A rapid detection method for microbial contamination of cell preparations based on qPCR technology

Abstract

The application discloses a kind of cell preparation microbial contamination rapid detection methods based on qPCR technology, belong to genetic engineering technical field, including obtaining to be detected live cell preparation sample, through microfiltration and ultrasonic vortex enrichment removes host large cell, obtains trace microorganism eluent, is rapidly prepared crude extraction nucleic acid template by one-step constant-temperature thermal lysis, it is connected into qPCR system containing targeted primer and runs amplification, according to the double determination matrix analysis curve of cycle number and specific melting peak constitution, signal is qualitatively and semi-quantitatively judged, determine whether live cell preparation exists microbial contamination, the present application realizes trace microorganism nondestructive concentration by microfiltration ultrasonic enrichment and one-step thermal lysis, overcomes the amplification inhibition of live cell complex matrix and host DNA false positive interference, relies on double determination matrix and realizes the accurate qualitative of trace contamination, provides a kind of short time-consuming, strong anti-interference, reliable aseptic rapid detection scheme.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and in particular relates to a rapid detection method for microbial contamination of cell preparations based on qPCR technology. Background Technology

[0002] With the rapid development of the biopharmaceutical industry, the application of cell preparations in personalized treatment is gradually expanding. Quality control of cell preparations is a key factor in ensuring their safety and efficacy. However, cell preparations are susceptible to microbial contamination during the production process, which can not only lead to preparation failure but also potentially trigger serious infections or immune responses in clinical treatment, posing fatal risks. Therefore, preventing microbial contamination is one of the most critical tasks in cell preparation production. Based on these risks, there is a greater demand for more efficient microbial testing of cell preparations. However, current pharmacopoeia methods rely on weeks of culture, a lengthy process that often significantly reduces or even completely destroys cell viability and efficacy, thus failing to meet the urgent clinical need for immediate reinfusion.

[0003] Existing aseptic rapid testing technologies are mainly divided into three categories: those based on microbial growth information, those based on live cell markers, and those based on microbial specific components. Although growth information-based detection technologies are more efficient than traditional methods, they still involve microbial culture and enrichment operations, resulting in a generally long detection cycle, which is difficult to match the short release window of cell preparations. On the other hand, counting and detection methods based on live cell markers are easily affected by the complex matrix environment of the cell preparation itself, leading to instability in alternative validation parameters such as specificity and detection limit of qualitative aseptic rapid tests. In contrast, nucleic acid amplification technology based on microbial specific components has the advantages of short cycle and high specificity. However, when directly applied to cell preparations, the large amount of human DNA from host cells or complex matrix excipients in the preparation can easily cause cross-interference and PCR amplification inhibition. Moreover, the potential microorganisms in the preparation are often at trace levels and are easily lost during routine nucleic acid extraction, resulting in false negatives. Summary of the Invention

[0004] The purpose of this invention is to address the problems of excessively long cycles in existing aseptic rapid detection technologies, as well as the susceptibility to complex matrix amplification inhibition, host background DNA interference, and difficulty in effectively enriching trace microorganisms when applied to cell preparations. Therefore, this invention proposes a rapid detection method for microbial contamination in cell preparations based on qPCR technology.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A rapid detection method for microbial contamination of cell preparations based on qPCR technology includes obtaining a sample of the live cell preparation to be tested, performing pretreatment on it by removing host cells, retaining it with a microporous membrane and eluting it with ultrasonic vortex, and obtaining an eluent containing trace amounts of microorganisms. The elution buffer was mixed with the microbial rapid lysis buffer, and a one-step isothermal thermal lysis was performed to prepare a crude nucleic acid template containing total microbial genomic DNA. Prepare a real-time quantitative PCR reaction system containing targeted amplification primers, add the crude extracted nucleic acid template, run amplification on a real-time quantitative PCR instrument and collect fluorescence signals to obtain amplification curves and melting curves; Based on a dual judgment matrix consisting of cycle number and specific melting peak, the signal is qualitatively and quantitatively judged to determine whether the live cell preparation is contaminated with microorganisms.

[0006] As a further description of the above technical solution: The specific steps of the microfiltration and ultrasonic vortex enrichment pretreatment are as follows: Centrifuge the cell preparation to be tested at 4℃ and 1000×g for 5 minutes, discard the host large cell precipitate, and collect the supernatant; The supernatant was microfiltered using a 0.22 μm hydrophilic polyethersulfone microporous filter. Remove the microporous membrane and place it into a centrifuge tube containing 50 μL of sterile nuclease-free water. Sonicate at 40 kHz for 3-5 minutes, followed by vigorous vortexing for 1 minute to remove trapped microorganisms and enrich them in the eluent.

[0007] As a further description of the above technical solution: The specific parameters for the one-step isothermal pyrolysis method are as follows: The enriched eluent was mixed with 2× microbial rapid lysis buffer at a volume ratio of 1:1 and vortexed for 30 seconds. Place at 85℃ and maintain the temperature for 15 minutes, then rapidly cool to 4℃ and maintain for 2 minutes; After a short period of centrifugation to allow air bubbles and cell debris to settle on the tube wall, the middle layer on the surface of the liquid is collected as a crude nucleic acid template for direct amplification.

[0008] As a further description of the above technical solution: In the one-step isothermal thermal lysis step, if the target contains Gram-positive bacteria with thick peptidoglycan cell walls, lysozyme at a working concentration of 20 mg / mL is added to the sample before adding the lysis buffer, pre-incubated at 37°C for 10-15 minutes, and then thermally lysed at 85°C.

