A rapid microbial contamination detection kit and application thereof in detection of microbial contamination of cell preparation

Abstract

The present application provides a kind of microbial contamination rapid detection kit and its application in cell preparation microbial contamination detection.The kit includes reagent A, reagent B, reagent C and reagent D;Reagent A includes first upstream primer, first downstream primer and first detection probe;Reagent B includes second upstream primer, second downstream primer and second detection probe;Reagent C includes third upstream primer, third downstream primer and third detection probe;Reagent D includes fourth upstream primer, fourth downstream primer and fourth detection probe.The kit provided by the present application can simultaneously detect bacterial, mycoplasma, chlamydia, fungi and other four types of microbial contamination with high sensitivity and high specificity, with a detection limit of up to 10 1 ~10 2 copies / reaction, only 100~1000 μL sample is needed for single detection, which can significantly reduce the consumption of sample, the detection period is short, and each primer and probe has high specificity for target microorganism without cross reaction.

Description

Technical Field

[0001] This invention relates to the field of molecular biology detection technology, specifically to a rapid detection kit for microbial contamination and its application in the detection of microbial contamination in cell preparations. Background Technology

[0002] As core products in cell therapy, regenerative medicine, and biopharmaceuticals, cell preparations are directly related to patients' health and lives in terms of quality and safety. Microbial contamination is one of the most common and serious quality problems in the production of cell preparations, mainly including bacteria, mycoplasma, chlamydia, and fungi. According to the Chinese Pharmacopoeia and relevant international regulations, cell preparations must undergo rigorous sterility and mycoplasma testing before release.

[0003] Currently, the detection of microbial contamination in cell preparations mainly employs traditional culture methods and single PCR detection methods. Traditional culture methods are the gold standard for microbial detection, but bacterial cultures require 7–14 days, and mycoplasma cultures require 21–28 days. Some difficult-to-culture or unculturable microorganisms are difficult to detect, thus traditional culture methods suffer from long detection cycles and limited sensitivity. Single PCR detection methods primarily target single pathogens, requiring multiple tests to detect all microorganisms. Cell preparation samples are often very valuable, and multiple tests result in sample waste. Furthermore, multiple tests increase operational complexity and the risk of contamination. Therefore, single PCR detection methods suffer from low throughput, high sample consumption, and cumbersome operation.

[0004] Although existing technologies have attempted to use multiplex PCR to detect bacterial, mycoplasma, chlamydia, and fungal microbial contamination in cell preparations, they generally suffer from problems such as mutual interference between multiple primers leading to decreased amplification efficiency and spectral crosstalk between fluorescence channels affecting detection accuracy.

[0005] Therefore, developing a rapid detection kit for microbial contamination using cell preparations that can simultaneously detect multiple microorganisms, has a short detection cycle, high sensitivity, and strong specificity has significant practical application value. Summary of the Invention

[0006] To address the problems of long detection cycles and limited sensitivity in traditional culture methods for detecting microbial contamination, low throughput, high sample consumption, and cumbersome operation in single PCR methods, and reduced amplification efficiency due to mutual interference between multiple primers and reduced detection accuracy due to spectral crosstalk between fluorescence channels in multiplex PCR methods, this invention provides a rapid microbial contamination detection kit and its application in the detection of microbial contamination in cell preparations.

[0007] According to a first aspect of the present invention, a rapid detection kit for microbial contamination is provided, the kit comprising reagent A, reagent B, reagent C and reagent D; Reagent A is used to detect bacteria. Reagent A includes a first upstream primer, a first downstream primer, and a first detection probe. The nucleotide sequence of the first upstream primer is shown in SEQ ID NO: 1, the nucleotide sequence of the first downstream primer is shown in SEQ ID NO: 2, and the nucleotide sequence of the first detection probe is shown in SEQ ID NO: 3. Reagent B is used to detect mycoplasma. Reagent B includes a second upstream primer, a second downstream primer, and a second detection probe. The nucleotide sequence of the second upstream primer is shown in SEQ ID NO: 4, the nucleotide sequence of the second downstream primer is shown in SEQ ID NO: 5, and the nucleotide sequence of the second detection probe is shown in SEQ ID NO: 6. Reagent C is used to detect Chlamydia. Reagent C includes a third upstream primer, a third downstream primer, and a third detection probe. The nucleotide sequence of the third upstream primer is shown in SEQ ID NO: 7, the nucleotide sequence of the third downstream primer is shown in SEQ ID NO: 8, and the nucleotide sequence of the third detection probe is shown in SEQ ID NO: 9. Reagent D is used to detect fungi. Reagent D includes a fourth upstream primer, a fourth downstream primer, and a fourth detection probe. The nucleotide sequence of the fourth upstream primer is shown in SEQ ID NO: 10, the nucleotide sequence of the fourth downstream primer is shown in SEQ ID NO: 11, and the nucleotide sequence of the fourth detection probe is shown in SEQ ID NO: 12.

[0008] The inventors of this application designed primers and TaqMan probes targeting bacterial 16S rDNA, mycoplasma 16S rDNA, chlamydia ompA gene, and fungal ITS2 region, respectively. After screening, they obtained a first upstream primer, a first downstream primer, and a first detection probe (reagent A) for detecting bacteria; a second upstream primer, a second downstream primer, and a second detection probe (reagent B) for detecting mycoplasma; a third upstream primer, a third downstream primer, and a third detection probe (reagent C) for detecting chlamydia; and a fourth upstream primer, a fourth downstream primer, and a fourth detection probe (reagent D) for detecting fungi. Using the above reagents A, B, C, and D as components of the kit, the rapid microbial contamination detection kit provided by this invention is obtained.

[0009] The rapid detection kit for microbial contamination provided by this invention enables highly sensitive and specific detection of four types of microbial contamination—bacteria, mycoplasma, chlamydia, and fungi—through multiplex real-time quantitative PCR and multi-channel fluorescence signals, with a detection limit as high as 10. 1 ~10 2The copies / reaction ratio (equivalent to a detection sensitivity of 1~10 CFU / mL) is significantly superior to traditional culture methods. A single detection requires only 100~1000 μL of sample, which can significantly reduce the consumption of precious samples. Furthermore, each primer and probe is highly specific to the target microorganism, and there is no cross-reaction between primers and probes. In addition, when using the kit provided by this invention to detect bacteria, mycoplasma, chlamydia, and fungi, the detection time is only 2~3 hours (including sample processing), which significantly shortens the detection cycle compared to traditional culture methods.

[0010] Preferably, the first detection probe, the second detection probe, the third detection probe, and the fourth detection probe each contain a fluorescent group and a quenching group. The fluorescent group is selected from Cy5, FAM, VIC, and ROX, and the quenching group is selected from BHQ1, BHQ3, and MGB.

