CN-121975960-A - Food microorganism fluorescence quantitative PCR detection and tracing system

CN121975960ACN 121975960 ACN121975960 ACN 121975960ACN-121975960-A

Abstract

The invention discloses a food microorganism fluorescent quantitative PCR detection and tracing system, which relates to the technical field of food analysis detection and comprises an integrated reaction carrier module, a dynamics data set module, a time-space tracing multidimensional map module and a time-space tracing multidimensional map module, wherein the integrated reaction carrier module is used for collecting an original food sample, realizing target microorganism cracking and nucleic acid separation and purification, extracting genome nucleic acid, establishing a real-time fluorescent quantitative PCR reaction system, generating an integrated reaction carrier for thermal cycle amplification of microorganisms, the dynamics data set module is used for executing a thermal cycle process in a closed system based on the integrated reaction carrier, exciting a multispecific fluorescent probe marking channel, capturing multispectral fluorescent signals in real time, generating an original fluorescent intensity-cycle number dynamics data set, and the time-space tracing multidimensional map module is used for carrying out feature extraction on a multispecific fluorescent amplification curve, coupling the time-space tracing coded molecule marking system and carrying out dynamic amplification efficiency self-adaptive calibration, and establishing a time-space tracing multidimensional map of microorganism quantitative-molecular typing. The invention improves the sensitivity of food microorganism detection.

Inventors

ZHENG JIAYU
FAN MINGHONG
MA RONG
ZHOU LU
LI HONGMEI

Assignees

宝应县有机食品质量监督检验中心

Dates

Publication Date: 20260505
Application Date: 20260206

Claims (10)