[0009] As a further description of the above technical solution: The targeted amplification primers include universal 16S rDNA-specific primers for bacteria and universal ITS region-specific primers for fungi that have been screened by Blast comparison of the human whole genome. The bacteria-specific primer sequences are as follows: Forward primer 515F: 5'-GTGYCAGCMGCCGCGGTAA-3'; Reverse primer 907R: 5'-CCGTCAATTCMTTTRAGTTT-3'; The sequences of the fungal-specific primers are as follows: Forward primer ITS1F: 5'-CTTGGTCATTTAGAGGAAGTAA-3'; Reverse primer ITS2: 5'-GCTGCGTTCTTCATCGATGC-3'.

[0010] As a further description of the above technical solution: The fluorescence quantitative qPCR reaction system is a 40 μL total reaction system per well, and the specific preparation ratio is as follows: Add 20.0 μL of 2×SYBR Green qPCR Master Mix, add the forward primer and the reverse primer to make the final concentration of each 0.2 μM, add the crude extracted nucleic acid template, the addition volume of which is strictly limited to 6.0 μL to dilute the concentration of residual inhibitors below the amplification inhibition threshold, and make up the remaining volume with deionized water.

[0011] As a further description of the above technical solution: The program parameters set for amplification operation on the real-time fluorescence quantitative PCR instrument are as follows: Pre-denaturation stage: 95°C, 3 minutes, run 1 cycle; PCR amplification stage: denaturation at 95°C for 10 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 20 seconds, for a total of 45 cycles, and fluorescence signals are collected at 72°C; Melting curve analysis stage: the temperature is slowly raised from 65°C to 95°C, the heating rate is set to 0.5°C / step, and fluorescence is continuously collected.

[0012] As a further description of the above technical solution: The signal judgment rules of the dual judgment matrix are as follows: Positive judgment: Ct ≤ 30 and the Tm value of the single melting peak falls within the specific interval; Weak positive judgment: 30 < Ct ≤ 35 and the Tm value is normal, which requires repeated verification in two duplicate wells; Negative judgment: Ct > 38 and no specific melting peak, or abnormal late peak appears at high cycles but with low and gentle Tm value, which is the signal of primer dimer; In the specific interval, the Tm value range of bacteria is defined as 83-87°C, and the Tm value range of fungi is defined as 80-84°C.

[0013] As a further description of the above technical solution: The live cell preparations include stem cells and CAR-T preparations, and the microorganisms cover Gram-positive bacteria, Gram-negative bacteria, facultative anaerobes, and filamentous fungi.

[0014] As a further description of the above technical solution: The live cell preparation is a complex matrix product containing host human DNA background and nutritional excipients. The method is specifically designed to eliminate PCR amplification inhibition of the complex cell preparation matrix, cross-interference of host human DNA and preparation excipients, as well as interference from non-specific amplification and primer dimers on the detection results, so as to improve the accuracy and reliability of microbial contamination determination.

[0015] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are: This invention integrates host cell removal, microporous membrane retention, ultrasonic vortex elution, one-step thermal lysis, and qPCR amplification and interpretation into a continuous process, eliminating cumbersome nucleic acid purification steps, simplifying the operation process, avoiding the loss of trace nucleic acids during extraction, and shortening the time required for traditional aseptic testing. It is suitable for live cell preparations with short release windows, such as stem cells and CAR-T. Furthermore, this invention establishes an anti-interference technology scheme for complex cell preparation matrices in the qPCR detection system. By strictly limiting the volume of crude extracted nucleic acid template to 6.0 μL, PCR inhibition caused by lysis residues is reduced. For Gram-positive bacteria whose targets contain thick peptidoglycan cell walls, lysozyme pretreatment is introduced to enhance nucleic acid release. A dual judgment matrix composed of Ct value and specific melting peak Tm value is used to reduce the interference of host human DNA, formulation excipients, non-specific amplification, and primer dimers on the detection results, thereby improving the accuracy and reliability of microbial contamination determination. Attached Figure Description

[0016] Figure 1 This is a flowchart of a rapid detection method for microbial contamination of cell preparations based on qPCR technology proposed in this invention; Figure 2 This is a standard curve of OD600 and CFU of Bacillus subtilis in Example 1 of the present invention; Figure 3 This is a standard curve of OD600 and CFU of Escherichia coli in Example 1 of the present invention; Figure 4 This is a standard curve of OD600 and CFU of Staphylococcus aureus in Example 1 of the present invention; Figure 5 This is a standard curve of OD600 and CFU of Pseudomonas aeruginosa in Example 1 of the present invention; Figure 6 This is a standard curve of Aspergillus niger at different OD620 and CFU in Example 1 of the present invention; Figure 7 This is a gel electrophoresis image used to verify the detection limit of bacteria in Example 3 of the present invention without microporous membrane concentration pretreatment, as verified by conventional PCR. Figure 8 This is a gel electrophoresis image of conventional PCR verification using the combination of bacterial universal primers 27 / 515F and 907R in the final result analysis of this invention. Figure 9 This is a gel electrophoresis image of conventional PCR verification for fungal universal primer combination amplification in the final result analysis of this invention; Figure 10 This is a melting curve of two primer pairs used in the final analysis of fungal quantitative qPCR in this invention. Figure 11 This is a diagram showing the amplification curves of two primer pairs used in the final analysis of fungal quantitative qPCR in this invention. Figure 12 This is the melting curve of bacterial fluorescence quantitative qPCR at 515F+907R in the final result analysis of this invention; Figure 13 This is the amplification curve of bacterial fluorescence quantitative qPCR at 515F+907R in the final result analysis of this invention. Detailed Implementation

[0017] 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.

