Rapid sterile detection method and kit based on 16S / 18S rRNA-cDNA digital PCR technology

Abstract

The invention discloses a rapid sterile detection method and kit based on a 16S / 18S rRNA-cDNA digital PCR technology, and the method comprises the following steps: S1, extracting total RNA in a to-be-detected sample to obtain bacteria 16S rRNA and fungi 18S rRNA; s2, carrying out reverse transcription on the bacterium 16S rRNA and the fungus 18S rRNA to obtain a stable cDNA (complementary deoxyribonucleic acid) fragment; and S3, carrying out digital PCR amplification by taking the cDNA fragment as a template, and judging whether bacterial contamination exists or not according to an amplification result so as to realize rapid sterile detection. According to the characteristics of high conservative and high copy of bacteria 16S rRNA / fungi 18S rRNA, 30 sets of universal primer probe combinations are verified, and gt can be covered by selecting the optimal two sets of primer probe combinations; 99.99% of common pollution bacteria / fungi in clinic and pharmacy, and no cross amplification with mitochondrial rRNA of mammals such as human, hamster, cattle and the like.

Description

Technical Field

[0001] This invention relates to a rapid sterile detection method and kit based on 16S / 18S rRNA-cDNA digital PCR (dPCR) technology. This invention belongs to the field of microbial detection technology. Background Technology

[0002] In the quality control of sterile pharmaceuticals, biologics, medical devices, and clinical samples, rapid sterility testing (RST) is a crucial step in ensuring product safety and efficacy. Traditional pharmacopoeia methods (such as the 2025 edition of the Chinese Pharmacopoeia, General Chapter 1101) rely on a 14-day culture period, which is insufficient to meet the urgent needs of modern biopharmaceuticals for rapid release, real-time process control, and closed-loop production. Especially for short-shelf-life products such as CAR-T cell therapy, mRNA vaccines, and gene therapy drugs, release delays not only lead to product spoilage and supply chain disruptions but also pose risks to clinical use. While some alternative methods exist, such as growth monitoring and ATP bioluminescence assays, they also require culture, have long cycles, and low sensitivity. Currently, there is no rapid method to distinguish between zero and one viable bacteria. Therefore, developing a broad-spectrum rapid sterility testing method that can be completed within 5 hours and has the sensitivity to distinguish between zero and one (absolute quantity, independent of volume) bacteria has become an urgent need in the industry.

[0003] Current mainstream rapid aseptic testing protocols still revolve around "genomic DNA extraction - qPCR / loop-mediated isothermal amplification": after sample lysis, amplification is performed using universal 16S / 18S rDNA primers, with results available in 3-4 hours. However, the high stability of DNA brings inherent drawbacks: ① Dead bacterial DNA can remain stable and be amplified and detected for several days, resulting in a high false positive rate; ② qPCR is easily inhibited by heme, heparin, polysaccharides, etc., and the detection limit remains at 10. 2 For low contamination levels (<10 CFU / mL), an additional overnight enrichment step is required; ③ The single-copy genome target signal is weak, and the Ct drift is large in complex matrices, resulting in a quantitative result deviation of up to an order of magnitude, which cannot meet the accuracy requirements of process control.

[0004] Compared to DNA extraction, RNA extraction offers advantages in rapid aseptic detection: Total bacterial RNA is extracted first, then reverse transcribed using random primers or 16S / 18S-specific primers to convert the abundant 16S / 18S rRNA into cDNA for amplification. In actively growing Gram-negative bacteria, the copy number of 16S / 18S rRNA is 10 times higher than that of a single copy of the genome. 3 ~10 4 Even against Gram-positive or dormant bacteria, it can maintain a concentration of >10.2 The copy number of 18S rRNA is higher for fungi, exceeding that of their single-copy genome by 10%. 4 ~10 5 The signal amplification effect increases the template amount by 100 to 1000 times at the same contamination level, thus eliminating the need for enrichment. Simultaneously, the RNA from dead bacteria rapidly degrades within 5 to 10 minutes due to the leakage of ribonuclease, naturally shielding the residual signal from dead bacteria and giving the detection "live bacteria selectivity." This method eliminates the need for chemical pretreatment such as PMA-photolysis to chelate dead bacterial DNA, shortening the operation time by nearly one hour and mitigating the potential environmental pollution from phototoxic reagents and the false positives caused by incomplete chelation of dead bacterial DNA.

[0005] Digital PCR (dPCR), as a third-generation PCR method, utilizes microfluidic technology to divide the amplification system into thousands or even tens of thousands of independent nanoliter-sized droplets. Each droplet contains either 0 or 1 template molecule, allowing for direct counting of positive droplets after endpoint amplification, achieving absolute quantification at the single-molecule level without the need for a standard curve. Compared to qPCR, this significantly reduces the risk of false negatives due to matrix inhibition or differences in amplification efficiency. Furthermore, dPCR exhibits higher tolerance to heme, cell lysates, and inhibitor concentrations than qPCR, making it particularly suitable for high-protein, high-nucleic acid background samples such as cell therapy final products. Secondly, dPCR interprets results solely based on the presence or absence of fluorescence signals during the reading phase, rather than relying on Ct values, thus avoiding the misjudgment of low-copy samples as negative because the qPCR amplification curve has not yet reached the plateau phase. In conclusion, the rapid, sterile detection strategy targeting 16S / 18S rRNA-cDNA and using dPCR as the analytical platform combines high sensitivity, viable cell specificity, and inhibitor tolerance, filling a gap in current technology. Summary of the Invention

[0006] The purpose of this invention is to provide a rapid sterile detection method and kit based on digital PCR technology using 16S / 18S rRNA-cDNA, with a detection limit of 1 (absolute amount, independent of volume) of bacteria / fungi.

[0007] To achieve the above objectives, the present invention employs the following technical means: A primer and probe for rapid aseptic detection based on 16S / 18S rRNA-cDNA digital PCR technology is disclosed, comprising a forward primer, a reverse primer, and a probe. Taking advantage of the highly conserved and high-copy-rate characteristics of bacterial 16S rRNA and fungal 18S rRNA, this invention validated 30 universal primer-probe combinations, selecting the two optimal combinations, which can cover >99.99% of common clinical and pharmaceutical contaminating bacteria / fungi, and exhibit no cross-amplification with mitochondrial rRNA from mammals such as humans, hamsters, and cattle.

