Multi-target fluorescent staining rapid sample preparation virus detection method

Abstract

The invention relates to the technical field of virus detection, and discloses a multi-target fluorescent staining rapid sample preparation virus detection method, which comprises the following steps: S1, sample pretreatment; s2, multi-target fluorescent dyeing: mixing the pretreated sample in S1 with a multi-target fluorescent dye combination to obtain a dyed sample; s3, purification after dyeing; s4, optical imaging acquisition; s5, image analysis and judgment: inputting the fluorescence image in S4 into an image analysis system, extracting fluorescence characteristic parameters of each target spot, and comparing the fluorescence characteristic parameters with a preset threshold value; and S6, outputting a result. By adopting the technical scheme of combining multi-target fluorescent staining and multi-target co-localization judgment, the technical effects of accurately identifying target viruses and avoiding non-specific interference are achieved, and compared with the technical scheme of single-target fluorescent staining or single-signal judgment in the prior art, the technical scheme of the invention has the advantages that the accuracy is high; the problems that the specificity is insufficient, and false positive and false negative occur due to interference of environmental impurities or similar pathogens are solved.

Description

Technical Field

[0001] This invention relates to the field of virus detection technology, specifically to a rapid sample preparation and virus detection method using multi-target fluorescent staining. Background Technology

[0002] Virus detection technology is a core support for the diagnosis of clinical infectious diseases, epidemic prevention and control, and public health security. Among them, fluorescence staining detection technology has been widely used in the field of rapid virus screening due to its advantages such as relatively simple operation, no need for complicated sample culture procedures, and rapid acquisition of detection signals. Most existing fluorescence staining detection schemes adopt a single-target design, that is, a specific fluorescent dye is selected to stain a single key structure or marker of the virus, and the virus is determined by detecting the presence or intensity of a single fluorescence signal. This type of technology has initially met the rapid screening needs in some low-interference sample detection scenarios.

[0003] However, the single-target fluorescence staining or single signal determination techniques commonly used in existing technologies lack multi-target cross-validation mechanisms. Relying solely on the binding and signal recognition of a single viral target makes it difficult to effectively distinguish between the target virus and environmental impurities, non-target pathogens, or structurally similar interfering substances in clinical samples. This results in insufficient detection specificity, a high likelihood of false positive or false negative results, and misleading clinical diagnosis, failing to meet the high requirements for accuracy and reliability in virus detection. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a rapid sample preparation and virus detection method using multi-target fluorescent staining. This method solves the problems of insufficient specificity and susceptibility to interference from environmental impurities or similar pathogens, which can lead to false positives and false negatives in existing single-target fluorescent staining or single-signal determination techniques.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a rapid sample preparation and virus detection method using multi-target fluorescent staining, comprising the following steps: S1. Sample pretreatment: Take the clinical sample to be tested, add an equal volume of sample lysis buffer to mix the sample with the lysis buffer, and incubate at 25-37℃ for 5-10 min to obtain the pretreated sample. The sample lysis buffer does not contain nuclease and retains the key structure of the virus and the specific binding sites of the markers. S2. Multi-target fluorescent staining: Mix the pretreated sample from S1 with the multi-target fluorescent dye combination and incubate at 30-37℃ in the dark for 8-15 minutes to obtain the stained sample; the multi-target fluorescent dye combination includes at least two specific fluorescent dyes, each of which specifically binds to different key structures or markers of the target virus, and the emission wavelengths of each fluorescent dye do not overlap. S3. Post-staining purification: The stained sample from S2 is centrifuged and filtered through a filter membrane to remove free fluorescent dye and sample impurities, resulting in a purified stained sample. S4. Optical Imaging Acquisition: Place the purified and stained sample from S3 into the optical imaging system, set the imaging conditions according to the spectral characteristics of the fluorescent dye, and acquire the fluorescence image. S5. Image Analysis and Judgment: Input the fluorescence image from S4 into the image analysis system, extract the fluorescence feature parameters of each target point and compare them with the preset threshold, and determine the detection result based on the co-localization of multiple targets. S6. Result Output: Automatically outputs the detection results and fluorescence signal characteristic report.

[0006] Preferably, in step S1, the sample lysis buffer includes 0.1%-1% nonionic surfactant, 5-20mM buffer, and pH 7.0-7.8. The sample and lysis buffer are mixed using a vortex oscillation method.

