A method for rapid site-specific analysis of biotin-labeled KRAS protein and a detection system

Abstract

The application discloses a kind of quick site analysis method and detection system of biotin labeled KRAS protein, belong to intelligent analysis technical field;Construct the site fluorescence intensity database under historical detection state, obtain the fluorescence intensity data of each amino acid site;Based on the data, construct the amino acid site-fluorescence intensity coordinate system of each sample, generate fluorescence intensity fluctuation curve;Extract the wave crest and wave trough of curve, construct fluorescence peak site pair set and valley site pair set;Obtain the mode of peak site pair and valley site pair in all samples, as high response site and low response site, calculate its probability of occurrence in new sample, and combine the peak value and valley value of real-time sample with preset threshold, output high response type, low response type or stable type classification result.The application realizes the quick, objective, intelligent discrimination of KRAS protein function state by historical data probabilistic modeling and fluctuation curve feature extraction.

Description

Technical Field

[0001] This invention relates to the field of intelligent analysis technology, specifically to a rapid site-specific analysis method and detection system for biotin-labeled KRAS protein. Background Technology

[0002] In recent years, with the rapid development of molecular marker technology and high-throughput detection methods, biotin-labeled fluorescence detection methods have been widely applied in the field of site-specific protein functional analysis, especially demonstrating significant value in the study of structure-function relationships of key signaling proteins such as KRAS. KRAS protein, as a key regulatory node in multiple signal transduction pathways, is closely related to the occurrence and development of various malignant tumors due to its mutational state. Therefore, researchers have gradually developed biotin-labeled site-specific fluorescence analysis technology, which infers protein conformational changes, interaction interfaces, and activity states by labeling specific sites and monitoring changes in fluorescence intensity. Traditional methods often rely on comparing the fluorescence intensity of single or small samples, using statistical mean or fold difference for site evaluation. In recent years, with the development of the Internet of Things and intelligent sensing technology, experimental data acquisition has achieved continuous acquisition in multiple batches and dimensions, providing a data foundation for constructing protein behavior pattern recognition based on large samples. However, existing technologies still largely rely on passive recording and offline analysis at the data processing level, lacking the ability to systematically model the dynamic fluctuations of fluorescence intensity and quantitatively predict site response tendencies.

[0003] Specifically, existing fluorescence analysis methods for biotin-labeled KRAS proteins typically focus only on the absolute value or simple difference of fluorescence intensity in a single sample, ignoring the site-specific patterns of fluorescence intensity fluctuations across multiple samples. In particular, they lack systematic extraction and cross-sample correlation analysis of sites corresponding to peaks and troughs. Such methods struggle to identify key sites that consistently exhibit high or low responses in multiple experiments, and cannot probabilistically infer the response type of unknown samples based on the distribution characteristics of historical samples. Furthermore, due to the lack of joint modeling of fluorescence peak and trough site pairs, existing technologies struggle to effectively distinguish between high-response, low-response, and stable samples, resulting in insufficient accuracy and generalization ability in determining the functional state of KRAS proteins. In summary, existing technologies suffer from insufficient utilization of historical fluorescence data, incomplete extraction of site response features, and a lack of probabilistic matching mechanisms. Summary of the Invention

[0004] The purpose of this invention is to provide a rapid site-specific analysis method and detection system for biotin-labeled KRAS protein, in order to solve the problems mentioned in the background art.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0006] A rapid site-specific analysis method for biotin-labeled KRAS protein includes the following steps: Step S1: Construct a site-specific fluorescence intensity database of biotin-labeled KRAS protein under historical detection conditions, and obtain site-specific fluorescence intensity data of amino acid sites in the biotin-labeled KRAS protein; Step S2: Based on the site-specific fluorescence intensity data, construct an amino acid site-fluorescence intensity coordinate system of biotin-labeled KRAS protein in a single sample, and construct a fluorescence intensity fluctuation curve; Step S3: Construct fluorescence peak site pairs and fluorescence trough site pairs corresponding to the fluorescence intensity fluctuation curve, and sort them sequentially according to the sample order to construct a set of fluorescence peak site pairs and a set of fluorescence trough site pairs; Step S4: Based on the set of fluorescence peak site pairs and the set of fluorescence trough site pairs, obtain the mode of the set of fluorescence peak site pairs and the set of fluorescence trough site pairs under all samples; calculate the probability of high-response sites and low-response sites appearing in the next sample to be tested, preset thresholds, analyze and perform type matching.

[0007] As a preferred embodiment of the rapid site-specific analysis method for biotin-labeled KRAS protein described in this invention, a site-specific fluorescence intensity database of biotin-labeled KRAS protein under historical detection conditions is constructed. This database records site-specific fluorescence intensity data for all amino acid sites contained in the biotin-labeled KRAS protein, with one amino acid site corresponding to one site-specific fluorescence intensity data record. Each site-specific fluorescence intensity data record includes site-specific fluorescence intensity data for all samples under the corresponding site under historical detection conditions. The historical detection conditions refer to the same experimental conditions (including buffer solution, temperature, excitation wavelength, and detector parameters).