[0008] A rapid sterility detection method based on 16S / 18S rRNA-cDNA digital PCR technology includes the following steps: S1: Extract total RNA from the sample to be tested to obtain bacterial 16S rRNA and fungal 18S rRNA; S2: Stable cDNA fragments were obtained by reverse transcription of bacterial 16S rRNA and fungal 18S rRNA. S3: Digital PCR amplification is performed using cDNA fragments as templates, and the presence or absence of bacterial contamination is determined based on the amplification results, enabling rapid aseptic detection.

[0009] In step S1, total RNA is extracted from the sample to be tested to obtain bacterial 16S rRNA and fungal 18S rRNA, specifically including: S1.1: First, add lysozyme buffer and lysozyme buffer to the sample to be tested; S1.2: Then add... β The guanidine isothiocyanate cleavage buffer of β-mercaptoethanol was used for cleavage. S1.3: After lysis, bacterial 16S rRNA and fungal 18S rRNA were obtained by centrifugation and elution using a silica membrane centrifuge column.

[0010] In step S2, stable cDNA fragments are obtained by reverse transcription of bacterial 16S rRNA and fungal 18S rRNA, specifically including: S2.1: Pre-denaturate the extracted RNA using 16S / 18S universal primers; S2.2: cDNA was synthesized using ProtoScript II reverse transcriptase after pre-denaturation; S2.3: Stable cDNA fragments are obtained after enzyme heat inactivation.

[0011] In step S2.1, the 16S / 18S universal primer is a universal primer designed based on the conserved regions of the bacterial 16S rRNA gene and the fungal 18S rRNA gene. Most preferably, the 16S / 18S universal primer uses 16S / 18S specific primers, which include: Bacterial forward primer FP: TACGGGAGGCAGCAGT; Bacterial reverse primer RP: GACTACCAGGGTATCTAATCCTGT; Fungal forward primer FP: GGCGATGGTTCATTCAAATTTCTG; Fungal reverse primer RP: CTGCTGGCACCAGACTT.

[0012] In step S2.1, pre-denaturation is performed at 55~75 ℃ for 4~10 min.

[0013] In step S2.2, cDNA is synthesized at 50-60 ℃ for 30-70 min.

[0014] In step S3, digital PCR amplification is performed using the cDNA fragment as a template, specifically including: Add the cDNA fragment, 16S / 18S universal primers, bacterial probe, fungal probe, and amplification premix to the sample plate of the digital PCR instrument, and then place the sample plate into the digital PCR instrument for digital PCR amplification. The reaction conditions for digital PCR amplification are as follows: initial denaturation at 90-100 ℃ for 1-4 min; followed by 30-50 cycles of denaturation at 90-100 ℃ for 15-60 s and annealing / extension at 50-70 ℃ for 15-120 s.

[0015] In step S3, the probe is a universal probe designed using conserved regions of the bacterial 16S rRNA gene and the fungal 18S rRNA gene. The bacterial probe is a universal probe designed using a conserved region of the bacterial 16S rRNA gene. The fungal probe is a universal probe designed using a conserved region of the fungal 18S rRNA gene. Most preferably, the bacterial probe is TGCCAGCAGCCGCGGTAATA, wherein the 5' end is labeled with a FAM fluorescent reporter group and the 3' end is labeled with a BHQ1 quencher group. The fungal probe is CATCCAAGGAAGGCAGCAGGC, with a BHQ1 quencher group labeled at the 5' end and a C3-FAM fluorescent reporter group labeled at the 3' end.

[0016] In step S3, the presence or absence of bacterial contamination is determined based on the amplification results, specifically including: When the copy number concentration of the negative control is < 2 copies / μL and the corrected copy number concentration of the sample is ≥ 1 copy / μL, it is determined to be contaminated with bacteria. The corrected copy number concentration of the sample is the measured copy number concentration of the sample minus the copy number concentration of the negative control. When the copy number concentration of the negative control is < 2 copies / μL and the corrected copy number concentration of the sample is < 1 copy / μL, it is considered to be sterile. When the copy number concentration of the negative control is ≥ 2 copies / μL, it is determined that the experimental environment is contaminated with bacteria.

[0017] A kit for implementing the rapid aseptic detection method based on 16S / 18S rRNA-cDNA digital PCR technology.

[0018] More specifically, a rapid sterile detection method based on 16S / 18S rRNA-cDNA digital PCR technology includes the following steps: (1) Steps for extracting total RNA from samples 1) Take 1 mL of the sample to be tested, centrifuge at 5000×g at 4 ℃ for 5 min, and discard the supernatant.

[0019] 2) Add 100 μL of lysozyme / lysozyme solution to the cell particles, vortex to resuspend, and incubate at 37°C for 15 min.

[0020] 3) Add 1% β 350 μL of lysis buffer containing mercaptoethanol was vortexed and resuspended.

[0021] 4) Transfer the lysis buffer to a homogenizer without nuclease tubes, centrifuge at 12000×g for 2 min at room temperature, and remove the homogenate.

[0022] 5) Add 250 μL of 100% ethanol to each volume of bacterial lysate.

[0023] 6) Thoroughly mix by vortexing to disperse any visible sediment.

[0024] 7) Transfer the sample (including any remaining precipitate) to the extraction column (with collection tube A).

[0025] 8) Centrifuge at 12000×g for 15 seconds at room temperature, discard the liquid in collection A, and reinsert the extraction column into collection tube A.

[0026] 9) Add 700 μL of washing buffer A to the extraction column. Centrifuge at 12000×g for 15 seconds at room temperature.

[0027] 10) Discard collection tube A and place the extraction column into collection tube B.

[0028] 11) Add 500 μL of washing buffer B to the extraction column and centrifuge at 12000×g for 15 seconds at room temperature.

[0029] 12) Discard the liquid in collection tube B and reinsert the extraction column into collection tube B.

[0030] 13) Add 500 μL of washing buffer B to the extraction column again and centrifuge at 12000×g for 15 seconds at room temperature.

[0031] 14) Discard the liquid in collection tube B and reinsert the extraction column into collection tube B.

[0032] 15) Centrifuge the extraction column with collection tube B at 12000×g for 1 min at room temperature.

[0033] 16) Discard collection tube B and insert the extraction column into collection tube C.

