CN114414542B

CN114414542B - Fluorescent signal detection method, device, medium and equipment for gene detection

Info

Publication number: CN114414542B
Application number: CN202111565858.9A
Authority: CN
Inventors: 韦祖健; 贾洵; 李�杰
Original assignee: Chengdu Hanchen Guangyi Bioengineering Co ltd; Chengdu Hanchen Guangyi Technology Co ltd
Current assignee: Chengdu Hanchen Guangyi Bioengineering Co ltd; Chengdu Hanchen Guangyi Technology Co ltd
Priority date: 2021-12-20
Filing date: 2021-12-20
Publication date: 2024-01-12
Anticipated expiration: 2041-12-20
Also published as: CN114414542A

Abstract

The invention discloses a fluorescent signal detection method, a fluorescent signal detection device, a fluorescent signal detection medium and fluorescent signal detection equipment for gene detection, and belongs to the technical field of gene detection. The method comprises the following steps: according to at least two preset fluorescent dyes, acquiring a first fluorescent signal of each fluorescent dye in the at least two fluorescent dyes under each fluorescent detection channel corresponding to the fluorescent dye respectively, and determining fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel respectively; obtaining a measured fluorescent signal of a sample to be measured under each fluorescent channel corresponding to each fluorescent dye respectively, wherein the sample to be measured contains at least two fluorescent dyes; and determining a target fluorescent signal corresponding to each fluorescent dye in the sample to be detected according to the measured fluorescent signal and fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel. According to the invention, the target fluorescent signal of the gene sample to be detected is determined through the fluorescent signal parameters of the fluorescent dye, so that the detection accuracy of the sample gene can be effectively improved.

Description

Fluorescent signal detection method, device, medium and equipment for gene detection

Technical Field

The invention relates to the technical field of gene detection, in particular to a fluorescent signal detection method, a fluorescent signal detection device, a fluorescent signal detection medium and fluorescent signal detection equipment for gene detection.

Background

In the gene detection, after a sample is amplified by PCR (Polymerase Chain Reaction ), the number of specific gene fragments is exponentially increased, at this time, a fluorescent signal in a reaction hole can be detected by some optical detection means, and whether the sample is a positive sample or not can be judged according to the intensity of the fluorescent signal, and even the concentration of the nucleic acid of the sample can be calculated.

The genotype of the sample may have three AA, AA and AA, where AA and AA are referred to as homozygotes and AA is referred to as heterozygote. Since the positive samples are all Aa heterozygotes, the optical detection is aimed at detecting a and a therein, thereby detecting Aa heterozygotes. To distinguish between a and a, two different fluorochromes are typically required for their separate detection in an optical assay, whereas different fluorochromes require channels of different wavelengths for detection. By channel is meant herein a filter system capable of allowing light of a specific wavelength band to pass. In optical detection, the sample needs to be irradiated by using excitation light with a specific wave band, and the reflected light is filtered by using a filter with the specific wave band, so that a fluorescent signal can be received. The excitation light bands of the five dyes FAM, HEX, t.r, cy5, Q705, the filter band of the detection filter (cylindrical color block) and the normalized light absorbance profile are shown in fig. 1.

As can be seen from fig. 1, in the lower detection wavelength chart, although several channels have different band distributions, the band with the best signal is selected for the channels as much as possible, but there is still a certain band overlap between them. That is, after passing through the filter of the FAM channel, the obtained fluorescence signal is not all FAM band signal, but is also mixed with signals overlapped by other bands according to the overlapping degree of the bands. That is, in the optical detection, the a-related signal and the a-related signal which can be directly collected are actually doped with each other, and in the case of a low sample concentration, the interference between the channels can seriously affect the judgment of the genotype, so that the detection result cannot be accurately given.

Disclosure of Invention

The embodiment of the invention provides a fluorescence signal detection method, a device, a medium and equipment for gene detection, which are used for solving the problem that the detected fluorescence signals are mutually doped and the genotype of a sample cannot be accurately judged.

In one aspect, the present invention provides a fluorescent signal detection method for gene detection, the method comprising:

acquiring a first fluorescent signal of each fluorescent dye in at least two fluorescent dyes under each fluorescent detection channel corresponding to the fluorescent dye respectively according to at least two preset fluorescent dyes, and determining fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel respectively;

Obtaining a measured fluorescent signal of a sample to be measured under each fluorescent channel corresponding to each fluorescent dye, wherein the sample to be measured contains at least two fluorescent dyes;

and determining a target fluorescent signal corresponding to each fluorescent dye in the sample to be detected according to the measured fluorescent signal and fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel.

In one possible implementation manner, the obtaining, according to at least two preset fluorescent dyes, a first fluorescent signal of each fluorescent dye of the at least two fluorescent dyes under each fluorescent detection channel corresponding to the fluorescent dye respectively includes:

acquiring a first initial fluorescence signal of each fluorescent dye in at least two fluorescent dyes under each fluorescence detection channel corresponding to the fluorescent dye respectively according to at least two preset fluorescent dyes;

acquiring a second fluorescent signal of a buffer solution with the same volume as the fluorescent dye under each fluorescent detection channel corresponding to the fluorescent dye respectively;

and respectively separating and removing the corresponding second fluorescent signals from the first initial fluorescent signals to obtain first fluorescent signals of each fluorescent dye under each corresponding fluorescent detection channel.

In one possible implementation, the determining a fluorescent signal parameter of each of the fluorescent dyes under each of the fluorescent detection channels, respectively, includes:

and determining a fluorescence signal intensity coefficient corresponding to each fluorescent dye under each fluorescent detection channel according to the first fluorescent signal of each fluorescent dye under each fluorescent detection channel.