[0011] Preferably, the fluorescent group is located at the 5' end of the first detection probe, the second detection probe, the third detection probe, and the fourth detection probe, and the quenching group is located at the 3' end of the first detection probe, the second detection probe, the third detection probe, and the fourth detection probe.

[0012] Preferably, the 5' end of the first detection probe is labeled with the fluorescent group Cy5, and the 3' end of the first detection probe is labeled with the quencher group BHQ3; the 5' end of the second detection probe is labeled with the fluorescent group FAM, and the 3' end of the second detection probe is labeled with the quencher group BHQ1; the 5' end of the third detection probe is labeled with the fluorescent group VIC, and the 3' end of the third detection probe is labeled with the quencher group BHQ1; the 5' end of the fourth detection probe is labeled with the fluorescent group ROX, and the 3' end of the fourth detection probe is labeled with the quencher group MGB.

[0013] The 5' ends of the first, second, third, and fourth detection probes are labeled with fluorescent groups Cy5, FAM, VIC, and ROX, respectively, while the 3' ends are quenched with BHQ series quenchers or MGB quenchers. During PCR extension, the 5'→3' exonuclease activity of Taq DNA polymerase hydrolyzes the detection probes bound to the template, physically separating the fluorescent groups from the quenchers and releasing fluorescent signals. By collecting the fluorescent signals in the detection system, the simultaneous detection of four types of microorganisms—bacteria, mycoplasma, chlamydia, and fungi—can be achieved. Furthermore, the emission peaks of the four fluorescent groups Cy5, FAM, VIC, and ROX are relatively far apart, resulting in good separation. During fluorescence signal acquisition, spectral overlap is minimized, allowing the signals from bacteria (Cy5), mycoplasma (FAM), chlamydia (VIC), and fungi (ROX) to be independently acquired and analyzed by the instrument without interference. This fundamentally avoids false positives and cross-interpretations, ensuring the specificity and accuracy of the quadruple detection. In addition, bacteria have high 16S rDNA copy numbers and complex backgrounds, so the Cy5 channel, which has a long wavelength and relatively less background interference, is used. Mycoplasma and Chlamydia generally have lower abundances, so the more sensitive FAM and VIC channels are used. Fungi use the ROX channel. This combination helps to obtain a more stable fluorescence signal during the detection process, thereby improving detection sensitivity and accuracy.

[0014] Preferably, the above kit also includes an internal control sample containing a human β-actin gene fragment.

[0015] Preferably, the above kit further includes reagent E, which is used to detect the internal control sample, and reagent F includes a fifth upstream primer, a fifth downstream primer and a fifth detection probe. The nucleotide sequence of the fifth upstream primer is shown in SEQ ID NO: 13, the nucleotide sequence of the fifth downstream primer is shown in SEQ ID NO: 14, and the nucleotide sequence of the fifth detection probe is shown in SEQ ID NO: 15.

[0016] β-actin is a housekeeping gene that is stably expressed in all human cells.

[0017] The kit provided in this protocol introduces an internal control sample and primers and probes targeting that internal control sample. The internal control sample can be a recombinant plasmid containing a human β-actin gene fragment or a synthetic DNA fragment. When using this kit to detect samples containing human cells, the internal control sample and primers and probes targeting it can effectively identify PCR reaction inhibition, nucleic acid extraction failure, sample degradation, and other situations, avoiding false negative test results and ensuring the reliability of the final test results.

[0018] Preferably, the above kit further includes positive control samples, which include a first plasmid, a second plasmid, a third plasmid, and a fourth plasmid. The first plasmid contains Escherichia coli ATCC 25922 genomic DNA or a 16S rDNA fragment, the second plasmid contains Mycoplasma stomatitis ATCC 23714 genomic DNA or a 16S rDNA fragment, the third plasmid contains Chlamydia trachomatis ATCCVR-902B genomic DNA or an ompA gene fragment, and the fourth plasmid contains Candida albicans ATCC 10231 genomic DNA or an ITS2 fragment.

[0019] The kit provided in this protocol introduces a first plasmid containing E. coli genomic DNA or a 16S rDNA fragment, a second plasmid containing Mycoplasma oralis genomic DNA or a 16S rDNA fragment, a third plasmid containing Chlamydia trachomatis genomic DNA or an ompA gene fragment, and a fourth plasmid containing Candida albicans genomic DNA or an ITS2 fragment. Using these four plasmids as positive control samples, the kit can not only detect the quality of the amplification reaction solution but also obtain the detection limit information of the detection reagents (i.e., the primers and probes in the kit).

[0020] Preferably, the kit further includes reagent F, which comprises a propidium azide bromide (PMA) solution with a concentration of 20-100 μM.

[0021] PMA is a nucleic acid intercalation dye that can penetrate the damaged cell membrane of dead cells and enter the cell interior to covalently cross-link with DNA. After activation by light, PMA forms a stable covalent bond with DNA, preventing DNA polymerase extension during subsequent PCR amplification and thus inhibiting the amplification of DNA from dead bacteria. However, because the cell membrane of living cells remains intact, PMA cannot enter, and therefore the DNA from living bacteria remains unaffected and can amplify normally.

[0022] Test samples contaminated with live microorganisms (e.g., cell preparations) pose an infection risk and should not be used, while test samples contaminated with dead microorganisms can guide subsequent processing by further assessing the risk of residual endotoxins / antigens.

[0023] This protocol introduces PMA into the kit to pretreat the test samples. By comparing the results of the PMA-treated group and the untreated group, the viability of microorganisms in the test samples can be quickly determined, which has important clinical significance for the application of cell preparations and other test samples.

[0024] Preferably, the above kit further includes reagent G, which comprises the following components: 0.02~0.05 U / μL hot-start Taq DNA polymerase, 200~400 μM dNTPs mixture, 40~60 mM KCl solution, 0.01~0.02 U / μL uracil DNA glycosylase (UNG enzyme), 200~400 μM dUTP, magnesium salt solution, Tris-HCl buffer, and stabilizer; the magnesium salt solution contains Mg 2+ The concentration is 2-4 mM; the pH of the Tris-HCl buffer is 8.3-8.5 and the concentration is 10-20 mM; the stabilizer includes the following components: 0.1-0.5 mg / mL bovine serum albumin (BSA) and 0.5-1.5 M betaine.

[0025] Introducing a PCR reaction premix containing the above components (i.e., reagent G) into the kit provides a suitable chemical environment for PCR amplification during the detection of the sample, ensuring the stability of the PCR amplification reaction and thus improving PCR amplification efficiency and microbial detection efficiency. Furthermore, the premixed form of reagent G reduces operational steps and minimizes human error.