[0034] 17) Add 30 μL to 3 × 100 μL of nuclease-free water to the center of the extraction column and incubate at room temperature for 1 min.

[0035] 18) Centrifuge at 12000×g for 2 min at room temperature, and collect the liquid in tube C, which is the total RNA of the sample.

[0036] (2) Specific reverse transcription steps of 16S / 18S rRNA in the sample 1) Template and primer pre-denaturation: Take 10 μL of total RNA extract from the sample and mix it with 0.1 μL of FP (100 μM) and 0.1 μL of RP (100 μM), add 1 μL of 10 mM dNTP, and bring the volume to 10 μL with nuclease-free water; heat shock at 65℃ for 5 min, centrifuge briefly and then immediately place on ice to break down the secondary structure of rRNA and improve reverse transcription efficiency.

[0037] 2) Reverse transcription reaction: Add 4 μL of 5×ProtoScript II Buffer, 2 μL of 0.1 M DTT, 1 μL of ProtoScript II RT (200 U / μL), 0.2 μL of RNase Inhibitor (40 U / μL), and 0.3 μL of nuclease-free water to an ice bath, for a total volume of 20 μL; heat-inactivate at 55℃ for 60 min, then at 70℃ for 15 min to terminate the reaction.

[0038] (3) Digital PCR operation Mix 2 μL of the sample reverse transcription product with 3 μL of 4×Probe PCR Mix, add 0.1 μL of FP (100 μM), 0.1 μL of RP (100 μM), and 0.05 μL of TM (100 μM), and bring the volume to 12 μL with nuclease-free water. Transfer the reaction mixture to 24-well 8.5k nanoplates, seal them, and analyze them using a digital PCR instrument. Each sample was analyzed three times in duplicate. A template-free control was used as a negative control. The thermal cycling program was 95℃ initial denaturation for 2 min, followed by 40 cycles including 95℃ denaturation for 30 seconds and 60℃ annealing / extension for 30 seconds. The copy number concentration was calculated based on the positive droplets. When the copy number concentration of the negative control is < 2 copies / μL and the corrected copy number concentration of the sample (sample measured copy number concentration - negative control copy number concentration) ≥ 1 copy / μL, bacterial contamination is considered present; when the copy number concentration of the negative control is < 2 copies / μL and the corrected copy number concentration of the sample is < 1 copy / μL, no bacterial contamination is considered present; when the copy number concentration of the negative control is ≥ 2 copies / μL, bacterial contamination is considered to have occurred in the testing environment or experimental procedures, and the source of bacterial contamination needs to be investigated and retested.

[0039] Compared to other existing sterility detection technologies, this invention offers advantages such as accuracy, sensitivity, and wide applicability. Compared to existing sterility detection technologies such as culture methods, qPCR, and ATP luminescence, this invention achieves simultaneous breakthroughs in multiple dimensions, including rapid sensitivity, live bacteria specificity, interference resistance, and quantification. Specific advantages are as follows: 1) Limit sensitivity: Utilizing the natural high copy number of 16S / 18S rRNA and dPCR single-molecule counting, the actual detection limit is 1 bacterium (absolute amount, independent of volume); it has a high positive detection rate for low-contamination samples with fewer than 10 bacteria, and the results can be obtained on the same day without overnight enrichment.

[0040] 2) Live bacteria specificity: RNA is the target, and the RNA of dead bacteria degrades within 5-10 minutes, naturally eliminating interference from free RNA, and the false positive rate is significantly reduced compared with traditional DNA-qPCR.

[0041] 3) Resistance to matrix interference: Droplet dPCR physically isolates inhibitors from the amplification system, making it resistant to interference from cells, serum, plasma, whole blood, vaccine adjuvants, chemical drugs, gelling agents, and nanopolymers. For complex biological samples, qPCR requires additional dilution or purification, while dPCR can be directly added to the instrument for detection without pretreatment.

[0042] 4) Absolute quantification and data compliance: The number of positive droplets (template copy number) is directly linearly correlated with the number of bacteria (CFU) and is not affected by fluctuations in amplification efficiency; the PDF report automatically generated by the instrument contains positive droplet plots and 95% confidence intervals, meeting the FDA 21CFR Part 11 electronic record requirements and facilitating GMP batch traceability.

[0043] Therefore, this invention is the first in the field of aseptic detection to seamlessly integrate the "16S / 18S rRNA-cDNA targeting strategy" with "digital PCR absolute quantification," breaking through the long-standing bottleneck of traditional methods in balancing speed, sensitivity, and viable bacteria specificity. The entire process takes less than 5 hours, with a detection limit of 1 bacterium (absolute quantity, independent of sample volume). This not only meets the same-day detection requirements for short-shelf-life products such as cell therapy drugs and mRNA vaccines, but also provides a standardized, verifiable, and auditable next-generation release solution for other pharmaceuticals (common chemical drugs, antibiotics, antibody drugs, ADC drugs, nanoparticles, gel drugs, biological products, etc.), possessing broad prospects for industrialization. Attached Figure Description

