A rapid detection method for salmonella in food based on fluorescent probe

Abstract

The application provides a kind of Salmonella rapid detection method in food based on fluorescent probe, it is related to food safety detection technical field, comprising the following steps: S1: obtain matrix interference characteristic parameter, calculate comprehensive interference index;S2: generate the washing number of pretreatment operation, elution volume and centrifugal speed;S3: in the food sample to be measured, add known amount of fluorescent internal standard, measure the initial fluorescence intensity of fluorescent internal standard before pretreatment, measure the residual fluorescence intensity of fluorescent internal standard after pretreatment is completed, calculate the measured internal standard recovery rate;S4: according to the dynamic determination threshold generated by comprehensive interference index, compare the measured internal standard recovery rate with dynamic determination threshold.The application can effectively improve the accuracy and detection efficiency of Salmonella fluorescent probe detection in complex food matrix by quantifying matrix interference, real-time monitoring internal standard recovery rate and combining dynamic threshold with automatic error correction.

Description

Technical Field

[0001] This invention relates to the field of food safety testing technology, specifically to a rapid detection method for Salmonella in food based on fluorescent probes. Background Technology

[0002] Salmonella is a common foodborne pathogen that can cause diseases such as typhoid fever, gastroenteritis, and sepsis. Statistics show that Salmonella poisoning accounts for 70%–80% of foodborne illnesses in my country, with over 90% of these cases caused by contaminated meat, eggs, and dairy products. Therefore, establishing rapid and sensitive methods for Salmonella detection is crucial for ensuring food safety. Fluorescent probe detection technology, due to its advantages of high sensitivity, strong specificity, and fast detection speed, has been widely used for the rapid detection of Salmonella in food. However, the current lack of differentiated pretreatment schemes and process quality control leads to a high risk of false negatives in complex matrix detection. Therefore, a method can be developed that quantifies matrix interference, monitors internal standard recovery in real time, and combines dynamic thresholds with automatic error correction to effectively improve the accuracy and efficiency of fluorescent probe detection of Salmonella in complex food matrices.

[0003] In the prior art, patent CN104975065B discloses a method for detecting Salmonella based on biochemical screening and fluorescent nanobioprobes. This technique includes: screening swabs that test positive for hydrogen sulfide by observing whether they produce black spots formed by ferrous sulfide; then incubating these swabs with a quantum dot nanoprobe solution conjugated with a specific antibody to achieve specific binding of the probe to the target bacteria. The target bacteria are detected by observing the fluorescence signal of the quantum dot conjugate on the swab. This method, combined with a stereomicroscope with a cutoff filter and a fluorescence excitation light source, enables visual judgment of results. It is highly operable and suitable for widespread application in the detection of large batches of samples. This method is simple to operate, highly specific, accurate, and efficient; initial screening can be completed within one day, and the isolation of the target bacteria within two days. The required swabs, culture media, and bioprobes are all easy to prepare, making it suitable for industrial production.

[0004] However, the aforementioned existing technologies lack specificity in their pretreatment schemes. They apply uniform operating parameters to different food matrices without considering the differences in the content of interfering substances such as fat, protein, and pigments. This leads to over-processing of pure samples and under-processing of complex samples. The pretreatment process lacks effective quality control methods. Internal standard technology is only applied to the quality control of the PCR amplification stage, which is result-based rather than process-based quality control, and its effectiveness cannot be evaluated in real time after pretreatment. The judgment threshold is singular and fixed, and is not dynamically adjusted according to the degree of interference from different food matrices. This results in a large number of misjudgments of complex samples because the recovery rate is objectively difficult to reach the fixed threshold. There is no automatic error correction mechanism after pretreatment failure, making it difficult for operators to accurately locate the root cause of the problem. They can only redo the entire batch, which is inefficient. The degree of interference from food matrices is only qualitatively described, lacking quantitative evaluation indicators, and cannot provide data support for the optimization of pretreatment schemes.