In one possible implementation manner, the determining, according to the measured fluorescent signal and the fluorescent signal parameter of each fluorescent dye under each fluorescent detection channel, the target fluorescent signal corresponding to each fluorescent dye in the sample to be measured specifically includes:

determining a target fluorescent signal corresponding to each fluorescent dye in the sample to be detected according to the measured fluorescent signals and fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel respectively through the following equation set,

Signal(A)＝a _A ×A _A +b _A ×B _B +…+n _A ×N _N

Signal(B)＝a _B ×A _A +b _B ×B _B +…+n _B ×N _N

…

Signal(N)＝a _N ×A _A +b _N ×B _B +…+n _N ×N _N

wherein Signal represents a measured fluorescence Signal of the sample to be detected under a fluorescence detection channel corresponding to one fluorescent dye, { a, B, … N } represents the type of fluorescent dye, { A, B, … N } represents a fluorescence detection channel corresponding to each of the fluorescent dyes { a, B, … N }, A _A Representing a target fluorescent signal of the fluorescent dye a in the sample to be detected under the fluorescent detection channel A, B _B Representing a target fluorescent signal of the fluorescent dye B in the sample to be detected under the fluorescent detection channel B, N _N Representing a target fluorescent signal, a, of the fluorescent dye N in the sample to be detected under the fluorescent detection channel N _A ,b _A ,n _A Respectively represent the fluorescence signal intensity coefficients of the fluorescent dye a, the fluorescent dye b and the fluorescent dye n under the fluorescent detection channel A, a _B ,b _B ,n _B Respectively represent the fluorescence signal intensity coefficients of the fluorescent dye a, the fluorescent dye B and the fluorescent dye n under the fluorescent detection channel B, a _N ,b _N ,n _N Respectively represent the fluorescence signal intensity coefficients of the fluorescent dye a, the fluorescent dye b and the fluorescent dye N under the fluorescence detection channel N.

In one possible implementation manner, after determining the target fluorescent signal corresponding to each fluorescent dye in the sample to be tested according to the measured fluorescent signal and the fluorescent signal parameter of each fluorescent dye under each fluorescent detection channel, the method further includes:

acquiring a second fluorescent signal of an equal-concentration mixture of at least two fluorescent dyes under each fluorescent detection channel corresponding to the fluorescent dyes respectively according to the preset at least two fluorescent dyes;

Determining a reference fluorescent signal corresponding to each fluorescent dye in the fluorescent dye mixture according to the second fluorescent signal and fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel respectively;

and calibrating a target fluorescent signal corresponding to each fluorescent dye in the sample to be tested according to the reference fluorescent signal.

In one possible implementation manner, the calibrating the target fluorescent signal corresponding to each fluorescent dye in the sample to be measured according to the reference fluorescent signal specifically includes:

and determining the ratio of the target fluorescent signal corresponding to each fluorescent dye in the sample to be detected to the reference fluorescent signal corresponding to the fluorescent dye, and obtaining a calibrated target fluorescent signal.

when the sample to be measured contains the reference fluorescent dye, target fluorescent signals corresponding to other fluorescent dyes in the sample to be measured are processed through normalization according to the target fluorescent signals corresponding to the reference fluorescent dye.

In one aspect, the present invention provides a fluorescent signal detection device for gene detection, the device comprising:

the signal acquisition module is used for acquiring a first fluorescent signal of each fluorescent dye in the at least two fluorescent dyes under each fluorescent detection channel corresponding to the fluorescent dye respectively according to at least two preset fluorescent dyes, and determining fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel respectively;

the parameter determining module is used for determining fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel respectively;

the signal acquisition module is further used for acquiring a measured fluorescent signal of a sample to be measured under each fluorescent channel corresponding to each fluorescent dye respectively, wherein the sample to be measured contains at least two fluorescent dyes;

and the analysis operation module is used for determining a target fluorescent signal corresponding to each fluorescent dye in the sample to be detected according to the measured fluorescent signal and fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel.

In one aspect, the invention provides a computer readable storage medium having stored therein at least one instruction that is loaded and executed by a processor to implement a fluorescence signal detection method for gene detection as described above.

In one aspect the present invention provides a genetic testing apparatus comprising a processor and a memory having stored therein at least one instruction loaded and executed by the processor to implement a fluorescence signal detection method for genetic testing as described above.

According to the fluorescent signal detection method, the fluorescent signal detection device, the fluorescent signal detection medium and the fluorescent signal detection equipment for gene detection, a first fluorescent signal of each fluorescent dye in at least two fluorescent dyes under each fluorescent detection channel corresponding to the fluorescent dye is obtained according to at least two preset fluorescent dyes, and fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel are determined; obtaining a measured fluorescent signal of a sample to be measured under each fluorescent channel corresponding to each fluorescent dye, wherein the sample to be measured contains at least two fluorescent dyes; and determining a target fluorescent signal corresponding to each fluorescent dye in the sample to be detected according to the measured fluorescent signal and fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel. Therefore, by means of the fluorescent signal parameters of various fluorescent dyes obtained in advance, when the gene sample to be detected is detected, the fluorescent signal detected by actual measurement is calibrated, so that a more accurate fluorescent detection signal is obtained, and the accuracy of a gene detection result is further effectively improved.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph of excitation light bands, filter bands and normalized absorption variation;

FIG. 2 is a gray scale plot of fluorescent signals for a single reaction well in a sample plate;

FIG. 3 is a gray scale plot of fluorescent signals from multiple reaction wells in a sample plate;

FIG. 4 is a schematic diagram of a genotype determination based on fluorescent signals;

FIG. 5 is a flow chart of a method for detecting fluorescent signals according to an embodiment of the present invention;

FIG. 6 is a flow chart of a method for detecting fluorescent signals according to another embodiment of the present invention;

FIG. 7 is a scatter plot of raw fluorescence signals;

FIG. 8 is a scatter plot of fluorescence signals after processing according to the related art;

FIG. 9 is a scatter plot of fluorescence signals processed according to the fluorescence signal detection method;

FIG. 10 is a scatter plot of the fluorescent signal of FIG. 9 normalized;

FIG. 11 is a block diagram of a fluorescent signal detection device according to an embodiment of the present invention;

fig. 12 is a block diagram of a fluorescent signal detection device according to another embodiment of the present invention.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the following detailed description of the embodiments of the present invention will be given with reference to the accompanying drawings.

In this embodiment, the fluorescent signal detection can be performed on the reaction hole containing the sample to be detected and the fluorescent dye in the sample plate before and after the PCR amplification reaction, as shown in fig. 2, the gray value of a center pixel point arbitrarily selected before the PCR amplification is shown in the left drawing, and the gray value is initially 6767; shown in the right panel is the gray value of the center pixel after PCR amplification, which increases to 12352. It is shown that the number of target genes in a sample is increased by a PCR amplification reaction, thereby realizing quantitative detection of the genes in the sample according to fluorescent signals generated by fluorescent dyes for identifying the target genes in the sample.

In practice, the fluorescent signal of only one reaction well shown in FIG. 2 is used to scan a plurality of reaction wells on a sample plate to obtain an example of the scan result shown in FIG. 3, in order to increase the gene detection energy.

