A microbial multiplex qPCR rapid detection method suitable for cell preparation

Abstract

The application discloses a kind of microbial multiplex qPCR rapid detection methods suitable for cell preparation, comprising the following steps: S1: sample receiving and kit preparation;S2: sample pretreatment;S3: qPCR reaction construction;S4: qPCR amplification procedure execution;S5: result analysis determination;S6: artificial intelligence assisted result verification, output final detection result.Belongs to the technical field of cell preparation, the structure can realize the synchronous rapid, accurate, high sensitivity detection of bacteria, fungi, mycoplasma in cell preparation, meet the quality control needs of cell preparation clinical rapid release.

Description

Technical Field

[0001] This invention relates to the field of cell preparation technology. Background Technology

[0002] Cell preparations are a type of live cell biological product. After production, these products cannot be processed using sterilization techniques. Microbial contamination can directly lead to product failure and may even cause serious safety risks such as shock and sepsis in users.

[0003] Currently, the microbial testing of cell preparations mainly relies on the aseptic culture method specified in the Chinese Pharmacopoeia. This method requires a culture period of at least 14 days, which cannot meet the needs of cell preparations for short-term use and rapid clinical release.

[0004] Among existing rapid detection technologies, real-time quantitative polymerase chain reaction (qPCR) has been attempted for microbial detection, but this type of technology has several technical drawbacks when applied to cell preparations: First, existing sample pretreatment protocols are not optimized for the high cell concentration and complex matrix characteristics of cell preparations. Proteins and polysaccharides in the cell matrix can interfere with nucleic acid extraction and amplification reactions, leading to false negatives in test results.

[0005] Second, most detection methods are difficult to achieve high-throughput simultaneous detection of multiple microorganisms. Although the existing SYBR Green dye method is low in cost, its non-specific luminescence characteristics cause the melting curves of different microorganisms to overlap easily during multiplex detection, making it impossible to effectively distinguish between bacteria, fungi, mycoplasma and viruses in the same reaction tube. Conventional probe methods often lack optimized multiplex detection system design, resulting in low detection throughput and long detection time.

[0006] Third, the current qPCR test results mainly rely on a single cycle threshold value, ignoring the morphological characteristics of the amplification curve itself; there is a lack of effective intelligent identification methods for "false positive jump lines" caused by probe degradation, bubble interference, or matrix autofluorescence drift, resulting in insufficient accuracy of the results.

[0007] Fourth, existing methods have high detection limits and lack targeted optimization of amplification procedures (such as annealing temperature control strategies), making it difficult to meet the need for accurate detection of low-concentration contamination in cell preparations and prone to non-specific amplification.

[0008] Therefore, there is an urgent need to develop a rapid detection method for microbial multiplex qPCR suitable for cell preparations to solve the problems in the existing technology. Summary of the Invention

[0009] The purpose of this invention is to provide a rapid multiplex qPCR detection method for microorganisms suitable for cell preparations, which can achieve simultaneous, rapid, accurate, and highly sensitive detection of bacteria, fungi, and mycoplasma in cell preparations, meet the quality control requirements for rapid clinical release of cell preparations, and has a simple structure and is easy to use, thereby solving the problems mentioned in the background art.

[0010] To achieve the above objectives, the present invention provides the following technical solution: A rapid multiplex qPCR detection method for microorganisms suitable for cell preparations includes the following steps: S1: Sample reception and kit preparation, including receiving qualified cell preparation samples and storing and labeling them as required, while preparing the necessary kits and consumables. The kits include microbial nucleic acid extraction kits and Taqman probe qPCR detection kits, and include dual-target gene-specific primer sets and probe sets for bacteria, fungi, mycoplasma and viruses. The dual-target gene primer sets are primer combinations corresponding to conserved genes and specific functional genes of each microorganism. S2: Sample pretreatment, including specific optimization processing of the qualified cell preparation samples received in S1, extracting bacterial and fungal nucleic acids, mycoplasma nucleic acids, and viral nucleic acids through three branch processes, including DNA viruses and RNA viruses; the branch processes use a complex of silica nanoparticles and polyvinylpyrrolidone as a matrix adsorbent, combined with a three-step optimization process of adsorption, washing, and elution to eliminate the interference of proteins and polysaccharides in the cell preparation, and obtain a purified microbial nucleic acid solution for downstream qPCR amplification reaction; S3: qPCR reaction construction, including constructing a dual-tube multiplex qPCR reaction system based on the purified microbial nucleic acid obtained in S2, including a first reaction tube and a second reaction tube; the first reaction tube targets dual target genes of bacteria and fungi, and the second reaction tube targets dual target genes of mycoplasma and viruses. The dual target gene primer set and probe in the system are designed for their respective target gene regions, and probes for different target genes in the same reaction tube are labeled with different fluorescent reporter groups to achieve channel differentiation; S4: qPCR amplification program execution, including executing a rapid amplification program combining Touchdown PCR and two-step method on the qPCR reaction system constructed in S3, improving amplification specificity through a temperature decrease strategy, and collecting and outputting cycle threshold and amplification curve data detected by the qPCR instrument; S5: Result analysis and judgment, including the cycle threshold, amplification curve and standard curve data obtained in S4, and outputting the qualitative and quantitative detection results of microorganisms through Ct value, P value, the judgment rules of the consistency of dual target gene amplification and statistical tests; S6: Artificial intelligence-assisted result verification, including using a trained, verified and tested convolutional neural network-long short-term memory network hybrid model to intelligently verify the raw qPCR data and judgment results, identify abnormal amplification, reaction inhibition, false positives and provide correction suggestions, and output the final detection results.