[0008] Obtain the site-specific fluorescence intensity data of the i-th amino acid site in the biotin-labeled KRAS protein, and record the site-specific fluorescence intensity data of the i-th amino acid site in the s-th sample as follows: .

[0009] As a preferred embodiment of the rapid site-directed analysis method for biotin-labeled KRAS protein described in this invention, based on site-directed fluorescence intensity data... An amino acid site-fluorescence intensity coordinate system for biotin-labeled KRAS protein in the s-th sample was constructed. The abscissa of the amino acid site-fluorescence intensity coordinate system is all the amino acid sites of the biotin-labeled KRAS protein arranged in sequence, and the ordinate of the amino acid site-fluorescence intensity coordinate system is the fixed-point fluorescence intensity data corresponding to all the amino acid sites of the biotin-labeled KRAS protein arranged in sequence.

[0010] Obtain the coordinates of the sites and their corresponding fixed-point fluorescence intensity data in the amino acid site-fluorescence intensity coordinate system; obtain all coordinate points and connect them sequentially to form a fluctuation curve, which is recorded as the fluorescence intensity fluctuation curve of biotin-labeled KRAS protein in the s-th sample. .

[0011] As a preferred embodiment of the rapid site-directed analysis method for biotin-labeled KRAS protein described in this invention, fluorescence intensity fluctuation curves are obtained respectively. The peaks and troughs correspond to the sites and fluorescence intensity data, which are then represented as fluorescence peak site pairs and denoted as... The sites corresponding to the troughs and the fluorescence intensity data are represented as fluorescence trough site pairs, and denoted as... ,in, This indicates the i-th amino acid site in the biotin-labeled KRAS protein corresponding to the peak. This represents the fixed-point fluorescence intensity data corresponding to the wave peak. This indicates the j-th amino acid site in the biotin-tagged KRAS protein corresponding to the trough. This represents the fixed-point fluorescence intensity data corresponding to the trough;

[0012] Obtain the fluorescence peak and trough pairs corresponding to the fluorescence intensity fluctuation curves for all samples, and sort them sequentially according to sample order to construct a set of fluorescence peak pairs and a set of fluorescence trough pairs, denoted as and respectively. and Where I represents the total number of amino acid sites in the biotin-labeled KRAS protein, and S represents the total number of samples in the historical detection state.

[0013] As a preferred embodiment of the rapid site-specific analysis method for biotin-labeled KRAS protein described in this invention, the method is based on fluorescence peak sites for... and fluorescence trough site pair set Obtain the mode of the set of fluorescence peak sites and the set of fluorescence valley sites for all samples;

[0014] The mode of the set of fluorescence peak sites is designated as the high-response site, and the mode of the set of fluorescence valley sites is designated as the low-response site. The probability of a high-response site appearing in the (S+1)th sample to be tested is calculated using the following formula:

[0015] ;

[0016] in, This indicates the probability of a high-response site appearing in the (S+1)th sample to be tested. This indicates a highly responsive site in the biotin-tagged KRAS protein. This indicates the high-response sites among all S samples in the historical detection state. The number of times;

[0017] The probability of a low-response site appearing in the (S+1)th sample to be tested is calculated using the following formula:

[0018] ;

[0019] in, This indicates the probability of a low-response site appearing in the (S+1)th sample to be tested. This indicates a low-response site in the biotin-tagged KRAS protein. This indicates low-response sites among all S samples in the historical detection state. The number of times.

[0020] As a preferred embodiment of the rapid site-specific analysis method for biotin-labeled KRAS protein described in this invention, real-time site-specific fluorescence intensity data of all amino acid sites in the (S+1)th sample to be tested are obtained, and a fluorescence intensity fluctuation curve of the (S+1)th sample to be tested is constructed. And extract the real-time fluorescence peak sites of the curve. and real-time fluorescence trough sites ;

[0021] If the real-time fluorescence peak site Compared with historical high response sites The same, and the real-time fluorescence intensity Falling into the range If so, the sample is determined to match the historical high-response characteristics at the peak. This represents the high-response sites among all S samples in the historical detection state. The mean fluorescence intensity, and These represent the preset fluctuation tolerances;

[0022] If the real-time fluorescence trough site Compared with historically low response sites The same, and the real-time fluorescence intensity Falling into the range If the sample matches a historical low response feature at a trough, then it is determined that the sample matches the trough feature. and These represent the preset fluctuation tolerances. This represents the low-response sites among all S samples in the historical detection state. The mean fluorescence intensity;

[0023] like And real-time peak and If a match is found, the sample is output as a high-response type; otherwise... And real-time valley value and If a match is found, the sample is output as a low-response type; otherwise... and Real-time peak and trough values ​​are respectively compared with and If a match is found, the output will be stable, where... and These represent the preset probability thresholds for high-response sites and low-response sites, respectively, which are determined by the statistical distribution of the historical sample set.