1. The utility model provides a food microorganism fluorescence ration PCR detects and traceability system which characterized in that includes: the system comprises an integrated reaction carrier module, a dynamics data set module and a space-time tracing multidimensional map module; the dynamic data set module is electrically connected with the integrated reaction carrier module, and the space-time tracing multidimensional mapping module is electrically connected with the dynamic data set module; The integrated reaction carrier module is used for collecting an original food sample, realizing target microorganism cracking and nucleic acid separation and purification, extracting target microorganism genome nucleic acid, establishing a real-time fluorescent quantitative PCR reaction system, and generating an integrated reaction carrier of the original food sample for thermal cycle amplification of target microorganisms; The dynamics data set module is used for executing a fluorescence quantitative PCR thermal cycle process in a closed system based on an integrated reaction carrier of original food sample target microorganisms for thermal cycle amplification, exciting a multi-specific fluorescent probe marking channel preset in the interior, capturing the whole process tracking and multi-spectrum fluorescent signals of exponential amplification dynamics of each target nucleic acid in real time, and generating an original fluorescence intensity-cycle number dynamics data set; The space-time tracing multidimensional atlas module utilizes a fusion convolutional neural network-attention mechanism algorithm to extract characteristics of a multi-target fluorescence amplification curve based on an original fluorescence intensity-cycle number dynamics data set, couples a space-time tracing coding molecular marker system and performs dynamic amplification efficiency self-adaptive calibration, and establishes a microorganism quantitative-molecular parting space-time tracing multidimensional atlas.
2. The system for fluorescence quantitative PCR detection and traceability of food microorganisms according to claim 1, wherein the integrated reaction carrier module comprises: The judging unit is used for obtaining a quantitative original food sample, automatically adjusting the dilution times according to the sample types, automatically calculating the optimal homogenizing time and intensity by combining the sample viscosity and the particle data, adding a homogenizing bag containing a lysis buffer and proteinase K, processing by a slapping type homogenizer, monitoring the turbidity change of the solution in real time, and judging the completion degree of the microbial cell lysis and the release degree of the genome nucleic acid.
3. The system for fluorescence quantitative PCR detection and tracing of food microorganisms according to claim 2, wherein the integrated reaction carrier module further comprises: The target microorganism nucleic acid separation and purification unit is used for automatically adding magnetic bead suspension based on the release degree of genome nucleic acid, dynamically adjusting the addition amount of the magnetic bead suspension according to the volume of the lysate and the impurity content, automatically completing the steps of nucleic acid magnetic bead combination, washing and elution, removing protein, fat and polysaccharide inhibitors in food matrixes, realizing the separation and purification of target microorganism nucleic acid, and obtaining high-purity target microorganism genome nucleic acid; And a generation unit of a real-time fluorescent quantitative PCR reaction system is formed by selecting a fully-closed reaction tube, automatically adding DNA polymerase, dNTPs and buffer solution to uniformly distribute components, and designing a targeted primer and a probe according to a target microorganism specific gene sequence.
4. A system for fluorescence quantitative PCR detection and traceability of food microorganisms according to claim 3, wherein the integrated reaction carrier module further comprises: The space-time tracing coding molecular marker system building unit designs a double-stranded DNA (deoxyribonucleic acid) tag with a unique coding sequence, carries out biotinylation modification on the double-stranded DNA tag, is covalently combined to a specific area on the inner wall of the totally-enclosed reaction tube, carries out concentration standardization treatment on the double-stranded DNA tag, and builds a target microorganism space-time tracing coding molecular marker system; The integrated reaction carrier generation unit is used for establishing an optimal freeze-drying curve of target microorganisms based on a real-time fluorescence quantitative PCR reaction system according to different reagent glass transition temperatures, converting a liquid reagent into a solid freeze-drying form stable at room temperature through a programmed freeze-drying process, and generating an integrated reaction carrier of original food sample target microorganisms for thermal cycle amplification.
5. The system for fluorescence quantitative PCR detection and tracing of food microorganisms according to claim 4, wherein the dynamic data set module comprises: the thermal cycle program unit is based on an integrated reaction carrier of the target microorganism of the original food sample for thermal cycle amplification, and an automatic re-dissolution real-time fluorescence quantitative PCR reaction system drives the Peltier element thermoelectric semiconductor thermal control module to automatically run the pre-denaturation, annealing and extension thermal cycle program according to the target microorganism amplification requirement.
6. The system for fluorescence quantitative PCR detection and tracing of food microorganisms according to claim 5, wherein the dynamic data set module further comprises: The monitoring data flow generating unit is used for setting a fixed sliding time window, continuously monitoring the real-time temperature of target microorganisms in the sliding time window, calculating the difference value between the real-time temperature of the target microorganisms and a preset target temperature to obtain target microorganism temperature proportional control, accumulating the temperature deviation of the past sliding time window to eliminate steady-state errors existing for a long time to obtain target microorganism temperature integral control, predicting the future trend of the temperature deviation and compensating the temperature fluctuation in advance through the temperature deviation change rate in the sliding time window to obtain target microorganism differential control, calculating the weighted sum of the target microorganism temperature proportional control, integral control and differential control, dynamically adjusting the heating/cooling power of the Peltier element thermoelectric semiconductor temperature control module, and generating a high-resolution time-temperature monitoring data flow; The real-time fluorescence quantitative PCR reaction system control circuit switches a multi-wavelength excitation light source according to a preset sequence aiming at each acquisition time point in a sliding time window based on the tail end of an annealing and extension thermal cycle process, the optimal excitation condition of the multi-specific fluorescent probe is preset in the matching carrier, the excitation light is purified into a light beam with specific wavelength through a light path system formed by a group of band-pass filters, and the light beam is vertically irradiated to a reaction hole to excite the multi-specific fluorescent probe marking channel.