[0011] By adopting the above technical solution and through the coordinated operation of six steps from S1 to S6, a closed-loop process from sample reception to final result output is achieved. The Taqman probe method combined with a dual-tube multiplex fluorescence channel allocation strategy solves the problem of the SYBR Green method's inability to distinguish between different detection methods in multiplex detection. At the same time, it overcomes the problem of excessive fluorescence channel limitation caused by dual-target gene design in single-tube detection. Touchdown PCR solves the contradiction of primer annealing temperature adaptation. Combined with dual-target gene design and AI intelligent verification, high specificity, high sensitivity, and simultaneous rapid detection of bacteria, fungi, mycoplasma, and viruses in cell preparations are achieved, effectively reducing the risk of false positives and false negatives.

[0012] As a further aspect of the present invention: the three branch processes of the S2 sample pretreatment are bacterial and fungal nucleic acid extraction process, mycoplasma nucleic acid extraction process, and viral nucleic acid extraction process, respectively. The input is the qualified cell preparation sample obtained in S1, and the output is the purified microbial nucleic acid solution for downstream qPCR amplification reaction. All three branch processes have been optimized to eliminate cell matrix interference.

[0013] By adopting the above technical solution, the branch extraction pathways for three types of microorganisms—bacteria / fungi, mycoplasma, and viruses—were clarified. Furthermore, the interference of complex matrices in cell preparations was eliminated through a dedicated optimized process, ensuring the output of high-quality purified nucleic acid solutions and providing a material basis for the accuracy of downstream qPCR amplification.

[0014] As a further aspect of the present invention: the qPCR reaction in S3 uses the purified microbial nucleic acid obtained in S2 as input and outputs a configured dual-tube qPCR reaction system, including a first reaction tube and a second reaction tube. The first reaction tube corresponds to the nucleic acid construction of bacteria and fungi, and the second reaction tube corresponds to the nucleic acid construction of mycoplasma and viruses. Both use a preset volume of total system. Each system includes a detection premix and a nucleic acid sample. The nucleic acid sample includes the nucleic acid of the sample to be tested, the standard nucleic acid, and the control nucleic acid.

[0015] By adopting the above technical solutions, the composition and construction method of the reaction system were standardized, providing a standardized reaction environment for multiplex qPCR detection, ensuring the accuracy of quantitative detection and the comparability of different batches of experiments.

[0016] As a further aspect of the present invention: the S4 qPCR amplification program takes the dual-tube qPCR reaction system constructed in S3 as input and outputs the cycle threshold and amplification curve data collected by the qPCR instrument. The amplification program is divided into a Touchdown stage and a two-step amplification stage, which are used to achieve nucleic acid denaturation, primer annealing and extension, probe hydrolysis to generate fluorescence signals, and simultaneously collect fluorescence signals.

[0017] By adopting the above technical solution, the coordination between the Touchdown stage and the two-step amplification stage is defined in detail. Fluorescence is generated by probe hydrolysis and collected in real time, which not only ensures the high efficiency of amplification, but also greatly improves the specificity of amplification through the temperature reduction strategy, providing high-quality data support for subsequent accurate Ct value calculation.

[0018] As a further aspect of the present invention: the S5 result analysis and judgment takes the cycle threshold, amplification curve and standard curve data of the first reaction tube and the second reaction tube obtained in S4 as input, merges and judges the data of the two tubes, and outputs the qualitative and quantitative detection results of microorganisms, including three parts: qualitative judgment, quantitative judgment and P-value calculation. The qualitative judgment is performed by the judgment rules of Ct value, P value and dual target gene amplification consistency. The quantitative judgment is performed by calculating the microbial copy number in the sample by standard curve regression logic. The P-value is calculated by statistical test logic to help determine the statistical significance of the detection results.

[0019] By adopting the above technical solutions, a set of scientific judgment criteria was established, which combined statistical tests of Ct value and P value with the consistency judgment of dual target genes, thus achieving accurate qualitative and quantitative analysis of microbial contamination, avoiding misjudgments that may be caused by a single Ct value judgment, and improving the reliability of the test results.

[0020] As a further aspect of the present invention: the S6 AI-assisted result verification takes the original qPCR amplification curve, cycle threshold and standard curve data as input, and outputs the final detection result after intelligent verification, including the identification of abnormal amplification, reaction inhibition and false positive. The module adopts a hybrid model with a preset structure. After the model is trained, verified and tested with the dataset, it is put into use and automatically identifies abnormal detection results and provides correction suggestions.

[0021] By adopting the above technical solution and using a hybrid deep learning model to perform secondary intelligent verification on the original amplified data, it is possible to automatically identify abnormal amplification, reaction inhibition, and false positives that are difficult to detect by conventional judgment logic, and provide correction suggestions, thereby significantly improving the authenticity and credibility of the final detection results.

[0022] As a further aspect of the present invention: the specific process of AI-assisted result verification in S6 is as follows: extract the feature vector of the amplification curve, including: the slope of the inflection point, the absolute value of the fluorescence plateau phase, and the noise variance of the baseline phase, wherein the slope of the inflection point is the maximum value of the first derivative, and input it into the convolutional neural network-long short-term memory network hybrid model; the AI ​​model accurately identifies and eliminates false positive inflection lines caused by probe degradation, bubble interference, and cell matrix autofluorescence drift, thereby achieving weak contamination identification.

[0023] By adopting the above technical solution, it was clarified how the AI ​​model can accurately eliminate false positive signals caused by physical factors such as probe degradation and bubble interference by analyzing deep curve morphology features such as the inflection point slope and the absolute value of the plateau period, thus achieving effective identification of weakly contaminated samples.

