained through the depth image, and the two-dimensional image is subjected to image analysis, which is not repeated.
Specifically, the invention is not limited to a specific manner of acquiring turbidity from a liquid level image,
In this embodiment, the average gray value can be obtained by converting the liquid level image into the gray image, and the turbidity is represented by the average gray value, which is not described herein.
In particular, for the relevant control adjustment logic of the optical read head, it is possible to introduce into the logic unit the corresponding hardware implementation by means of which the logic unit comprises a field programmable processor, a computer or a microprocessor in a computer.
Specifically, in step S1, optionally, when the reagent is disposed,
4 Compound holes are arranged on each sample, the whole procedure is operated on an ice plate, the front and the back are evenly mixed, and the total amount of reagents is as follows:
sample 9.2 μl (per well) =cdna: 0.4 μl+ddh20:8.8 μl;
Primer 10.8 μl (per well) =qpcr Mix10 μl+front primer 0.4 μl+rear primer 0.4 μl.
Specifically, the process of synthesizing cDNA by reverse transcription includes,
Adding 2 mu l of DNase into a 200 mu lEP tube without enzyme, adding pure water to 16 mu l, gently beating by a pipette for 5-10 times, and reacting for 5min at room temperature;
Adding 4 μl of 5× RTmix into each tube, gently beating for 10 times, mixing, reacting at 42deg.C for 15min, and freezing in-80deg.C.
Specifically, in the step S3, the scattering influence coefficient is calculated according to the formula (1),
In the formula (1), G represents a scattering influence coefficient, H represents a liquid level depth, H0 represents a liquid level depth threshold, L represents a turbidity, L0 represents a preset turbidity standard threshold, α represents a liquid level depth weight coefficient, and β represents a turbidity weight coefficient.
Specifically, the turbidity standard threshold is obtained by a preliminary measurement, wherein a plurality of identical reagents are used for an amplification cycle by a PCR instrument, liquid level images of the reagents are acquired, an average turbidity Δe of the liquid level images is acquired, and l0=Δe×g is set, wherein g represents a shift coefficient, and 0.85 < g < 0.9.
Specifically, the liquid level is the distance of the liquid level from the nozzle of the test tube, the liquid level depth threshold is in the interval [0.2LE,0.3LE ], LE represents the total length of the test tube.
In this example, α is 0.35 and β is 0.65.
According to the invention, the liquid level depth and the turbidity are obtained through the liquid level image of the reagent, the scattering influence coefficient is calculated, the optical scattering influence type of the reagent is further divided, in the practical situation, the test tube containing the sample has a certain depth, the optical path length from the optical reading head to the liquid level of the sample is short, the optical path difference exists, partial precipitation or slight turbidity of the reagent possibly exists after the product is increased along with the increase of the cycle times of amplification cycle, and certain scattering exists, and the phenomenon is aggravated when the optical path length is longer, so that the reality of the fluorescence intensity sampled by the optical reading head is influenced.
In particular, in said step S3, the process of classifying the optical scattering effect categories of the individual agents comprises,
Comparing the scattering influence coefficient of the single reagent with a preset scattering influence coefficient comparison threshold value,
If the scattering influence coefficient is greater than or equal to the scattering influence coefficient contrast threshold, judging that the reagent is in a strong optical scattering influence type;
And if the scattering influence coefficient is smaller than the scattering influence coefficient contrast threshold value, judging that the reagent is in a weak optical scattering influence type.
Specifically, the scattering effect coefficient contrast threshold is selected within the interval [1.15,1.3 ].
Specifically, the process of selecting the optical sampling mode in the step S4 includes,
If the reagent is in a category of strong optical scattering influence, selecting whether the optical sampling aiming at a single reagent is abnormal or not, and selecting an available sampling angle aiming at the reagent;
If the reagent is of the weak optical scattering influence type, then the initial sampling angle of the optical read head is selected to be maintained.
Specifically, in this embodiment, it is preferable that the initial sampling angle is perpendicular to the reagent level, and that the sampling reference point is located at the center of the reagent level.
Specifically, in the step S4, the process of adjusting the sampling angle of the optical pickup includes,
Adjusting the optical reading head to be vertical to the reagent liquid level, wherein the sampling datum point is positioned at the center of the reagent liquid level;
adjusting the sampling angle of the optical reading head so that the sampling reference points are respectively positioned on the preset offset points;
The offset points are positioned on a virtual circle with the liquid level center and the liquid level center as a reference and a preset radius, and the distances between adjacent offset points on the virtual circle are equal.
Preferably, the number of offset points is not excessive, and in this embodiment, it is recommended that the number of offset points on the virtual circle be set to 8.
The radius of the virtual circle should not be too large, which is at most 2/3 of the radius of the test tube.