[0005] The information disclosed in the background section is only intended to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide a rapid detection method for Salmonella in food based on fluorescent probes, thereby addressing the problems mentioned in the background section. This invention achieves personalized pretreatment scheme generation by quantifying matrix interference, utilizes real-time monitoring of recovery rate with fluorescent internal standards, and combines dynamic threshold determination and automatic error correction mechanisms to effectively reduce the risk of false negatives and significantly improve the accuracy and efficiency of detection.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A rapid detection method for Salmonella in food based on fluorescent probes includes the following steps: S1: Obtain matrix interference characteristic parameters, including: fat content, protein content and pigment content of the food sample to be tested; calculate the comprehensive interference index based on the fat content, protein content and pigment content; the comprehensive interference index is used to characterize the pretreatment difficulty of the food sample to be tested. S2: Based on the comprehensive interference index calculated in S1, generate the number of washes, elution volume and centrifugation speed of the pretreatment operation, wherein the number of washes, elution volume and centrifugation speed are all positively correlated with the comprehensive interference index. S3: Add a known amount of fluorescent internal standard to the food sample to be tested, and measure the initial fluorescence intensity of the fluorescent internal standard before pretreatment; perform sample pretreatment according to the number of washings, elution volume and centrifugation speed generated in S2; after pretreatment, measure the residual fluorescence intensity of the fluorescent internal standard, and calculate the measured internal standard recovery rate based on the initial fluorescence intensity and the residual fluorescence intensity. S4: Generate a dynamic judgment threshold based on the comprehensive interference index, wherein the dynamic judgment threshold is negatively correlated with the comprehensive interference index; compare the measured internal standard recovery rate with the dynamic judgment threshold: if the measured internal standard recovery rate is greater than or equal to the dynamic judgment threshold, the sample pretreatment is deemed effective and the obtained pretreatment product is used for subsequent fluorescent probe detection; if the measured internal standard recovery rate is less than the dynamic judgment threshold, the sample pretreatment is deemed invalid, and the number of washing cycles is increased before returning to S3 to re-execute the pretreatment.

[0008] Furthermore, the comprehensive interference index is calculated using the following formula: in: The comprehensive interference index; Fat content; Protein content; This refers to the pigment content; For fat items; For protein; For pigments; The fat interference coefficient; The protein interference coefficient; This represents the pigment interference coefficient.

[0009] Furthermore, the number of washes is calculated using the following formula: in: Number of washes; The baseline number of washes; The washing coefficient represents the increase in the number of washes required to increase the overall disturbance index F value per unit.

[0010] Furthermore, the elution volume is calculated using the following formula: in: This is the elution volume; Use the baseline elution volume; is the elution coefficient, used to control the response intensity of the elution volume as the comprehensive interference index F changes.

[0011] Furthermore, the centrifugal speed is calculated using the following formula. in: Centrifugal speed; The reference centrifugal speed; The centrifugal coefficient represents the increase in centrifugal speed required to increase the overall disturbance index F value per unit.

[0012] Furthermore, a known amount of fluorescent internal standard is added to the food sample to be tested. The fluorescent internal standard is a synthetically produced nucleic acid fragment labeled with a fluorescent dye, which does not participate in the amplification reaction of the target Salmonella nucleic acid and only serves as a tracer in the pretreatment process. After addition, the initial fluorescence intensity of the fluorescent internal standard before pretreatment is measured using a fluorescence detection device. Subsequently, sample pretreatment operations are performed according to the number of washes, elution volume, and centrifugation speed. The pretreatment operations include cell lysis, binding of nucleic acid to a solid-phase carrier, washing to remove impurities according to the number of washes, centrifugation according to the centrifugation speed, and elution to collect the target nucleic acid according to the elution volume. After pretreatment, the residual fluorescence intensity of the fluorescent internal standard in the treated solution is measured again using a fluorescence detection device.

[0013] Furthermore, the measured internal standard recovery rate is calculated using the following formula: in: This represents the measured internal standard recovery rate. The initial fluorescence intensity of the fluorescent internal standard before pretreatment; The residual fluorescence intensity of the fluorescent internal standard after pretreatment is given.

[0014] Furthermore, the dynamic determination threshold is calculated using the following formula: in: The threshold is determined dynamically. This is the preset lower threshold. The baseline recovery rate threshold; This is the threshold adjustment coefficient.