[0026] Preferably, the above kit further includes reagent H, which comprises the following components: lysis buffer, binding buffer, washing buffer I, washing buffer II, and elution buffer; the lysis buffer contains proteinase K, sodium dodecyl sulfate (SDS), and ethylenediaminetetraacetic acid (EDTA); the binding buffer contains 3-5 M first salt solution, 0.5-1 M second salt solution, and 20-50 mM Tris-HCl, the first salt solution contains guanidine hydrochloride or guanidine isothiocyanate, the second salt solution contains sodium chloride or sodium acetate, and the pH of Tris-HCl is 6.0-6.5; washing buffer I contains 20-35% (v / v) ethanol solution, 3-5 M first salt solution, 0.5-1 M second salt solution, and 20-50 mM Tris-HCl. The Tris-HCl solution has a first salt solution containing guanidine hydrochloride or guanidine isothiocyanate, and a second salt solution containing sodium chloride or sodium acetate. The pH of the Tris-HCl solution is 6.0–6.5. Washing buffer II is a 75% ethanol solution. The elution buffer is a low-salt Tris-EDTA buffer (low-salt TE buffer) or sterile water. The low-salt TE buffer contains 8–12 mM Tris-HCl solution and 0.1–1 mM ethylenediaminetetraacetic acid (EDTA) solution. The pH of the EDTA solution is 8.0–9.0.

[0027] According to a second aspect of the present invention, the application of the above-described rapid detection kit for microbial contamination in the detection of microbial contamination in cell preparations is provided.

[0028] The rapid detection kit for microbial contamination provided by this invention can be applied to the detection of microbial contamination in cell preparations. Through multiplex real-time quantitative PCR and multi-channel fluorescence signals, it can achieve high sensitivity and high specificity detection of four types of microbial contamination: bacteria, mycoplasma, chlamydia, and fungi. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the composition of the rapid detection kit for microbial contamination provided in Example 1.

[0030] Figure 2 The flowchart is for the microbial contamination detection method provided in Example 2.

[0031] Figure 3 This is a five-channel fluorescence detection spectral distribution diagram involved in the microbial contamination detection method provided in Example 2.

[0032] Figure 4 The qPCR standard curve and corresponding real-time amplification curve obtained from the detection of universal bacterial targets provided in Example 2 are shown.

[0033] Figure 5 The qPCR standard curve and corresponding real-time amplification curve obtained from the detection of universal mycoplasma targets provided in Example 2 are shown.

[0034] Figure 6 The qPCR standard curve and corresponding real-time amplification curve obtained from the detection of universal targets of Chlamydia provided in Example 2 are shown.

[0035] Figure 7 The qPCR standard curve and corresponding real-time amplification curve obtained from the detection of universal fungal targets provided in Example 2 are shown.

[0036] Figure 8 Amplification curves of samples with different proportions of viable bacteria provided for test example 3.

[0037] Figure 9 The curve showing the relationship between the ΔCt value and the proportion of viable bacteria provided for test example 3. Detailed Implementation

[0038] The technical features of the technical solution provided by the present invention will be further clearly and completely described below with reference to specific embodiments. 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.

[0039] Example 1 A rapid detection kit for microbial contamination, the composition of which is shown in the diagram below. Figure 1 As shown, the kit includes reagents A, B, C, D, E, F, G, H, internal control, and positive control. The specific components are shown in Table 1.

[0040] Table 1. Components of the rapid detection kit for microbial contamination in Example 1

[0041] Comparative Example 1 This comparative example provides a rapid detection kit for microbial contamination. Compared with Example 1, the difference in composition is that the nucleotide sequences of the primers and probes in Reagent A are different. Reagent A includes a sixth upstream primer, a sixth downstream primer, and a sixth detection probe. The specific nucleotide sequences are shown in Table 2.

[0042] Table 2. Primers and probes involved in reagent A of the kit for Comparative Example 1

[0043] Apart from the differences mentioned above, the materials, formulation ratios, and preparation procedures used in this comparative example are strictly consistent with those in Example 1.

[0044] Comparative Example 2 This comparative example provides a rapid detection kit for microbial contamination. Compared with Example 1, the difference in composition is that the nucleotide sequences of the primers and probes in Reagent A are different. Reagent A includes a seventh upstream primer, a seventh downstream primer, and a seventh detection probe. The specific nucleotide sequences are shown in Table 3.

[0045] Table 3 Primers and probes involved in reagent A of the kit for Comparative Example 2

[0046] Apart from the differences mentioned above, the materials, formulation ratios, and preparation procedures used in this comparative example are strictly consistent with those in Example 1.

[0047] Comparative Example 3 This comparative example provides a rapid detection kit for microbial contamination. Compared with Example 1, the difference in composition is that the nucleotide sequences of the primers and probes in Reagent B are different. Reagent B includes an eighth upstream primer, an eighth downstream primer, and an eighth detection probe. The specific nucleotide sequences are shown in Table 4.

[0048] Table 4. Primers and probes involved in reagent B of the kit for Comparative Example 3.

[0049] Apart from the differences mentioned above, the materials, formulation ratios, and preparation procedures used in this comparative example are strictly consistent with those in Example 1.

[0050] Comparative Example 4 This comparative example provides a rapid detection kit for microbial contamination. Compared with Example 1, the difference in composition is that the primers and probes in Reagent C have different nucleotide sequences. Reagent B includes a ninth upstream primer, a ninth downstream primer, and a ninth detection probe. The specific nucleotide sequences are shown in Table 5.

[0051] Table 5. Primers and probes involved in reagent C of the kit for Comparative Example 4.

[0052] Apart from the differences mentioned above, the materials, formulation ratios, and preparation procedures used in this comparative example are strictly consistent with those in Example 1.

[0053] Comparative Example 5 This comparative example provides a rapid detection kit for microbial contamination. Compared with Example 1, the difference in composition is that the primers and probes in Reagent E have different nucleotide sequences. Reagent B includes a tenth upstream primer, a tenth downstream primer, and a tenth detection probe. The specific nucleotide sequences are shown in Table 6.

[0054] Table 6. Primers and probes involved in reagent E of the kit for Comparative Example 5.

[0055] Apart from the differences mentioned above, the materials, formulation ratios, and preparation procedures used in this comparative example are strictly consistent with those in Example 1.