Specifically, the sampling reference point is the virtual intersection point of the irradiation vector of the optical pickup and the reagent liquid surface, and the irradiation vector is the virtual vector passing through the center point of the plane of the optical pickup mirror surface and having the direction perpendicular to the plane of the optical pickup mirror surface.
In particular, in the step S4, the process of determining whether the optical sampling is abnormal includes,
Comparing the fluorescence intensity discrete difference value with a preset standard fluorescence intensity discrete difference threshold value,
And if the fluorescence intensity discrete difference value is larger than the standard fluorescence intensity discrete difference threshold value, judging that the optical sampling is abnormal.
Specifically, the standard fluorescence intensity discrete difference threshold value C0 is obtained by a preliminary measurement, wherein a plurality of reagents are subjected to an amplification cycle in advance, the reagents belonging to the category of strong optical scattering influence are screened, the sampling angle of the optical reading head is adjusted, the adjustment method is consistent with step S4, the average value Δc of the fluorescence intensity discrete difference value is calculated, c0=r×Δc, r represents the precision coefficient, and 0.75 < r < 0.85.
Specifically, in the step S4, the process of calculating the discrete difference value of the fluorescence intensity includes,
In the process of adjusting the sampling angle of the optical reading head, recording the fluorescence intensity acquired by the optical reading head when the sampling reference point is at the offset point, calculating the fluorescence intensity discrete difference value according to the formula (2),
In the formula (2), C represents a fluorescence intensity discrete difference value, pi represents a fluorescence intensity sampled by the optical pickup when the sampling reference point is at the i-th offset point, Δp represents an average value of fluorescence intensities sampled by the optical pickup when the sampling reference point is at each offset point, n represents the number of offset points, and i is an integer greater than 0.
In particular, in said step S4, selecting an available sampling angle for said reagent comprises,
In the step S4, selecting an available sampling angle for the reagent includes,
Comparing the fluorescence intensities acquired by the optical reading head when the sampling datum points are at each offset point so as to solve the fluorescence intensity difference corresponding to each offset point;
if the fluorescence intensity difference corresponding to the offset point is smaller than the preset fluorescence intensity difference threshold, judging that the sampling angle corresponding to the offset point is available;
the fluorescence intensity difference is the average difference value of the fluorescence intensity corresponding to the single offset point and the fluorescence intensity corresponding to the rest offset points.
Similarly, after determining that the optical sampling is abnormal and determining the available sampling angle, it can be verified whether the sampling angle initially set is the available sampling angle, if not, the sampled data obtained by the optical sampling can be marked, and further, data support is provided when data analysis is applied subsequently.
In the invention, the sampling angle of the optical reading head is considered to be adjusted, the discrete difference values of the fluorescence intensities of different offset points in the adjustment process are verified, whether the optical sampling is abnormal or not is judged, in the actual situation, if the reagent is in the category of strong optical scattering influence, the reliability of the fluorescence intensity sampled by the optical reading head is easy to influence, especially, under the influence of the condition that the scattering phenomenon is strong and the superimposed optical path is long, in a sampling area above a test tube, different sampling subareas can exist in the data sampled by different sampling angles, so that the sampling reliability of the optical reading head is doubtful, therefore, a plurality of offset points encircling the center of the reagent liquid level are considered to be arranged, the sampling reference point is positioned on the offset points by adjusting the angle of the optical reading head, so that the optical reading head verifies the fluorescence intensity sampled by the optical reading head after the original sampling angle is verified, the reliability of the optical reading head is verified, and support is provided for data choosing and further, the accuracy and the reliability of experiments are improved.
In the step S1, the centrifugation temperature is 4 ℃, the centrifugation rotating speed is 3000rpm, and the centrifugation is maintained for 5min.
Specifically, in the step S2, the amplification cycle includes,
Pre-denaturation for 2min15s at 95 ℃;
Denaturation for 15s at 95 ℃;
annealing for 30s at 60 ℃;
extending for 30s at 72 ℃.
Specifically, in the step S5, the relative expression level of GDF-15 is calculated by the 2-DeltaCt method.
The 2-delta Ct method is a simple method for analyzing the relative change and the relative expression amount of the gene in a real-time quantitative PCR experiment, and is the prior art and is not repeated.
Thus far, the technical solution of the present invention has been described in connection with the preferred embodiments shown in the drawings, but it is easily understood by those skilled in the art that the scope of protection of the present invention is not limited to these specific embodiments. Equivalent modifications and substitutions for related technical features may be made by those skilled in the art without departing from the principles of the present invention, and such modifications and substitutions will be within the scope of the present invention.