[0024] As a further aspect of the present invention: the matrix adsorbent in S2 includes a complex of silica nanoparticles and polyvinylpyrrolidone, and the amount added is 5%-8% of the sample volume. It specifically adsorbs non-nucleic acid impurities in cell preparations during nucleic acid extraction and does not affect the recovery rate of microbial nucleic acids.

[0025] By adopting the above technical solution, and using a specific ratio of complex adsorbent, inhibitors such as proteins and polysaccharides are specifically removed during the extraction process, while nucleic acids are not adsorbed. This solves the problem of inhibition of PCR amplification by cell preparation matrix components and ensures the sensitivity of detection.

[0026] As a further aspect of the present invention: the dual-target gene combination specifically includes: bacteria corresponding to the 16S rRNA gene and the gyrB gene, fungi corresponding to the 18S rRNA gene and the CYP51 gene, mycoplasma corresponding to the 16S rRNA gene and the gap gene, among viruses, HBV corresponding to the S gene and the X gene, HCV corresponding to the NS5B gene and the Core gene, HIV-1 corresponding to the gag gene and the env gene, and HCMV corresponding to the UL83 gene and the UL122 gene.

[0027] By adopting the above technical solution, a combination of conserved genes and specific functional genes was selected for each microorganism. Through the simultaneous amplification of dual targets, false negatives caused by single gene mutations were effectively prevented, further improving the robustness and accuracy of the detection method.

[0028] As a further aspect of the present invention: the Touchdown PCR combined with the two-step rapid amplification program specifically includes: after pre-denaturation, entering the Touchdown cycling stage, the annealing temperature starts at 65°C, decreasing by 0.5°C in each cycle until reaching 55°C, for a total of 20 cycles; then performing the two-step amplification cycle, denaturing at 95°C for 10 seconds, annealing and extending at 60°C for 30 seconds, for a total of 30 cycles, and collecting fluorescence signals at the 60°C stage.

[0029] By adopting the above technical solution, a precise temperature control scheme is provided, ensuring the feasibility of the Touchdown PCR strategy in actual operation. By gradually changing the temperature gradient, the optimal balance between amplification specificity and efficiency is achieved at the experimental level.

[0030] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention constructs a dual-tube multiplex qPCR reaction system, optimizing the allocation of channels to address the limitations of conventional qPCR instruments. The first reaction tube contains specific probes labeled with different fluorescent groups targeting conserved and functional genes of bacteria and fungi, while the second reaction tube contains specific probes labeled with different fluorescent groups targeting conserved and functional genes of mycoplasma and viruses. Both tubes run synchronously on the same PCR plate, and the data are merged for evaluation. This not only solves the technical challenge of overlapping and indistinguishable melting curves in the SYBR Green method for multiplex detection but also overcomes the problem of excessive fluorescence channel limitations caused by dual-target gene design in single-tube multi-target detection. While ensuring specificity, it significantly increases detection throughput, shortens the detection cycle, and meets the rapid clinical release requirements of cell preparations.

[0031] 2. This invention uses a Touchdown strategy to set a gradually decreasing annealing temperature in the early stages of the reaction, ensuring that the primers preferentially and specifically bind to the template at high temperatures. This effectively inhibits the amplification of non-specific products and the formation of primer dimers, solving the problem of insufficient specificity of traditional fixed annealing temperature programs in complex matrices. It also eliminates the need for tedious pre-experiment annealing temperature optimization.

[0032] 3. This invention extracts deep feature vectors such as the inflection point slope of the amplification curve, the absolute value of the fluorescence plateau phase, and the noise variance of the baseline phase. Using a CNN-LSTM model, it can accurately identify and eliminate false positive inflection lines caused by probe degradation, bubble interference, and cell matrix autofluorescence drift. This enables the identification of weak contamination that cannot be judged by the naked eye, raising the accuracy and reliability of the detection results to a new level.

[0033] 4. This invention optimizes the sample pretreatment process and utilizes a composite matrix adsorbent of silica nanoparticles and polyvinylpyrrolidone to specifically adsorb impurities such as proteins and polysaccharides in cell preparations, significantly improving the purity of nucleic acid extraction. Combined with the synergistic verification of dual-target gene primer sets, it effectively reduces the false negative rate and achieves accurate detection of low-concentration contamination. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the overall structure of a rapid microbial multiplex qPCR detection method applicable to cell preparations, as described in an embodiment of the present invention. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] This invention integrates sample pretreatment, multiplex qPCR reaction, and artificial intelligence verification to achieve rapid and simultaneous detection of multiple microorganisms in cell preparations.

[0037] The principle is to amplify the target microbial nucleic acid in a dual-tube qPCR reaction system using specific primers and Taqman probes. Simultaneous and independent detection of bacteria / fungi and mycoplasma / viruses is achieved through multiple fluorescence channels in different reaction tubes. The data from the two tubes are combined for analysis, and quantitative analysis is performed using standard curves and statistical methods. Furthermore, an artificial intelligence model is used to extract the morphological features of the amplification curve to identify abnormal results, thereby improving the reliability and efficiency of the detection.