7. The system for fluorescence quantitative PCR detection and tracing of food microorganisms according to claim 6, wherein the dynamic data set module further comprises: The capture unit is used for capturing the fluorescence intensity Rn value of each cycle of each food sample under each fluorescence channel in real time by utilizing a photoelectric detector based on the fluorescent signal released after the multi-specific fluorescent probe is excited, generating high-resolution dynamic data of each food sample, capturing the whole process tracking and multi-spectrum fluorescent signal of exponential amplification dynamic of each target nucleic acid in real time, and reading the sample number automatically associated with the double-stranded DNA label arranged in the carrier; The data set establishing unit automatically collects and arranges fixed time points of each thermal cycle to synchronously record fluorescence intensity Rn value data, food sample numbers, cycle numbers and fluorescence channels through a rapid sampling mechanism, and combines high-resolution time-temperature monitoring data streams to generate an original fluorescence intensity-cycle number dynamics data set.
8. The system for fluorescence quantitative PCR detection and tracing of food microorganisms according to claim 7, wherein the space-time tracing multidimensional mapping module comprises: The data preprocessing unit is used for selecting fluorescent intensity Rn values of 3 rd to 15 th cycles for the original signal of each fluorescent probe marking channel to perform polynomial fitting as a base line based on the original fluorescent intensity-cycle number dynamics data set, performing base line correction on the original fluorescent signal, performing noise smoothing processing by using an exponential weighted moving average, and performing data preprocessing on the data set; The characteristic extraction unit is used for establishing a multi-target fluorescence amplification curve with the length of m according to n fluorescence probe marking channels after pretreatment, establishing a multi-channel time sequence image of n multiplied by m two-dimensional tensors through channel dimension stacking, scanning by utilizing one-dimensional convolution kernels with 3-5 cycle widths, learning the high correlation characteristic of the local first derivative of the multi-target fluorescence amplification curve, identifying an exponential growth inflection point of an original fluorescence signal from a stable baseline, compressing the multi-channel time sequence image length through a pooling layer, polymerizing global information, and learning the global morphological characteristic of the multi-target fluorescence amplification curve to obtain the average amplification efficiency of the exponential growth period, the saturated fluorescence intensity Rn value of a platform period and the integral bending degree of the multi-target fluorescence amplification curve; And the focusing characteristic unit is used for introducing a multi-head attention mechanism based on the global morphological characteristics of the multi-target fluorescent amplification curve, calculating the query, key and value matrix of each channel in the global morphological characteristics of the multi-target fluorescent amplification curve, dynamically calculating the attention weight of each channel to the current characteristics, and carrying out weighted summation on the attention weight of the current characteristics of each channel to obtain the focusing characteristic vector of the multi-target fluorescent amplification curve.
9. The system for fluorescence quantitative PCR detection and tracing of food microorganisms according to claim 8, wherein the space-time tracing multidimensional mapping module further comprises: The initial template copy number calculation unit extracts a corresponding multi-target fluorescent amplification curve focusing characteristic vector, average amplification efficiency in an exponential amplification period and the integral bending degree of a multi-target fluorescent amplification curve by utilizing an original fluorescent intensity-cycle number dynamic data set generated by amplifying standard substances with known initial template copy numbers under the same condition, learns a mapping relation between the multi-target fluorescent amplification curve focusing characteristic vector and the multi-target fluorescent amplification curve focusing characteristic efficiency under the specific condition, predicts the multi-target fluorescent amplification curve focusing characteristic dynamic amplification efficiency, establishes a dynamic amplification efficiency self-adaptive calibration model and calculates the target microorganism initial template copy number of each food sample.
10. The system for fluorescence quantitative PCR detection and tracing of food microorganisms according to claim 9, wherein the space-time tracing multidimensional mapping module further comprises: The classification unit is used for training a target microorganism classifier network based on labeled food sample data by taking a multi-target fluorescence amplification curve focusing feature vector as input, setting a plurality of Softmax output layers, labeling and classifying the food sample target microorganisms, and taking the prediction of each target microorganism category and the probability of confidence as output; further, the labeled food sample data include salmonella positive/negative, escherichia coli positive/negative, listeria monocytogenes positive/negative; The information reporting unit reads the space-time traceability coding molecular markers of the integrated reaction carrier, binds the initial template copy number, the category prediction, the existence confidence probability and the traceability codes of the target microorganisms of each food sample, and generates a structured complete information report of each food sample; The spectrum construction unit is used for carrying out grid division by taking sample batch and acquisition date sequence as rows and taking target microorganism types as columns on the basis of each food sample structured complete information report, wherein the numerical value of each cell is the initial template copy number of the sample-microorganism pair, the number of each cell is mapped to the cell by blue-yellow-red colors according to the numerical value of each cell, a microorganism quantitative heat map is generated, target flora combinations with high co-occurrence frequency of pollution patterns are identified, characteristic pollution patterns with statistical significance are marked, and the characteristic pollution patterns are coupled with the microorganism quantitative map and space-time traceability coding molecular markers of the sample in a multi-layer manner to establish a space-time traceability multidimensional spectrum of microorganism quantitative-molecular typing.