[0056] Example 2 A method for detecting microbial contamination is proposed based on the rapid microbial contamination detection kit provided in Example 1, wherein the reagents used are those in the rapid microbial contamination detection kit provided in Example 1. The flowchart of the microbial contamination detection method provided in this embodiment is as follows: Figure 2 As shown, it includes the following steps: 1. Sample pretreatment (1) Weigh 12.5 mg of PMA dry powder, dissolve it in 250 μL of dimethyl sulfoxide (DMSO) to prepare a 50 mM solution, dispense it into single-use portions, and store it at -20℃ protected from light; (2) Take 100~1000 μL of the sample to be tested (e.g., cell preparation) and divide it into two parts, A and B, as needed. Part A of the sample to be tested is not treated with PMA solution (used to detect total microbial DNA), and part B of the sample to be tested is treated with PMA solution (only to detect viable bacterial DNA). The process of treating sample B with PMA solution is as follows: PMA solution is added to sample B to achieve a final concentration of 20 μM. The sample is incubated at 25°C in the dark for 5-10 minutes, gently inverting and mixing every 2 minutes. Then, sample B treated with PMA solution is placed on ice, and a 500 W halogen lamp is placed 15-20 cm away from sample B for 5-15 minutes of light treatment (or a biological LED light source with a wavelength of 460-470 nm is used to irradiate sample B at a distance of 15-20 cm for 10-15 minutes). During the irradiation process, the sample is gently inverted and mixed every 3 minutes to obtain the pretreated sample.

[0057] 2. Nucleic acid extraction Add an equal volume of lysis buffer to the pretreated sample, mix thoroughly, and incubate at 56°C for 10 minutes. Add 1.5 times the volume of binding buffer and an appropriate amount of magnetic beads, and incubate at room temperature for 5 minutes to allow the nucleic acid to bind to the magnetic beads. Then, use a magnetic rack to adsorb the magnetic beads, discard the supernatant, and wash the magnetic beads 1-2 times each with washing buffer I and washing buffer II, 300 μL each time. After washing, dry the magnetic beads at room temperature for 5-10 minutes, then add 50-100 μL of elution buffer, incubate at room temperature for 2-5 minutes, and then adsorb the magnetic beads using a magnetic rack. Collect the supernatant containing nucleic acid. The extracted nucleic acid can be used immediately for detection or stored at -20°C for later use.

[0058] 3. Preparation of quality control materials (1) Positive control: Mix the first plasmid containing Escherichia coli ATCC 25922 genomic DNA, the second plasmid containing Mycoplasma oralis ATCC 23714 genomic DNA, the third plasmid containing Chlamydia trachomatis ATCC VR-902B genomic DNA, and the fourth plasmid containing Candida albicans ATCC 10231 genomic DNA in equal proportions, and dilute to 10 μL with TE buffer. 4 copies / μL; (2) Negative control: sterile TE buffer; (3) Internal control: Recombinant plasmid containing human β-actin gene fragment, diluted to 105 copies / μL.

[0059] 4. Preparation of PCR reaction system (1) Preparation of primer-probe mixture Table 7 Preparation of primer-probe mixture

[0060] Primer and probe sequences were synthesized separately. Each primer and probe was dissolved in sterile TE buffer and prepared into a 100 μM stock solution. The primer-probe mixture (1 mL) with a final concentration of 10× was prepared according to the proportions shown in Table 7. After mixing, the mixture was dispensed and stored at -20℃ in the dark. (2) Preparation of PCR reaction premix Table 8 Preparation of PCR reaction premix

[0061] Prepare 2×PCR reaction premix (10 mL) according to the proportions shown in Table 8, mix well, aliquot, and store at -20℃ in the dark. (3) Preparation of PCR reaction system Table 9 PCR Reaction System

[0062] Prepare a 25 μL PCR reaction system according to the components and volumes shown in Table 9. At the same time, add the following positive control group, negative control group and blank control group. In the positive control group, the positive quality control material is used to replace the nucleic acid template to be tested; in the negative control group, the negative quality control material is used to replace the nucleic acid template to be tested; and in the blank control group, sterile deionized water is used to replace the nucleic acid template to be tested.

[0063] 5. Detection using an ABI 7500 real-time quantitative PCR instrument (1) Instrument setup Select the ABI 7500 Fast real-time quantitative PCR instrument, set it to simultaneous detection across five channels, and the fluorescence spectral distribution of the five channels is shown below. Figure 3 As shown, the details are as follows: Channel 1 (Cy5): Bacterial detection, excitation wavelength 649 nm, emission wavelength 670 nm; Channel 2 (FAM): Mycoplasma detection, excitation wavelength 495 nm, emission wavelength 520 nm; Channel 3 (VIC): Chlamydia detection, excitation wavelength 538 nm, emission wavelength 554 nm; Channel 4 (ROX): Fungal detection, excitation wavelength 586 nm, emission wavelength 610 nm; Channel 5 (CY3): Internal reference detection, excitation wavelength 550nm, emission wavelength 570nm; (2) PCR reaction procedure Table 10 PCR reaction procedure

[0064] The nucleic acids extracted from the test samples were detected according to the PCR reaction procedure shown in Table 10. The total reaction time was approximately 90-120 min.

[0065] 6. Result Interpretation and Analysis (1) Interpretation of internal reference channels (validity determination) Interpret the results of the internal parameter channel (CY3) according to the following rules: a. The Ct value of the internal control sample is within the expected range (usually 25~35): the sample is valid and can be analyzed further; b. Abnormal Ct value or no amplification curve in the internal control sample: The sample is invalid and nucleic acid needs to be extracted and tested again; (2) Interpretation of each pathogen channel Table 11 Interpretation rules for each pathogen channel

[0066] The detection results of each pathogen (microorganism) are interpreted according to the interpretation rules shown in Table 11; (3) Determination of live and dead bacteria Table 12 Rules for determining live and dead bacteria

[0067] Compare the Ct values ​​of the same PMA-treated group (B test samples) Ct B Ct value of the untreated group (A test samples) A Difference (ΔCt = Ct) B - Ct A ), and determine the liveness or deadness of each pathogen (microorganism) according to the rules for determining live and dead bacteria in Table 12; (4) Quantitative analysis Quantitative detection can be achieved by establishing standard curves for detecting various pathogens, as detailed below: a. Use positive control samples with a 10-fold serial dilution (10 1 -10 7 Establish a standard curve using copies / μL; b. Standard curve linear range: 10 1 -10 7 copies / reaction c. Amplification efficiency requirement: 90%-110%; d. Correlation coefficient R 2 ≥0.99.