[0024] A rapid site-specific detection system for biotin-labeled KRAS protein, comprising: a database construction and data acquisition module, a coordinate system and curve construction module, a pair construction module, and a probability calculation and analysis module;

[0025] The database construction and data acquisition module: constructs a database of the fixed-point fluorescence intensity of biotin-labeled KRAS protein in historical detection states, and acquires the fixed-point fluorescence intensity data of amino acid sites in the biotin-labeled KRAS protein;

[0026] The coordinate system and curve construction module: Based on the fixed-point fluorescence intensity data, it constructs an amino acid site-fluorescence intensity coordinate system for biotin-labeled KRAS protein in a single sample, and constructs a fluorescence intensity fluctuation curve;

[0027] The pair construction module constructs pairs of fluorescence peak sites and pairs of fluorescence valley sites corresponding to the fluorescence intensity fluctuation curves, and sorts them sequentially according to the sample order to construct pairs of fluorescence peak sites and pairs of fluorescence valley sites.

[0028] The probability calculation and analysis module: based on the fluorescence peak site pair set and the fluorescence valley site pair set, obtains the mode of the fluorescence peak site pair set and the fluorescence valley site pair set in all samples; calculates the probability of high response sites and low response sites appearing in the next sample to be tested, presets thresholds, analyzes and performs type matching.

[0029] Furthermore, the database construction and data acquisition module includes a database construction and data acquisition unit;

[0030] The database construction and data acquisition unit: constructs a database of site-specific fluorescence intensity of biotin-labeled KRAS protein under historical detection conditions. The database records site-specific fluorescence intensity data for all amino acid sites in the biotin-labeled KRAS protein, with one amino acid site corresponding to one site-specific fluorescence intensity data record. The site-specific fluorescence intensity data record contains site-specific fluorescence intensity data for the corresponding site under all samples in historical detection conditions. The unit also acquires site-specific fluorescence intensity data for amino acid sites in the biotin-labeled KRAS protein.

[0031] Furthermore, the coordinate system and curve construction module includes coordinate system and curve construction units;

[0032] The coordinate system and curve construction unit: Based on the fixed-point fluorescence intensity data, an amino acid site-fluorescence intensity coordinate system of biotin-labeled KRAS protein in a single sample is constructed. The abscissa of the amino acid site-fluorescence intensity coordinate system represents all amino acid sites of the biotin-labeled KRAS protein arranged in sequence, and the ordinate of the amino acid site-fluorescence intensity coordinate system represents the fixed-point fluorescence intensity data corresponding to all amino acid sites of the biotin-labeled KRAS protein arranged in sequence. The coordinate points of the sites and the corresponding fixed-point fluorescence intensity data in the amino acid site-fluorescence intensity coordinate system are obtained. All coordinate points are obtained and connected sequentially to form a fluctuation curve, which is recorded as the fluorescence intensity fluctuation curve of biotin-labeled KRAS protein in a single sample.

[0033] Furthermore, the set construction module includes a set construction unit;

[0034] The pair construction unit: acquires the sites and fluorescence intensity data corresponding to the peaks and troughs of the fluorescence intensity fluctuation curves respectively, and describes the sites and fluorescence intensity data corresponding to the peaks as fluorescence peak site pairs, and the sites and fluorescence intensity data corresponding to the troughs as fluorescence trough site pairs; acquires the fluorescence peak site pairs and fluorescence trough site pairs corresponding to the fluorescence intensity fluctuation curves of all samples, and sorts them in order according to the sample sequence to construct the fluorescence peak site pair set and the fluorescence trough site pair set.

[0035] Compared with existing technologies, the beneficial effects achieved by this invention are as follows: This invention provides a rapid site-specific analysis method and detection system for biotin-labeled KRAS proteins. By constructing a database of site-specific fluorescence intensity under historical detection conditions, it integrates discrete single-detection data into a statistically significant full-sample space, laying a data foundation for longitudinal comparison in subsequent analysis. This effectively avoids random errors and achieves site-level data indexing, thus solving the problem of idle historical data. Furthermore, mapping the fluorescence intensity of each site to a fluorescence intensity fluctuation curve according to the amino acid sequence not only reveals the spatial coupling response pattern between sites but also preserves protein domain information, making the identification of response hotspots easier than manual methods. The threshold was upgraded to objective waveform morphology analysis; the peaks and troughs of each fluctuation curve were extracted and peak site pairs and trough site pairs were constructed, compressing continuous curves into key feature pairs, significantly reducing data dimensionality. At the same time, a cross-sample correspondence between site and response type was established, providing an accurate counting unit for subsequent probability statistics; the frequency of occurrence of high and low response sites in historical samples was calculated as the predicted probability, and the peak / trough site matching and fluorescence intensity range of real-time samples were used for dual verification. Finally, the classification results of high response type, low response type or stable type were output based on the probability threshold, which significantly improved the objectivity, robustness and intelligence of KRAS protein functional state discrimination. Attached Figure Description