[0007] Preferably, in S2, the key viral structures or markers include at least two of the following: viral capsid proteins, envelope proteins, nucleic acid proteins, or enzyme proteins.

[0008] Preferably, in step S2, the fluorescent dye is selected from at least two of fluorescein, rhodamine, cyanine dyes or quantum dots, and the difference in emission wavelength between the dyes is ≥50nm; The volume ratio of the multi-target fluorescent dye combination to the pretreated sample is 1:5-1:10, and after mixing, it is gently oscillated continuously at a frequency of 500-800 rpm for 30-60 s.

[0009] Preferably, in step S3, the filter membrane pore size is 0.22-0.45 μm, the centrifugation speed is 8000-10000 rpm, and the centrifugation time is 1-2 min.

[0010] Preferably, in step S4, the optical imaging system is a fluorescence microscope or a laser confocal imaging system, which can simultaneously or sequentially acquire fluorescence signals from different target points; The exposure time of the optical imaging system is set to 0.1-1.0s, and the imaging area covers the entire effective area of ​​the sample drop, with an effective area coverage rate of ≥95%.

[0011] Preferably, in step S5, the fluorescence characteristic parameters include fluorescence intensity, signal distribution density, and multi-target co-localization rate, and the preset threshold is obtained by calibration using standard positive samples.

[0012] Preferably, in step S5, the determination rule includes: if the fluorescence characteristic parameters of at least two target points meet the preset threshold and the co-localization rate is ≥70%, then it is determined to be positive; otherwise, it is negative.

[0013] Preferably, the clinical samples to be tested include: blood, sputum, throat swabs, nasopharyngeal swabs, or stool samples.

[0014] Preferably, in step S1, during the vortex oscillation process, the temperature of the mixture of sample and lysis solution is maintained at 25-30°C, the frequency of vortex oscillation is 1000-1500 rpm, and the oscillation time is 10-20 s.

[0015] This invention provides a rapid sample preparation and virus detection method using multi-target fluorescent staining. It offers the following advantages: 1. This invention achieves the technical effect of accurately identifying target viruses and avoiding non-specific interference by adopting a technical solution that combines multi-target fluorescent staining with multi-target co-localization determination. Compared with the existing single-target fluorescent staining or single signal determination technical solutions, it solves the problems of insufficient specificity and susceptibility to interference from environmental impurities or similar pathogens, resulting in false positives and false negatives.

[0016] 2. The present invention adopts a technical solution of centrifugation purification of filter membrane after staining, which achieves the technical effect of effectively separating free fluorescent dye and bound virus and optimizing the quality of fluorescence imaging. Compared with the existing fluorescent staining detection scheme that lacks a targeted impurity removal step, it solves the problems of high background noise, low fluorescence signal recognition, and the impact on the accuracy of subsequent image analysis.

[0017] 3. This invention employs a rapid pretreatment with nuclease-free lysis buffer, active oscillation mixing, and synergistic adaptation parameters for each step. This achieves the technical effects of simplifying the sample preparation process, improving detection efficiency, and enhancing result consistency. Compared with existing technologies that rely on complex sample culture, cumbersome pretreatment processes, or lack synergistic design of parameters for each step, this invention solves the problems of complex operation, lengthy processing time, poor result stability, and difficulty in meeting the needs of rapid clinical screening and automated promotion. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the process flow for a rapid sample preparation and virus detection method using multi-target fluorescent staining according to the present invention. Detailed Implementation