According to the principle of genetics, under ideal conditions, after two genotypes of target gene fragments of a sample to be detected are marked by adopting two different fluorescent dyes, the genotypes of the target fragments of the sample to be detected can be judged by the intensity of the two fluorescent signals by respectively acquiring fluorescent signals of the two fluorescent dyes under the corresponding fluorescent detection channels. As shown in the scatter diagram of fig. 4, the vertical axis represents fluorescence signals under the FAM channel, and the detection target is genotype a; the horizontal axis represents the fluorescence signal in HEX channel, and the detection target is genotype a. The signal analysis shows that the 4 samples at the upper right corner have stronger signals under two channels, and are Aa-type, namely the samples are positive; the lower left 4 samples were negative with no significant signal in both channels. Similarly, the two samples in the lower right hand corner are AA type and AA type is not detected. In order to obtain the best quality data as shown in fig. 4, it is necessary to process the fluorescence signal by the fluorescence signal detection method described below.

Example 1

The method for detecting a fluorescent signal for gene detection according to an embodiment of the present invention shown in fig. 5 is a flowchart, and the method for detecting a fluorescent signal can be applied to a gene detection apparatus. The fluorescence signal detection method may include:

Step 501, obtaining a first fluorescent signal of each fluorescent dye in at least two fluorescent dyes under each fluorescent detection channel corresponding to the fluorescent dye respectively according to at least two preset fluorescent dyes, and determining fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel.

In this embodiment, each fluorescent dye corresponds to a specific fluorescent detection channel, and the fluorescent signal of the fluorescent dye can be detected under the fluorescent detection channel corresponding to the fluorescent dye by irradiation of the excitation light source corresponding to the fluorescent dye. According to two or more than two fluorescent dyes involved in detection of a gene sample to be detected and corresponding fluorescent detection channels, a first fluorescent signal of each fluorescent dye under each fluorescent detection channel is obtained. Specifically, a sample plate or a sample tube may be set in advance according to the number of kinds of fluorescent dyes, for example, a corresponding number of sample plates are set for the kinds of fluorescent dyes involved in a sample to be detected, each sample plate is filled with one fluorescent dye, and the concentration and volume of the fluorescent dye contained in each sample plate are the same, the sample plate is irradiated by an excitation light source corresponding to the fluorescent dye in the sample plate, and a fluorescent signal of the sample plate is detected under a fluorescent detection channel corresponding to the fluorescent dye in the sample plate. Further, when a plurality of reaction wells are included in the sample plate, each fluorescent dye is respectively loaded into the plurality of reaction wells in each sample plate, the concentration and volume of the fluorescent dye loaded in each reaction well of the plurality of sample plates are the same, each reaction well of the sample plate is irradiated by an excitation light source corresponding to the fluorescent dye in the sample plate, and a fluorescent signal in each reaction well in the sample plate is detected under a fluorescent detection channel corresponding to the fluorescent dye in the sample plate.

For a sample plate filled with a first fluorescent dye, respectively placing the sample plate under a fluorescent detection channel corresponding to each fluorescent dye in two or more preset fluorescent dyes, irradiating the sample plate through an excitation light source corresponding to the fluorescent detection channel, and receiving a fluorescent signal of the sample plate under the fluorescent detection channel, thereby obtaining the fluorescent signal of the sample plate filled with the first fluorescent dye under each fluorescent detection channel. By analogy, through the above steps, the fluorescence signal of the sample plate with the second fluorescent dye under each fluorescent detection channel, the fluorescence signal of the sample plate with the third fluorescent dye under each fluorescent detection channel, and so on can be further obtained, and finally the fluorescence signal of each sample plate with different fluorescent dyes under each fluorescent detection channel is obtained as the first fluorescence signal. Of course, the fluorescence detection of each sample plate containing different fluorescent dyes under each of the remaining fluorescent detection channels may be sequentially completed by separately detecting each sample plate containing different fluorescent dyes under the first fluorescent detection channel, so that the fluorescence signal of each sample plate containing different fluorescent dyes under each fluorescent detection channel is finally obtained, which is not limited herein, as the first fluorescence signal.

Further, according to the first fluorescence signal, determining a fluorescence signal parameter of each fluorescent dye under each fluorescence detection channel. Specifically, according to the first fluorescent signal, fluorescent signal parameters corresponding to different fluorescent dyes under a certain fluorescent detection channel are determined through normalization, wherein the fluorescent signal parameters comprise fluorescent signal intensity coefficients, and further the fluorescent signal parameters corresponding to different fluorescent dyes under each fluorescent detection channel are determined. Particularly, when a plurality of reaction holes are included in the sample plate, any one reaction hole in the sample plate can be selected, and the fluorescent signal parameters corresponding to different fluorescent dyes in the reaction holes under each fluorescent detection channel are determined and used as the fluorescent signal parameters corresponding to the sample plate for target fluorescent signal detection during sample detection; two or more than two reaction holes in the sample plate can be selected, and the average value of fluorescent signal parameters corresponding to different fluorescent dyes in the reaction holes under each fluorescent detection channel can be determined and used as the fluorescent signal parameter corresponding to the sample plate for target fluorescent signal detection during sample detection; the corresponding fluorescent signal parameters of different fluorescent dyes in each reaction well of the sample plate under each fluorescent detection channel can also be determined for target fluorescent signal detection during sample detection.

Step 502, obtaining a measured fluorescent signal of a sample to be measured under each fluorescent channel corresponding to each fluorescent dye, wherein the sample to be measured contains at least two fluorescent dyes.

In this embodiment, two or more fluorescent dyes are added to the gene sample to be detected before the PCR amplification reaction, and after the PCR amplification reaction is completed, a measured fluorescent signal of the gene sample to be detected under each fluorescent detection channel is obtained. Specifically, a sample plate filled with a gene sample to be detected and two or more fluorescent dyes is firstly placed on a fluorescent detection channel corresponding to a first fluorescent dye, the sample plate is irradiated by an excitation light source corresponding to the first fluorescent dye, and a fluorescent signal is detected and obtained under the fluorescent detection channel corresponding to the fluorescent dye; then, a sample plate filled with a gene sample to be detected and two or more fluorescent dyes is firstly placed on a fluorescent detection channel corresponding to the second fluorescent dye, the sample plate is irradiated by an excitation light source corresponding to the second fluorescent dye, and a fluorescent signal is detected and obtained under the fluorescent detection channel corresponding to the fluorescent dye. And the like, obtaining a measured fluorescent signal of the gene sample to be detected under the fluorescent detection channel corresponding to each fluorescent dye.