[0036] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0037] Figure 1 This is a schematic diagram of the steps of a rapid site-directed analysis method for biotin-labeled KRAS protein according to the present invention;

[0038] Figure 2 This is a schematic diagram of the structure of a rapid site-specific detection system for biotin-labeled KRAS protein according to the present invention. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Please see Figure 1 In this first embodiment, a rapid site-directed analysis method for biotin-labeled KRAS protein is provided, which includes the following steps:

[0041] Step S1: Construct a database of site-specific fluorescence intensity of biotin-labeled KRAS protein in historical detection states, and obtain site-specific fluorescence intensity data of amino acid sites in the biotin-labeled KRAS protein.

[0042] Specifically, a database of site-specific fluorescence intensity of biotin-labeled KRAS protein under historical detection conditions is constructed. This database records site-specific fluorescence intensity data for all amino acid sites of the biotin-labeled KRAS protein, with one amino acid site corresponding to one site-specific fluorescence intensity data record. Each site-specific fluorescence intensity data record contains site-specific fluorescence intensity data for the corresponding site under all samples in the historical detection conditions. The historical detection conditions refer to identical experimental conditions (including buffer solution, temperature, excitation wavelength, and detector parameters).

[0043] Obtain the site-specific fluorescence intensity data of the i-th amino acid site in the biotin-labeled KRAS protein, and record the site-specific fluorescence intensity data of the i-th amino acid site in the s-th sample as follows: .

[0044] It should be noted that by systematically collecting fluorescence intensity data of each amino acid site of biotin-labeled KRAS protein in all historical samples under the same experimental conditions (buffer, temperature, excitation light, etc.), a structured database containing all sites and full sample dimensions is constructed. This step integrates the originally discrete and isolated single detection data into a traceable and statistically significant historical sample space. Compared with traditional methods that only use instantaneous data from a single sample or a small number of control groups, this step, by establishing a historical database under the same conditions, enables subsequent analysis to utilize the distribution characteristics of a large sample, effectively avoiding the interference of random errors and instantaneous fluctuations in single measurements on the determination of site response.

[0045] Step S2: Based on the fixed-point fluorescence intensity data, construct an amino acid site-fluorescence intensity coordinate system for biotin-labeled KRAS protein in a single sample, and construct a fluorescence intensity fluctuation curve.

[0046] Specifically, based on point-to-point fluorescence intensity data An amino acid site-fluorescence intensity coordinate system for biotin-labeled KRAS protein in the s-th sample was constructed. The abscissa of the amino acid site-fluorescence intensity coordinate system is all the amino acid sites of the biotin-labeled KRAS protein arranged in sequence, and the ordinate of the amino acid site-fluorescence intensity coordinate system is the fixed-point fluorescence intensity data corresponding to all the amino acid sites of the biotin-labeled KRAS protein arranged in sequence.

[0047] Obtain the coordinates of the sites and their corresponding fixed-point fluorescence intensity data in the amino acid site-fluorescence intensity coordinate system; obtain all coordinate points and connect them sequentially to form a fluctuation curve, which is recorded as the fluorescence intensity fluctuation curve of biotin-labeled KRAS protein in the s-th sample. .

[0048] It should be noted that, for each sample, a coordinate system is generated with all amino acid sites as the x-axis (arranged sequentially) and the fluorescence intensity of the corresponding sites as the y-axis. Connecting the coordinate points sequentially creates a unique fluorescence intensity fluctuation curve for that sample. This curve transforms abstract site fluorescence data into a concrete waveform. Traditional methods only focus on the absolute value of a single site, while this step visualizes the fluorescence intensity changes between adjacent sites through continuous broken lines, explicitly expressing the "regional fluctuation characteristics" caused by protein conformational changes or differences in labeling efficiency. Using the native amino acid sequence as the x-axis preserves the correlation information between the protein's primary structure and potential tertiary structure folding, allowing the waveform fluctuations (peaks / troughs) to directly correspond to the response hotspots of specific functional or structural domains.

[0049] Step S3: Construct pairs of fluorescence peak sites and pairs of fluorescence valley sites corresponding to the fluorescence intensity fluctuation curves, and sort them sequentially according to the sample order to construct sets of fluorescence peak site pairs and sets of fluorescence valley site pairs.