[0019] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0020] Please see the appendix Figure 1This invention provides a rapid sample preparation method for virus detection using multi-target fluorescent staining, comprising the following steps: S1. Sample pretreatment: Take the clinical sample to be tested, add an equal volume of sample lysis buffer to mix the sample with the lysis buffer, and incubate at 25-37℃ for 5-10 min to obtain the pretreated sample. The sample lysis buffer does not contain nuclease and retains the key structure of the virus and the specific binding sites of the markers. In S1, the sample lysis buffer includes 0.1%-1% nonionic surfactant and 5-20mM buffer, with a pH of 7.0-7.8. The sample and lysis buffer are mixed using a vortex oscillation method. In S1, during the vortex oscillation process, the temperature of the mixture of sample and lysis buffer is maintained at 25-30℃, the frequency of vortex oscillation is 1000-1500rpm, and the oscillation time is 10-20s. Clinical samples to be tested include: blood, sputum, throat swabs, nasopharyngeal swabs, or stool samples; S2, Multi-target fluorescent staining: Mix the pretreated sample from S1 with the multi-target fluorescent dye combination and incubate at 30-37℃ in the dark for 8-15 minutes to obtain the stained sample; the multi-target fluorescent dye combination includes at least two specific fluorescent dyes, each of which specifically binds to different key structures or markers of the target virus, and the emission wavelengths of each fluorescent dye do not overlap. In S2, the key viral structures or markers include at least two of the following: viral capsid protein, envelope protein, nucleic acid, or enzyme protein. In S2, the fluorescent dye is selected from at least two of the following: fluorescein, rhodamine, cyanine dyes, or quantum dots, and the difference in emission wavelength between the dyes is ≥50nm. The volume ratio of the multi-target fluorescent dye combination to the pretreated sample is 1:5-1:10, and after mixing, it is gently oscillated continuously at a frequency of 500-800 rpm for 30-60 s. S3. Post-staining purification: The stained sample from S2 is centrifuged and filtered through a filter membrane to remove free fluorescent dye and sample impurities, resulting in a purified stained sample. In S3, the filter membrane pore size is 0.22-0.45μm, the centrifugation speed is 8000-10000rpm, and the centrifugation time is 1-2min; S4. Optical Imaging Acquisition: Place the purified and stained sample from S3 into the optical imaging system, set the imaging conditions according to the spectral characteristics of the fluorescent dye, and acquire the fluorescence image. In S4, the optical imaging system is a fluorescence microscope or a laser confocal imaging system, which can simultaneously or sequentially acquire fluorescence signals from different target points. The exposure time of the optical imaging system is set to 0.1-1.0 s, and the imaging area covers the entire effective area of ​​the sample drop, with an effective area coverage rate of ≥95%. S5. Image Analysis and Judgment: Input the fluorescence image from S4 into the image analysis system, extract the fluorescence feature parameters of each target point and compare them with the preset threshold, and determine the detection result based on the co-localization of multiple targets. In S5, the fluorescence characteristic parameters include fluorescence intensity, signal distribution density, and multi-target co-localization rate. The preset threshold is obtained by calibration using standard positive samples. In S5, the judgment rules include: if the fluorescence characteristic parameters of at least two targets meet the preset threshold and the co-localization rate is ≥70%, then it is judged as positive; otherwise, it is negative. S6. Result Output: Automatically outputs the detection results and fluorescence signal characteristic report.

[0021] Tris-HCl buffer is a tris(hydroxymethyl)aminomethane hydrochloride buffer, designed for sample lysis buffers containing 5-20 mM buffer at pH 7.0-7.8. The following preparation method for Tris-HCl buffer is provided: Preparation Example 1 Accurately weigh 0.6057g of Tris base using an electronic balance and place it in a 500mL beaker; Add 400 mL of deionized water and stir on a magnetic stirrer until completely dissolved; Use a pipette to draw concentrated hydrochloric acid and slowly add it dropwise to the above solution while stirring. At the same time, monitor the pH in real time with a calibrated pH meter until the pH of the solution stabilizes at 7.0. Transfer the pH-adjusted solution to a 1000mL volumetric flask, wash the beaker three times with deionized water, transfer all the washings into the volumetric flask, and finally add deionized water to the mark and shake well. Aliquot the adjusted buffer solution into sterile reagent bottles, seal and store at 4°C. Shelf life is 6 months.

[0022] Preparation Example 2 Accurately weigh 1.4537g of Tris base and place it in a 500mL beaker; Add 400 mL of deionized water and stir until completely dissolved; Slowly add concentrated hydrochloric acid, stirring and monitoring the pH until the solution pH stabilizes at 7.4; Transfer to a 1000mL volumetric flask, wash the beaker and dilute to the mark, then shake well; Store in a sealed container at 4℃. Shelf life is 6 months.

[0023] Preparation Example 3 Accurately weigh 2.4228g of Tris base and place it in a 500mL beaker; Add 400 mL of deionized water and stir until completely dissolved; Slowly add concentrated hydrochloric acid, stirring and monitoring the pH until the solution pH stabilizes at 7.8; Transfer to a 1000mL volumetric flask, wash the beaker and dilute to the mark, then shake well; Store in a sealed container at 4℃. Shelf life is 6 months.