[0044] Figure 1 This is a flowchart outlining the main operations and methods of the present invention for verification. Figure 2 This refers to the 96-well plate and the corresponding bacterial count per well under different dilution factors in the "Preparation and Counting of Bacterial Suspensions with Different Bacterial Counts" step of Example 1 of the present invention. Figure 3 This is a 4x fluorescence microscope image of 50 bacteria in 1 μL of bacterial solution during the "fluorescence microscopy counting" step in Example 1 of this invention; Figure 4 This is a 4x fluorescence microscope image of 500 bacteria in 1 μL of bacterial solution during the "fluorescence microscopy counting" step in Example 1 of this invention; Figure 5 This is a 4x fluorescence microscope image of 5 bacteria in 1 μL of bacterial solution during the "fluorescence microscopy counting" step in Example 1 of the present invention. Figure 6 This is a 40x fluorescence microscope image of 50 bacteria in 1 μL of bacterial solution during the "fluorescence microscopy counting" step in Example 1 of this invention; Figure 7 This is a 4x fluorescence microscope image showing that 1 μL of bacterial solution contains 1 bacterial count in the "fluorescence microscopy counting" step of Example 1 of the present invention; Figure 8 This is a 40x fluorescence microscope image showing that 1 μL of bacterial solution contains 1 bacterial count in the "fluorescence microscopy counting" step of Example 1 of the present invention; Figure 9This is a linear fitting curve of the bacterial count and copy number corresponding to the three sets of primers in Escherichia coli in Example 1 of the present invention; Figure 10 This is a linear fitting curve of Escherichia coli count and copy number at 6 concentration points in Example 1 of the present invention; Figure 11 This is a linear fitting curve of Bacillus subtilis bacterial count and copy number at 7 concentration points in Example 1 of the present invention; Figure 12 This is a linear fitting curve of Staphylococcus aureus bacterial count and copy number at 5 concentration points in Example 1 of the present invention; Figure 13 This is a linear fitting curve of Pseudomonas aeruginosa bacterial count and copy number at 5 concentration points in Example 1 of the present invention; Figure 14 This is a bar chart showing the copy number concentration of four samples each of bacterial count 1 and bacterial count 0 in Example 2 of the present invention; Figure 15 This is a bar chart showing the average copy number concentration of four samples each of bacterial count 1 and bacterial count 0 in Example 2 of the present invention; Figure 16 This is a curve fitting the Escherichia coli count and copy number at 7 concentration points in Example 3 of the present invention; Figure 17 This is a comparison of the bacterial count and copy number fitting curves between the direct DNA extraction group and the RNA reverse transcriptome in Example 3 of the present invention; Figure 18 This is a bar chart showing the negative control, positive control, and sample copy number concentration after sterilization using the four sterilization methods in Example 4 of this invention; Figure 19 This is a linear fitting curve of bacterial count and copy number after adding CAR-T cell matrix in Example 5 of the present invention; Figure 20 This is a bar chart showing the copy number concentration of CAR-T cells in actual samples from Example 6 of this invention. Figure 21 This is a linear fitting curve of the number of bacteria and the copy number corresponding to the three sets of primers in Candida albicans in Example 7 of the present invention; Figure 22 This is a linear fitting curve of the number of Candida albicans bacteria and copy number at 7 concentration points in Example 7 of the present invention; Figure 23 This is a linear fitting curve of Aspergillus nidulans number and copy number at 5 concentration points in Example 7 of the present invention. Detailed Implementation

[0045] The technical solution of the present invention will be clearly and completely described below in conjunction with the implementing regulations. Obviously, the described implementing regulations are only a part of the implementing regulations of the present invention, and not all of the implementing regulations. Based on the implementing regulations of the present invention, all other implementing regulations obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] The strains used in the embodiments of this invention are existing strains, as detailed below: Example 1: Investigation into the relationship between bacterial 16S rRNA-cDNA sequence count and copy number (1) Design and screening of primers and probes The 16S rRNA gene sequence is shown in SEQ ID NO: 1. The full-length bacterial 16S rRNA gene is approximately 1500 bp, and 16S rRNA accounts for more than 80% of the total RNA in bacteria. The 16S rRNA gene can be divided into multiple variable regions and conserved regions. The variable regions (V1~V9) differ among different bacteria and can be used to distinguish different bacterial species, while the conserved regions are very similar in all bacteria and can be used to design universal primers for PCR amplification. Through reviewing relevant literature and independent design, 16 sets of universal primer-probe combinations for bacteria were designed and screened targeting highly conserved fragments of the bacterial 16S rRNA gene. All probes are labeled with a FAM fluorescent reporter group at the 5' end and a BHQ1 quencher group at the 3' end. The specific sequences are as follows: Table 1 Primers and probes for bacterial 16S rRNA gene (2) Preparation and counting of bacterial suspensions with different bacterial counts 1) Strains and plasmids Gram-negative bacteria used are Escherichia coli: E. coli DH5 α The engineered bacteria (pUC19-Kan-GFP) carry a kanamycin resistance marker and an IPTG-inducible GFP gene in their plasmids. Gram-positive bacteria are *Bacillus subtilis*. The engineered bacteria *Bacillus subtilis* 168 (pUB110-Spec-GFP) carry a spectinomycin resistance marker and express green fluorescent protein. Both exhibit stable green fluorescence signals, making them suitable for low-concentration viable bacterial counts using fluorescence microscopy. Standard strains of *Staphylococcus aureus* and *Pseudomonas aeruginosa* were also used for plate counts to calculate bacterial concentrations.

[0047] 2) Culture and Induction ① Take 100 μL of the -80℃ culture medium and inoculate it into 5 mL of LB liquid medium (containing 50 μg / mL). -1 ① Add kanamycin), culture at 37℃ and 220 rpm for 4 h with shaking; ② Add IPTG to a final concentration of 1 mM, and continue to culture overnight at 37℃ and 220 rpm (16 h) to allow GFP to be fully expressed.

[0048] 3) Collection and washing of bacterial cells ① Take 1 mL of overnight bacterial culture, centrifuge at 2000×g and 4℃ for 5 min, and discard the supernatant; ② Add 1 mL of sterile PBS (pH 7.4) to resuspend, and centrifuge and wash once under the same conditions; ③ Finally, mix well with 1 mL of PBS to obtain the "stock solution".

[0049] 4) Serial dilution of bacterial culture: Dilute the stock solution 10-fold serially to obtain 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 There are a total of 6 basic dilutions; to further refine the low bacterial count range, further dilutions were performed at 10... -4 10 -5 10 -6 Intermediate dilutions of 2x and 4x were performed, resulting in a total of 11 concentration points: 1×10 -1 1×10 -2 1×10 -3 4×10 -4 2×10 -4 1×10 -4 4×10 -5 2×10 -5 1×10 -5 5×10 -6 1×10 -6 .

[0050] 5) Fluorescence microscopy counting ① Take a 96-well transparent plate and add 1 μL of the bacterial solution of each gradient to the bottom of the corresponding well; ② Observe using a fluorescence microscope (FITC channel, excitation / emission 488 / 520 nm, 10× objective); ③ When the number of fluorescent spots in the bacterial solution is less than 100, manually count the number of green fluorescent spots in each well; ④ When the number of fluorescent spots in the bacterial solution is greater than 100, calculate the count by multiplying the measured value of the adjacent low dilution by the dilution factor to avoid counting errors.

[0051] Table 2. Number of Escherichia coli in each well of a 96-well plate at different dilutions. Table 2 shows that the average bacterial count at each dilution factor is directly proportional to the dilution factor. Therefore, the dilution factor can be calculated to be 4 × 10⁻⁶. -4 The bacterial count was 160, and the dilution factor was 1×10⁻⁶. -3 The bacterial count is 400.