[0015] Furthermore, the obtained measured internal standard recovery rate Compare with the dynamic judgment threshold T generated based on the comprehensive interference index F; if This indicates that the retention rate of the fluorescent internal standard during the pretreatment process has reached an acceptable level commensurate with the complexity of the food sample matrix. In other words, the loss of the target nucleic acid and the interference from residual inhibitors during pretreatment are both within acceptable limits. Therefore, the sample pretreatment is deemed effective, and the resulting pretreatment product can be directly used for subsequent fluorescent probe detection. This indicates that the fluorescent internal standard was lost too much during the pretreatment process or the fluorescence signal was excessively quenched, meaning that the pretreatment effect did not reach an acceptable level. Therefore, the sample pretreatment was deemed invalid. In this case, the number of washes was increased by 1, and the process was returned to S3 to re-execute the pretreatment.

[0016] Furthermore, the washing, centrifugation, and elution collection operations in S3 are returned to be performed, without repeating the cell lysis and nucleic acid binding to the solid-phase carrier operations; then, the residual fluorescence intensity of the fluorescent internal standard in the treated solution is measured again, the new measured internal standard recovery rate is calculated, and compared again with the dynamic judgment threshold; the cycle is repeated until... Or until the preset error correction limit is reached; if the preset error correction limit is reached... Still smaller If the interference from the sample matrix is ​​too strong, an alarm signal will be issued, indicating that the sample matrix interference is too strong and suggesting that the sample be diluted and retested or that the detection method be changed.

[0017] Compared with existing technologies, the advantages of this invention are: personalized generation of pretreatment schemes by quantifying matrix interference, eliminating reliance on experience; forward quality control by real-time monitoring of internal standard recovery rate, reducing the risk of false negatives; avoiding misjudgments and increasing throughput through dynamic thresholds; and achieving precise remediation through automatic error correction. This invention provides reliable quality assurance for fluorescent probe detection of Salmonella, significantly improving the accuracy and efficiency of detection. Attached Figure Description

[0018] Figure 1 This is a schematic flowchart of a rapid detection method for Salmonella in food based on fluorescent probes according to the present invention. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0020] It should be noted that, unless otherwise defined, the technical or scientific terms used in this invention should have the ordinary meaning understood by one of ordinary skill in the art to which this invention pertains. The terms "first," "second," and similar terms used in this invention do not indicate any order, quantity, or importance, but are merely used to distinguish different components. Terms such as "comprising" or "including" mean that the element or object preceding the word encompasses the elements or objects listed following the word and their equivalents, without excluding other elements or objects. Terms such as "connected" or "linked" are not limited to physical or mechanical connections, but can include electrical connections, whether direct or indirect. Terms such as "upper," "lower," "left," and "right" are used only to indicate relative positional relationships; when the absolute position of the described object changes, the relative positional relationship may also change accordingly.

[0021] Example: Please see Figure 1 The present invention provides the following technical solutions: A rapid detection method for Salmonella in food based on fluorescent probes includes the following steps: S1: Obtain the matrix interference characteristic parameters of the food sample to be tested. These parameters include fat content, protein content, and pigment content. The fat content is determined by near-infrared spectroscopy or Soxhlet extraction; the protein content is determined by near-infrared spectroscopy or Kjeldahl method; and the pigment content is obtained by measuring absorbance at 410 nm or 280 nm using UV-Vis spectrophotometry. Fat, protein, and pigment are the three main interfering substances in food samples. Fat interferes with PCR amplification by inhibiting Taq enzyme activity; protein reduces the binding efficiency of nucleic acids to the solid-phase carrier by increasing solution viscosity; and pigment directly interferes with fluorescence detection by absorbing or scattering fluorescence signals. These three substances act on different stages of the detection process and together constitute the core indicator for evaluating the difficulty of food matrix pretreatment. After obtaining the above three parameters, they are substituted into the formula for calculating the comprehensive interference index.