[0018] Please see Figure 1 The present invention provides a technical solution: a rapid detection method for microbial contamination of cell preparations based on qPCR technology, comprising obtaining a live cell preparation sample to be tested, performing pretreatment on it by removing host cells, retaining it with a microporous membrane and eluting it with ultrasonic vortex, and obtaining an eluent containing trace amounts of microorganisms; The elution buffer was mixed with the microbial rapid lysis buffer, and a one-step isothermal thermal lysis was performed to prepare a crude nucleic acid template containing total microbial genomic DNA. Prepare a real-time quantitative PCR reaction system containing targeted amplification primers, add crude extracted nucleic acid template, run amplification on a real-time quantitative PCR instrument and collect fluorescence signals to obtain amplification curves and melting curves; Based on a dual judgment matrix consisting of cycle number and specific melting peak, the signal is qualitatively and quantitatively judged to determine whether the live cell preparation is contaminated with microorganisms.

[0019] The specific steps for microfiltration and ultrasonic vortex enrichment pretreatment are as follows: Centrifuge the cell preparation to be tested at 4℃ and 1000×g for 5 minutes, discard the host large cell precipitate, and collect the supernatant; The supernatant was microfiltered and retained using a 0.22 μm hydrophilic polyethersulfone microporous filter; Remove the microporous membrane and place it into a centrifuge tube containing 50 μL of sterile nuclease-free water. Sonicate at 40 kHz for 3-5 minutes, followed by vigorous vortexing for 1 minute to remove trapped microorganisms and enrich them in the eluent.

[0020] The specific parameters for one-step isothermal pyrolysis are as follows: The enriched eluent was mixed with 2× microbial rapid lysis buffer at a volume ratio of 1:1 and vortexed for 30 seconds. Place at 85℃ and maintain the temperature for 15 minutes, then rapidly cool to 4℃ and maintain for 2 minutes; After a short period of centrifugation to allow air bubbles and cell debris to settle on the tube wall, the middle layer on the surface of the liquid is collected as a crude nucleic acid template for direct amplification.

[0021] In the one-step isothermal thermal lysis step, if the target contains Gram-positive bacteria with thick peptidoglycan cell walls, lysozyme at a working concentration of 20 mg / mL should be added to the sample before adding the lysis buffer, pre-incubated at 37°C for 10-15 minutes, and then thermally lysed at 85°C.

[0022] Targeted amplification primers include universal 16S rDNA-specific primers for bacteria and universal ITS region-specific primers for fungi that have been screened by Blast comparison of the human whole genome; The bacteria-specific primer sequences are as follows: Forward primer 515F: 5'-GTGYCAGCMGCCGCGGTAA-3'; Reverse primer 907R: 5'-CCGTCAATTCMTTTRAGTTT-3'; The fungal-specific primer sequences are: Forward primer ITS1F: 5'-CTTGGTCATTTAGAGGAAGTAA-3'; Reverse primer ITS2: 5'-GCTGCGTTCTTCATCGATGC-3'.

[0023] The quantitative qPCR reaction system consists of a 40 μL total reaction volume per well, with the following specific proportions: Add 20.0 μL of 2×SYBR Green qPCR Master Mix, add forward primer and reverse primer to make their final concentrations both 0.2 μM, add the crude nucleic acid template with its addition volume strictly limited to 6.0 μL, so as to dilute the residual inhibitor concentration to below the amplification inhibition threshold, and make up the remaining volume with deionized water.

[0024] The program parameter settings for amplification run on the real-time fluorescent quantitative PCR instrument are as follows: Pre-denaturation stage: 95°C, 3 minutes, run 1 cycle; PCR amplification stage: denaturation at 95°C for 10 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 20 seconds, set a total of 45 cycles, and collect fluorescent signals at 72°C; Melting curve analysis stage: slowly heat up from 65°C to 95°C, set the heating rate to 0.5°C / step, and continuously collect fluorescence.

[0025] The signal judgment rules of the dual judgment matrix are as follows: Positive judgment: Ct ≤ 30 and the Tm value of the single melting peak falls within the specific interval; Weak positive judgment: 30 < Ct ≤ 35 with normal Tm value, which requires repeated verification in duplicate wells; Negative judgment: Ct > 38 with no specific melting peak, or an abnormally late peak appears at high cycles with low Tm value and gentle signal, which is the signal of primer dimer; In the specific interval, the Tm value range of bacteria is defined as 83-87°C, and the Tm value range of fungi is defined as 80-84°C.

[0026] The live cell preparations include stem cell and CAR-T preparations, and the microorganisms cover Gram-positive bacteria, Gram-negative bacteria, facultative anaerobes and filamentous fungi.

[0027] The live cell preparation is a complex matrix product containing host human DNA background and nutritional excipients. The method is specially used to eliminate the interference of PCR amplification inhibition of the complex cell preparation matrix, cross-interference from host human DNA and preparation excipients, as well as non-specific amplification and primer dimers on the detection results, so as to improve the accuracy and reliability of microbial contamination judgment.