[0038] Example 1 In this embodiment of the invention, a rapid detection method for microbial multiplex qPCR suitable for cell preparations is described, see [link to relevant documentation]. Figure 1 As shown, the system comprises six modules: sample reception and reagent kit preparation, sample pretreatment, qPCR reaction construction, qPCR amplification program execution, result analysis and judgment, and AI-assisted result verification. The inputs and outputs of each module are strictly integrated, and all steps are specifically optimized for the characteristics of cell preparations. The specific technical solution is as follows: I. Sample Receiving and Reagent Kit Preparation (1) Sample input and processing requirements The input sample should be a mesenchymal stem cell preparation or a natural killer cell preparation, and the sample volume should be greater than 2 mL. The sample should be stored in a sterile, sealed centrifuge tube.

[0039] The storage conditions for samples within 0 to 12 hours are 4 degrees Celsius. If the storage time exceeds 12 hours, the storage conditions must be adjusted to -20 degrees Celsius. All samples must be labeled with a unique sample number to distinguish different test objects.

[0040] (2) Kit preparation The kits used in this method include a microbial nucleic acid extraction kit and a Taqman probe-based qPCR detection kit. All reagents must be stored and used in accordance with the specified conditions.

[0041] The nucleic acid extraction kit uses the Novizan Microbial DNA Rapid Extraction Kit. It should be stored at room temperature. After opening, the kit should be placed in a biosafety cabinet. The shelf life is 12 months.

[0042] The nucleic acid extraction kit also includes a kit for extracting novizan virus DNA and RNA. The storage environment is room temperature. After opening, the kit should be placed in a biosafety cabinet. The shelf life is 12 months.

[0043] The qPCR detection kit is a self-developed, sterile, rapid detection kit for cell preparations. It contains specific primers and probe combinations for bacteria, fungi, mycoplasma, and viruses. The probes for different microorganisms are labeled with different fluorescent reporter groups (such as FAM, HEX, ROX, Cy5, etc.). The storage environment is -20 degrees Celsius. The kit needs to be equilibrated to room temperature before use. Repeated freeze-thaw cycles should be avoided during use. The shelf life is 6 months.

[0044] During the experiment, you need to bring your own anhydrous ethanol, as well as consumables such as eight-tube strips, qPCR plates, qPCR sealing films, pipette tips, and 1.5mL nuclease-free centrifuge tubes.

[0045] II. Sample Preprocessing Sample pretreatment is divided into three branches: bacterial and fungal nucleic acid extraction, mycoplasma nucleic acid extraction, and viral nucleic acid extraction. The input is a qualified cell preparation sample from Module 1, and the output is a purified microbial nucleic acid solution, which can be directly used for downstream qPCR amplification reactions. The specific steps are as follows: (1) Bacterial and fungal nucleic acid extraction steps Add 1 mL of mesenchymal stem cell preparation or natural killer cell preparation sample to a lysis tube, add 50 µL of matrix adsorbent, wherein the mass ratio of silica nanoparticles to polyvinylpyrrolidone is 1:3, vortex at room temperature for 5 minutes, and then centrifuge at 12000 times the acceleration of gravity for 3 minutes. Use a pipette to collect the culture supernatant, which will be used for subsequent processing. Repeat this step when collecting samples multiple times.

[0046] Add 400µL of lysis buffer, 30µL of proteinase K, and 160µL of binding buffer to the lysis tube in sequence. Place the lysis tube on a vortex mixer and vortex at maximum speed for 10 minutes to fully lyse the microbial cells.

[0047] Place the lysis tube in a 70°C water bath or metal bath and heat for 5 minutes. Centrifuge at 12,000 times the acceleration of gravity for 1 minute to remove foam. Transfer the supernatant after centrifugation to a new 1.5 mL nuclease-free centrifuge tube.

[0048] Add 240µL of anhydrous ethanol to the centrifuge tube obtained in step 3, shake to mix, and then briefly centrifuge to collect the residual liquid on the inner wall of the tube cap.

[0049] Transfer the entire mixture obtained in step 4 to a nucleic acid adsorption column, centrifuge at 12,000 times the gravitational acceleration for 1 minute, discard the filtrate, and repeat the transfer operation until the entire mixture is processed.

[0050] Add 500µL of washing buffer WP along the wall of the adsorption column, centrifuge at 12000 times gravity acceleration for 1 minute, discard the filtrate, and use it to remove impurities.

[0051] Add 600µL of washing buffer (WB) along the wall of the adsorption column. Before use, ensure that anhydrous ethanol has been added to the washing buffer (WB). Centrifuge at 12000 times gravity for 1 minute, discard the filtrate, and use it for further purification of nucleic acids.

[0052] The adsorption column was placed in a collection tube and centrifuged for 1 minute at 12,000 times the acceleration of gravity to remove residual liquid from the adsorption column.

[0053] Transfer the adsorption column to a new 1.5 mL elution tube, and add 50 µL of elution buffer EB to the center of the adsorption column. The elution buffer EB needs to be preheated to 55 degrees Celsius. Preheating is used to improve the efficiency of nucleic acid elution. After standing at room temperature for 2 to 5 minutes, centrifuge at 12,000 times the acceleration of gravity for 1 minute.

[0054] Discard the adsorption column, and the elution product obtained is the bacterial and fungal genomic DNA, which can be directly used for downstream bacterial and fungal qPCR detection.

[0055] The preparation method of the matrix adsorbent is as follows: Take silica nanoparticles with a particle size of 50-100nm and polyvinylpyrrolidone with a molecular weight of 10000-20000, add them to sterile water at a mass ratio of 1:3, stir magnetically for 30 minutes until completely dispersed, freeze dry and grind into powder for later use. When using, add 5%-8% of the sample volume and vortex at room temperature for 5 minutes. It can specifically adsorb non-nucleic acid impurities such as proteins and polysaccharides, and the microbial nucleic acid recovery rate is ≥90%.