[0052] Plate culture counting Take 10 μL of each grade of bacterial suspension from LB solid medium and spread it evenly on the surface of the medium using a spreader. After spreading, invert the medium and place it in a 37°C incubator. After incubation for 3 days, count the colonies in the medium and use the count results to calculate the concentration of each grade of bacterial suspension.

[0053] (3) Extraction of total RNA from the sample Add 10 μL of lysozyme solution to a 96-well transparent plate and incubate at 37°C for 15 min.

[0054] Adding 1% β 10 μL of lysis buffer containing β-mercaptoethanol.

[0055] Add 10 μL of 70% ethanol.

[0056] Transfer the sample (including any remaining precipitate) to the extraction column (with collection tube A).

[0057] Centrifuge at 12000×g for 15 seconds at room temperature, discard the liquid in collection A, and reinsert the extraction column into collection tube A.

[0058] Add 140 μL of washing buffer A to the extraction column. Centrifuge at 12000×g for 15 seconds at room temperature.

[0059] Discard collection tube A and place the extraction column into collection tube B.

[0060] Add 100 μL of washing buffer B to the extraction column and centrifuge at 12000×g for 15 seconds at room temperature.

[0061] Discard the liquid in collection tube B and reinsert the extraction column into collection tube B.

[0062] Add 100 μL of washing buffer B to the extraction column again and centrifuge at 12000×g for 15 seconds at room temperature.

[0063] Discard the liquid in collection tube B and reinsert the extraction column into collection tube B.

[0064] At room temperature, the extraction column with collection tube B was centrifuged at 12000×g for 1 min.

[0065] Discard collection tube B and insert the extraction column into collection tube C.

[0066] Add 30 μL of nuclease-free water to the center of the extraction column and incubate at room temperature for 1 min.

[0067] Centrifuge at 12000×g for 2 min at room temperature, and the liquid in collection tube C is the total RNA of the sample.

[0068] (4) Specific reverse transcription of 16S rRNA in the sample ① Template and primer pre-denaturation: Take 10 μL of total RNA extract from the sample and mix it with 0.1 μL of FP (100 μM) and 0.1 μL of RP (100 μM), add 1 μL of 10 mM dNTP, and bring the volume to 12.5 μL with nuclease-free water; heat shock at 65℃ for 5 min, centrifuge briefly, and immediately place on ice to unravel the secondary structure of rRNA and improve reverse transcription efficiency.

[0069] ② Reverse transcription reaction: Add the following to an ice bath in sequence: 4 μL of 5×ProtoScript II Buffer, 2 μL of 0.1 MDTT, 1 μL of ProtoScript II RT (200 U / μL), 0.2 μL of RNase Inhibitor (40 U / μL), and 0.3 μL of nuclease-free water, for a total volume of 20 μL; heat-inactivate at 55℃ for 60 min, then at 70℃ for 15 min to terminate the reaction.

[0070] (5) Digital PCR operation Mix 2 μL of the sample reverse transcription product with 3 μL of 4×Probe PCR Mix, add 0.1 μL of FP (100 μM), 0.1 μL of RP (100 μM), and 0.05 μL of TM (100 μM), and bring the volume to 12 μL with nuclease-free water. Transfer the reaction mixture to 24-well 8.5k nanoplates, seal them, and analyze them using a digital PCR instrument. Each sample is analyzed three times in duplicate. The non-fluorescent wells are used as blank controls. The thermal cycling program is 95℃ initial denaturation for 2 min, followed by 40 cycles including 95℃ denaturation for 30 seconds and 60℃ annealing / extension for 30 seconds. The copy number concentration is calculated based on the positive droplets. When the copy number concentration of the negative control is < 2 copies / μL and the corrected copy number concentration of the sample (sample measured copy number concentration - negative control copy number concentration) ≥ 1 copy / μL, bacterial contamination is considered present; when the copy number concentration of the negative control is < 2 copies / μL and the corrected copy number concentration of the sample is < 1 copy / μL, no bacterial contamination is considered; when the copy number concentration of the negative control is ≥ 2 copies / μL, bacterial contamination is considered to have been introduced into the testing environment or during the experimental procedure, and the source of bacterial contamination needs to be eliminated before retesting. Results are shown in Tables 3-8 and... Figures 9-13 As shown.

[0071] Table 3. Relationship between bacterial count and copy number concentration for different primers in Escherichia coli Table 3 shows that 16 universal primer-probe combinations targeting a highly conserved fragment of the bacterial 16S rRNA gene were designed and screened, all of which showed significant copy number concentration signals against 1 CFU and 10 CFU bacteria, confirming the feasibility of using 16S rRNA as the detection target. Subsequent experiments randomly selected three primer-probe combinations (numbered Bacteria_fwd / rev-1, Bacteria_fwd / rev-2, and Bacteria_fwd / rev-3) for linearity validation.

[0072] Table 4. Relationship between bacterial count and copy number for three random primer-probe combinations in Escherichia coli. Table 4 and Figure 9 The results showed a good linear relationship between bacterial count and copy number using the three random primer sets in *E. coli*, further indicating that the universal primer-probe combination designed and screened based on a highly conserved fragment of the bacterial 16S rRNA gene is suitable for this method. After comprehensive evaluation, the *Bacteria_fwd / rev-1* primers were selected for subsequent experiments.

[0073] Table 5. Relationship between bacterial count and copy number of Escherichia coli 16S rRNA-cDNA genome. Table 6. Relationship between Bacillus subtilis 16S rRNA-cDNA sequence count and copy number Table 7 Relationship between Staphylococcus aureus 16S rRNA-cDNA genome count and copy number Table 8. Relationship between bacterial count and copy number of Pseudomonas aeruginosa 16S rRNA-cDNA genome. Tables 4-8 and Figures 10-13 The results showed a good linear relationship between the number of bacteria and the copy number of the bacterial 16S rRNA-cDNA genome, and the copy number of one bacterium could be detected.

[0074] Example 2: Stability test for single bacterial count detection (1) Preparation and counting of bacterial suspensions with different bacterial counts This step is the same as the operation in Example 1, “(2) Preparation and counting of bacterial solutions with different bacterial counts”.