[0028] 1. Strains; In accordance with the pharmacopoeia and the guiding principles for methodological alternative validation, 5 representative standard strains covering Gram-positive bacteria, Gram-negative bacteria, facultative anaerobes and filamentous fungi are selected in this application: (1) Staphylococcus aureus, Bacteria / Gram-positive bacteria, aerobic; (2) Bacillus subtilis, Bacteria / Gram-positive bacteria, facultative anaerobic; (3) Escherichia coli, Bacteria / Gram-negative bacteria, aerobic; (4) Pseudomonas aeruginosa, bacteria / Gram-negative bacteria, aerobic; (5) Aspergillus niger, a filamentous fungus, aerobic.

[0029] 2. Reagents: (1) Culture medium and diluent: LB medium, PDA medium, 10% glycerol wash solution, sterile PBS buffer, lysozyme powder (purity >20,000 U / mg), deionized water and electrophoresis agarose.

[0030] (2) Nucleic acid release and amplification reagents: Rapid lysis buffer: Takara microbial lysis buffer (catalog number: 9164); Standard PCR reaction solution: Taq enzyme mixture (Polymerex, catalog number: MF002-PIUS-01), PrimeSTAR HS high-fidelity polymerase (Takara, catalog number: R010A, specifically for amplification of Staphylococcus aureus crude extract); qPCR detection reagent: SYBR Green qPCR Master Mix (Aikerui kit).

[0031] (3) Specific amplification primer sequences: After screening using universally conserved sequence prediction and human whole-genome BLAST alignment, the following bacterial and fungal core primer pairs for qPCR amplification were identified: (1) Universal 16S rDNA-specific primers for bacteria: Forward primer 515F: 5'-GTGYCAGCMGCCGCGGTAA-3'; Reverse primer 907R: 5'-CCGTCAATTCMTTTRAGTTT-3'; (2) Fungal universal ITS region-specific primers: Forward primer ITS1F: 5'-CTTGGTCATTTAGAGGAAGTAA-3'; Reverse primer ITS2: 5'-GCTGCGTTCTTCATCGATGC-3'; (3) Universal amplification candidate primers for fungi: Forward primer ITS1: 5'-TCCGTAGGTGAACCTGCGG-3'; Reverse primer ITS4: 5'-TCCTCCGCTTATTGATATGC-3'.

[0032] 3. Equipment: Real-time quantitative PCR instrument, ordinary gradient PCR instrument, agarose gel electrophoresis instrument and gel imaging instrument, micro spectrophotometer, high-speed refrigerated centrifuge, ultrasonic cleaner, vortex shaker, and sterile ultra-clean workbench.

[0033] 4. Preparation of core reagents and culture media: (1) Sterile 0.1M PBS buffer (pH 7.4): NaCl 8.0g, KCl 0.2g, Na2HPO4 1.44g, KH2PO4 0.24g Weigh the above reagents, add them to 800 mL of ultrapure water and stir thoroughly to dissolve. Use 1 M HCl or 1 M NaOH to precisely adjust the pH of the solution to 7.4, bring the volume to 1000 mL, dispense into glass reagent bottles, autoclave at 121 °C for 20 minutes, cool and store at room temperature for later use.

[0034] (2) Microbial activation and counting culture medium; LB liquid culture medium (for bacteria): Weigh 10g of tryptone, 5g of yeast extract, and 10g of NaCl, dissolve them in 1000mL of ultrapure water, stir to dissolve, and then autoclave at 121℃ for 20 minutes. PDA solid culture medium (for fungi / Aspergillus niger): Peel and cut 200g of potatoes into chunks, add 1000mL of water and boil for 30 minutes. Filter the liquid through gauze, add 20g of glucose and 20g of agar, add water to make up to 1000mL, autoclave at 115℃ for 20 minutes, and pour into plates while hot.

[0035] (3) Lysozyme working solution (20 mg / mL): Weigh 20 mg of lysozyme powder (purity > 20,000 U / mg), dissolve it in 1.0 mL of sterile nuclease-free water, gently pipette until completely dissolved, filter sterilize using a 0.22 μm sterile syringe filter membrane, dispense into sterile tubes at 50 μL / tube, and store at -20°C, avoiding repeated freeze-thaw cycles.

[0036] (4) Specific primer working solution (10 μM): Centrifuge the dry primers (515F / 907R and ITS1F / ITS2) at 4000 rpm for 1 minute to allow the dry powder to settle to the bottom of the tube. According to the synthesis amount (nmol) on the primer synthesis sheet, add the corresponding volume of nuclease-free water, shake to mix, take 10 μL of the stock solution, add 90 μL of nuclease-free water and mix well, aliquot and store at -20℃ in the dark.

[0037] All preparation processes must be completed in the preparation room, and only sterilized reagents can be brought into the laminar flow hood.