Ideally, only the fluorescent signal emitted by the fluorescent dye after being excited can be detected under the fluorescent detection channel corresponding to the fluorescent dye, and the fluorescent signals emitted by other fluorescent dyes cannot be detected. However, in practice, since the excitation light source and the fluorescence detection channel are not absolutely ideal, when two or more kinds of fluorescent dyes are added to the gene sample to be detected, after the gene sample to be detected is irradiated by the excitation light source corresponding to one of the fluorescent dyes, the fluorescent signal detected under the fluorescence detection channel corresponding to the fluorescent dye generally includes both the target fluorescent signal emitted by the fluorescent dye and the interference fluorescent signal emitted by other fluorescent dyes.

It should be noted that, in order to improve the gene detection flux, the sample plate may include a plurality of reaction holes, and each reaction hole is added with a different gene sample to be detected, and correspondingly, the fluorescent light signals measured by each reaction hole in the sample plate are detected respectively, so as to realize the detection of a plurality of gene samples to be detected simultaneously.

Step 503, determining a target fluorescent signal corresponding to each fluorescent dye in the sample to be tested according to the measured fluorescent signal and the fluorescent signal parameter of each fluorescent dye under each fluorescent detection channel.

In this embodiment, two or more kinds of fluorescent dyes are added to the sample to be tested to identify the target gene, and the measured fluorescent signals of the sample to be tested under each fluorescent detection channel include the target fluorescent signals emitted by the fluorescent dyes corresponding to the fluorescent channels in the sample to be tested and also include the interference fluorescent signals emitted by other fluorescent dyes, and the target fluorescent signals corresponding to each fluorescent dye in the sample to be tested are determined by calculating according to the fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel determined in step 501.

Further, when the sample plate includes a plurality of reaction holes, each reaction hole is filled with a different sample to be measured, a measured fluorescent signal of the sample to be measured in the reaction hole under each fluorescent detection channel is obtained, and a target fluorescent signal corresponding to each fluorescent dye in the sample to be measured is determined according to the fluorescent signal parameters of each fluorescent dye in the reaction hole under each fluorescent detection channel determined in step 501. Specifically, the target fluorescent signal corresponding to each fluorescent dye in the sample to be tested in each corresponding reaction well in the sample plate may be determined according to the fluorescent signal parameters corresponding to the different fluorescent dyes in each reaction well under each fluorescent detection channel determined in step 501.

Example two

The method flow chart of the fluorescence signal detection method for gene detection according to one embodiment of the present invention as shown in fig. 6 can be applied to a gene detection apparatus. The fluorescence signal detection method may include:

step 601, obtaining a first fluorescent signal of each fluorescent dye in at least two fluorescent dyes under each fluorescent detection channel corresponding to the fluorescent dye respectively according to at least two preset fluorescent dyes, and determining fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel.

Specifically, the step of obtaining the first fluorescent signal of each of the at least two fluorescent dyes under each fluorescent detection channel corresponding to the fluorescent dye according to the preset at least two fluorescent dyes may include the following substeps:

(1) According to at least two preset fluorescent dyes, acquiring a first initial fluorescent signal of each fluorescent dye in the at least two fluorescent dyes under each fluorescent detection channel corresponding to the fluorescent dye respectively.

In this embodiment, according to the detection purpose or sample type, the fluorescent dyes involved in detecting the gene sample to be detected are determined, each fluorescent dye is respectively loaded into a sample plate or a sample tube, and the concentration and the volume of the fluorescent dye loaded into each sample plate or sample tube are the same. When a plurality of reaction wells are included in the sample plate, the concentration and volume of the fluorescent dye loaded in the reaction well of each sample plate are the same. Generally, the number of fluorescent dyes used for sample gene detection is not less than two, and the present embodiment can be applied to a sample gene detection scenario in which the number of fluorescent dyes is two or more.

In order to facilitate understanding of the technical solution of this embodiment, the detection of the gene sample to be detected needs to be described by taking 3 kinds of fluorescent dyes, namely FAM fluorescent dye, HEX fluorescent dye and ROX fluorescent dye as examples, and the fluorescent detection channels corresponding to the 3 kinds of fluorescent dyes are respectively FAM fluorescent detection channel, HEX fluorescent detection channel and ROX fluorescent detection channel. It will be appreciated that the fluorescent dye in this example may be specifically a combination of any three of FAM, HEX, ROX, TAMRA, CY or CY3 fluorescent dyes used in a PCR amplification reaction, and is not limited herein.

Ideally, only the fluorescent signal emitted by a certain fluorescent dye can be detected under the fluorescent detection channel corresponding to the certain fluorescent dye, but the fluorescent signal emitted by other fluorescent dyes cannot be detected, that is, ideally, under the FAM fluorescent detection channel, the fluorescent signal can be obtained only when the sample plate filled with the FAM fluorescent dye is detected, and the fluorescent signal cannot be obtained when the sample plate filled with the HEX fluorescent dye or the ROX fluorescent dye is detected; similarly, only fluorescent signals of a sample plate containing HEX fluorescent dye or ROX fluorescent dye can be detected correspondingly under the HEX fluorescent detection channel or the ROX fluorescent detection channel. Specifically, if the fluorescence signal detected under each fluorescence detection channel is normalized, so that the intensity of the fluorescence signal is represented by a closed interval of 0-1, when the excitation light source corresponding to the FAM fluorescent dye irradiates the sample plate containing the FAM fluorescent dye, the HEX fluorescent dye and the ROX fluorescent dye, respectively, the intensity of the signal detected under the FAM fluorescent detection channel is 1, 0 and 0, respectively; similarly, after the excitation light sources corresponding to the HEX fluorescent dyes respectively irradiate the sample plates filled with the FAM fluorescent dyes, the HEX fluorescent dyes and the ROX fluorescent dyes, the signal intensities detected under the HEX fluorescent detection channels are respectively 0, 1 and 0; after the sample plate filled with FAM fluorescent dye, HEX fluorescent dye and ROX fluorescent dye is respectively irradiated by the excitation light source corresponding to the ROX fluorescent dye, the signal intensities detected under the ROX fluorescent detection channel are respectively 0, 0 and 1. As shown in table 1, wherein the excitation light source corresponding to the fluorescent dye is denoted as in, and the sample plate detection signal with the fluorescent dye is denoted as out.

TABLE 1

However, in practice, since the excitation light source and the fluorescence detection channel are not absolutely ideal, when the excitation light source corresponding to the FAM fluorescent dye irradiates the sample plate containing the FAM fluorescent dye, the HEX fluorescent dye and the ROX fluorescent dye, respectively, the sample plate containing the HEX fluorescent dye and the ROX fluorescent dye is detected under the FAM fluorescent detection channel, and a certain fluorescent signal can still be detected. This is also the case when performing fluorescence detection in the HEX fluorescence detection channel and the ROX fluorescence detection channel, as shown in FIG. 1. Therefore, when two or more kinds of fluorescent dyes are added into the gene sample to be detected, after the gene sample to be detected is irradiated by an excitation light source corresponding to one of the fluorescent dyes, the fluorescent signals detected under the fluorescent detection channel corresponding to the fluorescent dye generally include both the target fluorescent signals emitted by the fluorescent dye and the interference fluorescent signals emitted by other fluorescent dyes.