[0068] The qPCR standard curve and corresponding real-time amplification curve obtained by using the rapid microbial contamination detection kit provided in Example 1 and referring to the method provided in this example to detect the universal bacterial target (E. coli 16S rDNA fragment) are shown below. Figure 4 As shown, where, Figure 4 Figure A in the figure represents the qPCR standard curve obtained by detecting a universal bacterial target. The horizontal axis, Quantity, represents the initial template amount of the bacterial target sequence in each qPCR reaction system (copies / reaction), which increases in a 10-fold gradient from left to right. The vertical axis, Ct (Cycle threshold), represents the number of PCR cycles required for the fluorescence signal to first cross the set threshold. The red squares in the figure are the measured Ct values ​​of the standards at each dilution (i.e., the universal bacterial target), and the black line is its linear regression fitting line. Figure 4 The key regression parameters output by the instrument are shown below Figure A. Target: Bac—the name of the detection channel, indicating that the standard curve was obtained from the Cy5 channel corresponding to "Bacterial Universal Target (Bac)". Slope: 3.469—Slope of the standard curve, Inter: 35.201—Intercept of the regression line on the Y-axis, reflecting the instrument's minimum detection sensitivity, R 2 0.992 — Coefficient of determination, characterizing the goodness of linear fit between Ct value and log10 (Quantity). The closer to 1, the better the linear relationship. Eff%: 94.216 — PCR amplification efficiency; Figure 4 Figure B in the figure represents the real-time amplification curve obtained by detecting a universal bacterial target. The horizontal axis Cycle represents the PCR cycle number from 1 to 40, and the vertical axis ΔRn represents the normalized value of the Reporter channel fluorescence signal after subtracting the baseline, which is used to characterize the relative amount of bacterial target amplification products in each cycle reaction tube.

[0069] The qPCR standard curve and corresponding real-time amplification curve obtained by using the rapid microbial contamination detection kit provided in Example 1 and referring to the method provided in this example to detect the universal target of mycoplasma (mycoplasma 16S rDNA) are shown below. Figure 5 As shown, where, Figure 5 Figure A in the figure represents the qPCR standard curve obtained by detecting the universal target of mycoplasma. The horizontal axis Quantity represents the initial template amount of mycoplasma target sequence in each qPCR reaction system (copies / reaction), which increases in a 10-fold gradient from left to right. The vertical axis Ct (Cycle threshold) represents the number of PCR cycles required for the fluorescence signal to first cross the set threshold. The red squares in the figure are the measured Ct values ​​of the standards at each dilution, and the black line is its linear regression fitting line. Figure 5The key regression parameters output by the instrument are shown below Figure A. Target: Myco—the detection channel name, indicating that the standard curve was obtained from the FAM channel corresponding to "Mycoplasma Universal Target (Mycoplasma)". Slope: 3.548—Slope of the standard curve, Inter: 43.036—Intercept of the regression line on the Y-axis, reflecting the instrument's minimum detection sensitivity, R 2 : 0.99 — coefficient of determination, characterizing the goodness of linear fit between Ct value and log10 (Quantity), the closer to 1, the better the linear relationship; Eff%: 91.359 — PCR amplification efficiency; Figure 5 Figure B in the figure represents the real-time amplification curve obtained by detecting the universal target of mycoplasma. The horizontal axis Cycle represents the number of PCR cycles, from 1 to 40, and the vertical axis ΔRn represents the normalized value of the fluorescence signal in the Reporter channel after subtracting the baseline, which is used to characterize the relative amount of mycoplasma target amplification products in each cycle reaction tube.

[0070] The qPCR standard curve and corresponding real-time amplification curve obtained by using the rapid microbial contamination detection kit provided in Example 1 and referring to the method provided in this example to detect the universal target of Chlamydia (Chlamydia ompA gene fragment) are shown below. Figure 6 As shown, where, Figure 6 Figure A in the figure represents the qPCR standard curve obtained by detecting the universal target of Chlamydia. The horizontal axis Quantity represents the initial template amount of Chlamydia target sequence in each qPCR reaction system (copies / reaction), which increases in a 10-fold gradient from left to right. The vertical axis Ct (Cycle threshold) represents the number of PCR cycles required for the fluorescence signal to first cross the set threshold. The red squares in the figure are the measured Ct values ​​of the standards at each dilution, and the black line is its linear regression fitting line. Figure 6 The key regression parameters output by the instrument are shown below Figure A. Target: Chla—the name of the detection channel, indicating that the standard curve was obtained from the VIC channel corresponding to "Chlamydia universal target (Chla)". Slope: 3.545—Slope of the standard curve, Inter: 35.03—Intercept of the regression line on the Y-axis, reflecting the instrument's minimum detection sensitivity, R 2 0.995 — coefficient of determination, characterizing the goodness of linear fit between Ct value and log10 (Quantity). The closer to 1, the better the linear relationship. Eff%: 91.475 — PCR amplification efficiency. Figure 6Figure B in the figure represents the real-time amplification curve obtained by detecting the universal target of Chlamydia. The horizontal axis Cycle represents the PCR cycle number from 1 to 40, and the vertical axis ΔRn represents the normalized value of the Reporter channel fluorescence signal after subtracting the baseline, which is used to characterize the relative amount of Chlamydia target amplification product in each cycle reaction tube.

[0071] The qPCR standard curve and corresponding real-time amplification curve obtained by using the rapid microbial contamination detection kit provided in Example 1 and referring to the method provided in this example to detect the universal fungal target (Candida albicans ITS2 fragment) are shown below. Figure 7 As shown, where, Figure 7 Figure A in the figure represents the qPCR standard curve obtained by detecting universal fungal targets. The horizontal axis Quantity represents the initial template amount of fungal target sequence in each qPCR reaction system (copies / reaction), which increases in a 10-fold gradient from left to right. The vertical axis Ct (Cycle threshold) represents the number of PCR cycles required for the fluorescence signal to first cross the set threshold. The red squares in the figure are the measured Ct values ​​of the standards at each dilution, and the black line is their linear regression fitting line. Figure 7 The key regression parameters output by the instrument are shown below Figure A. Target: Fungi-18S—the detection channel name, indicating that the standard curve was obtained from the FAM channel corresponding to "Fungi Universal Target (ROX)". Slope: 3.356—Slope of the standard curve, Inter: 20.903—Intercept of the regression line on the Y-axis, reflecting the instrument's minimum detection sensitivity, R 2 0.994 — Coefficient of determination, characterizing the goodness of linear fit between Ct value and log10 (Quantity). The closer to 1, the better the linear relationship. Eff%: 98.606 — PCR amplification efficiency. Figure 7 Figure B in the figure represents the real-time amplification curve obtained by detecting a universal fungal target. The horizontal axis Cycle represents the PCR cycle number from 1 to 40, and the vertical axis ΔRn represents the normalized value of the Reporter channel fluorescence signal after subtracting the baseline, which is used to characterize the relative amount of fungal target amplification products in each cycle reaction tube.