[0075] (2) Extraction of total RNA from the sample Four samples were selected: one containing 1 bacterial cell and one containing no bacteria. The total RNA extraction procedure was the same as that in Example 1, "(3) Extraction of total RNA from the sample".

[0076] (3) Specific reverse transcription of 16S rRNA in the sample This step is the same as the operation of “16S rRNA specific reverse transcription in sample” in Example 1 (4).

[0077] (4) Digital PCR operation This step is the same as the step in Example 1, “(5) Digital PCR Operation”. The results are shown in Table 9 and Figures 14-15 As shown.

[0078] Table 9 Comparison of copy number concentrations between bacterial count 1 and bacterial count 0 Table 9 and Figures 14-15 The results showed that the copy number concentrations of bacteria 0 and 1 were stable in the four samples. The total average copy number concentration of bacteria 0 was 0.812 copies / μL, and the total average copy number concentration of bacteria 1 was 2.180 copies / μL, with a signal-to-noise ratio of 2.683.

[0079] Example 3: Investigation into the relationship between bacterial count and copy number in directly extracted DNA sequences (1) Preparation and counting of bacterial suspensions with different bacterial counts This step is the same as the steps "1) strains and plasmids, 2) culture and induction, 3) cell collection and washing, and 5) fluorescence microscopy counting" in Example 1 "(2) preparation and counting of bacterial solutions with different bacterial counts". In step "4) gradient dilution of bacterial solution", the dilution gradient is modified to: the original solution is serially diluted 10 times to obtain 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 There are a total of 8 basic concentrations.

[0080] (2) Extraction of total bacterial DNA from the sample Genomic DNA was extracted from bacterial lysates using a genomic DNA column kit.

[0081] (3) Digital PCR operation This step is the same as the step in Example 1, “(5) Digital PCR Operation”. The results are shown in Table 10 and Figures 16-17 As shown.

[0082] Table 10 Relationship between bacterial count and copy number in directly extracted DNA sequences Table 10 and Figure 16 The results showed that when the bacterial count was less than 50, the DNA copy number could not be detected significantly, and the limit of quantitative detection was higher than 50 bacterial count. Figure 17 The results showed that compared with the method of directly extracting DNA, the method of first extracting RNA and then reverse transcribing it into cDNA has higher detection sensitivity.

[0083] Example 4: Detection of dead bacteria under multiple sterilization methods (1) Preparation and counting of bacterial suspensions with different bacterial counts This step is the same as the operation in Example 1, “(2) Preparation and counting of bacterial solutions with different bacterial counts”.

[0084] (2) Multiple sterilization methods for treating bacterial solutions A bacterial suspension containing 10^10 bacteria per 1 μL was dropped into the center of the bottom of several vials. The samples were sterilized using four methods: moist heat sterilization, dry heat sterilization, ultraviolet (UV) sterilization, and antibiotic sterilization. Moist heat sterilization was performed at 121°C for 30 min in a high-temperature steam autoclave; dry heat sterilization was performed at 160°C for 2 h in an oven; UV sterilization was performed by irradiating the samples under a 254 nm UV lamp in a biosafety cabinet for 4 h; and antibiotic sterilization involved adding chloramphenicol and incubating for 6 h. Negative samples were blank bacterial suspensions with 0 bacteria, and positive samples were bacterial suspensions containing 10^10 bacteria per 1 μL, which were not sterilized.

[0085] (3) Extraction of total RNA from the sample This step is the same as the operation in Example 1, “(3) Extraction of total RNA from the sample”.

[0086] (4) Specific reverse transcription of 16S rRNA in the sample This step is the same as the operation of “16S rRNA specific reverse transcription in sample” in Example 1 (4).

[0087] (5) Digital PCR operation This step is the same as the step in Example 1, “(5) Digital PCR Operation”. The results are shown in Table 11 and Figure 18 As shown.

[0088] Table 11 Results of dead bacteria detection under various sterilization methods Table 11 and Figure 18The results showed that the mean copy number concentration of samples after moist heat sterilization was not significantly different from the negative control, but significantly different from the positive control (P=0.0025); the mean copy number concentration of samples after dry heat sterilization was not significantly different from the negative control, but significantly different from the positive control (P=0.0017); the mean copy number concentration of samples after moist heat sterilization was not significantly different from the negative control, but significantly different from the positive control (P=0.0016); and the mean copy number concentration of samples after moist heat sterilization was not significantly different from the negative control, but significantly different from the positive control (P=0.0029). In summary, after processing samples using various sterilization methods, this method does not detect dead bacteria, thus eliminating interference from dead bacteria in actual sample testing.

[0089] Example 5: The Influence of CAR-T Cell Matrix on Detection Methods (1) Preparation and counting of bacterial suspensions with different bacterial counts This step is the same as the operation in Example 1, “(2) Preparation and counting of bacterial solutions with different bacterial counts”.

[0090] Incorporation of CAR-T cell matrix Take 10 μL of a concentration of 1×10 7 / mL of CAR-T cells were added to the counted bacterial culture in a 96-well plate and mixed. The room temperature group (CAR-T+E.coli-1) proceeded directly to the next step of total RNA extraction from the sample; the cryopreservation group (CAR-T+E.coli-2) had the 96-well plate sealed and stored at -80℃ for 4 h before the total RNA extraction step was performed, to simulate the state of actual samples frozen before testing.

[0091] (3) Extraction of total RNA from the sample This step is the same as the operation in Example 1, “(3) Extraction of total RNA from the sample”.

[0092] (4) Specific reverse transcription of 16S rRNA in the sample This step is the same as the operation of “16S rRNA specific reverse transcription in sample” in Example 1 (4).

[0093] (5) Digital PCR operation This step is the same as the step in Example 1, “(5) Digital PCR Operation”. The results are shown in Tables 12-13 and Figure 19 As shown.

[0094] Table 12 Relationship between bacterial count and copy number in the ambient temperature group (CAR-T+E.coli-1) Table 13 Relationship between bacterial count and copy number in the cryopreserved group (CAR-T+E.coli-2) Tables 12-13 and Figure 19 The results showed that after incorporating CAR-T cell matrix, both the cryopreserved group and the room temperature group exhibited a good linear relationship in bacterial count and copy number, and copies of one bacterium could be detected. The cryopreserved group, having undergone -80℃ freezing for 4 hours, experienced partial loss of RNA in the bacterial cells, resulting in a lower linear slope than the room temperature group, but this did not affect the detection of a single bacterium.