[0050] Specifically, fluorescence intensity fluctuation curves were obtained separately. The peaks and troughs correspond to the sites and fluorescence intensity data, which are then represented as fluorescence peak site pairs and denoted as... The sites corresponding to the troughs and the fluorescence intensity data are represented as fluorescence trough site pairs, and denoted as... ,in, This indicates the i-th amino acid site in the biotin-labeled KRAS protein corresponding to the peak. This represents the fixed-point fluorescence intensity data corresponding to the wave peak. This indicates the j-th amino acid site in the biotin-tagged KRAS protein corresponding to the trough. This represents the fixed-point fluorescence intensity data corresponding to the trough;

[0051] Obtain the fluorescence peak and trough pairs corresponding to the fluorescence intensity fluctuation curves for all samples, and sort them sequentially according to sample order to construct a set of fluorescence peak pairs and a set of fluorescence trough pairs, denoted as and respectively. and Where I represents the total number of amino acid sites in the biotin-labeled KRAS protein, and S represents the total number of samples in the historical detection state.

[0052] It should be noted that the local maxima (peaks) and local minima (troughs) on each fluctuation curve are extracted separately. The site index is paired with the extreme values ​​of fluorescence intensity, and arranged in sample order to form peak-site pairs and valley-site pairs. The original curve contains continuous information of all sites, and direct use for cross-sample comparison is computationally intensive and greatly affected by noise. This step compresses the curve into several key feature pairs (peak / valley values), which significantly reduces the data dimensionality while retaining the core biological significance. By recording which site in each sample exhibits the strongest response (peak) and the weakest response (valley), the functional predisposition of the same physical site between different samples can be directly counted and compared.

[0053] Step S4: Based on the fluorescence peak site pairs and fluorescence valley site pairs, obtain the mode of the fluorescence peak site pairs and fluorescence valley site pairs in all samples; calculate the probability of high response sites and low response sites appearing in the next sample to be tested, preset the threshold, analyze and perform type matching.

[0054] Specifically, based on the fluorescence peak sites, the set and fluorescence trough site pair set Obtain the mode of the set of fluorescence peak sites and the set of fluorescence valley sites for all samples;

[0055] The mode of the set of fluorescence peak sites is designated as the high-response site, and the mode of the set of fluorescence valley sites is designated as the low-response site. The probability of a high-response site appearing in the (S+1)th sample to be tested is calculated using the following formula:

[0056] ;

[0057] in, This indicates the probability of a high-response site appearing in the (S+1)th sample to be tested. This indicates a highly responsive site in the biotin-tagged KRAS protein. This indicates the high-response sites among all S samples in the historical detection state. The number of times;

[0058] The probability of a low-response site appearing in the (S+1)th sample to be tested is calculated using the following formula:

[0059] ;

[0060] in, This indicates the probability of a low-response site appearing in the (S+1)th sample to be tested. This indicates a low-response site in the biotin-tagged KRAS protein. This indicates low-response sites among all S samples in the historical detection state. The number of times.

[0061] Furthermore, real-time fixed-site fluorescence intensity data of all amino acid sites in the (S+1)th sample to be tested are obtained, and the fluorescence intensity fluctuation curve of the (S+1)th sample to be tested is constructed. And extract the real-time fluorescence peak sites of the curve. and real-time fluorescence trough sites ;

[0062] If the real-time fluorescence peak site Compared with historical high response sites The same, and the real-time fluorescence intensity Falling into the range If so, the sample is determined to match the historical high-response characteristics at the peak. This represents the high-response sites among all S samples in the historical detection state. The mean fluorescence intensity, and These represent the preset fluctuation tolerances;

[0063] If the real-time fluorescence trough site Compared with historically low response sites The same, and the real-time fluorescence intensity Falling into the range If the sample matches a historical low response feature at a trough, then it is determined that the sample matches the trough feature. and These represent the preset fluctuation tolerances. This represents the low-response sites among all S samples in the historical detection state. The mean fluorescence intensity;

[0064] like And real-time peak and If a match is found, the sample is output as a high-response type; otherwise... And real-time valley value and If a match is found, the sample is output as a low-response type; otherwise... and Real-time peak and trough values ​​are respectively compared with and If a match is found, the output will be stable, where... and These represent the preset probability thresholds for high-response sites and low-response sites, respectively, which are determined by the statistical distribution of the historical sample set.

[0065] It should be noted that the frequencies of high-response sites (mode of peak site pairs) and low-response sites (mode of valley site pairs) in the historical sample set are calculated as probabilities. Then, a real-time curve is constructed for the sample to be tested (the (S+1)th sample), and its real-time peak site pairs and valley site pairs are extracted and compared with... and Site matching and fluorescence intensity range matching (mean ± tolerance) are performed. Finally, the sample type (high-response / low-response / stable) is determined by combining probability thresholds.