[0022] The comprehensive interference index is calculated using the following formula: in: The comprehensive interference index; Fat content; Protein content; This refers to the pigment content; The item is for fat, and its fat content is... It is used in calculations in logarithmic form to characterize the physical law that the inhibitory effect of fat on PCR amplification decreases marginally with increasing concentration; The item is protein, including protein content. It is used in calculations in the form of square roots to characterize the sublinear growth characteristics of proteins that increase solution viscosity and decrease nucleic acid binding efficiency; For pigments, the pigment content is... It participates in the calculation in a linear form to characterize the photophysical properties of the quenching effect of pigments on fluorescence signals, which are strictly proportional to their concentration. The fat interference coefficient is used to adjust the contribution weight of fat content to the overall interference index. Its value is positively correlated with the degree of inhibition of PCR polymerase activity by fat. The protein interference coefficient is used to adjust the contribution weight of protein content to the overall interference index. Its value is positively correlated with the efficiency of protein in increasing solution viscosity and hindering the binding of nucleic acids to the solid-phase carrier. The pigment interference coefficient is used to adjust the contribution weight of pigment content to the comprehensive interference index. Its value is positively correlated with the absorption or quenching intensity of the fluorescence signal by the pigment. The three coefficients mentioned above are all dimensionless constants, pre-calibrated through standard interference spike recovery experiments. Their function is to unify the measured indicators of fat, protein, and pigment—three different dimensions and physical mechanisms—to the same scale, ensuring that the contribution weight of each factor to the overall interference index matches its actual interference intensity. Since different fluorescent probe detection platforms have varying tolerances to interference, orthogonal experiments can be used to refine the coefficients for specific detection systems. , , The value of is optimized and adjusted to achieve the best match between the comprehensive interference index and the preprocessing difficulty level of the corresponding detection platform.

[0023] S2: Based on the comprehensive interference index calculated by S1, generate the number of washes, elution volume and centrifugation speed for the pretreatment operation respectively; The number of washes is calculated using the following formula: in: The number of washes is the final number of times the washing operation is repeated, expressed as a positive integer. The baseline number of washes represents the minimum number of washes required to effectively separate nucleic acids from interfering substances under interference-free conditions. Using standard samples free of interfering substances, 1 to 5 washes were set, and the recovery rate of the internal standard under each condition was measured. The minimum number of washes required for the recovery rate to reach ≥70% and tend to stabilize was taken as the value of W0. The washing coefficient represents the increase in the number of washes required to compensate for a unit increase in the overall disturbance index F value. A higher F-value indicates greater sensitivity of the pretreatment operation to the complexity of the sample matrix. A series of standard interfering samples containing different concentrations of fat, protein, and pigment were prepared. With a recovery rate greater than or equal to 70% and a detection sensitivity of 10 CFU / mL as the target, an orthogonal experimental design was used to determine the optimal number of washes for each interfering concentration. The optimal number of washes required to increase by a unit F-value was fitted to determine the optimal number of washes. The value of .

[0024] The elution volume is calculated using the following formula: in: This is the elution volume; The baseline elution volume represents the minimum volume of elution buffer required to fully release the target nucleic acid from the solid support under interference-free conditions. When the sample matrix does not contain interfering substances such as fats, proteins, or pigments, the elution efficiency is limited only by the physicochemical properties of the solid support surface. The baseline elution volume is determined by measuring the nucleic acid recovery rate at different volumes, based on ensuring that the elution buffer can completely immerse the solid support and form a continuous liquid film. η is the elution coefficient, characterizing the response strength of the elution volume as the comprehensive interference index F increases. A larger η value indicates a greater increase in elution volume corresponding to a unit increase in F value, meaning a higher degree of compensation for sample matrix complexity by the pretreatment operation. The η value was determined by constraining the nucleic acid concentration of the eluted product to be no less than the detection sensitivity requirement, and was obtained through recovery experiments on different matrix samples.

[0025] The centrifugal speed is calculated using the following formula. in: Centrifugal speed; The reference centrifuge speed represents the minimum centrifuge speed required to achieve effective separation of the solid carrier and the liquid matrix under interference-free conditions. When the food sample does not contain thickening or interfering substances such as fat, protein, and pigments, the solution viscosity is close to that of pure water, and the sedimentation resistance of the solid carrier is minimal. The reference centrifuge speed is determined based on ensuring that the solid carrier completely precipitates to a clear supernatant within a set centrifugation time. It is determined by measuring the turbidity of the supernatant and the recovery rate of the solid carrier at different speeds. The centrifugal coefficient represents the increase in centrifugal speed required to increase the overall disturbance index F value per unit. A higher F-value indicates a greater sensitivity of the centrifugation operation to the complexity of the sample matrix; that is, for every unit increase in the F-value, the centrifugation speed should increase accordingly. rpm; The determination of the separation efficiency was based on the constraint that the solid carrier effectively precipitates in each centrifugation step and the target nucleic acid is not pressed into the precipitate. It was obtained by fitting the separation efficiency of different matrix samples through experiments, ensuring that complex samples can still be effectively separated within a limited centrifugation time.