[0095] Example 6: Detection of actual CAR-T cell samples (1) Comparison of plate coating counting method Take 3 samples from different batches, each with a cell concentration of 1×10⁻⁶. 7 CAR-T cells were collected at a density of / mL and labeled C-1, C-2, and C-3. 10 μL of each of the C-1, C-2, and C-3 cell samples were evenly spread onto LB agar plates. An unspread LB agar plate served as a blank control. The spread LB agar plates and the blank control were incubated at 37°C for 14 days. After incubation, the LB agar plates were removed for colony counting.

[0096] (2) Extraction of total RNA from the sample Take 3 samples from different batches, each with a cell concentration of 1×10⁻⁶. 7 / mL of CAR-T cells, one copy of each batch of samples, numbered C-1-1, C-1-2, C-2-1, C-2-2, C-3-1 and C-3-2 respectively. The extraction steps of total RNA from CAR-T cell samples are the same as those in Example 1 "(3) Extraction of total RNA from samples".

[0097] (3) Specific reverse transcription of 16S rRNA in the sample This step is the same as the operation of “16S rRNA specific reverse transcription in sample” in Example 1 (4).

[0098] (4) Digital PCR operation This step is the same as the step in Example 1, “(5) Digital PCR Operation”. The results are shown in Table 14 and Figure 20 As shown.

[0099] Table 14 Detection results of actual CAR-T cell samples Table 14 and Figure 20The results showed that none of the 6 CAR-T cell samples were contaminated with bacteria. In addition, none of the 6 samples in the plate count control group grew colonies after 14 days of incubation, indicating that the results of this detection method were consistent with the results of the plate count control group.

[0100] Example 7: Investigation into the relationship between fungal 18S rRNA-cDNA genome number and copy number (1) Design and screening of primers and probes The 18S rRNA gene is a component of the large subunit of the eukaryotic ribosome, and its gene sequence is relatively conserved in eukaryotes. The 18S rRNA gene can be divided into several variable regions and conserved regions. Variable regions (V1~V9, excluding V6) differ among different fungi and can be used to distinguish different fungal species, while conserved regions are very similar in all fungi and can be used to design universal primers for PCR amplification. Through literature review and independent design, 14 specific primer-probe combinations were screened targeting highly conserved fragments of the fungal 18S rRNA gene. All probes are labeled with a BHQ1 quencher at the 5' end and a C3-FAM fluorescent reporter at the 3' end. The specific sequences are as follows: Table 15 Primers and probes for fungal 18S rRNA genes (2) Preparation and counting of bacterial suspensions with different bacterial counts 1) Strains and plasmids Using standard Candida albicans and Aspergillus nidulans spores, both bacteria have clear outlines under a microscope, making them suitable for counting viable bacteria at low concentrations.

[0101] 2) Cultivation and Induction: Take 100 μL of Candida albicans culture at -80℃ and inoculate it into 5 mL of YPD liquid medium. Incubate overnight (16 h) at 30℃ and 220 rpm with shaking. Inoculate Aspergillus nidulans onto PDA plates and incubate in the dark at 37℃ for 3 days until the surface turns dark green. Add 5 mL of sterile water containing 0.01% Tween-80, gently scrape to collect, filter to remove hyphae, centrifuge at 3000×g for 5 min, and resuspend to obtain a high-purity spore suspension.

[0102] 3) Collection and washing of bacterial cells ① Take 1 mL of overnight bacterial culture, centrifuge at 2000×g and 4℃ for 5 min, and discard the supernatant; ② Add 1 mL of sterile PBS (pH 7.4) to resuspend, and centrifuge and wash once under the same conditions; ③ Finally, mix well with 1 mL of PBS to obtain the "stock solution".

[0103] 4) Serial dilution of bacterial culture: Dilute the stock solution 10-fold serially to obtain 10-1 10 -2 10 -3 10 -4 There are a total of 6 basic dilutions; to further refine the low bacterial count range, further dilutions were performed at 10... -1 10 -2 10 -3 Intermediate dilutions of 5-fold and 2.5-fold were performed, resulting in a total of 10 concentration points: 1×10⁻⁶. -1 5×10 -2 2.5×10 -2 1×10 -2 5×10 -3 2.5×10 -3 1×10 -3 5×10 -4 2.5×10 -4 1×10 -4 .

[0104] 5) Fluorescence microscopy counting ① Take a 96-well transparent plate and add 1 μL of the bacterial solution of each gradient to the bottom of the corresponding well; ② Observe using a bright-field fluorescence microscope; ③ When the number of bacteria in the bacterial solution is less than 150, visually count the number of bacteria in each well; ④ When the number of bacteria in the bacterial solution is greater than 150, calculate the count by multiplying the measured value of the adjacent low dilution by the dilution factor to avoid counting errors.

[0105] Table 16. Bacterial counts in each well of a 96-well plate at different dilutions. Table 16 shows that the average bacterial count at each dilution factor is directly proportional to the dilution factor.

[0106] (3) Extraction of total RNA 1) Add 10 μL of lysozyme solution to a 96-well transparent plate and incubate at 37°C for 15 min.

[0107] 2) Add 1% β 10 μL of lysis buffer containing β-mercaptoethanol.

[0108] 3) Add 10 μL of 70% ethanol.

[0109] 4) Transfer the sample (including any remaining precipitate) to the extraction column (with collection tube A).

[0110] 5) Centrifuge at 12000×g for 15 seconds at room temperature, discard the liquid in collection A, and reinsert the extraction column into collection tube A.

[0111] 6) Add 140 μL of washing buffer A to the extraction column. Centrifuge at 12000×g for 15 seconds at room temperature.

[0112] 7) Discard collection tube A and place the extraction column into collection tube B.

[0113] 8) Add 100 μL of washing buffer B to the extraction column and centrifuge at 12000×g for 15 seconds at room temperature.

[0114] 9) Discard the liquid in collection tube B and reinsert the extraction column into collection tube B.

[0115] 10) Add 100 μL of washing buffer B to the extraction column again and centrifuge at 12000×g for 15 seconds at room temperature.

[0116] 11) Discard the liquid in collection tube B and reinsert the extraction column into collection tube B.