[0024] The following is a description with reference to specific embodiments: Among luciferins, FITC-S targets the S protein, while Cy3-N targets the nucleocapsid protein N. Example

[0025] A rapid sample preparation and virus detection method using multi-target fluorescent staining includes the following steps: S1. Sample pretreatment: Take 100 μL of clinical pharyngeal swab sample and add an equal volume of 100 μL of sample lysis buffer. The sample lysis buffer includes 0.1% nonionic surfactant, 5 mM Tris-HCl buffer, and pH 7.0. When mixing the sample and lysis buffer, use vortexing at a frequency of 1000 rpm for 10 s. During the vortexing process, maintain the temperature of the mixture at 25℃. Then incubate at 25℃ for 5 min to obtain the pretreated sample. S2. Multi-target fluorescent staining: Add a multi-target fluorescent dye combination to the pretreated sample obtained in S1. The volume ratio of the dye combination to the pretreated sample is 1:10. The dye combination includes two specific fluorescent dyes: FITC-S (525nm emission wavelength) against the spike protein of the novel coronavirus and Cy3-N (575nm emission wavelength) against the nucleocapsid protein. The difference in emission wavelength between the two dyes is 50nm. After mixing, gently oscillate at a frequency of 500rpm for 30s. Then incubate at 30℃ in the dark for 8min to obtain the stained sample. S3. Post-staining purification: Transfer the stained sample obtained in S2 into a filter membrane centrifuge tube with a filter membrane pore size of 0.22 μm. Centrifuge at 8000 rpm for 1 min to remove free fluorescent dye and sample impurities, and obtain the purified stained sample. S4. Optical Imaging Acquisition: The stained sample purified in S3 is dropped onto the imaging slide and placed in a fluorescence microscope. Fluorescence signals of different target points are acquired sequentially. The exposure time is set to 0.1s. The imaging area covers the entire effective area of ​​the sample drop, with an effective area coverage rate of ≥95%. Fluorescence images of dual target points are acquired. S5. Image Analysis and Judgment: Input the fluorescence image acquired in S4 into the image analysis system to extract three types of fluorescence characteristic parameters: fluorescence intensity, signal distribution density, and multi-target co-localization rate. The preset threshold is obtained by calibration using standard positive samples of COVID-19. The judgment rule is that if the fluorescence characteristic parameters of both targets meet the preset threshold and the co-localization rate is ≥70%, it is judged as positive; otherwise, it is negative. S6. Result Output: The image analysis system automatically outputs the detection results and fluorescence signal feature report to complete the virus detection. Example

[0026] A rapid sample preparation and virus detection method using multi-target fluorescent staining includes the following steps: S1. Sample pretreatment: Take 100 μL of clinical pharyngeal swab sample and add an equal volume of 100 μL of sample lysis buffer. The sample lysis buffer includes 0.5% nonionic surfactant, 12 mM Tris-HCl buffer, and pH 7.4. When mixing the sample and lysis buffer, use vortexing at a frequency of 1200 rpm for 15 s. During the vortexing process, maintain the temperature of the mixture at 28 ℃. Then incubate at 37 ℃ for 8 min to obtain the pretreated sample. S2. Multi-target fluorescent staining: A multi-target fluorescent dye combination was added to the pretreated sample obtained in S1. The volume ratio of the dye combination to the pretreated sample was 1:8. The dye combination included two specific fluorescent dyes: FITC-S (525nm emission wavelength) against the SARS-CoV-2 spike protein and Cy3-N (585nm emission wavelength) against the nucleocapsid protein. The difference in emission wavelength between the two dyes was 60nm. After mixing, the sample was gently shaken continuously at a frequency of 650 rpm for 45 seconds. Then, it was incubated at 37°C in the dark for 10 minutes to obtain the stained sample. S3. Post-staining purification: Transfer the stained sample obtained in S2 into a filter membrane centrifuge tube with a filter membrane pore size of 0.3 μm. Centrifuge at 9000 rpm for 1.5 min to remove free fluorescent dye and sample impurities, and obtain the purified stained sample. S4. Optical Imaging Acquisition: The stained sample purified in S3 is dropped onto the imaging slide and placed into the laser confocal imaging system. Fluorescence signals of different target points are acquired simultaneously. The exposure time is set to 0.5s. The imaging area covers the entire effective area of ​​the sample drop, with an effective area coverage rate of ≥95%. Dual-target fluorescence images are acquired. S5. Image Analysis and Judgment: Input the fluorescence image acquired in S4 into the image analysis system to extract three types of fluorescence characteristic parameters: fluorescence intensity, signal distribution density, and multi-target co-localization rate. The preset threshold is obtained by calibration using standard positive samples of COVID-19. The judgment rule is that if the fluorescence characteristic parameters of both targets meet the preset threshold and the co-localization rate is ≥70%, it is judged as positive; otherwise, it is negative. S6. Result Output: The image analysis system automatically outputs the detection results and fluorescence signal feature report to complete the virus detection. Example