[0056] In one feasible embodiment, the matrix adsorbent comprises an optimized three-step process of adsorption, washing, and elution: ①Adsorption: After adding the matrix adsorbent at room temperature, vortex for 5 minutes and let stand for 2 minutes to ensure that non-nucleic acid impurities are fully adsorbed; ② Washing: Use gradient concentration washing buffer. The first wash uses 500 μL of washing buffer containing 0.5% Tween-20, and the second wash uses 600 μL of washing buffer without Tween-20. Centrifuge at 12000 times gravity acceleration for 1 minute to remove residual adsorbent and impurities. ③ Elution: Use elution buffer EB preheated to 55℃, add it in two drops, 25μL each time. After each addition, let it stand at room temperature for 3 minutes, centrifuge to collect the eluent, improve the nucleic acid recovery rate to more than 95%, which is 20% higher than the conventional single elution recovery rate.

[0057] (2) Mycoplasma nucleic acid extraction steps Take 200µL of the culture supernatant collected in step 2.1, mix it with 500µL of lysis buffer VL, and vortex for 15 to 30 seconds to lyse mycoplasma cells.

[0058] Place the RNA adsorption column in a 2 mL collection tube, transfer the above mixture into the RNA adsorption column, centrifuge at 12000 times gravity acceleration for 1 minute, and discard the filtrate.

[0059] Add 600 µL of wash buffer RW to the RNA adsorption column, centrifuge at 12000 times gravity for 1 minute, discard the filtrate, and repeat this step once.

[0060] The RNA adsorption column was centrifuged at 12,000 times the acceleration of gravity for 1 minute to remove residual liquid from the adsorption column.

[0061] Transfer the RNA adsorption column to a new 1.5 mL RNase-free centrifuge tube, add 50 µL of RNase-free ultrapure water to the center of the adsorption membrane, incubate at room temperature for 1 minute, and then centrifuge at 12000 times the acceleration of gravity for 1 minute.

[0062] Discard the adsorption column, and the resulting elution product is mycoplasma RNA, which can be directly used for downstream mycoplasma qPCR detection.

[0063] (3) Viral nucleic acid extraction steps Take 1 mL of cell preparation sample, add 50 µL of matrix adsorbent, vortex at room temperature for 5 minutes, centrifuge at 12000 times gravity acceleration for 3 minutes, and collect the supernatant; Mix 200µL of supernatant with 500µL of lysis buffer (VL) and vortex for 15-30 seconds; Transfer the mixture to an RNA adsorption column, centrifuge at 12000 times gravity for 1 minute, and discard the filtrate; Add 600µL of washing buffer RW to the adsorption column, centrifuge for 1 minute, discard the filtrate, and repeat once. Centrifuge the empty column for 1 minute to remove residual liquid; Transfer the adsorption column to a new RNase-free centrifuge tube, add 50 µL of RNase-free ultrapure water to the center of the membrane, incubate at room temperature for 1 minute, then centrifuge for 1 minute. The elution product is the viral nucleic acid, including DNA and RNA.

[0064] For RNA viruses such as HCV and HIV, subsequent reverse transcription processing is required during the extraction process.

[0065] III. Construction of qPCR reaction system The input to this module is the purified microbial nucleic acid obtained from module 2, and the output is the configured multiplex qPCR reaction system.

[0066] To address the issue of overlapping melting curves in the SYBR Green method, which makes differentiation difficult in multiplex detection, this embodiment employs the TaqMan probe method, utilizing multiple fluorescence channels to achieve simultaneous detection in a dual-tube system. The reaction mixture uses a total volume of 20 µL, with the specific configuration as follows: (1) Construction of multiplex qPCR reaction system The reaction system used the TaqMan probe method, with a total volume of 20 µL. 15 µL of the premixed detection solution (containing Taq enzyme, dNTPs, and Mg) was taken. 2+ Add specific primers and probes for the corresponding microorganisms (such as buffer solutions) and mix with 5 µL of sample nucleic acid.

[0067] Multiple detection strategy: To achieve "simultaneous and rapid" detection and adapt to the fluorescence channel limitations of conventional qPCR instruments, this invention adopts a two-tube reaction system.

[0068] The first reaction tube is used for bacterial / fungal detection, for example: bacterial conserved gene probe labeled FAM, bacterial functional gene probe labeled HEX, fungal conserved gene probe labeled ROX, and fungal functional gene probe labeled Cy5; the second reaction tube is used for mycoplasma / virus detection, for example: mycoplasma conserved gene probe labeled FAM, mycoplasma functional gene probe labeled HEX, viral conserved gene probe labeled ROX, and viral functional gene probe labeled Cy5.

[0069] All probes were labeled with the corresponding quencher group, such as BHQ1 or TAMRA, at their 3' ends. Both tubes were run simultaneously on a PCR plate, and the data were combined for final interpretation.

[0070] (2) RNA virus detection and treatment For RNA viruses such as HCV and HIV, reverse transcriptase can be added to the reaction system or a one-step RT-qPCR premix can be used to complete reverse transcription and qPCR amplification in a two-tube system.

[0071] (3) Primer and probe design requirements Primers are 18-25 bp in length and contain 40%-60% GC; probes are 20-30 bp in length and have a Tm value 5-10°C higher than the primers. The final primer concentration is 0.4 µmol / L, and the final probe concentration is 0.2 µmol / L.

[0072] No melting curve analysis is required for the amplification products; the focus is on optimizing primer and probe specificity to ensure no cross-reaction.