Taking the above three fluorescent dyes as an example, the fluorescent signals FFAM, FHEX, FROX of the sample plates with three fluorescent dyes detected through the FAM fluorescent detection channel, the fluorescent signals HFAM, HHEX, HROX of the sample plates with three fluorescent dyes detected through the HEX fluorescent detection channel, and the fluorescent signals RFAM, RHEX, RROX of the sample plates with three fluorescent dyes detected through the ROX fluorescent detection channel are recorded, respectively, as shown in the following table 2, that is, the first initial fluorescent signals.

TABLE 2

(2) And obtaining a second fluorescent signal of the buffer solution with the same volume as the fluorescent dye under each fluorescent detection channel corresponding to the fluorescent dye respectively.

In this embodiment, the same volume of buffer as the fluorochrome is added separately to the sample plate, and accordingly, when a plurality of reaction wells are included in the sample plate, the buffer is filled in the plurality of reaction wells in each sample plate, respectively, and the concentration and volume of the buffer filled in each reaction well are the same as those of the fluorochrome in sub-step (1). Further, detecting the fluorescent signals of the sample plate filled with the buffer solution through the FAM fluorescent detection channel, the HEX fluorescent detection channel and the ROX fluorescent detection channel respectively, and correspondingly obtaining the fluorescent signals of the buffer solution under each fluorescent detection channel, namely BFAM (binary fluorescent amplification) of the fluorescent signals under the FAM fluorescent detection channel, BHEX (fluorescent amplification) of the fluorescent signals under the HEX fluorescent detection channel and BROX (fluorescent amplification) of the fluorescent signals under the ROX fluorescent detection channel.

(3) And respectively separating and removing the corresponding second fluorescent signals from the first initial fluorescent signals to obtain first fluorescent signals of each fluorescent dye under each corresponding fluorescent detection channel.

In this embodiment, when the sample to be detected is detected, the sample to be detected, the fluorescent dyes and the buffer solution are required to be mixed, so as to obtain more accurate fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel, thereby obtaining more accurate detection results when the sample to be detected is detected, and optimizing the first initial fluorescent signal, that is, separating and removing the second fluorescent signal detected under the corresponding fluorescent detection channel from the first initial fluorescent signal, so as to finally obtain the first fluorescent signal of each fluorescent dye under the corresponding fluorescent detection channel. Specifically, taking the above three fluorescent dyes as examples, the first fluorescent signals of the FAM fluorescent dye, the HEX fluorescent dye, and the ROX fluorescent dye in the FAM fluorescent detection channel, the HEX fluorescent detection channel, and the ROX fluorescent detection channel, respectively, are finally obtained, as shown in table 3.

TABLE 3 Table 3

Wherein ffamb=ffam-BFAM, fhexb=fhex-BHEX, froxb=frox-BROX, and so on.

Further, step 601 further comprises: and determining fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel respectively according to the first fluorescent signals.

In this embodiment, according to the first fluorescence signal shown in table 3, the fluorescence signal parameters corresponding to different fluorescent dyes under each fluorescence detection channel are determined by normalization, wherein the fluorescence signal parameters include fluorescence signal intensity coefficients. Specifically, as shown in table 4, taking the above three fluorescent dyes as examples, the fluorescent signal intensity coefficients C (FFAMB), C (FHEXB) and C (frame) of the FAM fluorescent dye, the HEX fluorescent dye and the ROC fluorescent dye detected under the FAM fluorescent detection channel are determined by normalization according to the first fluorescent signals FFAMB, FHEXB and frame corresponding to the FAM fluorescent detection channel, wherein C (FFAMB), C (FHEXB), C (frame) e [0,1]; likewise, the fluorescence signal intensity coefficients C (HFAMB), C (HHEXB) and C (HROXB) of the FAM fluorescent dye, the HEX fluorescent dye and the ROC fluorescent dye which are correspondingly detected under the HEX fluorescent detection channel are determined through normalization according to the first fluorescence signals HFAMB, HHEXB and HROXB corresponding to the HEX fluorescent detection channel, wherein C (HFAMB), C (HHEXB), C (HROXB) E [0,1]; and determining fluorescence signal intensity coefficients C (RFAMB), C (RHEXB) and C (RROXB) of the FAM fluorescent dye, the HEX fluorescent dye and the ROC fluorescent dye which are detected correspondingly under the ROX fluorescent detection channel through normalization according to the first fluorescence signals RFAMB, RHEXB and RROXB which correspond to the ROX fluorescent detection channel, wherein C (RFAMB), C (RHEXB) and C (RROXB) E [0,1].

TABLE 4 Table 4

Step 602, obtaining a measured fluorescent signal of a sample to be measured under each fluorescent channel corresponding to each fluorescent dye, wherein the sample to be measured contains at least two fluorescent dyes.

Specifically, taking the three fluorescent dyes as examples, a measured fluorescent signal UFAM of a sample to be detected under a FAM fluorescent detection channel, a measured fluorescent signal UHEX under a HEX fluorescent detection channel, and a measured fluorescent signal UROX under a ROX fluorescent detection channel are respectively obtained.

And step 603, determining a target fluorescent signal corresponding to each fluorescent dye in the sample to be detected according to the measured fluorescent signal and fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel.

In this embodiment, taking the above three fluorescent dyes as examples, according to the measured fluorescent signal of the sample under test under each fluorescent detection channel and the fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel, the target fluorescent signal corresponding to each fluorescent dye in the sample under test is determined by the following equation set, with ufamb=c (FFAMB) ×rfam+c (HFAMB) ×rhex+c (RFAMB) ×rrox

UHEXB＝C(FHEXB)×RFAM+C(HHEXB)×RHEX+C(RHEXB)×RROX

UROXB＝C(FROXB)×RFAM+C(HROXB)×RHEX+C(RROXB)×RROX

Wherein, ufamb=ufam-BFAM, i.e. BFAM separating and removing the fluorescent signal of the buffer solution under the FAM fluorescent detection channel from the fluorescent detection signal of the sample to be detected under the FAM fluorescent detection channel; uhexb=uhex-BHEX, i.e. BHEX separating and removing the fluorescent signal of the buffer solution under the HEX fluorescent detection channel from the fluorescent detection signal of the sample to be detected under the HEX fluorescent detection channel; UROXB = UROX-BROX, i.e., BROX that separates and removes the fluorescent signal of the buffer under the ROX fluorescent detection channel from the fluorescent detection signal of the sample under test under the ROX fluorescent detection channel; RFAM is a target fluorescent signal of a sample to be detected under a FAM fluorescent detection channel; RHEX is a target fluorescent signal of the sample to be detected under the HEX fluorescent detection channel; RROX is the target fluorescent signal of the sample to be detected under the ROX fluorescent detection channel.