[0026] The number of washes is positively correlated with the comprehensive interference index. A higher comprehensive interference index indicates a higher content of interfering substances such as fat, protein, or pigments in the food sample, requiring more washes to fully remove residual interfering substances. The elution volume is also positively correlated with the comprehensive interference index. A higher comprehensive interference index indicates a more complex sample matrix and a tighter binding between the target nucleic acid and the solid-phase carrier, requiring a larger elution volume to ensure that the target nucleic acid is fully released and collected. However, the increase in elution volume is limited by linear structure control to avoid excessive dilution. The centrifugation speed is also positively correlated with the comprehensive interference index. A higher comprehensive interference index indicates a higher content of thickening substances in the sample, a higher solution viscosity, and a greater sedimentation resistance of the solid-phase carrier, requiring a higher centrifugation speed to generate sufficient centrifugal force to ensure effective precipitation of the solid-phase carrier in each centrifugation separation step.

[0027] The number of washing cycles is used to characterize the adequacy of impurity removal during pretreatment. The more washing cycles, the more thoroughly the non-specific adsorbed fats, proteins, pigments, and other interfering substances on the solid-phase carrier surface are removed, resulting in higher product purity. However, excessive washing cycles can also lead to partial elution of the bound target nucleic acids, causing a decrease in recovery rate. Therefore, the number of washing cycles needs to be precisely set according to the comprehensive interference index.

[0028] The elution volume is used to characterize the completeness of the release and recovery of the target nucleic acid from the solid support. The larger the elution volume, the higher the proportion of the target nucleic acid remaining on the surface and in the pores of the solid support is dissolved and collected. However, excessively increasing the elution volume will also dilute the concentration of the final product and affect the detection sensitivity. Therefore, it needs to be controlled within an appropriate range.

[0029] The centrifugation speed is used to characterize the effectiveness of the separation between the solid support and the liquid matrix in each pretreatment step. The higher the speed, the greater the centrifugal force, the faster the sedimentation rate of the solid support and the denser the precipitate. However, excessive speed may push the target nucleic acid into the precipitate and cause loss. Therefore, it is necessary to select an appropriate centrifugation force according to the complexity of the sample matrix.

[0030] The number of washes, elution volume, and centrifugation speed work together to form a complete operating parameter system that matches the difficulty of sample pretreatment. These parameters ensure the purity, recovery rate, and separation efficiency of the target nucleic acid at different stages of the pretreatment process, and together ensure that the quality of the pretreatment product meets the requirements of subsequent fluorescent probe detection.

[0031] S3: Add a known amount of fluorescent internal standard to the food sample to be tested. The fluorescent internal standard is a synthetic nucleic acid fragment labeled with a fluorescent dye. Its sequence has no homology with the Salmonella target gene. It does not participate in the amplification reaction of the target Salmonella nucleic acid, nor does it specifically bind to the food matrix components. It is only used as a tracer in the entire pretreatment process to monitor the loss of the target nucleic acid during the pretreatment process.

[0032] After adding a known amount of fluorescent internal standard to the food sample to be tested and mixing thoroughly, the fluorescence is measured using a fluorescence detection device under preset excitation and emission wavelength conditions. The obtained fluorescence signal value is the initial fluorescence intensity, in relative fluorescence units, which serves as the benchmark reference value for calculating the recovery rate.

[0033] Subsequently, sample pretreatment operations were performed according to the number of washes, elution volume, and centrifugation speed. The pretreatment operations included, in sequence: cell lysis to release nucleic acid, selective binding of nucleic acid to a solid-phase carrier, multiple washes according to the number of washes generated to remove residual impurities, centrifugation according to the centrifugation speed generated, and elution and collection of target nucleic acid according to the elution volume generated.