[0038] 5. Experimental steps: (1) Microfiltration and enrichment pretreatment of the test samples; In a clean bench, aspirate 1.0 mL of the cell preparation to be tested into a sterile 2.0 mL EP tube. Set the parameters of the refrigerated centrifuge to 4°C, 1000×g, and centrifuge for 5 min. After centrifugation, the host cells have settled to the bottom of the tube. Carefully aspirate all the supernatant with a pipette and transfer it to a new sterile tube. Discard the cell pellet. Aspirate the supernatant using a 2mL sterile syringe, attach a 0.22μm hydrophilic polyethersulfone (PES) microporous filter to the tip, and push the syringe evenly to allow all the liquid to pass through the filter membrane. Microorganisms will be trapped on the surface of the filter membrane. Carefully remove the microporous membrane from inside the filter using sterile forceps sterilized by burning with an alcohol lamp flame, and place it into a 1.5 mL sterile centrifuge tube containing 50 μL of sterile nuclease-free water; Place the centrifuge tubes in an ultrasonic cleaner, set the frequency to 40 kHz, and ultrasonically vibrate for 3 to 5 minutes. Then place them on a vortex mixer and vortex vigorously for 1 minute to allow the trace bacteria or fungal spores trapped on the filter membrane to be fully detached and enriched in 50 μL of elution buffer.

[0039] (2) One-step rapid lysis preparation of template nucleic acid; Take 27 μL of the enriched eluent, add 27 μL of 2× microbial rapid lysis buffer (volume ratio 1:1), place on a vortex shaker and shake vigorously for 30 seconds to ensure that the lysed components are in full contact with the microorganisms; Place the mixed tube into the PCR instrument and run the isothermal lysis program. The reaction is kept at 85°C for 15 minutes, then rapidly cooled to 4°C and held for 2 minutes. After lysis, place the centrifuge tube on a centrifuge and centrifuge briefly at 3000 rpm for 10 seconds to allow air bubbles and condensate on the tube wall to settle. At this time, there may be trace amounts of cell debris precipitated at the bottom of the tube. The supernatant is the crude template containing total genomic DNA of the microorganism and can be used directly for subsequent amplification. When aspirating the lysis supernatant, the pipette tip should not touch the bottom of the tube; it should be aspirated from the upper layer of the liquid surface. The precipitate at the bottom of the tube is rich in incompletely disintegrated proteins and cell wall fragments, which will inhibit polymerase activity when introduced into the qPCR system. Furthermore, since Gram-positive bacteria have tough and thick peptidoglycan cell walls, relying solely on 85°C thermal lysis may result in incomplete nucleic acid release, leading to an overestimation of the Ct value for quantitative detection. If the target contains such bacteria, before adding the lysis buffer, add 2 μL of lysozyme (working concentration 20 mg / mL) to 27 μL of sample, pre-incubate at 37°C for 10-15 min, and then perform thermal lysis at 85°C.

[0040] (3) Preparation of fluorescent quantitative qPCR amplification system and parameter setting; Perform operations on ice to prevent cross-contamination, separate the reagent preparation area from the template loading area, and prepare the bacterial detection (515F / 907R primers) and fungal detection (ITS1F / ITS2 primers) separately in different tubes; Prepare the total system for a single reaction well on a sterile ice box in sequence. The total system configuration for a single reaction well is as follows: 2×SYBR Green qPCR Master Mix: 20.0μL; 10μM Forward Primer (10μM): 0.8μL (final concentration 0.2μM); 10μM Reverse Primer (10μM): 0.8μL (final concentration 0.2μM); Crude nucleic acid template obtained by lysis: 6.0μL; Finally, add 12.4μL of deionized water to make up to 40μL; Gently pipette up and down 5 to 8 times with a pipette to mix thoroughly, avoid generating violent bubbles, then flick the tube wall gently, place it on a microcentrifuge for a short centrifugation to ensure that all liquid sinks to the bottom of the tube; Place the prepared qPCR 8-strip tube smoothly into the reaction tank of the quantitative PCR instrument, make sure that each well is tightly capped, lock the hot lid, and set the running program parameters as follows: The pre-denaturation stage is set at 95°C for 3min (1 cycle); The PCR amplification stage is set to denature at 95°C for 10s, anneal at 55°C for 30s, extend at 72°C for 20s (a total of 45 cycles are set, and fluorescence signals are collected at 72°C); The melting curve analysis stage is set to slowly heat up from 65°C to 95°C (heating rate 0.5°C / step) and continuously collect fluorescence; Obtain the amplification curve and melting curve, and the signal judgment criteria are as follows: The negative control (water) has no Ct value; Positive: Ct ≤ 30 and the Tm value of the single melting peak falls within the specific interval, where the bacterial Tm value is in the range of 83-87°C, and the fungal Tm value is in the range of 80-84°C; Weakly positive: 30 < Ct ≤ 35 and the Tm value is normal, which requires repeated verification in duplicate wells; Negative: Ct > 38 and no specific melting peak. If an abnormal late peak appears at high cycles but with a low and gentle Tm, it is judged as primer dimer, and the result is still recorded as negative.

[0041] Furthermore, the amount of nucleic acid template added to each well must be strictly controlled at 6.0 μL. SYBR Green dye is extremely sensitive to light, and the prepared system tubes must be wrapped in aluminum foil or placed in a dark box while waiting to be used on the instrument.