According to the target fluorescent signals corresponding to each fluorescent dye in the sample to be detected, the number of target genes marked by each fluorescent dye can be determined, so that the genotype of the sample to be detected is determined.

Example III

On the basis of the second embodiment, in order to obtain the target fluorescent signal more accurately when detecting the gene sample to be detected, the fluorescent signal detection method for gene detection provided in this embodiment further includes, after step 603:

step 604, obtaining a second fluorescent signal of the mixture of the at least two fluorescent dyes under each fluorescent detection channel corresponding to the fluorescent dye according to the preset at least two fluorescent dyes.

In this embodiment, according to the detection purpose or sample type, the fluorescent dye involved in detecting the gene sample to be detected is determined, and, still taking the above FAM fluorescent dye, HEX fluorescent dye and ROX fluorescent dye as examples, an equal concentration mixture of the three fluorescent dyes is loaded into the sample plate, where the volume of the mixture is the same as the volume of each fluorescent dye in step 601, and the concentration of each fluorescent dye in the mixture is the same as the concentration of each fluorescent dye in step 601. Likewise, when a plurality of reaction wells are contained in the sample plate, the volume of the mixture charged in each reaction well is the same. Further, a second fluorescent signal of the sample plate containing the mixture is obtained under the FAM fluorescent detection channel, the HEX fluorescent detection channel and the ROX fluorescent detection channel, respectively.

Step 605, determining a reference fluorescent signal corresponding to each fluorescent dye in the fluorescent dye mixture according to the second fluorescent signal and fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel respectively;

in this embodiment, according to the second fluorescent signals of the sample plate with the mixture under the FAM fluorescent detection channel, the HEX fluorescent detection channel and the ROX fluorescent detection channel, respectively, and the fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel determined in step 601, respectively, the reference fluorescent signal corresponding to each fluorescent dye in the fluorescent dye mixture is determined in step 603. Specifically, taking the three fluorescent dyes of the FAM fluorescent dye, the HEX fluorescent dye and the ROX fluorescent dye as examples, respectively obtaining second initial fluorescent signals of the sample plate filled with the three fluorescent dye mixtures under the FAM fluorescent detection channel, the HEX fluorescent detection channel and the ROX fluorescent detection channel, and separating and removing fluorescent signals of the buffer solution under the corresponding fluorescent detection channel from the second initial fluorescent signals to obtain second fluorescent signals of the sample plate filled with the three fluorescent dye mixtures under the FAM fluorescent detection channel, the HEX fluorescent detection channel and the ROX fluorescent detection channel. According to the second fluorescent signal and the fluorescent signal parameter of each fluorescent dye under each fluorescent detection channel determined in step 601, referring to step 603, the reference fluorescent signals STDRFAM, STDRHEX and STDRROX corresponding to FAM fluorescent dye, HEX fluorescent dye and ROX fluorescent dye in the fluorescent dye mixture are determined.

Step 606, determining a target fluorescent signal corresponding to each fluorescent dye in the sample to be tested according to the reference fluorescent signal.

In this embodiment, the target fluorescent signal corresponding to each fluorescent dye in the sample to be measured is calibrated according to the reference fluorescent signal, so as to improve the accuracy of the detection result of the sample to be measured. Specifically, taking the above three fluorescent dyes of FAM fluorescent dye, HEX fluorescent dye and ROX fluorescent dye as examples, according to the target fluorescent signals RFAM, RHEX and RROX corresponding to each fluorescent dye, and the reference fluorescent signals STDRFAM, STDRHEX and STDRROX corresponding to each fluorescent dye in the sample to be tested determined in step 603, the target fluorescent signal corresponding to each fluorescent dye in the calibrated sample to be tested is determined by the following formula,

RFAM_STD＝RFAM/STDRFAM

RHEX_STD＝RHEX/STDRHEX

RROX_STD＝RROX/STDRROX

the RFAM_STD, RHEX_STD and RROX_STD are respectively target fluorescent signals corresponding to each fluorescent dye in the calibrated sample to be tested.

It should be noted that, step 604 and step 605 may also be performed before step 603.

In this embodiment, multiple fluorescent dyes related to the sample to be detected are mixed to obtain a reference fluorescent signal of the mixture under each fluorescent detection channel, and the target fluorescent signal in the sample to be detected is calibrated, so that the detection result of the sample to be detected is more accurate.

Example IV

On the basis of the second embodiment and the third embodiment, in order to obtain the target fluorescent signal more accurately when detecting the gene sample to be detected, the fluorescent signal detection method for gene detection provided by the embodiment further includes, after obtaining the target fluorescent signal corresponding to each fluorescent dye in the sample to be detected through the technical scheme described in the second embodiment or the third embodiment:

in step 607, when the sample to be measured includes the reference fluorescent dye, the target fluorescent signals corresponding to other fluorescent dyes in the sample to be measured are processed by normalization according to the target fluorescent signals corresponding to the reference fluorescent dye.

In general, in order to eliminate objective errors caused by hardware devices on detection results, a reference fluorescent dye is added into a sample to be detected during gene sample detection, the reference fluorescent dye does not participate in a PCR reaction, and the fluorescent signal of the reference fluorescent dye does not increase along with the PCR amplification reaction, that is, the fluorescent signal of the reference fluorescent dye remains substantially unchanged during the reaction. Therefore, when detecting the gene sample, the fluorescence signal detection error is caused by the factors such as uneven illumination of an excitation light source, sample bubbles or evaporation of a reaction system, and the like, and the accuracy of a sample detection result can be improved by carrying out homogenization treatment on the factors through reference fluorescence signals.