[0034] After pretreatment, the residual fluorescence intensity of the fluorescent internal standard in the treated solution was measured again using the same fluorescence detection equipment under the same wavelength conditions. The measured internal standard recovery rate was calculated by comparing the fluorescence intensity before and after pretreatment, so as to quantitatively evaluate the retention ratio of the fluorescent internal standard in the whole pretreatment process.

[0035] The measured internal standard recovery rate was calculated using the following formula: in: This represents the measured internal standard recovery rate. The initial fluorescence intensity of the fluorescent internal standard before pretreatment; The residual fluorescence intensity of the fluorescent internal standard after pretreatment is given.

[0036] S4: Generate a dynamic judgment threshold based on the comprehensive interference index. The dynamic judgment threshold is calculated using the following formula: in: The threshold is determined dynamically. The preset threshold represents the lowest acceptable internal standard recovery rate of this invention. This threshold is set based on the basic requirements of the food safety testing field: if the internal standard recovery rate is less than 50%, it indicates that more than half of the target nucleic acid was lost during the pretreatment process, and the reliability of the results cannot be guaranteed if the test is continued. Therefore, the threshold should not be further lowered.

[0037] The baseline recovery threshold represents the minimum internal standard recovery rate required to determine the effectiveness of sample pretreatment under interference-free conditions. The value of is determined as follows: using standard samples that do not contain interfering substances such as fat, protein and pigment as the test object, the internal standard recovery rate is determined under the baseline pretreatment conditions, and a recovery rate of not less than 70% is used as the criterion for acceptance. As the upper limit of the judgment threshold, when the sample does not contain interfering substances, the measured internal standard recovery rate must reach [a certain percentage]. The decision can be passed; , k, is the threshold adjustment coefficient used to control the rate at which the dynamic judgment threshold decreases as the comprehensive interference index F increases. A larger k value indicates a greater decrease in the threshold with increasing sample matrix complexity, meaning a higher tolerance for complex samples; a smaller k value indicates less influence of matrix complexity on the threshold, resulting in a stricter judgment standard. k is determined by balancing the recovery rate and detection accuracy for different matrix samples, and the optimal value maximizing the overall accuracy is selected through ROC curve analysis.

[0038] The dynamic judgment threshold is negatively correlated with the comprehensive interference index. A higher comprehensive interference index indicates a higher content of interfering substances such as fat, protein, or pigments in the food sample, a greater risk of physical loss of the target nucleic acid during pretreatment, and a higher possibility of fluorescence signal quenching. Objectively, the internal standard recovery rate is unlikely to reach the level of a pure sample, thus the dynamic judgment threshold is correspondingly lowered. When the F value is low, the sample matrix is ​​pure, pretreatment interference is small, and the dynamic judgment threshold is close to the baseline value. The judgment criteria are stringent; as the F-value increases, the sample matrix becomes more complex, and the dynamic judgment threshold is appropriately lowered to provide reasonable tolerance for complex samples. This avoids unnecessary error correction operations caused by a large number of valid pretreatments being misjudged as invalid due to an overly strict fixed threshold. This negative correlation design allows the judgment criteria to adaptively adjust with the difficulty of sample pretreatment, ensuring strict quality control for pure samples while avoiding excessive demands on complex samples. It minimizes invalid repeated processing while maintaining detection accuracy, thereby improving detection throughput and method economy.

[0039] like This indicates that the retention rate of the fluorescent internal standard after undergoing the complete pretreatment process, including cell lysis, nucleic acid binding, washing to remove impurities, centrifugation, and elution collection, has reached an acceptable level commensurate with the complexity of the food sample matrix. At this point, the degree of physical loss of the target nucleic acid during pretreatment and the interference of residual inhibitors on subsequent PCR amplification are within acceptable limits. The purity and concentration of the pretreatment product meet the requirements for downstream fluorescent probe detection. Therefore, the sample pretreatment is deemed effective, and the obtained pretreatment product can be directly used in subsequent fluorescent probe detection steps.

[0040] like This indicates that the fluorescent internal standard was excessively lost during pretreatment or that the fluorescence signal was excessively quenched by residual pigments or other substances in the sample, meaning the pretreatment effect was not acceptable. Continuing detection under these conditions would face a high risk of false negatives. Therefore, the sample pretreatment was deemed invalid. The number of washes (W) was increased by one, and the process was returned to step S3 to re-execute the pretreatment.