Claims (9)
1. A method for detecting expression of a growth differentiation factor 15 gene, comprising:
step S1, preparing a reagent containing a gene primer, adding the reagent into a pore plate, and putting the pore plate into a precooled high-speed centrifuge for centrifugation, wherein the gene primer is GDF-15;
S2, carrying out amplification circulation on the centrifuged reagent through a PCR instrument, and arranging an independent image acquisition unit on an optical reading head of the PCR instrument to acquire liquid level images of the reagents;
S3, acquiring liquid level depth and turbidity according to the liquid level image, and calculating a scattering influence coefficient to divide the optical scattering influence category of the single reagent;
Step S4, selecting an optical sampling mode of the optical reading head for a single reagent according to the optical scattering influence category of the reagent, including,
If the reagent is of a strong optical scattering influence type, selecting and adjusting a sampling angle of an optical reading head to continuously change the position of a sampling datum point on the liquid surface of the reagent, acquiring fluorescence intensities acquired by different sampling angles in real time, judging whether optical sampling for a single reagent is abnormal or not based on a fluorescence intensity discrete difference value, and selecting an available sampling angle for the reagent, wherein the sampling datum point is a virtual intersection point of an irradiation vector of the optical reading head and the liquid surface of the reagent;
if the reagent is of a weak optical scattering influence type, selecting an initial sampling angle of the optical reading head to be maintained;
and S5, constructing an amplification cycle curve according to the data obtained by optical sampling to calculate the relative expression quantity of the GDF-15.
2. The method for detecting gene expression of growth differentiation factor 15 according to claim 1, wherein in the step S3, the scattering influence coefficient is calculated according to the formula (1),
；
In the formula (1), G represents a scattering influence coefficient, H represents a liquid level depth, H0 represents a liquid level depth threshold, L represents a turbidity, L0 represents a preset turbidity standard threshold, α represents a liquid level depth weight coefficient, and β represents a turbidity weight coefficient.
3. The method for detecting gene expression of growth differentiation factor 15 according to claim 1, wherein the step S3 of classifying the optical scattering effect type of the individual agents comprises,
Comparing the scattering influence coefficient of the single reagent with a preset scattering influence coefficient comparison threshold value,
If the scattering influence coefficient is greater than or equal to the scattering influence coefficient contrast threshold, judging that the reagent is in a strong optical scattering influence type;
And if the scattering influence coefficient is smaller than the scattering influence coefficient contrast threshold value, judging that the reagent is in a weak optical scattering influence type.
4. The method for detecting gene expression of growth differentiation factor 15 according to claim 1, wherein the step S4 of adjusting the sampling angle of the optical pick-up comprises,
Adjusting the sampling angle of the optical reading head so that the sampling reference points are respectively positioned on the preset offset points;
The offset points are positioned on a virtual circle with the liquid level center and the liquid level center as a reference and a preset radius, and the distances between adjacent offset points on the virtual circle are equal.
5. The method for detecting gene expression of growth differentiation factor 15 according to claim 1, wherein the step S4 of determining whether the optical sampling is abnormal comprises,
Comparing the fluorescence intensity discrete difference value with a preset standard fluorescence intensity discrete difference threshold value,
And if the fluorescence intensity discrete difference value is larger than the standard fluorescence intensity discrete difference threshold value, judging that the optical sampling is abnormal.
6. The method for detecting gene expression of growth differentiation factor 15 according to claim 5, wherein in step S4, the process of calculating the discrete difference value of fluorescence intensity comprises,
In the process of adjusting the sampling angle of the optical reading head, recording the fluorescence intensity acquired by the optical reading head when the sampling reference point is at the offset point, calculating the fluorescence intensity discrete difference value according to the formula (2),
；
In the formula (2), C represents a fluorescence intensity discrete difference value, pi represents a fluorescence intensity sampled by the optical pickup when the sampling reference point is at the i-th offset point, Δp represents an average value of fluorescence intensities sampled by the optical pickup when the sampling reference point is at each offset point, n represents the number of offset points, and i is an integer greater than 0.
7. The method for detecting gene expression of growth differentiation factor 15 according to claim 1, wherein in step S4, selecting an available sampling angle for the reagent comprises,
Comparing the fluorescence intensities acquired by the optical reading head when the sampling datum points are at each offset point so as to solve the fluorescence intensity difference corresponding to each offset point;
if the fluorescence intensity difference corresponding to the offset point is smaller than the preset fluorescence intensity difference threshold, judging that the sampling angle corresponding to the offset point is available;
the fluorescence intensity difference is the average difference value of the fluorescence intensity corresponding to the single offset point and the fluorescence intensity corresponding to the rest offset points.
8. The method for detecting gene expression of growth differentiation factor 15 according to claim 1, wherein in the step S1, the centrifugation temperature is 4℃and the centrifugation speed is 3000rpm, and the centrifugation is maintained for 5 minutes.
9. The method for detecting gene expression of growth differentiation factor 15 according to claim 1, wherein in the step S2, the amplification cycle comprises,
Pre-denaturation for 2min15s at 95 ℃;
Denaturation for 15s at 95 ℃;
annealing for 30s at 60 ℃;
extending for 30s at 72 ℃.
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