[0042] 6. Implementation Example Verification and Data Analysis: (1) Example 1: Preparation of microbial standard solution and establishment of CFU standard curve; Representative bacterial strains used for activation verification: Bacillus subtilis, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa were inoculated into 10 mL of prepared LB liquid medium and cultured overnight at 37°C with shaking at 220 rpm until the logarithmic growth phase. The absorbance of the suspension was measured using a spectrophotometer. The four bacterial suspensions were serially diluted to OD600 of 0.2, 0.4, 0.6, 0.8, and 1.0 (approximately...). (CFU / mL), take the bacterial suspension at each OD value, dilute it and spread it on a plate, and count the number of CFUs; Aspergillus niger was spread onto prepared PDA solid plates and incubated at 28°C for 3 days. The plates were then washed with 2 mL of sterile PBS buffer to remove fungal spores. The spore solution was filtered to remove hyphae, and diluted to OD620 values ​​of 0.2, 0.4, 0.6, 0.8, and 1.0. Each OD value was used to further dilute the solution and spread it onto plates. Cell counting was performed using a cell counting chamber. Because the CFU of Aspergillus niger spore solution at low concentrations (OD620 = 1.0) was much lower than... Therefore, the Aspergillus niger spore solution was adjusted to OD620=3.0 (approximately 3×107 CFU / mL), and the high-concentration spore solution with OD620 of 3.0 was directly selected for counting.

[0043] CFU Standard Curve Plotting: Bacterial solutions with different OD gradients were serially diluted 10-fold and plated. Based on the number of colonies grown (CFU), a standard curve was plotted relating OD values ​​to CFU concentrations. The CFU standard curves for the four bacteria are shown below. Figures 2-5 As shown, the CFU standard curve of Aspergillus niger is as follows: Figure 6 As shown; Since the linear relationship between OD and CFU is not clear, OD600 was set to 1.0 for all bacteria in subsequent experiments (Bacillus subtilis: 2×). CFU / mL, E. coli: 1× CFU / mL, Staphylococcus aureus: 2× CFU / mL, Pseudomonas aeruginosa: 4× CFU / mL), Aspergillus niger OD620 was 3.0 (3× (CFU / mL), then collect CFU cells.

[0044] (2) Example 2: Validation of the specificity and applicability of aseptic rapid detection based on cell preparation matrix: Single clones of four bacteria were picked and resuspended in LB medium and in the matrix of three sterile cell preparation products: peripheral blood NK preparation (hereinafter referred to as PB), umbilical cord mesenchymal preparation (hereinafter referred to as MS) and PB+MS. After lysis, 6 μL of template was taken and qPCR was performed according to the 515F+907R system to evaluate its specificity. In applicability verification, data is collected. Four bacterial strains from CFU were resuspended in PB / MS / PB+MS formulations, and qPCR was performed using an optimized 6 μL template volume.

[0045] The experimental results of dual verification of specificity and applicability are shown in Tables 1 and 2; Table 1: Ct values ​​from specificity validation experiments (18 μL amplification template);

[0046] Table 2: Ct value data of applicability validation experiment (real cell preparation resuspension + 6μL optimized template amount);

[0047] The data above show that this method has significant detection specificity for stem cells and reinfused immunotherapy products, and the host cell human DNA or nutritional excipients in the preparation will not cause cross-false positive reactions. In LB medium, the complex organic color and PCR blocking substances caused the system to be completely inhibited. In pure water system, the Ct value of Staphylococcus aureus showed skipped wells or late peaks due to the tolerance of the hard peptidoglycan cell wall to thermal lysis. After changing the sample volume to 6 μL and enzyme pretreatment, as shown in Table 2, the microorganisms incorporated into all preparation samples showed strong and stable amplification signals. The Ct value difference between the negative control and the strain template was significant (ΔCt>8), which proved the clinical applicability of this technology to complex live cell products.

[0048] (3) Example 3: Evaluation of the robustness and detection limit of the qPCR detection method; In the robustness study, the performance of the qPCR core component was assessed by varying the proportions (with the Master Mix concentration in the system subject to reasonable fluctuations of ±10%: normal concentration 20 μL, increased by 10% to 22 μL, and decreased by 10% to 18 μL). The anti-interference capability of microbial genome amplification performance; In the detection limit assessment, representative strains were diluted and aliquoted as follows: , , The quantitative gradient was resuspended in a PB+MS composite formulation for direct lysis and amplification (without microporous membrane concentration pretreatment).

[0049] The experimental data are recorded in Tables 3 and 4. Table 3: Durability test results (Ct values) when critical reagent concentrations fluctuate during operation;

[0050] Table 4: qPCR detection limit (LOD) results (Ct values) of strains directly lysed in the product (without membrane concentration);

[0051] The robustness assessment results (Table 3) show that, under ±10% operational error perturbation of the key amplification components, the Ct values ​​of the detection system for the four major bacteria did not drift significantly, and the Tm values ​​of the melting peak remained completely consistent, demonstrating the industrial robustness of the detection system. The detection limit assessment of the direct lysis method without microporous membrane concentration (Table 4) exhibits an anomalous data structure: When the bacterial culture input volume is to During this period, due to the inhibition of residual substances in the crude extract of the sample or the interference of non-specific amplification under high cycle number, its amplification signal was significantly lagging behind the negative control. The blank negative control was subjected to frequent opening and sealing during the later verification stage, which inevitably led to environmental aerosol or trace bacterial contamination, and its background Ct value was abnormally inverted to around 30. The above data structure was also confirmed in the electrophoresis results of conventional PCR. Figure 7 middle: 1:2000Marker; 2-4: 515F+907R primers, template E. coli 10^6 CFU, 3 replicates per template; 5-7: 515F+907R primers, template E. coli 10^5 CFU, 3 replicates per template; 8-10: 515F+907R primers, template E. coli 10^4 CFU, 3 replicates per template; 11-13: 515F+907R primers, template PB+MS, 3 replicates per template; 14-16: 515F+907R primers, template water, 3 replicates per template; like Figure 7 As shown, only at extremely high concentrations (such as 10) 6 Weak amplification bands can only be observed under CFU, while at 10 4 At CFU and lower concentrations, the amplification signal is extremely weak; The results indicate that if the microporous membrane pretreatment is omitted, the sensitivity of the method will be significantly reduced, and the absolute number of bacteria must not be less than [a certain value]. By forcibly running a microporous membrane retention and ultrasonic detachment enrichment pretreatment procedure, the effective enrichment factor of the sample can be increased by more than 20 times, avoiding degradation of the crude extract, and finally achieving a true system detection (LOD) of 100 CFU / mL.