[0041] Perform the washing, centrifugation, and elution collection steps in S3, without repeating the cell lysis and nucleic acid binding steps. The rationale for this design is that cell lysis and nucleic acid binding have already been completed in the initial pretreatment, and the target nucleic acid has been immobilized on the surface of the solid carrier. Repeating the above steps will not only fail to remove the bound interfering substances, but will also cause unnecessary loss of the captured nucleic acid. Therefore, the washing, centrifugation, and elution steps should be enhanced only to selectively remove interfering substances such as fats, proteins, and pigments remaining on the surface of the solid carrier.

[0042] After repeating the above steps, measure the residual fluorescence intensity of the fluorescent internal standard in the treated solution again, recalculate the measured internal standard recovery rate, and compare it with the dynamic judgment threshold again. Repeat the above cyclic operation until the measured internal standard recovery rate reaches or exceeds the dynamic judgment threshold, or the cumulative number of error corrections reaches the preset upper limit of error corrections.

[0043] If the measured internal standard recovery rate is still lower than the dynamic judgment threshold after reaching the preset error correction limit, it indicates that the concentration of interfering substances in the current sample matrix exceeds the carrying capacity limit of the pretreatment system of this method. At this time, the system will issue an alarm signal to remind the operator that the current matrix interference is too strong and it is recommended to dilute and re-detect or change to a suitable detection method to avoid false negative results caused by insufficient pretreatment.

[0044] The above formulas are all dimensionless calculations. The formulas are derived from software simulations based on a large amount of collected data to obtain the most recent real-world results. The preset parameters in the formulas are set by those skilled in the art according to the actual situation.

[0045] The above embodiments can be implemented, in whole or in part, by software, hardware, firmware, or any other combination thereof. When implemented in software, the above embodiments can be implemented, in whole or in part, as a computer program product. Those skilled in the art will recognize that the units and algorithm steps of the various examples described in conjunction with the embodiments disclosed herein can be implemented by electronic hardware, or a combination of computer software and electronic hardware. Whether these functions are implemented in hardware or software depends on the specific application and design constraints of the technical solution.

[0046] The units described as separate components may or may not be physically separate. The components shown as units may or may not be physical units; they may be located in one place or distributed across multiple network units. Some or all of the units can be selected to achieve the purpose of this embodiment, depending on actual needs.

[0047] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application.

Claims

1. A rapid detection method for Salmonella in food based on fluorescent probes, characterized in that, Includes the following steps: S1: Obtain matrix interference characteristic parameters, including: fat content, protein content and pigment content of the food sample to be tested; calculate the comprehensive interference index based on the fat content, protein content and pigment content; the comprehensive interference index is used to characterize the pretreatment difficulty of the food sample to be tested. S2: Based on the comprehensive interference index calculated in S1, generate the number of washes, elution volume and centrifugation speed of the pretreatment operation, wherein the number of washes, elution volume and centrifugation speed are all positively correlated with the comprehensive interference index. S3: Add a known amount of fluorescent internal standard to the food sample to be tested, and measure the initial fluorescence intensity of the fluorescent internal standard before pretreatment; perform sample pretreatment according to the washing number, elution volume and centrifugation speed generated in S2; After pretreatment, the residual fluorescence intensity of the fluorescent internal standard is measured, and the measured internal standard recovery rate is calculated based on the initial fluorescence intensity and the residual fluorescence intensity. S4: Generate a dynamic judgment threshold based on the comprehensive interference index, wherein the dynamic judgment threshold is negatively correlated with the comprehensive interference index; compare the measured internal standard recovery rate with the dynamic judgment threshold: if the measured internal standard recovery rate is greater than or equal to the dynamic judgment threshold, the sample pretreatment is deemed effective and the obtained pretreatment product is used for subsequent fluorescent probe detection; if the measured internal standard recovery rate is less than the dynamic judgment threshold, the sample pretreatment is deemed invalid, and the number of washing cycles is increased before returning to S3 to re-execute the pretreatment.

2. The rapid detection method for Salmonella in food based on fluorescent probes according to claim 1, characterized in that: The comprehensive interference index is calculated using the following formula: in: The comprehensive interference index; Fat content; Protein content; This refers to the pigment content; For fat items; For protein; For pigments; The fat interference coefficient; The protein interference coefficient; This represents the pigment interference coefficient.