[0072] 7. Quality Control Standards (1) Requirements for positive control All five channels should show effective amplification, and the Ct values ​​should be within the expected range; (2) Requirements for negative controls Amplification should not occur in any of the five pathogen detection channels (Ct value > 40 or no Ct value); The internal control channel should show normal amplification; (3) Blank control requirements No amplification should occur in any channel. (4) Batch quality control Each batch of tests must include one positive control, one negative control, and one blank control. If the quality control fails, the results for that batch are invalid and must be retested.

[0073] Comparative Examples 6-10 Comparative Examples 6-10 provide a method for detecting microbial contamination. Compared with Example 2, the difference in their components is that different reagent kits are used. Specifically, the microbial contamination detection method provided in Comparative Example 6 uses the rapid microbial contamination detection kit provided in Comparative Example 1, the microbial contamination detection method provided in Comparative Example 7 uses the rapid microbial contamination detection kit provided in Comparative Example 2, the microbial contamination detection method provided in Comparative Example 8 uses the rapid microbial contamination detection kit provided in Comparative Example 3, the microbial contamination detection method provided in Comparative Example 9 uses the rapid microbial contamination detection kit provided in Comparative Example 4, and the microbial contamination detection method provided in Comparative Example 10 uses the rapid microbial contamination detection kit provided in Comparative Example 5.

[0074] Apart from the differences mentioned above, the materials, formulation ratios, and preparation operations used in Comparative Examples 6-10 were strictly consistent with those in Example 2.

[0075] Test Example 1 This test case aims to verify the specificity of the primers and probes in the kits of Examples 1 and 1-5 by using the rapid microbial contamination detection kits provided in Examples 1 and 1-5, respectively, and by referring to the microbial contamination detection methods provided in Examples 2 and 6-10. The specific experimental procedures are as follows: 1. Experimental Materials Standard bacterial strains: Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853, Staphylococcus epidermidis ATCC 12228; Standard strains of mycoplasma: Mycoplasma stomatitis ATCC 23714, Mycoplasma pneumoniae ATCC 29342, and Mycoplasma fermentans ATCC 19989; Chlamydia standard strains: Chlamydia trachomatis ATCC VR-902B, Chlamydia pneumoniae ATCC VR-1310; Standard strains of fungi: Candida albicans ATCC 10231, Aspergillus niger ATCC 16404, and Saccharomyces cerevisiae ATCC 9763.

[0076] 2. Experimental Methods (1) Extract genomic DNA from each standard strain; (2) The DNA of each strain was detected using the rapid detection kits for microbial contamination provided in Example 1 and Comparative Examples 1-5, respectively; (3) Verify the specificity of each primer and probe in the rapid detection kit for microbial contamination provided in Example 1 and Comparative Examples 1-5.

[0077] 3. Experimental Results Table 13 Specific detection results

[0078] Note: In Table 13, "+" indicates that Ct≤38, and the test result is positive; "-" indicates that there is no amplification, and the test result is negative.

[0079] Different strains were tested using the rapid detection kits for microbial contamination provided in Example 1 and Comparative Examples 1-5, respectively, and the detection methods for microbial contamination provided in Example 2 and Comparative Examples 6-10, respectively. The results are shown in Table 13.

[0080] As shown in Table 13, when the kits of Example 1 and Comparative Examples 1-4 were used to detect the target microorganisms, the primers and probes could detect the corresponding target microorganisms (bacteria, mycoplasma, chlamydia and fungi) with high specificity and no cross-reactivity. However, when the kit of Comparative Example 5 was used to detect fungi, it could not be detected, resulting in false negative results.

[0081] Test Example 2 This test case aims to verify the detection sensitivity of the primers and probes in the kits of Examples 1 and 1-5 by using the rapid microbial contamination detection kits provided in Examples 1 and 1-5, respectively, and by referring to the microbial contamination detection methods provided in Examples 2 and 6-10, respectively, to detect positive control samples with different gradient dilutions corresponding to different pathogens. The specific experimental procedures are as follows: 1. Experimental Methods (1) Positive control samples for each pathogen (i.e., the first plasmid containing Escherichia coli ATCC 25922 genomic DNA, the second plasmid containing Mycoplasma stomatitis ATCC 23714 genomic DNA, the third plasmid containing Chlamydia trachomatis ATCC VR-902B genomic DNA, and the fourth plasmid containing Candida albicans ATCC 10231 genomic DNA) were serially diluted 10-fold (10 6 ~10 0 (copies / μL) (2) Eight replicates were performed for each dilution; (3) Determine the detection limit (LOD, the lowest concentration corresponding to a 95% detection rate) for each pathogen.

[0082] 2. Experimental Results Table 14 Sensitivity Detection Results

[0083] Note: In Table 14, "Detection Limit (copies / reaction)" refers to the lowest number of target nucleic acids that can be stably detected in a single PCR reaction system; "Detection Limit (copies / μL)" refers to the lowest number of target nucleic acids contained per microliter of nucleic acid extraction stock solution. The two satisfy the following conversion relationship: Detection Limit (copies / reaction) = Detection Limit (copies / μL) × Volume of nucleic acid template added in a single reaction (μL).

[0084] In the sensitivity detection process of this embodiment, 5 μL of nucleic acid template was added to each reaction. Therefore, the detection limit (copies / reaction) = detection limit (copies / μL) × 5. The two columns of values ​​are converted accordingly. This relationship only changes with the sample volume and does not affect the relative comparison of sensitivity between different embodiments.

[0085] "8 / 8 detection concentration" refers to the lowest template concentration (copies / reaction) that can be stably detected in 8 parallel qPCR reactions.

[0086] The rapid detection kits for microbial contamination provided in Example 1 and Comparative Examples 1-5 were used to detect positive control samples of different dilution ratios corresponding to different pathogens, and the detection methods for microbial contamination provided in Example 2 and Comparative Examples 6-10 were used respectively. The results are shown in Table 14.

[0087] As shown in Table 14, compared with Comparative Examples 1-5, the detection limit of the kit provided in Example 1 for various pathogens reached 25-100 copies / reaction, which shows high sensitivity.

[0088] Test Example 3 This test case aims to verify the effectiveness of the kit in distinguishing between live and dead bacteria by using the rapid microbial contamination detection kit provided in Example 1 and referring to the microbial contamination detection method provided in Example 2. The specific experimental operation steps are as follows: 1. Experimental Materials Freshly cultured E. coli (live bacteria), E. coli sterilized at 121℃ for 30 minutes (dead bacteria), and samples of live and dead bacteria mixed in different proportions.