[0073] IV. Execution of qPCR amplification program This module takes as input the qPCR reaction system configured in Module 3 and outputs the cycle threshold and amplification curve data collected by the qPCR instrument. To improve amplification specificity and resolve the incompatibility of annealing temperatures for different primers, this embodiment employs a Touchdown PCR combined with a two-step rapid amplification program, eliminating the need for melting curve analysis.

[0074] The specific procedure is as follows: Phase 1 (Pre-denaturation): 95℃ for 3 minutes, 1 cycle, used to activate enzyme activity and perform initial denaturation.

[0075] The second stage (Touchdown amplification): 95℃ for 10 seconds, 65℃ for 30 seconds (decreasing by 0.5℃ per cycle), for a total of 20 cycles. In this stage, the annealing temperature decreases from 65℃ to 55℃. The high-temperature initiation ensures specific primer binding, while the gradual decrease in temperature improves amplification efficiency and effectively eliminates non-specific amplification.

[0076] The third stage (two-step amplification): 95℃ for 10 seconds, 60℃ for 30 seconds, for a total of 30 cycles. Annealing and extension are performed in this stage, and fluorescence signals are acquired at the 60℃ step. The temperature is kept constant in this stage to ensure the efficiency of exponential amplification and the stability of fluorescence signal acquisition.

[0077] V. Result Analysis and Judgment This module takes as input the qPCR cycle threshold, amplification curve, and standard curve data obtained from module 4, and outputs the qualitative and quantitative detection results of microorganisms. Since a probe method is used, the melting curve determination is no longer employed; the determination rules are optimized as follows: (1) Qualitative result determination If a sample simultaneously meets the following conditions: Ct value ≤ control Ct value, P value ≤ 0.05, and consistency of dual-target gene amplification (difference between dual-channel Ct values ​​≤ 2), the sample is considered to have corresponding microbial contamination; otherwise, it is considered to have no corresponding microbial contamination.

[0078] (2) Quantitative result determination The regression coefficient of the standard curve must be greater than 0.95, and the limit of quantitation must be less than 10 copies / mL. Substitute the sample Ct value into the standard curve equation to calculate the actual copy number.

[0079] (3) Calculation of P-value The sample Ct value and the control Ct value are substituted into the T-test formula for calculation, which helps to determine the statistical significance of the test results.

[0080] VI. Artificial Intelligence-Assisted Result Verification Model This module takes as input the raw qPCR amplification curve, cycle threshold, and standard curve data, and outputs the final detection result after intelligent validation. It is used to identify abnormal amplification, reaction inhibition, false positives, etc. The model training and application process is as follows: (1) Model dataset construction The dataset comprises three categories: positive sample data, including amplification curves, cycle thresholds, and copy number data for microbial contamination samples; negative sample data, including amplification curves and cycle threshold data for sterile cell preparation samples; and anomalous sample data, including data caused by reaction inhibition, nonspecific amplification, and reagent failure. The dataset contains 100,000 records, all derived from actual cell preparation testing samples.

[0081] (2) Dataset partitioning The dataset was divided into training, validation, and test sets in a 7:2:1 ratio. The training set contained 70,000 data points and was used for model training.

[0082] The validation set contains 20,000 data points and is used to tune the model parameters. The test set contains 10,000 data points and is used to evaluate the final performance of the model.

[0083] (3) Model feature extraction and structure construction A hybrid model structure combining convolutional neural networks and long short-term memory networks is adopted. For the Taqman amplification curve, feature vectors such as the inflection point slope (maximum value of the first derivative), the absolute value of the fluorescence plateau period, and the noise variance during the baseline period are extracted and input into the CNN-LSTM model.

[0084] (4) Model training parameter settings The batch size is set to 32 to control the number of samples per training round. The number of training rounds is 100 to ensure the model fully learns the data features. The learning rate is 0.001 to control the model training speed.

[0085] The cross-entropy loss function is used to measure the difference between the model's predicted values ​​and the actual values. The Adam optimizer is used to update the model parameters.

[0086] (5) Model Validation and Application The model's accuracy was evaluated using a validation set after each training round. The final model can accurately identify and eliminate "false positive thresholds" caused by probe degradation, bubble interference, and cell matrix autofluorescence drift, enabling the identification of subtle contamination that is indistinguishable to the naked eye and improving the accuracy of detection results.

[0087] Example 2 The difference between this embodiment and Embodiment 1 is as follows: Sample pretreatment stage: Based on the bacterial and fungal nucleic acid extraction and mycoplasma nucleic acid extraction procedures, a new viral nucleic acid extraction branch procedure is added. Specifically, take 1 mL of cell preparation sample, add 50 µL of matrix adsorbent, in which the mass ratio of silica nanoparticles to polyvinylpyrrolidone is 1:3, vortex for 5 min, centrifuge at 12000 times gravity acceleration for 3 min, take the supernatant and operate according to the instructions of the Novozymes Virus DNA Extraction Kit to obtain purified viral nucleic acid; qPCR reaction system construction: Construct a two-tube qPCR reaction system using the TaqMan probe method, with 20µL of total volume in each reaction tube.

[0088] The first reaction tube contains 15 µL of bacterial / fungal detection premix and 5 µL of corresponding nucleic acid; the second reaction tube contains 15 µL of mycoplasma / virus detection premix and 5 µL of purified viral nucleic acid / standard / control. The initial concentration of the standard is copies / µL, serially diluted to copies / µL. The detection premix in the second reaction tube includes mycoplasma and HBV, HCV, HIV-1, and HCMV specific primers and probes; it also includes Taq enzyme, dNTPs, etc. Result interpretation rules: A triple interpretation is adopted, that is, when the Ct value is ≤ the Ct value of the control and the P value is ≤ 0.05, it is judged as positive for the corresponding microorganism; if either condition is not met, it is judged as negative. Validation results: Effective against bacteria containing 10 CFU / mL, mycoplasma containing 5 CFU / mL, The simulant contamination assay for HBV copies / mL was accurate, and the false negative rate due to matrix interference was reduced to below 0.3%.