[0066] Traditional techniques can only provide retrospective descriptions of existing samples. This new step, however, extrapolates the likelihood of specific response sites in new samples based on historical frequencies, achieving a leap from descriptive statistics to inferential statistics. A match is only considered complete when the real-time peak site matches a historical high-response site, and the real-time fluorescence intensity falls within a controllable tolerance range of the historical mean. This mechanism effectively eliminates false matches caused by random noise or experimental drift, improving classification specificity. Based on the criteria of high probability + peak matching → high response type; low probability + valley matching → low response type; low probability in both aspects but simultaneous peak and valley matching → stable type, this multi-criteria decision framework can characterize the functional heterogeneity of KRAS proteins in different samples (e.g., activated state, inhibited state, homeostatic state), overcoming the limitation of existing methods that can only output continuous intensity values ​​and cannot provide functional labels.

[0067] This step provides a rapid and automated qualitative / semi-quantitative analysis tool for high-throughput screening of KRAS protein mutation status, drug response prediction, and conformational dynamic monitoring, significantly improving the decision-making intelligence level of biomarker detection systems in the Internet of Things environment.

[0068] Please see Figure 2 In this second embodiment: a rapid site-specific detection system for biotin-labeled KRAS protein is provided. The system includes: a database construction and data acquisition module, a coordinate system and curve construction module, a set construction module, and a probability calculation and analysis module.

[0069] The database construction and data acquisition module: constructs a database of the fixed-point fluorescence intensity of biotin-labeled KRAS protein in historical detection states, and acquires the fixed-point fluorescence intensity data of amino acid sites in the biotin-labeled KRAS protein;

[0070] The coordinate system and curve construction module: Based on the fixed-point fluorescence intensity data, it constructs an amino acid site-fluorescence intensity coordinate system for biotin-labeled KRAS protein in a single sample, and constructs a fluorescence intensity fluctuation curve;

[0071] The pair construction module constructs pairs of fluorescence peak sites and pairs of fluorescence valley sites corresponding to the fluorescence intensity fluctuation curves, and sorts them sequentially according to the sample order to construct pairs of fluorescence peak sites and pairs of fluorescence valley sites.

[0072] The probability calculation and analysis module: based on the fluorescence peak site pair set and the fluorescence valley site pair set, obtains the mode of the fluorescence peak site pair set and the fluorescence valley site pair set in all samples; calculates the probability of high response sites and low response sites appearing in the next sample to be tested, presets thresholds, analyzes and performs type matching.

[0073] Furthermore, the database construction and data acquisition module includes a database construction and data acquisition unit;

[0074] The database construction and data acquisition unit: constructs a database of site-specific fluorescence intensity of biotin-labeled KRAS protein under historical detection conditions. The database records site-specific fluorescence intensity data for all amino acid sites in the biotin-labeled KRAS protein, with one amino acid site corresponding to one site-specific fluorescence intensity data record. The site-specific fluorescence intensity data record contains site-specific fluorescence intensity data for the corresponding site under all samples in historical detection conditions. The unit also acquires site-specific fluorescence intensity data for amino acid sites in the biotin-labeled KRAS protein.

[0075] Furthermore, the coordinate system and curve construction module includes coordinate system and curve construction units;

[0076] The coordinate system and curve construction unit: Based on the fixed-point fluorescence intensity data, an amino acid site-fluorescence intensity coordinate system of biotin-labeled KRAS protein in a single sample is constructed. The abscissa of the amino acid site-fluorescence intensity coordinate system represents all amino acid sites of the biotin-labeled KRAS protein arranged in sequence, and the ordinate of the amino acid site-fluorescence intensity coordinate system represents the fixed-point fluorescence intensity data corresponding to all amino acid sites of the biotin-labeled KRAS protein arranged in sequence. The coordinate points of the sites and the corresponding fixed-point fluorescence intensity data in the amino acid site-fluorescence intensity coordinate system are obtained. All coordinate points are obtained and connected sequentially to form a fluctuation curve, which is recorded as the fluorescence intensity fluctuation curve of biotin-labeled KRAS protein in a single sample.

[0077] Furthermore, the set construction module includes a set construction unit;

[0078] The pair construction unit: acquires the sites and fluorescence intensity data corresponding to the peaks and troughs of the fluorescence intensity fluctuation curves respectively, and describes the sites and fluorescence intensity data corresponding to the peaks as fluorescence peak site pairs, and the sites and fluorescence intensity data corresponding to the troughs as fluorescence trough site pairs; acquires the fluorescence peak site pairs and fluorescence trough site pairs corresponding to the fluorescence intensity fluctuation curves of all samples, and sorts them in order according to the sample sequence to construct the fluorescence peak site pair set and the fluorescence trough site pair set.