Description

Food microorganism fluorescence quantitative PCR detection and tracing system Technical Field The invention relates to the technical field of food analysis and detection, in particular to a food microorganism fluorescent quantitative PCR detection and tracing system. Background The traditional food microorganism PCR detection method generally relies on single-channel fluorescent signal interpretation and fixed threshold cycle number analysis, is difficult to accurately correct baseline drift and nonspecific signal interference, so that detection sensitivity to low-concentration or complex substrate samples is insufficient, false negative/false positive risks are high, the prior art mostly adopts independent analysis modes of each fluorescent channel, ignores the associated dynamic characteristics of a multi-target amplification curve, so that the estimation deviation of amplification efficiency is large, the quantitative result is inaccurate, detection data and tracing information are disjointed, automatic and intelligent multidimensional data fusion analysis capability is lacking, microbial load, type and space-time sources cannot be effectively associated, quick identification and visual tracing of pollution modes are difficult to realize, and the accuracy and timeliness of food safety risk early warning and process control are restricted. Disclosure of Invention In order to solve the technical problems, the technical scheme provides a food microorganism fluorescence quantitative PCR detection and tracing system, which solves the problems of accuracy and timeliness of food safety risk early warning and process control. In order to achieve the above purpose, the invention adopts the following technical scheme: A food microorganism fluorescent quantitative PCR detection and traceability system, comprising: the system comprises an integrated reaction carrier module, a dynamics data set module and a space-time tracing multidimensional map module; the dynamic data set module is electrically connected with the integrated reaction carrier module, and the space-time tracing multidimensional mapping module is electrically connected with the dynamic data set module; The integrated reaction carrier module is used for collecting an original food sample, realizing target microorganism cracking and nucleic acid separation and purification, extracting target microorganism genome nucleic acid, establishing a real-time fluorescent quantitative PCR reaction system, and generating an integrated reaction carrier of the original food sample for thermal cycle amplification of target microorganisms; The dynamics data set module is used for executing a fluorescence quantitative PCR thermal cycle process in a closed system based on an integrated reaction carrier of original food sample target microorganisms for thermal cycle amplification, exciting a multi-specific fluorescent probe marking channel preset in the interior, capturing the whole process tracking and multi-spectrum fluorescent signals of exponential amplification dynamics of each target nucleic acid in real time, and generating an original fluorescence intensity-cycle number dynamics data set; The space-time tracing multidimensional atlas module utilizes a fusion convolutional neural network-attention mechanism algorithm to extract characteristics of a multi-target fluorescence amplification curve based on an original fluorescence intensity-cycle number dynamics data set, couples a space-time tracing coding molecular marker system and performs dynamic amplification efficiency self-adaptive calibration, and establishes a microorganism quantitative-molecular parting space-time tracing multidimensional atlas. Preferably, the integrated reaction carrier module comprises: The judging unit is used for obtaining a quantitative original food sample, automatically adjusting the dilution times according to the sample types, automatically calculating the optimal homogenizing time and intensity by combining the sample viscosity and the particle data, adding a homogenizing bag containing a lysis buffer and proteinase K, processing by a slapping type homogenizer, monitoring the turbidity change of the solution in real time, and judging the completion degree of the microbial cell lysis and the release degree of the genome nucleic acid. Preferably, the integrated reaction carrier module further comprises: The target microorganism nucleic acid separation and purification unit is used for automatically adding magnetic bead suspension based on the release degree of genome nucleic acid, dynamically adjusting the addition amount of the magnetic bead suspension according to the volume of the lysate and the impurity content, automatically completing the steps of nucleic acid magnetic bead combination, washing and elution, removing protein, fat and polysaccharide inhibitors in food matrixes, realizing the separation and purification of target microorganism nucleic acid, and obtaining high-pu