[0089] Example 3 The difference between this embodiment and Embodiment 1 is as follows: Primer design: Bacterial primers target conserved sequences in the V3-V4 region of the 16S rRNA gene, fungal primers target the V5-V6 region of the 18S rRNA gene, mycoplasma primers target the V2 region of the 16S rRNA gene, and viral primers target their respective functional genes. All primers were confirmed to have no risk of cross-hybridization by BLAST comparison. Specificity verification: Using human amniotic mesenchymal stem cell DNA, human polyomavirus JCV DNA, and human normal tissue DNA as control templates, qPCR detection showed no amplification signal, proving that the primers only specifically bind to the nucleic acid of the target microorganism. Sensitivity validation: The detection sensitivity for Mycoplasma pneumoniae reached 2.5 CFU / mL, and the detection sensitivity for HBV reached 50 copies / mL, both of which are better than the detection limits published in the comparative documents.

[0090] Example 4 The difference between this embodiment and Embodiment 1 is as follows: Amplification was performed using a dual-target gene primer set. Taking bacteria (Escherichia coli) as an example, the 16S rRNA gene primer and the gyrB gene primer were amplified simultaneously. The amplification program used a two-step Touchdown PCR method, with the annealing temperature decreasing from 65℃ to 55℃. The result determination adopts a triple determination rule; Validation results: The simulated contaminated cell preparation containing 1 copy / µL of Escherichia coli and 0.5 copies / µL of HBV was tested. The simulated contaminated cell preparation included a high concentration of protein and polysaccharide matrix. Both target genes were successfully amplified, the Ct value difference was 1.2, and the triple judgment met the positive criteria, indicating accurate detection. The conventional method using a single target gene, fixed annealing temperature, and double determination resulted in one false negative due to matrix interference and low template concentration, with HBV not detected. This demonstrates that the sensitivity and anti-interference ability of this method are significantly superior to the conventional method.

[0091] The design of the dual-target gene primer set is based on the following: for each microorganism, housekeeping genes (such as bacterial 16S rRNA) with a conservation rate of ≥98% are screened through the NCBI database, along with species-specific functional genes (such as bacterial gyrB, which encodes the DNA gyrase B subunit and exists only in bacteria). The primer amplification fragment length difference between the two types of genes is 30-50 bp (probe design avoids cross-reaction), and BLAST comparison confirms that there is no risk of cross-hybridization. Simultaneous amplification of dual-target genes can achieve a synergistic effect of broad coverage of conserved genes and accurate verification of functional genes, avoiding missed detection due to gene mutation of a single target gene.

[0092] In the triple judgment rule, the consistency judgment standard for dual target gene amplification is: the difference in Ct values ​​between the amplification of two types of target genes of the same microorganism is ≤2. If it is not met, it is judged as amplification abnormality, and further analysis is required in conjunction with the artificial intelligence-assisted verification module. This rule can effectively eliminate false positives caused by non-specific amplification and single target gene contamination, and the false positive rate is reduced by 60% compared with the double judgment rule.

[0093] This invention provides a rapid multiplex qPCR detection method for microorganisms suitable for cell preparations, which can achieve simultaneous, rapid, accurate and highly sensitive detection of bacteria, fungi and mycoplasma in cell preparations, meeting the quality control requirements for rapid clinical release of cell preparations.

[0094] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0095] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment includes only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A rapid multiplex qPCR detection method for microorganisms suitable for cell preparations, characterized in that, Includes the following steps: S1: Sample reception and kit preparation, including receiving qualified cell preparation samples and storing and labeling them as required, while preparing the necessary kits and consumables. The kits include microbial nucleic acid extraction kits and Taqman probe qPCR detection kits, and include dual-target gene-specific primer sets and probe sets for bacteria, fungi, mycoplasma and viruses. The dual-target gene primer sets are primer combinations corresponding to conserved genes and specific functional genes of each microorganism. S2: Sample pretreatment, including specific optimization processing of the qualified cell preparation samples received in S1, extracting bacterial and fungal nucleic acids, mycoplasma nucleic acids, and viral nucleic acids through three branch processes, including DNA viruses and RNA viruses; the branch processes use a complex of silica nanoparticles and polyvinylpyrrolidone as a matrix adsorbent, combined with a three-step optimization process of adsorption, washing, and elution to eliminate the interference of proteins and polysaccharides in the cell preparation, and obtain a purified microbial nucleic acid solution for downstream qPCR amplification reaction; S3: qPCR reaction construction, including constructing a dual-tube multiplex qPCR reaction system based on the purified microbial nucleic acid obtained in S2, including a first reaction tube and a second reaction tube; the first reaction tube targets dual target genes of bacteria and fungi, and the second reaction tube targets dual target genes of mycoplasma and viruses. The dual target gene primer set and probe in the system are designed for their respective target gene regions, and probes for different target genes in the same reaction tube are labeled with different fluorescent reporter groups to achieve channel differentiation; S4: qPCR amplification program execution, including executing a rapid amplification program combining Touchdown PCR and two-step method on the qPCR reaction system constructed in S3, improving amplification specificity through a temperature decrease strategy, and collecting and outputting cycle threshold and amplification curve data detected by the qPCR instrument; S5: Result analysis and judgment, including the cycle threshold, amplification curve and standard curve data obtained in S4, and outputting the qualitative and quantitative detection results of microorganisms through Ct value, P value, the judgment rules of the consistency of dual target gene amplification and statistical tests; S6: Artificial intelligence-assisted result verification, including using a trained, verified and tested convolutional neural network-long short-term memory network hybrid model to intelligently verify the raw qPCR data and judgment results, identify abnormal amplification, reaction inhibition, false positives and provide correction suggestions, and output the final detection results.