[0079] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0080] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A rapid site-directed analysis method for biotin-labeled KRAS protein, characterized in that, The method includes the following steps: Step S1: Construct a database of site-specific fluorescence intensity of biotin-labeled KRAS protein in historical detection states, and obtain site-specific fluorescence intensity data of amino acid sites in the biotin-labeled KRAS protein; Step S2: Based on the fixed-point fluorescence intensity data, construct an amino acid site-fluorescence intensity coordinate system for biotin-labeled KRAS protein in a single sample, and construct a fluorescence intensity fluctuation curve; Step S3: Construct pairs of fluorescence peak sites and pairs of fluorescence valley sites corresponding to the fluorescence intensity fluctuation curves, and sort them sequentially according to the sample order to construct sets of fluorescence peak site pairs and sets of fluorescence valley site pairs; Step S4: Based on the fluorescence peak site pairs and fluorescence valley site pairs, obtain the mode of the fluorescence peak site pairs and fluorescence valley site pairs in all samples; calculate the probability of high response sites and low response sites appearing in the next sample to be tested, preset the threshold, analyze and perform type matching.

2. The rapid site-directed analysis method for biotin-labeled KRAS protein according to claim 1, characterized in that, The specific implementation process of step S1 includes: A database of site-specific fluorescence intensity of biotin-labeled KRAS protein under historical detection conditions was constructed. The database records site-specific fluorescence intensity data for all amino acid sites of the biotin-labeled KRAS protein, with one amino acid site corresponding to one site-specific fluorescence intensity data record. The site-specific fluorescence intensity data record contains site-specific fluorescence intensity data for the corresponding site under all samples in historical detection conditions. Obtain the site-specific fluorescence intensity data of the i-th amino acid site in the biotin-labeled KRAS protein, and record the site-specific fluorescence intensity data of the i-th amino acid site in the s-th sample as follows: .

3. The rapid site-directed analysis method for biotin-labeled KRAS protein according to claim 2, characterized in that, The specific implementation process of step S2 includes: Based on site-specific fluorescence intensity data An amino acid site-fluorescence intensity coordinate system for biotin-labeled KRAS protein in the s-th sample was constructed. The abscissa of the amino acid site-fluorescence intensity coordinate system is all the amino acid sites of the biotin-labeled KRAS protein arranged in sequence, and the ordinate of the amino acid site-fluorescence intensity coordinate system is the fixed-point fluorescence intensity data corresponding to all the amino acid sites of the biotin-labeled KRAS protein arranged in sequence. Obtain the coordinates of the sites and their corresponding fixed-point fluorescence intensity data in the amino acid site-fluorescence intensity coordinate system; obtain all coordinate points and connect them sequentially to form a fluctuation curve, which is recorded as the fluorescence intensity fluctuation curve of biotin-labeled KRAS protein in the s-th sample. .

4. The rapid site-directed analysis method for biotin-labeled KRAS protein according to claim 3, characterized in that, The specific implementation process of step S3 includes: Obtain fluorescence intensity fluctuation curves respectively The peaks and troughs correspond to the sites and fluorescence intensity data, which are then represented as fluorescence peak site pairs and denoted as... The sites corresponding to the troughs and the fluorescence intensity data are represented as fluorescence trough site pairs, and denoted as... ,in, This indicates the i-th amino acid site in the biotin-labeled KRAS protein corresponding to the peak. This represents the fixed-point fluorescence intensity data corresponding to the wave peak. This indicates the j-th amino acid site in the biotin-tagged KRAS protein corresponding to the trough. This represents the fixed-point fluorescence intensity data corresponding to the trough; Obtain the fluorescence peak and trough pairs corresponding to the fluorescence intensity fluctuation curves for all samples, and sort them sequentially according to sample order to construct a set of fluorescence peak pairs and a set of fluorescence trough pairs, denoted as and respectively. and Where I represents the total number of amino acid sites in the biotin-labeled KRAS protein, and S represents the total number of samples in the historical detection state.

5. The rapid site-directed analysis method for biotin-labeled KRAS protein according to claim 4, characterized in that, The specific implementation process of step S4 includes: Based on fluorescence peak site pairs and fluorescence trough site pair set Obtain the mode of the set of fluorescence peak sites and the set of fluorescence valley sites for all samples; The mode of the set of fluorescence peak sites is designated as the high-response site, and the mode of the set of fluorescence valley sites is designated as the low-response site. The probability of a high-response site appearing in the (S+1)th sample to be tested is calculated using the following formula: ； in, This indicates the probability of a high-response site appearing in the (S+1)th sample to be tested. This indicates a highly responsive site in the biotin-tagged KRAS protein. This indicates the high-response sites among all S samples in the historical detection state. The number of times; The probability of a low-response site appearing in the (S+1)th sample to be tested is calculated using the following formula: ； in, This indicates the probability of a low-response site appearing in the (S+1)th sample to be tested. This indicates a low-response site in the biotin-tagged KRAS protein. This indicates low-response sites among all S samples in the historical detection state. The number of times.