[0052] 6. Final Result Analysis; (1) In response to the problem that the golden survival window of stem cells, CAR-T and other live biological agents is only 24 to 72 hours and the time for clinical release is urgent, the efficient sterile detection methodology developed in this invention breaks through the limitations of the traditional culture method (membrane filtration method) which relies on the spontaneous division and reproduction of microorganisms and requires a long observation period of 7 to 14 days. By integrating micro-concentration enrichment pretreatment technology (using 0.22μm PES microporous membrane to intercept trace microorganisms and supplemented by ultrasonic and vortex elution) and one-step rapid thermal lysis process (mixing sample and lysis buffer 1:1 and reacting at 85℃ for 15 min, skipping the conventional nucleic acid column purification step), the total time of the whole process from sample receiving, centrifugation enrichment, lysis and cell wall breaking to qPCR fluorescence quantitative result determination is precisely controlled within 2 to 4 hours, thereby improving the timeliness of microbial quality control of cell preparations.

[0053] (2) To address the problem of PCR amplification inhibition caused by complex matrices in live cell preparations, this invention solves the problem of total amplification inhibition caused by the addition of high-volume crude extracts through cross-optimization of multiple sample loading. The amount of nucleic acid template in the qPCR reaction system is scientifically anchored at the golden ratio of 6.0 μL, ensuring that the concentration of residual inhibitors is successfully diluted below the amplification inhibition threshold. Furthermore, for Gram-positive Firmicutes such as Staphylococcus aureus, this method combines 20 mg / mL lysozyme with pre-incubation at 37°C for 10-15 min to improve the cell wall disruption process, ensuring 100% full release of nucleic acids from a broad spectrum of bacteria and fungi. 10 mg / mL lysozyme is incorporated into the PB, MS, and PB+MS real cell preparation matrices. 7 In the CFU representative strain suitability verification, all samples showed strong and stable amplification signals (negative control and strain template ΔCt>8), reducing the risk of false negatives and missed detections.

[0054] (3) In accordance with the pharmacopoeia and the guidelines for alternative validation of methods, this method selected five representative standard strains covering Gram-positive bacteria, Gram-negative bacteria, facultative anaerobes and filamentous fungi for comprehensive validation. The universal 16S rDNA specific primers for bacteria (515F / 907R) and the universal ITS region specific primers for fungi (ITS1F / ITS2) used in the method showed excellent species broadness and target specificity. The targeting specificity of primers can be achieved through... Figure 8 and Figure 9 It was confirmed by electrophoresis results that after amplification using the above primer combination, the products all showed clear, single bands of the expected size (around 400 bp for bacteria and around 350 bp for fungi), and no non-specific bands were observed throughout the process. In terms of detection limits, the detection limit of this system consistently reaches 10 for direct pyrolysis without concentration. 3 In the detection of large volumes of drug solutions combined with microfiltration pretreatment, the limit of detection (LOD) at the CFU level can be significantly reduced to 100 CFU / mL. Combined with a dual judgment matrix consisting of 45 cycle parameters and specific melting peak Tm values ​​(83℃ to 87℃ for bacteria and 80℃ to 84℃ for fungi), accurate qualitative and qualitative determination of trace contamination can be achieved. The instrument output data for establishing this dual-determination matrix is ​​as follows: Figures 10-13 (The graph shows the amplification and melting curves of bacterial and fungal quantitative qPCR. The graph clearly shows the typical and main specific melting peaks formed by each representative strain in the later stage of amplification. The peak values ​​fall entirely within the Tm value range defined above, which proves the reliability of the judgment matrix in screening out non-specific interference and improving the accurate qualitative analysis of trace contamination.

[0055] (4) Considering the operational errors in the actual production environment, this method underwent a rigorous robustness assessment. By changing the ratio of the core components of qPCR, that is, subjecting the standard amount of Master Mix (20 μL) in the reaction system to a large range of artificial spotting error fluctuations of ±10% (18 μL to 22 μL), the results showed that, compared with the negative control, all bacteria could be detected stably at 100%, and the minor environmental differences caused by operational errors did not have a substantial impact on the detection conclusions, amplification effect and quantitative output.

[0056] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A rapid detection method for microbial contamination in cell preparations based on qPCR technology, characterized in that, include: Obtain the live cell preparation sample to be tested, and perform pretreatment such as host cell removal, microporous membrane retention and ultrasonic vortex elution to obtain the eluent containing trace amounts of microorganisms. The elution buffer was mixed with the microbial rapid lysis buffer, and a one-step isothermal thermal lysis was performed to prepare a crude nucleic acid template containing total microbial genomic DNA. Prepare a real-time quantitative PCR reaction system containing targeted amplification primers, add the crude extracted nucleic acid template, run amplification on a real-time quantitative PCR instrument and collect fluorescence signals to obtain amplification curves and melting curves; Based on a dual judgment matrix consisting of cycle number and specific melting peak, the signal is qualitatively and quantitatively judged to determine whether the live cell preparation is contaminated with microorganisms.