2. The rapid detection method for microbial multiplex qPCR suitable for cell preparations according to claim 1, characterized in that, The three branches of the S2 sample preprocessing process are bacterial and fungal nucleic acid extraction process, mycoplasma nucleic acid extraction process, and viral nucleic acid extraction process. The input is the qualified cell preparation sample obtained in S1, and the output is the purified microbial nucleic acid solution for downstream qPCR amplification reaction. All three branches have been optimized to eliminate cell matrix interference.

3. The rapid detection method for microbial multiplex qPCR suitable for cell preparations according to claim 1, characterized in that, The qPCR reaction in S3 uses the purified microbial nucleic acid obtained in S2 as input and outputs a configured dual-tube qPCR reaction system, including a first reaction tube and a second reaction tube. The first reaction tube corresponds to the nucleic acid construction of bacteria and fungi, and the second reaction tube corresponds to the nucleic acid construction of mycoplasma and viruses. Both use a preset volume of total system. Each system includes a detection premix and a nucleic acid sample. The nucleic acid sample includes the nucleic acid of the sample to be tested, the standard nucleic acid, and the control nucleic acid.

4. The rapid detection method for microbial multiplex qPCR suitable for cell preparations according to claim 1, characterized in that, The S4 qPCR amplification program takes the dual-tube qPCR reaction system constructed in S3 as input and outputs the cycle threshold and amplification curve data collected by the qPCR instrument. The amplification program is divided into a Touchdown stage and a two-step amplification stage, which are used to achieve nucleic acid denaturation, primer annealing and extension, probe hydrolysis to generate fluorescence signals, and simultaneously collect fluorescence signals.

5. The rapid detection method for microbial multiplex qPCR suitable for cell preparations according to claim 1, characterized in that, The S5 result analysis and judgment takes the cycle threshold, amplification curve, and standard curve data of the first and second reaction tubes obtained in S4 as input, merges and judges the data of the two tubes, and outputs the qualitative and quantitative detection results of microorganisms, including three parts: qualitative judgment, quantitative judgment, and P-value calculation. The qualitative judgment is performed by the judgment rules of Ct value, P value, and dual-target gene amplification consistency. The quantitative judgment is performed by calculating the microbial copy number in the sample by standard curve regression logic. The P-value is calculated by statistical test logic to help determine the statistical significance of the detection results.

6. The rapid detection method for microbial multiplex qPCR in cell preparations according to claim 1, characterized in that, The S6 AI-assisted result verification takes the original qPCR amplification curve, cycle threshold, and standard curve data as input and outputs the final detection result after intelligent verification, including the identification of abnormal amplification, reaction inhibition, and false positives. The module adopts a hybrid model with a preset structure. This model is put into use after being trained, verified, and tested on the dataset, and automatically identifies abnormal detection results and provides correction suggestions.

7. The rapid detection method for microbial multiplex qPCR suitable for cell preparations according to claim 6, characterized in that, The specific process of AI-assisted result verification in S6 is as follows: extract the feature vector of the amplification curve, including: the slope of the inflection point, the absolute value of the fluorescence plateau phase, and the noise variance of the baseline phase, wherein the slope of the inflection point is the maximum value of the first derivative, and input it into the convolutional neural network-long short-term memory network hybrid model; the AI ​​model accurately identifies and eliminates false positive inflection lines caused by probe degradation, bubble interference, and cell matrix autofluorescence drift, thereby achieving weak contamination identification.

8. The rapid detection method for microbial multiplex qPCR in cell preparations according to claim 1, characterized in that, The matrix adsorbent in S2 comprises a complex of silica nanoparticles and polyvinylpyrrolidone, and is added at 5%-8% of the sample volume. It specifically adsorbs non-nucleic acid impurities in cell preparations during nucleic acid extraction without affecting the recovery rate of microbial nucleic acids.

9. The rapid detection method for microbial multiplex qPCR suitable for cell preparations according to claim 1, characterized in that, The specific dual-target gene combinations are as follows: bacteria corresponding to the 16S rRNA gene and the gyrB gene, fungi corresponding to the 18S rRNA gene and the CYP51 gene, mycoplasma corresponding to the 16S rRNA gene and the gap gene, viruses such as HBV corresponding to the S gene and the X gene, HCV corresponding to the NS5B gene and the Core gene, HIV-1 corresponding to the gag gene and the env gene, and HCMV corresponding to the UL83 gene and the UL122 gene.

10. The rapid detection method for microbial multiplex qPCR in cell preparations according to claim 1, characterized in that, The Touchdown PCR combined with the two-step rapid amplification program specifically includes: after pre-denaturation, entering the Touchdown cycling stage, the annealing temperature starts at 65℃ and decreases by 0.5℃ in each cycle until it reaches 55℃, for a total of 20 cycles; then performing the two-step amplification cycle, denaturing at 95℃ for 10 seconds and annealing and extending at 60℃ for 30 seconds, for a total of 30 cycles, and collecting fluorescence signals at the 60℃ stage.

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