[0027] A rapid sample preparation and virus detection method using multi-target fluorescent staining includes the following steps: S1. Sample pretreatment: Take 100 μL of clinical pharyngeal swab sample and add an equal volume of 100 μL of sample lysis buffer. The sample lysis buffer includes 1% nonionic surfactant, 20 mM Tris-HCl buffer, and pH 7.8. When mixing the sample and lysis buffer, use vortexing at a frequency of 1500 rpm for 20 s. During the vortexing process, maintain the temperature of the mixture at 30℃. Then incubate at 37℃ for 10 min to obtain the pretreated sample. S2. Multi-target fluorescent staining: Add a multi-target fluorescent dye combination to the pretreated sample obtained in S1. The volume ratio of the dye combination to the pretreated sample is 1:5. The dye combination includes two specific fluorescent dyes: FITC-S (525nm emission wavelength) against the spike protein of the novel coronavirus and Cy3-N (605nm emission wavelength) against the nucleocapsid protein. The difference in emission wavelength between the two dyes is 80nm. After mixing, gently oscillate at a frequency of 800rpm for 60s. Then incubate at 37℃ in the dark for 15min to obtain the stained sample. S3. Post-staining purification: Transfer the stained sample obtained in S2 into a filter membrane centrifuge tube with a pore size of 0.45 μm. Centrifuge at 10,000 rpm for 2 min to remove free fluorescent dye and sample impurities, and obtain the purified stained sample. S4. Optical Imaging Acquisition: The stained sample purified in S3 is dropped onto the imaging slide and placed into the laser confocal imaging system. Fluorescence signals of different target points are acquired simultaneously. The exposure time is set to 1.0s. The imaging area covers the entire effective area of ​​the sample drop, with an effective area coverage rate of ≥95%. Dual-target fluorescence images are acquired. S5. Image Analysis and Judgment: Input the fluorescence image acquired in S4 into the image analysis system to extract three types of fluorescence characteristic parameters: fluorescence intensity, signal distribution density, and multi-target co-localization rate. The preset threshold is obtained by calibration using standard positive samples of COVID-19. The judgment rule is that if the fluorescence characteristic parameters of both targets meet the preset threshold and the co-localization rate is ≥70%, it is judged as positive; otherwise, it is judged as negative. S6. Result Output: The image analysis system automatically outputs the detection results and fluorescence signal feature report to complete the virus detection.

[0028] Comparative Example 1 The purification step after staining in step S3 is omitted; the remaining steps are the same as in Example 2.

[0029] Comparative Example 2 The multi-target fluorescent staining process of S2 is replaced with single-target fluorescent staining, retaining only one specific fluorescent dye. S2 Single-target fluorescent staining: Add a single specific fluorescent dye to the pretreated sample of S1, retaining only FITC-S which is effective against the spike protein of the novel coronavirus. The emission wavelength is 525 nm. The dye to sample volume ratio is still 1:8. After mixing, gently shake at 650 rpm for 45 s and incubate at 37°C in the dark for 10 min to obtain the stained sample. The remaining steps are the same as in Example 2.

[0030] Comparative Example 3 Replace the vortex oscillation mixing in S1 with static mixing, and replace the gentle oscillation mixing in S2 with static mixing; S1 Sample Pretreatment: Without vortexing, the sample and lysis buffer were gently inverted 3 times and then allowed to stand. The temperature of the mixture was maintained at 28℃ and incubated at 37℃ for 8 minutes to obtain the pretreated sample. S2 Multi-target Fluorescent Staining: After gently inverting the sample 3 times, let it stand and incubate at 37°C in the dark for 10 minutes to obtain the stained sample. The remaining steps are the same as in Example 2.

[0031] Comparative Example 4 The multi-target co-localization determination in S5 is replaced with determination based on the single target signal meeting the standard. S5 Image Analysis and Judgment: Extract the fluorescence intensity and signal distribution density parameters of the two targets and compare them with the preset threshold. As long as the characteristic parameters of any target meet the preset threshold, it is judged as positive. There is no need to detect the co-localization rate of multiple targets. The remaining steps are the same as in Example 2.