3. The rapid detection method for Salmonella in food based on fluorescent probes according to claim 2, characterized in that: The number of washes is calculated using the following formula: in: Number of washes; The baseline number of washes; The washing coefficient represents the increase in the number of washes required to increase the overall disturbance index F value per unit.

4. The rapid detection method for Salmonella in food based on fluorescent probes according to claim 2, characterized in that: The elution volume is calculated using the following formula: in: This is the elution volume; Use the baseline elution volume; is the elution coefficient, used to control the response intensity of the elution volume as the comprehensive interference index F changes.

5. The rapid detection method for Salmonella in food based on fluorescent probes according to claim 2, characterized in that: The centrifugal speed is calculated using the following formula. in: Centrifugal speed; The reference centrifugal speed; The centrifugal coefficient represents the increase in centrifugal speed required to increase the overall disturbance index F value per unit.

6. The rapid detection method for Salmonella in food based on fluorescent probes according to claim 5, characterized in that: A known amount of fluorescent internal standard is added to the food sample to be tested. The fluorescent internal standard is a synthetic nucleic acid fragment labeled with a fluorescent dye, which does not participate in the amplification reaction of the target Salmonella nucleic acid, but only serves as a tracer in the pretreatment process. After addition, the initial fluorescence intensity of the fluorescent internal standard before pretreatment is measured using a fluorescence detection device. Subsequently, sample pretreatment operations were performed according to the number of washes, elution volume, and centrifugation speed. The pretreatment operations included cell lysis, binding of nucleic acids to a solid-phase carrier, washing to remove impurities according to the number of washes, centrifugation separation according to the centrifugation speed, and elution collection of target nucleic acids according to the elution volume. After the pretreatment was completed, the residual fluorescence intensity of the fluorescent internal standard in the treated solution was measured again using a fluorescence detection device.

7. The rapid detection method for Salmonella in food based on fluorescent probes according to claim 6, characterized in that: The measured internal standard recovery rate was calculated using the following formula: in: This represents the measured internal standard recovery rate. The initial fluorescence intensity of the fluorescent internal standard before pretreatment; The residual fluorescence intensity of the fluorescent internal standard after pretreatment is given.

8. The rapid detection method for Salmonella in food based on fluorescent probes according to claim 7, characterized in that: The dynamic determination threshold is calculated using the following formula: in: The threshold is determined dynamically. This is the preset lower threshold. The baseline recovery rate threshold; This is the threshold adjustment coefficient.

9. The rapid detection method for Salmonella in food based on fluorescent probes according to claim 8, characterized in that: The measured internal standard recovery rate obtained Compare with the dynamic judgment threshold T generated based on the comprehensive interference index F; if This indicates that the retention rate of the fluorescent internal standard during the pretreatment process has reached an acceptable level commensurate with the complexity of the food sample matrix. In other words, the loss of the target nucleic acid and the interference from residual inhibitors during pretreatment are both within acceptable limits. Therefore, the sample pretreatment is deemed effective, and the resulting pretreatment product can be directly used for subsequent fluorescent probe detection. This indicates that the fluorescent internal standard was lost too much during the pretreatment process or the fluorescence signal was excessively quenched, meaning that the pretreatment effect did not reach an acceptable level. Therefore, the sample pretreatment was deemed invalid. In this case, the number of washes was increased by 1, and the process was returned to S3 to re-execute the pretreatment.

10. A rapid detection method for Salmonella in food based on a fluorescent probe according to claim 9, characterized in that: Return to the washing, centrifugation, and elution collection operations in S3, without repeating the cell lysis and nucleic acid binding to the solid-phase carrier operations; then, measure the residual fluorescence intensity of the fluorescent internal standard in the treated solution again, calculate the new measured internal standard recovery rate, and compare it again with the dynamic judgment threshold. Repeat the loop until... Or until the preset limit for the number of error corrections is reached; If the preset error correction limit is reached... Still smaller If the interference from the sample matrix is ​​too strong, an alarm signal will be issued, indicating that the sample matrix interference is too strong and suggesting that the sample be diluted and retested or that the detection method be changed.

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