[0089] 2. Experimental Methods (1) Adjust the concentration of live bacteria and dead bacteria to 10. 6 CFU / mL; (2) Prepare mixed samples according to the concentration ratio of live bacteria: dead bacteria = 100:0, 75:25, 50:50, 25:75, 10:90, 0:100; (3) Each mixed sample was treated with PMA (+PMA) and not treated (-PMA). (4) DNA was extracted and then qPCR was performed.

[0090] 3. Experimental Results Table 15 Results of the verification of the effectiveness in distinguishing between live and dead bacteria

[0091] Samples with different ratios of live to dead bacteria were prepared and treated with PAM, while samples without PAM treatment served as a control (untreated group). Amplification was performed according to the method provided in Example 2, yielding the following results: Figure 8 The amplification curve shown is fitted to obtain the following result: Figure 9 The curve showing the relationship between the ΔCt value and the proportion of viable bacteria is shown.

[0092] The samples containing live and dead bacteria mixed in different proportions were tested using the rapid detection kit for microbial contamination provided in Example 1 and the detection method for microbial contamination provided in Example 2. The results are shown in Table 15.

[0093] As shown in Table 15, the PMA in the kit provided in Example 1 can effectively distinguish between live and dead bacteria when the sample is treated. The ΔCt value has a good correspondence with the proportion of live bacteria, which verifies the effectiveness of the kit provided in Example 1 in distinguishing between live and dead bacteria.

[0094] Test Example 4 This test case aims to verify the detection efficacy of the kits provided in Example 1 and Comparative Examples 1-5 by using the rapid detection kits for microbial contamination provided in Example 1 and Comparative Examples 1-5, and by referring to the microbial contamination detection methods provided in Example 2 and Comparative Examples 6-10, respectively. The results are compared with those obtained by traditional culture methods for detecting cell preparation samples. The results are shown in Table 16.

[0095] The detection of cell preparation samples using traditional culture methods includes the following steps: 1. Bacterial and fungal detection (sterility test). 1. Culture medium preparation Pancreatic soybean peptone liquid medium: used for the culture of aerobic and anaerobic bacteria.

[0096] Sabouraud dextrose liquid medium: used for the culture of fungi (yeasts, molds).

[0097] Control: Each batch of tests should include a negative control (sterile culture medium) and a positive control (inoculated with <100 CFU of Staphylococcus aureus, Pseudomonas aeruginosa, Staphylococcus epidermidis, Candida albicans, Aspergillus niger, etc.).

[0098] 2. Sample processing and inoculation Under aseptic conditions, a certain amount of cell preparation (usually 1% of the total preparation or no less than 1 mL) is directly inoculated into the above liquid culture medium.

[0099] 3. Cultivation and Observation Bacteria: Incubate in incubators at 30-35℃ and 20-25℃ respectively.

[0100] Fungi: Incubate in an incubator at 20-25℃.

[0101] Observation period: at least 14 days, with observations on days 3, 5, 7, and 14.

[0102] Positive result determination: The culture medium shows turbidity, precipitation, bacterial film, gas production, etc., and the presence of microbial growth is confirmed by microscopic examination and transculturing plates.

[0103] Negative report: After 14 days, all culture media were clear and were deemed to have passed the sterility test.

[0104] II. Mycoplasma Detection 1. Culture medium preparation Mycoplasma liquid culture medium and mycoplasma solid culture medium are commonly used commercial culture media, and growth promotion experiments are required for verification.

[0105] Positive control: Use known low-generation (<15 passages) mycoplasma strains, such as Mycoplasma pneumoniae and Mycoplasma oralis.

[0106] 2. Sample processing and inoculation At least 1 mL of cell preparation sample was directly inoculated into mycoplasma liquid culture medium (usually diluted at a ratio of 1:10 or 1:20), and positive control and negative control (uninoculated culture medium) were set up at the same time.

[0107] 3. Cultivation and Observation Liquid culture: Incubate at 35~37℃ under anaerobic or microaerobic (5% CO2, 95% N2) conditions.

[0108] Blind transfer: On days 3, 7, and 14 of culture, take 0.2–0.5 mL of liquid culture and transfer it to fresh liquid and solid culture media, respectively.

[0109] Solid culture observation: After solid plates are cultured under the same conditions, observe under an inverted microscope (100~400x) whether there are typical "fried egg-like" colonies (dense in the center and transparent around the periphery).

[0110] Positive result determination: The liquid culture medium changes color (e.g., phenol red indicator turns yellow) and typical colonies appear on the solid culture medium.

[0111] Negative report: If all passaged cultures show no positive signs by day 28 (or according to the pharmacopoeia), they are considered negative for mycoplasma.

[0112] III. Chlamydia Detection 1. Cell preparation Use cell lines that are sensitive to Chlamydia, such as McCoy cells and HeLa-229 cells.

[0113] A dense monolayer is formed by culturing in 24-well plates or cell culture flasks.

[0114] 2. Sample processing and inoculation The samples were diluted with transport medium or cell maintenance medium containing antibiotics (to inhibit contaminating bacteria, such as gentamicin, vancomycin, and amphotericin B); the treated samples were inoculated onto a monolayer of cells and then centrifuged at 35°C and 3000×g for 1 hour to promote the adsorption of chlamydia into the cells; the supernatant was removed and cell maintenance medium containing actinomycin (to inhibit host cell protein synthesis and promote chlamydia proliferation) was added.

[0115] 3. Cultivation and Observation Incubate at 35℃ in a 5% CO2 incubator for 48-72 hours.

[0116] Fixation and staining: After culturing, discard the culture medium, fix the cells with methanol, and stain them with a Chlamydia-specific fluorescent antibody (targeting the major outer membrane protein MOMP).

[0117] Microscopic examination: Observe under a fluorescence microscope whether there are bright green fluorescent inclusion bodies in the cytoplasm.

[0118] Positive result determination: The observation of typical inclusion bodies indicates a positive result.

[0119] Negative report: No inclusion bodies indicate a negative result, but sometimes blind passage is required for confirmation.

[0120] Table 16 Detection Results of CAR-T Cell Preparation Samples

[0121] The actual samples (30 batches of CAR-T cell preparation samples collected from a cell therapy institution) were tested using the rapid detection kits for microbial contamination provided in Example 1 and Comparative Examples 1-5, respectively, and the detection methods for microbial contamination provided in Example 2 and Comparative Examples 6-10, respectively. The results are shown in Table 16.

[0122] Table 16 shows that compared with the rapid microbial contamination detection kits provided in Comparative Examples 1-5, the rapid microbial contamination detection kit provided in Example 1, combined with the microbial contamination detection methods provided in Examples 2 and Comparative Examples 6-10, has a higher positive detection rate for bacteria, mycoplasma, chlamydia, and fungi, and the detection time is only 3 hours. Furthermore, analysis of the samples that tested positive using the kit provided in Example 1 revealed that among the bacterial positive samples, 4 were contaminated with live bacteria, and 1 was a residual dead bacteria. These results demonstrate that the kit provided in Example 1 is completely consistent with the detection results of the traditional culture method, but the detection time is shortened to 3 hours, and it can distinguish between live and dead bacteria, exhibiting significant application advantages.