[0032] Table 1. Performance Indicator Comparison Data Table

[0033] By combining Examples 1-3 and Comparative Examples 1-4, and by comparing the performance index data, it can be seen that the key influencing factors in this invention, such as multi-target fluorescence staining design, post-staining purification process, active oscillation mixing method, multi-target co-localization judgment logic, and parameter adaptability of each step, have a significant impact on the specificity, sensitivity, false positive rate, false negative rate, image signal-to-noise ratio, and result consistency of virus detection. Moreover, these factors have a good synergistic effect in targeted binding, interference removal, and signal verification.

[0034] Comparative Example 1, due to the removal of the post-staining purification process and the absence of a step to remove free dye and impurities, suffered severe interference with fluorescence imaging, resulting in an image signal-to-noise ratio of only 1.6, a decrease of 72.4% compared to Example 25.8. The false positive rate increased to 11.7%, a 22.4-fold increase compared to Example 20.5, and the detection specificity was only 88.3%, a decrease of 11.3% compared to Example 299.5%. Comparative Example 2, by using single-target fluorescent staining instead of multi-target staining, could not distinguish interfering substances through multi-target synergy. The detection specificity was only 82.6% and the sensitivity was 85.8%, which were 17.0% and 13.8% lower than those of Example 2, respectively. The false positive rate was 17.4% and the false negative rate was 14.2%, which were 33.8 times and 20.3 times higher than those of Example 2, respectively. Comparative Example 3, due to the use of static mixing instead of active oscillation mixing, resulted in uneven sample lysis and insufficient staining binding, with a detection sensitivity of only 89.2%, a decrease of 10.2% compared to Example 2. However, the consistency CV value reached 9.5%, an increase of 4.3 times compared to Example 2's 11.8%. Comparative Example 4, due to the use of single-target determination instead of multi-target co-localization determination, lacked signal cross-validation, resulting in a false positive rate of 14.9%, which was 28.8 times higher than that of Example 2, and a detection specificity of only 85.1%, which was 14.5% lower than that of Example 2.

[0035] In the examples, the virus detection specificity was between 96.2% and 99.5%, the sensitivity was between 95.8% and 99.3%, the false positive rate was between 0.5% and 3.8%, the false negative rate was between 0.7% and 4.2%, the image signal-to-noise ratio was between 3.2 and 5.8, and the consistency CV value was between 1.8% and 4.5%. All of these were significantly better than the comparative example, which had a specificity of 82.6% and 91.5%, a sensitivity of 85.8% and 92.3%, a false positive rate of 8.5% and 17.4%, a false negative rate of 7.7% and 14.2%, an image signal-to-noise ratio of 1.6 and 4.0, and a consistency CV value of 5.1% and 9.5%. Example 2 showed the best performance, with a specificity of 99.5%, sensitivity of 99.3%, false positive rate of 0.5%, false negative rate of 0.7%, image signal-to-noise ratio of 5.8, and result consistency CV value of 1.8%. The detection efficiency and accuracy were outstanding in synergy. Although the minimum parameter of Example 1 and the maximum parameter of Example 3 were slightly inferior to those of Example 2 due to parameter boundary effects, they still maintained good detection performance, which proves the rationality and adaptability of the parameter range of the present invention.

[0036] In Examples 1-3, S1 sample pretreatment, S2 multi-target fluorescent staining, S3 post-staining purification, and S5 multi-target co-localization determination are matched with each other. In Example 2, for the detection of the novel coronavirus, a dye-to-sample volume ratio of 1:8 and a gentle oscillation parameter of 650 rpm are used, along with incubation at 37°C in the dark for 10 min to ensure that the fluorescent dye binds specifically to the viral S and N proteins. Then, the free dye is removed by centrifugation through a 0.3 μm filter membrane, which improves the image signal-to-noise ratio to 5.8. Finally, combined with the determination rule of co-localization rate ≥70%, the optimal detection effect of 99.5% specificity and 99.3% is achieved, which is due to this method. Although Example 1 uses the minimum parameters of a dye volume ratio of 1:10 and an oscillation frequency of 500 rpm, it still maintains good performance with a specificity of 96.2% and a sensitivity of 95.8% due to the continued synergistic logic of specific binding, interference removal, and cross-validation. This demonstrates the flexible adaptability of parameters under a unified synergistic standard. At the same time, the temperature-controlled oscillation of 25-30℃ and 1000-1500 rpm in Example S1 is compatible with the staining parameters of 30-37℃ and 8-15 min incubation in Example S2, avoiding viral target denaturation and inactivation and ensuring dye binding efficiency. The filter membrane pore size of 0.22-0.45 μm in Example S3 matches the size of viral particles, removing small molecule impurities while retaining the virus, providing a clear signal basis for the 0.1-1.0s exposure time of optical imaging in Example S4. In contrast, Comparative Example 1 lacks a post-staining purification process, resulting in a sharp increase in background noise due to free dye, with an image signal-to-noise ratio of only 1.6, which directly affects the accuracy of subsequent image analysis and significantly increases the false positive rate.