Specifically, taking the three fluorescent dyes of the FAM fluorescent dye, the HEX fluorescent dye and the ROX fluorescent dye as examples, assuming that the ROX fluorescent dye is a reference fluorescent dye, determining target fluorescent signals RFAM, RHEX and RROX corresponding to the FAM fluorescent dye, the HEX fluorescent dye and the ROX dye in a sample to be tested respectively based on the technical scheme of the second embodiment, carrying out normalization processing on the target fluorescent signals RFAM and RHEX according to the reference fluorescent signal RROX by the following formula to obtain normalized target fluorescent signals NorRFAM and NorRHEX,

NorRFAM＝RFAM/RROX

NorRHEX＝RHEX/RROX。

further, in some cases, since the reference fluorescent dye is not added to the sample to be measured, or the amount of the added reference fluorescent dye is too small, the value of the target fluorescent signal RROX is extremely low, so that accurate results cannot be obtained or even result errors occur according to the calculation of the formula. Therefore, a first preset threshold value is introduced, whether a target fluorescent signal corresponding to the reference fluorescent dye is larger than the first preset threshold value is judged, and if yes, the target fluorescent signal value corresponding to each other fluorescent dye in the sample to be detected is determined through the formula; if not, the target fluorescence signals RFAM and RHEX are not normalized, and the RFAM and RHEX are taken as final target fluorescence signals. The first preset threshold may be an empirical value, or a numerical value calculated by a specific algorithm, which is not limited in this embodiment.

Similarly, based on the technical solution of the third embodiment, after determining the calibrated target fluorescent signals rfam_std, rhex_std, and rrox_std corresponding to the FAM fluorescent dye, the HEX fluorescent dye, and the ROX dye in the sample to be tested, if the ROX fluorescent dye is the reference fluorescent dye, determining whether the target fluorescent signal rrox_std corresponding to the ROX fluorescent dye is greater than the second preset threshold, if so, normalizing the target fluorescent signals rfam_std and rhex_std according to the reference fluorescent signal rrox_std by the following formula to obtain normalized target fluorescent signals rfam_std_r and rhex_std_r,

RFAM_STD_R＝RFAM_STD/RROX_STD

RHEX_STD_R＝RHEX_STD/RROX_STD

if not, the target fluorescence signals rfam_std and rhex_std are not normalized, and the final target fluorescence signals rfam_std and rhex_std are obtained.

In this embodiment, when the sample to be measured includes the reference fluorescent dye, the target fluorescent signals corresponding to other fluorescent dyes in the sample to be measured are normalized according to the target fluorescent signals corresponding to the reference fluorescent dye, so as to eliminate the detection error of the fluorescent signals and improve the accuracy of the sample detection result.

In summary, according to the fluorescence signal detection method for gene detection provided by the embodiment of the present invention, first, according to at least two preset fluorescent dyes, a first fluorescence signal of each of the at least two fluorescent dyes under each fluorescence detection channel corresponding to the fluorescent dye is obtained, and a fluorescence signal parameter of each of the fluorescent dyes under each fluorescence detection channel is determined; then, obtaining a measured fluorescent signal of a sample to be measured under each fluorescent channel corresponding to each fluorescent dye, wherein the sample to be measured contains at least two fluorescent dyes; and finally, determining a target fluorescent signal corresponding to each fluorescent dye in the sample to be detected according to the measured fluorescent signal and fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel. Therefore, by means of the fluorescent signal parameters of various fluorescent dyes obtained in advance, when the gene sample to be detected is detected, the fluorescent signal detected by actual measurement is calibrated, so that a more accurate fluorescent detection signal is obtained, and the accuracy of a gene detection result is further effectively improved.

According to the detection data of the gene detection experiment, the beneficial effects of the embodiment of the invention are further described, firstly, the sample plate filled with the sample to be detected and the fluorescent dye is subjected to fluorescent detection, and a scatter diagram shown in fig. 7 is obtained according to the original fluorescent signals respectively obtained under each fluorescent detection channel, wherein the vertical axis represents the fluorescent signals under the HEX channel, and the horizontal axis represents the fluorescent signals under the FAM channel. It can be seen that the scattered points corresponding to each sample data to be tested in fig. 7 are clustered together, so that it is difficult to perform effective data analysis. Next, according to the technical solution of the embodiment of the present invention, the fluorescence data of the sample board to be tested is calculated by referring to the fluorescence signal, and the scatter diagram corresponding to the sample to be tested is updated, as shown in fig. 8, it can be seen that the data that is disordered before begins to converge, but the genotype still cannot be distinguished. Further, according to the technical scheme of the embodiment of the invention, the fluorescent data of the sample plate to be tested is calibrated through the fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel, and the scatter diagram corresponding to the sample to be tested is updated, as shown in fig. 9. It can be seen that the distribution of the scattered points corresponding to the sample to be measured in fig. 9 is optimized, and the samples can be distinguished to a certain extent. Finally, according to the technical scheme of the embodiment of the invention, the fluorescence data of the sample plate to be tested is normalized through the reference fluorescence signal after the calibration processing, so as to obtain a scatter diagram as shown in fig. 10. At this time, compared with the original data, the degree of distinction between the negative sample and the positive sample is enough to be subjected to clustering analysis, so that the accuracy of sample analysis is effectively improved, and the design expectation is met.

Example five

The embodiment of the invention provides a structural block diagram of a fluorescent signal detection device for gene detection, and as shown in fig. 11, the fluorescent signal detection device can be applied to gene detection equipment. The fluorescence signal detection device may include:

a signal obtaining module 1110, configured to obtain, according to at least two preset fluorescent dyes, a first fluorescent signal of each of the at least two fluorescent dyes under each fluorescent detection channel corresponding to the fluorescent dye respectively;

a parameter determining module 1120, configured to determine a fluorescent signal parameter of each fluorescent dye under each fluorescent detection channel;

the signal obtaining module 1110 is further configured to obtain a measured fluorescent signal of a sample to be measured under each fluorescent channel corresponding to each fluorescent dye, where the sample to be measured includes the at least two fluorescent dyes;

and the analysis operation module 1130 is configured to determine a target fluorescent signal corresponding to each fluorescent dye in the sample to be measured according to the measured fluorescent signal and fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel.

In an alternative embodiment, the signal acquisition module 1110 is further configured to: acquiring a first initial fluorescence signal of each fluorescent dye in at least two fluorescent dyes under each fluorescence detection channel corresponding to the fluorescent dye respectively according to at least two preset fluorescent dyes; and obtaining a second fluorescent signal of the buffer solution with the same volume as the fluorescent dye under each fluorescent detection channel corresponding to the fluorescent dye respectively.

The parameter determining module 1120 is further configured to: and respectively separating and removing the second fluorescent signals from the first initial fluorescent signals to obtain first fluorescent signals of each fluorescent dye under each corresponding fluorescent detection channel.