6. The rapid site-directed analysis method for biotin-labeled KRAS protein according to claim 5, characterized in that, The specific implementation process of step S4 also includes: Real-time fixed-site fluorescence intensity data of all amino acid sites in the (S+1)th sample to be tested were obtained, and the fluorescence intensity fluctuation curve of the (S+1)th sample to be tested was constructed. And extract the real-time fluorescence peak sites of the curve. and real-time fluorescence trough sites ; If the real-time fluorescence peak site Compared with historical high response sites The same, and the real-time fluorescence intensity Falling into the range If so, the sample is determined to match the historical high-response characteristics at the peak. This represents the high-response sites among all S samples in the historical detection state. The mean fluorescence intensity, and These represent the preset fluctuation tolerances; If the real-time fluorescence trough site Compared with historically low response sites The same, and the real-time fluorescence intensity Falling into the range If the sample matches a historical low response feature at a trough, then it is determined that the sample matches the trough feature. and These represent the preset fluctuation tolerances. This represents the low-response sites among all S samples in the historical detection state. The mean fluorescence intensity; like And real-time peak and If a match is found, the sample is output as a high-response type; otherwise... And real-time valley value and If a match is found, the sample is output as a low-response type; otherwise... and Real-time peak and trough values ​​are respectively compared with and If a match is found, the output will be of stable type, where... and These represent the preset probability thresholds for high-response sites and low-response sites, respectively.

7. A rapid site-directed detection system for biotin-labeled KRAS protein, performing a rapid site-directed analysis method for biotin-labeled KRAS protein as described in any one of claims 1-6, characterized in that, The system includes: a database construction and data acquisition module, a coordinate system and curve construction module, a set construction module, and a probability calculation and analysis module; The database construction and data acquisition module: constructs a database of the fixed-point fluorescence intensity of biotin-labeled KRAS protein in historical detection states, and acquires the fixed-point fluorescence intensity data of amino acid sites in the biotin-labeled KRAS protein; The coordinate system and curve construction module: Based on the fixed-point fluorescence intensity data, it constructs an amino acid site-fluorescence intensity coordinate system for biotin-labeled KRAS protein in a single sample, and constructs a fluorescence intensity fluctuation curve; The pair construction module constructs pairs of fluorescence peak sites and pairs of fluorescence valley sites corresponding to the fluorescence intensity fluctuation curves, and sorts them sequentially according to the sample order to construct pairs of fluorescence peak sites and pairs of fluorescence valley sites. The probability calculation and analysis module: based on the fluorescence peak site pair set and the fluorescence valley site pair set, obtains the mode of the fluorescence peak site pair set and the fluorescence valley site pair set in all samples; calculates the probability of high response sites and low response sites appearing in the next sample to be tested, presets thresholds, analyzes and performs type matching.

8. The rapid site-specific detection system for biotin-labeled KRAS protein according to claim 7, characterized in that: The database construction and data acquisition module includes a database construction and data acquisition unit; The database construction and data acquisition unit: constructs a database of site-specific fluorescence intensity of biotin-labeled KRAS protein under historical detection conditions. The database records site-specific fluorescence intensity data for all amino acid sites in the biotin-labeled KRAS protein, with one amino acid site corresponding to one site-specific fluorescence intensity data record. The site-specific fluorescence intensity data record contains site-specific fluorescence intensity data for the corresponding site under all samples in historical detection conditions. The unit also acquires site-specific fluorescence intensity data for amino acid sites in the biotin-labeled KRAS protein.

9. The rapid site-specific detection system for biotin-labeled KRAS protein according to claim 8, characterized in that: The coordinate system and curve construction module includes coordinate system and curve construction units; The coordinate system and curve construction unit: Based on the fixed-point fluorescence intensity data, an amino acid site-fluorescence intensity coordinate system of biotin-labeled KRAS protein in a single sample is constructed. The abscissa of the amino acid site-fluorescence intensity coordinate system is all the amino acid sites of the biotin-labeled KRAS protein arranged in sequence, and the ordinate of the amino acid site-fluorescence intensity coordinate system is the fixed-point fluorescence intensity data corresponding to all the amino acid sites of the biotin-labeled KRAS protein arranged in sequence. Obtain the coordinates of the site and the corresponding fixed-point fluorescence intensity data in the amino acid site-fluorescence intensity coordinate system; All coordinate points are obtained and connected sequentially to form a fluctuation curve, which is recorded as the fluorescence intensity fluctuation curve of biotin-labeled KRAS protein in a single sample.

10. A rapid site-specific detection system for biotin-labeled KRAS protein according to claim 9, characterized in that: The set construction module includes a set construction unit; The pair construction unit: respectively acquires the sites and fluorescence intensity data corresponding to the peaks and troughs of the fluorescence intensity fluctuation curve, and describes the sites and fluorescence intensity data corresponding to the peaks as fluorescence peak site pairs, and the sites and fluorescence intensity data corresponding to the troughs as fluorescence trough site pairs; Obtain the fluorescence peak site pairs and fluorescence valley site pairs corresponding to the fluorescence intensity fluctuation curves of all samples, and sort them sequentially according to the sample order to construct the fluorescence peak site pair set and the fluorescence valley site pair set.

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