[0037] The multi-target co-localization determination in Example S5 and the multi-target staining design in Example S2 form a core synergy. In Example 2, the presence of the target virus is double-verified by extracting the fluorescence intensity, signal distribution density, and co-localization parameters of the two targets, effectively eliminating the interference of single-target binding and controlling the false positive rate at 0.5%. In contrast, the single-target staining in Comparative Example 2 lacks target cross-verification and cannot distinguish non-specific binding signals, resulting in a false positive rate of 17.4%. Although the single-target determination in Comparative Example 4 retains multi-target staining, it does not utilize co-localization parameters, and the false positive rate is still as high as 14.9%, directly demonstrating the necessity of multi-target synergistic design.

[0038] Furthermore, the vortex oscillation temperature control design of Example S1 at 25-30℃, combined with the nuclease-free characteristics of the sample lysis buffer, ensures the integrity of the specific binding sites of key viral structures, providing a basis for multi-target staining in S2. Without the temperature control design, viral target denaturation may occur, resulting in decreased binding efficiency and further affecting detection sensitivity.

[0039] Comparative Example 1, due to the removal of the purification process after staining, suffered severe interference with fluorescence imaging, resulting in a detection specificity of 88.3% and a sensitivity of 90.5%, which were 11.3% and 8.9% lower than those of Example 2, respectively. Comparative Example 2, with its single-target staining design, achieved a specificity of 82.6% and a sensitivity of 85.8%, which were 17.0% and 13.8% lower than those of Example 2, respectively. Comparative Example 3, due to insufficient binding caused by static mixing, had a sensitivity of 89.2% and a result consistency CV value of 9.5%, which were 10.2% and 4.3 times lower than those of Example 2, respectively. Comparative Example 4, with its single-target judgment logic, had a false positive rate of 14.9% and a specificity of 85.1%, which were 28.8 times higher and 14.5% lower than those of Example 2, respectively.

[0040] Example 1, using the minimum parameters of lysis buffer surfactant 0.1%, dye volume ratio 1:10, and oscillation frequency 1000 rpm, resulted in slightly lower sample lysis and staining efficiency, leading to a specificity of 96.2%, sensitivity of 95.8%, and image signal-to-noise ratio of 3.2, which were 3.3%, 3.5%, and 44.8% lower than Example 2, respectively. Example 3, using the maximum parameters of lysis buffer surfactant 1%, dye volume ratio 1:5, and oscillation frequency 1500 rpm, resulted in slightly non-specific binding due to excessively high reagent concentrations, with a specificity of 97.1% and image signal-to-noise ratio of 4.1, which were 2.4% and 29.3% lower than Example 2, respectively. Although the detection performance was close to that of Example 2, the increased reagent usage may lead to higher costs, making its long-term cost-effectiveness slightly inferior to Example 2.

[0041] In summary, the key processes in this invention, such as multi-target fluorescent staining (S1-S6), post-staining purification, active oscillation mixing, and multi-target co-localization determination, work together synergistically to effectively solve the core problems of insufficient specificity, significant interference signals, poor result consistency, and long detection cycles in traditional virus detection. This significantly improves the accuracy, stability, and efficiency of virus detection. In particular, Example 2 achieves an optimal balance of high specificity (99.5%), high sensitivity (99.3%), low false positive rate (0.5%), and low false negative rate (0.7%), with the entire process taking ≤40 minutes, making it easy to automate and promote. Examples 1-3 further verify the rationality and adaptability of the parameter range. Together, these examples demonstrate that this invention is fully adaptable to the rapid screening and identification needs of various clinical virus samples, such as blood, sputum, and throat swabs, providing a replicable and scalable technical solution for the timely diagnosis and treatment of virus-related diseases.