In an alternative embodiment, as shown in fig. 12, the signal acquisition module 1110 is further configured to: acquiring a second fluorescent signal of an equal-concentration mixture of at least two fluorescent dyes under each fluorescent detection channel corresponding to the fluorescent dyes respectively according to the preset at least two fluorescent dyes;

and, a parameter determination module 1120, further configured to: determining a reference fluorescent signal corresponding to each fluorescent dye in the fluorescent dye mixture according to the second fluorescent signal and fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel respectively;

the fluorescence signal detection device for gene detection according to the embodiment of the invention further comprises: the signal calibration module 1140 is configured to calibrate a target fluorescent signal corresponding to each fluorescent dye in the sample to be tested according to the reference fluorescent signal.

In an alternative embodiment, the fluorescent signal detection apparatus for gene detection according to the embodiment of the present invention further includes: normalization module 1150 for:

In summary, according to the fluorescence signal detection device for gene detection provided by the embodiment of the present invention, first, according to at least two preset fluorescent dyes, a first fluorescence signal of each fluorescent dye in the at least two fluorescent dyes under each fluorescence detection channel corresponding to the fluorescent dye is obtained, and a fluorescence signal parameter of each fluorescent dye under each fluorescence detection channel is determined; then, obtaining a measured fluorescent signal of a sample to be measured under each fluorescent channel corresponding to each fluorescent dye, wherein the sample to be measured contains at least two fluorescent dyes; and finally, determining a target fluorescent signal corresponding to each fluorescent dye in the sample to be detected according to the measured fluorescent signal and fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel. Therefore, by means of the fluorescent signal parameters of various fluorescent dyes obtained in advance, when the gene sample to be detected is detected, the fluorescent signal detected by actual measurement is calibrated, so that a more accurate fluorescent detection signal is obtained, and the accuracy of a gene detection result is further effectively improved.

One embodiment of the present invention provides a computer-readable storage medium having stored therein at least one instruction that is loaded and executed by a processor to implement a fluorescence signal detection method for gene detection as described above.

An embodiment of the present invention provides a gene assaying device including a processor and a memory having at least one instruction stored therein, the instruction being loaded and executed by the processor to implement a fluorescence signal assaying method for gene assaying as described above.

It should be noted that: in the fluorescence signal detection device provided in the above embodiment, only the division of the above functional modules is used for illustration, and in practical application, the above functional allocation may be performed by different functional modules according to needs, that is, the internal structure of the fluorescence signal detection device is divided into different functional modules, so as to complete all or part of the functions described above. In addition, the fluorescence signal detection device and the fluorescence signal detection method provided in the foregoing embodiments belong to the same concept, and specific implementation processes thereof are detailed in the method embodiments and are not described herein again.

It will be understood by those skilled in the art that all or part of the steps for implementing the above embodiments may be implemented by hardware, or may be implemented by a program for instructing relevant hardware, where the program may be stored in a computer readable storage medium, and the storage medium may be a read-only memory, a magnetic disk or an optical disk, etc.

The above description should not be taken as limiting the embodiments of the invention, but rather should be construed to cover all modifications, equivalents, improvements, etc. that may fall within the spirit and principles of the embodiments of the invention.

Claims

1. A fluorescent signal detection method for gene detection, comprising the steps of:

Determining a target fluorescent signal corresponding to each fluorescent dye in the sample to be detected according to the measured fluorescent signal and fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel;

when the fluorescent signal parameter is a fluorescent signal intensity coefficient, the measured fluorescent signal under one fluorescent channel is equal to the weighted sum of the fluorescent signal intensity coefficient of each fluorescent dye under the fluorescent detection channel and the target fluorescent signal of each fluorescent dye in the sample to be detected under the fluorescent detection channel;

after the step of determining the target fluorescent signal corresponding to each fluorescent dye in the sample to be measured according to the measured fluorescent signal and the fluorescent signal parameter of each fluorescent dye under each fluorescent detection channel, the method further comprises:

2. The method according to claim 1, wherein the step of obtaining the first fluorescent signal of each of the at least two fluorescent dyes under each fluorescent detection channel to which the fluorescent dye corresponds, according to at least two preset fluorescent dyes, comprises:

3. The method for detecting a fluorescent signal for gene detection according to claim 1 or 2, wherein the step of determining a fluorescent signal parameter for each of the fluorescent dyes under each of the fluorescent detection channels, respectively, comprises:

4. The method for detecting fluorescent signals for gene detection according to claim 3, wherein the step of determining the target fluorescent signal corresponding to each fluorescent dye in the sample to be detected according to the measured fluorescent signals and the fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel respectively comprises the following steps:

Signal(A)=a _A ×A _A +b _A ×B _B +…+n _A ×N _N

Signal(B)=a _B ×A _A +b _B ×B _B +…+n _B ×N _N

…

Signal(N)=a _N ×A _A +b _N ×B _B +…+n _N ×N _N

5. The method for detecting fluorescent signals for gene detection according to claim 1, wherein said step of calibrating the target fluorescent signal corresponding to each fluorescent dye in the sample to be detected based on the reference fluorescent signal comprises:

6. The method for detecting fluorescent signals for gene detection according to claim 4 or 5, wherein after the step of determining a target fluorescent signal corresponding to each fluorescent dye in the sample to be detected according to the measured fluorescent signals and fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel, the method further comprises:

7. A fluorescent signal detection device for gene detection, the device comprising:

the analysis operation module is used for determining a target fluorescent signal corresponding to each fluorescent dye in the sample to be detected according to the measured fluorescent signal and fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel respectively;

the signal acquisition module is further used for acquiring a second fluorescent signal of the equal-concentration mixture of the at least two fluorescent dyes under each fluorescent detection channel corresponding to the fluorescent dyes respectively according to the at least two preset fluorescent dyes;

the parameter determining module is further configured to determine a reference fluorescent signal corresponding to each fluorescent dye in the fluorescent dye mixture according to the second fluorescent signal and fluorescent signal parameters of each fluorescent dye under each fluorescent detection channel respectively;

and the signal calibration module is used for calibrating a target fluorescent signal corresponding to each fluorescent dye in the sample to be tested according to the reference fluorescent signal.

8. A computer-readable storage medium having stored therein at least one instruction that is loaded and executed by a processor to implement the fluorescence signal detection method for gene detection according to any one of claims 1 to 6.

9. A genetic testing apparatus comprising a processor and a memory having stored therein at least one instruction that is loaded and executed by the processor to implement the fluorescence signal detection method for genetic testing of any one of claims 1 to 6.