[0123] Test Example 5 This test case aims to verify the stability of the rapid microbial contamination detection kit provided in Example 1, referring to the microbial contamination detection method provided in Example 2. The specific experimental operation steps are as follows: 1. Experimental Methods The reagent kits were stored at -20℃ (recommended storage conditions), 4℃, and room temperature (25℃), and samples were taken at 0, 1, 3, 6, and 12 months to evaluate the reagent performance.

[0124] 2. Detection indicators (1) Changes in Ct value of positive quality control samples (ΔCt<1 is acceptable); (2) Status of detection sensitivity maintenance; (3) Detection specificity of each channel.

[0125] 3. Experimental Results Table 17 Results of reagent kit stability test

[0126] The stability test results of the rapid detection kit for microbial contamination provided in Example 1 are shown in Table 17.

[0127] As shown in Table 17, the kit provided in Example 1 has a shelf life of more than 12 months when stored at -20℃, and about 6 months at 4℃. Therefore, it is not recommended to store it at room temperature.

[0128] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention, but such modifications or substitutions are all within the scope of protection of the present invention.

Claims

1. A rapid detection kit for microbial contamination, characterized in that: The kit includes reagent A, reagent B, reagent C, and reagent D; The reagent A is used to detect bacteria. The reagent A includes a first upstream primer, a first downstream primer, and a first detection probe. The nucleotide sequence of the first upstream primer is shown in SEQ ID NO: 1, the nucleotide sequence of the first downstream primer is shown in SEQ ID NO: 2, and the nucleotide sequence of the first detection probe is shown in SEQ ID NO:

3. The reagent B is used to detect mycoplasma. The reagent B includes a second upstream primer, a second downstream primer, and a second detection probe. The nucleotide sequence of the second upstream primer is shown in SEQ ID NO: 4, the nucleotide sequence of the second downstream primer is shown in SEQ ID NO: 5, and the nucleotide sequence of the second detection probe is shown in SEQ ID NO:

6. The reagent C is used to detect Chlamydia. The reagent C includes a third upstream primer, a third downstream primer, and a third detection probe. The nucleotide sequence of the third upstream primer is shown in SEQ ID NO: 7, the nucleotide sequence of the third downstream primer is shown in SEQ ID NO: 8, and the nucleotide sequence of the third detection probe is shown in SEQ ID NO:

9. The reagent D is used to detect fungi. The reagent D includes a fourth upstream primer, a fourth downstream primer, and a fourth detection probe. The nucleotide sequence of the fourth upstream primer is shown in SEQ ID NO: 10, the nucleotide sequence of the fourth downstream primer is shown in SEQ ID NO: 11, and the nucleotide sequence of the fourth detection probe is shown in SEQ ID NO:

12.

2. The rapid detection kit for microbial contamination as described in claim 1, characterized in that: The first detection probe, the second detection probe, the third detection probe, and the fourth detection probe each contain a fluorescent group and a quenching group. The fluorescent group is selected from Cy5, FAM, VIC, and ROX, and the quenching group is selected from BHQ1, BHQ3, and MGB.

3. The rapid detection kit for microbial contamination as described in claim 2, characterized in that: The fluorescent group is located at the 5' end of the first detection probe, the second detection probe, the third detection probe, and the fourth detection probe, and the quenching group is located at the 3' end of the first detection probe, the second detection probe, the third detection probe, and the fourth detection probe.

4. The rapid detection kit for microbial contamination as described in claim 3, characterized in that: The 5' end of the first detection probe is labeled with the fluorescent group Cy5, and the 3' end of the first detection probe is labeled with the quenching group BHQ3. The 5' end of the second detection probe is labeled with the fluorescent group FAM, and the 3' end of the second detection probe is labeled with the quenching group BHQ1; The 5' end of the third detection probe is labeled with the fluorescent group VIC, and the 3' end of the third detection probe is labeled with the quenching group BHQ1. The 5' end of the fourth detection probe is labeled with the fluorescent group ROX, and the 3' end of the fourth detection probe is labeled with the quenching group MGB.

5. The rapid detection kit for microbial contamination as described in claim 1, characterized in that: The kit also includes an internal control containing a human β-actin gene fragment.

6. The rapid detection kit for microbial contamination as described in claim 5, characterized in that: The kit also includes reagent E, which is used to detect the internal control sample. Reagent E includes a fifth upstream primer, a fifth downstream primer, and a fifth detection probe. The nucleotide sequence of the fifth upstream primer is shown in SEQ ID NO: 13, the nucleotide sequence of the fifth downstream primer is shown in SEQ ID NO: 14, and the nucleotide sequence of the fifth detection probe is shown in SEQ ID NO:

15.

7. The rapid detection kit for microbial contamination as described in claim 1, characterized in that: The kit also includes positive controls comprising a first plasmid, a second plasmid, a third plasmid, and a fourth plasmid. The first plasmid contains Escherichia coli ATCC 25922 genomic DNA or a 16S rDNA fragment. The second plasmid contains Mycoplasma oralis ATCC23714 genomic DNA or a 16S rDNA fragment. The third plasmid contains Chlamydia trachomatis ATCC VR-902B genomic DNA or an ompA gene fragment. The fourth plasmid contains Candida albicans ATCC 10231 genomic DNA or an ITS2 fragment.

8. The rapid detection kit for microbial contamination as described in claim 1, characterized in that: The kit also includes reagent F, which comprises a propidium azide bromide solution with a concentration of 20–100 μM.

9. The rapid detection kit for microbial contamination as described in claim 1, characterized in that, The kit also includes reagent G, which comprises the following components: 0.02–0.05 U / μL hot-start Taq DNA polymerase, 200–400 μM dNTPs mixture, 40–60 mM KCl solution, 0.01–0.02 U / μL UNG enzyme, 200–400 μM dUTP, magnesium salt solution, Tris-HCl buffer, and stabilizer; Mg in magnesium salt solution 2+ The concentration is 2~4 mM; The Tris-HCl buffer solution has a pH of 8.3-8.5 and a concentration of 10-20 mM. The stabilizer comprises the following components: 0.1~0.5 mg / mL bovine serum albumin and 0.5~1.5 M betaine.

10. The application of the rapid detection kit for microbial contamination as described in any one of claims 1 to 9 in the detection of microbial contamination in cell preparations.

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