[0042] Table 2, Performance Indicators (GB Standard)

[0043] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A rapid sample preparation and virus detection method using multi-target fluorescent staining, characterized in that, Includes the following steps: S1. Sample pretreatment: Take the clinical sample to be tested, add an equal volume of sample lysis buffer to mix the sample with the lysis buffer, and incubate at 25-37℃ for 5-10 min to obtain the pretreated sample. The sample lysis buffer does not contain nuclease and retains the key structure of the virus and the specific binding sites of the markers. S2. Multi-target fluorescent staining: Mix the pretreated sample from S1 with the multi-target fluorescent dye combination and incubate at 30-37℃ in the dark for 8-15 minutes to obtain the stained sample; the multi-target fluorescent dye combination includes at least two specific fluorescent dyes, each of which specifically binds to different key structures or markers of the target virus, and the emission wavelengths of each fluorescent dye do not overlap. S3. Post-staining purification: The stained sample from S2 is centrifuged and filtered through a filter membrane to remove free fluorescent dye and sample impurities, resulting in a purified stained sample. S4. Optical Imaging Acquisition: Place the purified and stained sample from S3 into the optical imaging system, set the imaging conditions according to the spectral characteristics of the fluorescent dye, and acquire the fluorescence image. S5. Image Analysis and Judgment: Input the fluorescence image from S4 into the image analysis system, extract the fluorescence feature parameters of each target point and compare them with the preset threshold, and determine the detection result based on the co-localization of multiple targets. S6. Result Output: Automatically outputs the detection results and fluorescence signal characteristic report.

2. The method for rapid sample preparation and virus detection using multi-target fluorescent staining according to claim 1, characterized in that: In S1, the sample lysis buffer includes 0.1%-1% nonionic surfactant and 5-20mM buffer solution with a pH of 7.0-7.

8. The sample and lysis buffer are mixed using a vortex oscillation method.

3. The method for rapid sample preparation and virus detection using multi-target fluorescent staining according to claim 1, characterized in that: In S2, the key viral structures or markers include at least two of the following: viral capsid proteins, envelope proteins, nucleic acid proteins, or enzyme proteins.

4. The method for rapid sample preparation and virus detection using multi-target fluorescent staining according to claim 1, characterized in that: In S2, the fluorescent dye is selected from at least two of fluorescein, rhodamine, cyanine dyes or quantum dots, and the difference in emission wavelength between each dye is ≥50nm. The volume ratio of the multi-target fluorescent dye combination to the pretreated sample is 1:5-1:10, and after mixing, it is gently oscillated continuously at a frequency of 500-800 rpm for 30-60 s.

5. The method for rapid sample preparation and virus detection using multi-target fluorescent staining according to claim 1, characterized in that: In S3, the filter membrane pore size is 0.22-0.45μm, the centrifugation speed is 8000-10000rpm, and the centrifugation time is 1-2min.

6. The method for rapid sample preparation and virus detection using multi-target fluorescent staining according to claim 1, characterized in that: In S4, the optical imaging system is a fluorescence microscope or a laser confocal imaging system, which can synchronously or sequentially acquire fluorescence signals from different target points; The exposure time of the optical imaging system is set to 0.1-1.0s, and the imaging area covers the entire effective area of ​​the sample drop, with an effective area coverage rate of ≥95%.

7. The method for rapid sample preparation and virus detection using multi-target fluorescent staining according to claim 1, characterized in that: In S5, the fluorescence characteristic parameters include fluorescence intensity, signal distribution density, and multi-target co-localization rate, and the preset threshold is obtained by calibration using standard positive samples.

8. The method for rapid sample preparation and virus detection using multi-target fluorescent staining according to claim 1, characterized in that: In S5, the determination rules include: if the fluorescence characteristic parameters of at least two target points meet the preset threshold and the co-localization rate is ≥70%, then it is determined to be positive; otherwise, it is negative.

9. The method for rapid sample preparation and virus detection using multi-target fluorescent staining according to claim 1, characterized in that: The clinical samples to be tested include: blood, sputum, throat swabs, nasopharyngeal swabs, or stool samples.

10. The method for rapid sample preparation and virus detection using multi-target fluorescent staining according to claim 2, characterized in that: In step S1, during the vortex oscillation process, the temperature of the mixture of sample and lysis solution is maintained at 25-30℃, the frequency of vortex oscillation is 1000-1500rpm, and the oscillation time is 10-20s.

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