2. The rapid detection method for microbial contamination of cell preparations based on qPCR technology according to claim 1, characterized in that, The specific steps of the microfiltration and ultrasonic vortex enrichment pretreatment are as follows: Centrifuge the cell preparation to be tested at 4℃ and 1000×g for 5 minutes, discard the host large cell precipitate, and collect the supernatant; The supernatant was microfiltered using a 0.22 μm hydrophilic polyethersulfone microporous filter. Remove the microporous membrane and place it into a centrifuge tube containing 50 μL of sterile nuclease-free water. Sonicate at 40 kHz for 3-5 minutes, followed by vigorous vortexing for 1 minute to remove trapped microorganisms and enrich them in the eluent.

3. The rapid detection method for microbial contamination of cell preparations based on qPCR technology according to claim 1, characterized in that, The specific parameters for the one-step isothermal pyrolysis method are as follows: The enriched eluent was mixed with 2× microbial rapid lysis buffer at a volume ratio of 1:1 and vortexed for 30 seconds. Place at 85℃ and maintain the temperature for 15 minutes, then rapidly cool to 4℃ and maintain for 2 minutes; After a short period of centrifugation to allow air bubbles and cell debris to settle on the tube wall, the middle layer on the surface of the liquid is collected as a crude nucleic acid template for direct amplification.

4. The rapid detection method for microbial contamination of cell preparations based on qPCR technology according to claim 1, characterized in that, In the one-step isothermal thermal lysis step, if the target contains Gram-positive bacteria with thick peptidoglycan cell walls, lysozyme at a working concentration of 20 mg / mL is added to the sample before adding the lysis buffer, pre-incubated at 37°C for 10-15 minutes, and then thermally lysed at 85°C.

5. The rapid detection method for microbial contamination of cell preparations based on qPCR technology according to claim 1, characterized in that, The targeted amplification primers include universal 16S rDNA-specific primers for bacteria and universal ITS region-specific primers for fungi that have been screened by Blast comparison of the human whole genome. The bacteria-specific primer sequences are as follows: Forward primer 515F: 5'-GTGYCAGCMGCCGCGGTAA-3'; Reverse primer 907R: 5'-CCGTCAATTCMTTTRAGTTT-3'; The fungal-specific primer sequences are: Forward primer ITS1F: 5'-CTTGGTCATTTAGAGGAAGTAA-3'; Reverse primer ITS2: 5'-GCTGCGTTCTTCATCGATGC-3'.

6. The rapid detection method for microbial contamination of cell preparations based on qPCR technology according to claim 1, characterized in that, The quantitative qPCR reaction system is a 40 μL total reaction volume per well, with the specific preparation ratio as follows: Add 2×SYBR Green qPCR Master Mix 20.0 μL, add forward primer and reverse primer to make their final concentrations both 0.2 μM, add the crude extracted nucleic acid template, whose added volume is strictly limited to 6.0 μL, so as to dilute the residual inhibitor concentration to below the amplification inhibition threshold, and make up the remaining volume with deionized water.

7. The rapid detection method for microbial contamination of cell preparations based on qPCR technology according to claim 1, characterized in that, The program parameters for amplification run on the real-time fluorescent quantitative PCR instrument are set as follows: Pre-denaturation stage: 95℃, 3 minutes, run 1 cycle; PCR amplification stage: denature at 95℃ for 10 seconds, anneal at 55℃ for 30 seconds, extend at 72℃ for 20 seconds, set a total of 45 cycles, and collect fluorescence signals at 72℃; Melting curve analysis stage: slowly heat up from 65℃ to 95℃, set the heating rate to 0.5℃ / step, and continuously collect fluorescence.

8. The rapid detection method for microbial contamination of cell preparations based on qPCR technology according to claim 1, characterized in that, The signal judgment rules of the dual judgment matrix are as follows: Positive judgment: Ct ≤ 30 and the Tm value of the single melting peak falls within the specific interval; Weak positive judgment: 30 < Ct ≤ 35 and the Tm value is normal, which requires repeated verification in duplicate wells; Negative judgment: Ct > 38 and no specific melting peak, or an abnormal late peak appears at high cycles with a low Tm value and flat primer dimer signal; In the specific interval, the Tm value range of bacteria is defined as 83-87℃, and the Tm value range of fungi is defined as 80-84℃.

9. The rapid detection method for microbial contamination of cell preparations based on qPCR technology according to claim 1, characterized in that, The viable cell preparations include stem cell and CAR-T preparations, and the microorganisms cover Gram-positive bacteria, Gram-negative bacteria, facultative anaerobes and filamentous fungi.

10. The rapid detection method for microbial contamination of cell preparations based on qPCR technology according to claim 1, characterized in that, The viable cell preparation is a complex matrix product containing host human DNA background and nutrient excipients, and the method is specifically used to eliminate the interference of PCR amplification inhibition of the complex cell preparation matrix, cross-interference from host human DNA and preparation excipients, as well as non-specific amplification and primer dimers on the detection results, so as to improve the accuracy and reliability of microbial contamination judgment.

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