[0117] 12) Centrifuge the extraction column with collection tube B at 12000×g for 1 min at room temperature.

[0118] 13) Discard collection tube B and insert the extraction column into collection tube C.

[0119] 14) Add 30 μL of nuclease-free water to the center of the extraction column and incubate at room temperature for 1 min.

[0120] 15) Centrifuge at 12000×g for 2 min at room temperature, and collect the liquid in tube C, which is the total RNA of the sample.

[0121] (4) Specific reverse transcription of 18S rRNA in the sample ① Template and primer pre-denaturation: Take 10 μL of total RNA extract from the sample and mix it with 0.1 μL of FP (100 μM) and 0.1 μL of RP (100 μM), add 1 μL of 10 mM dNTP, and bring the volume to 12.5 μL with nuclease-free water; heat shock at 65℃ for 5 min, centrifuge briefly, and immediately place on ice to unravel the secondary structure of rRNA and improve reverse transcription efficiency.

[0122] ② Reverse transcription reaction: Add the following to an ice bath in sequence: 4 μL of 5×ProtoScript II Buffer, 2 μL of 0.1 MDTT, 1 μL of ProtoScript II RT (200 U / μL), 0.2 μL of RNase Inhibitor (40 U / μL), and 0.3 μL of nuclease-free water, for a total volume of 20 μL; heat-inactivate at 55℃ for 60 min, then at 70℃ for 15 min to terminate the reaction.

[0123] (5) Digital PCR operation Mix 2 μL of the sample reverse transcription product with 3 μL of 4×Probe PCR Mix, add 0.1 μL of FP (100 μM), 0.1 μL of RP (100 μM), and 0.05 μL of TM (100 μM), and bring the volume to 12 μL with nuclease-free water. Transfer the reaction mixture to 24-well 8.5k nanoplates, seal them, and analyze them using a digital PCR instrument. Each sample is analyzed three times in duplicate. The non-fluorescent wells are used as blank controls. The thermal cycling program is 95 °C for initial denaturation for 2 min, followed by 40 cycles including 95 °C denaturation for 30 seconds and 60 °C annealing / extension for 30 seconds. The copy number concentration is calculated based on the positive droplets. When the copy number concentration of the negative control is < 2 copies / μL and the corrected copy number concentration of the sample (sample measured copy number concentration - negative control copy number concentration) ≥ 1 copy / μL, bacterial contamination is considered present; when the copy number concentration of the negative control is < 2 copies / μL and the corrected copy number concentration of the sample is < 1 copy / μL, no bacterial contamination is considered present; when the copy number concentration of the negative control is ≥ 2 copies / μL, bacterial contamination is considered to have been introduced into the testing environment or during the experimental procedure, and the source of bacterial contamination needs to be eliminated before retesting. Results are shown in Tables 17-20 and... Figures 21-23 As shown.

[0124] Table 17 Relationship between bacterial count and copy number concentration for different primers in Candida albicans Table 17 shows that 14 universal primer-probe combinations for fungi were designed and screened, targeting a highly conserved fragment of the fungal 18S rRNA gene. These combinations all showed significant copy number concentration signals against both 1 CFU and 10 CFU fungi, confirming the feasibility of using 18S rRNA as the detection target. Subsequent experiments randomly selected three primer-probe combinations (Fungi_fwd / rev-1, Fungi_fwd / rev-2, and Fungi_fwd / rev-3) for linearity validation.

[0125] Table 18 Relationship between bacterial count and copy number for different primers in Candida albicans Table 18 and Figure 21 The results showed a good linear relationship between the number of bacteria and the copy number of the three random primer sets in Candida albicans, indicating that the specific primer-probe combination designed and screened based on the highly conserved fragment of the fungal 18S rRNA gene is suitable for this method. After comprehensive evaluation, the Fungi_fwd / rev-1 primers were selected for subsequent experiments.

[0126] Table 19 Relationship between Candida albicans 18S rRNA-cDNA genome count and copy number Table 20 Relationship between Aspergillus nidulans 18S rRNA-cDNA sequence count and copy number Tables 19-20 and Figures 22-23 The results showed a good linear relationship between the bacterial count and copy number in the 18S rRNA-cDNA group, and the copy number of 1 CFU of Candida albicans could be detected.

[0127] sequence list 16S rRNA gene sequence: Escherichia coli strain U 5 / 41 16S ribosomal RNA 1 agtttgatca tggctcagat tgaacgctgg cggcaggcct aacacatgca agtcgaacgg 61 taacaggaag cagcttgctg ctttgctgac gagtggcgga cgggtgagta atgtctggga 121 aactgcctga tggaggggga taactactgg aaacggtagc taataccgca taacgtcgca 181 agcacaaaga gggggacctt agggcctctt gccatcggat gtgcccagat gggattagct 241 agtaggtggg gtaacggctc acctaggcga cgatccctag ctggtctgag aggatgacca 301 gcaacactgg aactgagaca cggtccagac tcctacggga ggcagcagtg gggaatattg 361 cacaatgggc gcaagcctga tgcagccatg cngcgtgtat gaagaaggcc ttcgggttgt 421 aaagtacttt cagcggggag gaagggagta aagttaatac ctttgctcat tgacgttacc 481 541 gttaatcgga attactgggc gtaaagcgca cgcaggcggt ttgttaagtc agatgtgaaa 601 tccccgggct caacctggga actgcatctg atactggcaa gcttgagtct cgtagagggg 661 ggtagaattc caggtgtagc ggtgaaatgc gtagagatct ggaggaatac cggtggcgaa 721 ggcggccccc tggacgaaga ctgacgctca ggtgcgaaag cgtggggagac aaacaggatt 781 agataccctg gtagtccacg ccgtaaacga tgtcgacttg gaggttgtgc ccttgaggcg 841 tggcttccgg anntaacgcg ttaagtcgac cgcctgggga gtacggccgc aaggttaaaa 901 ctcaaatgaa ttgacgggg ccgcacaagc ggtggagcat gtggtttaat tcgatgcaac 961 gcgaagaacc ttacctggtc ttgacatcca cggaagttt cagagatgag aatgtgcctt 1021 cgggaaccgt gagacaggtg ctgcatggct gtcgtcagct cgtgttgtgaaatgttgggt 1081 taagtcccgc aacgagcgca acccttatcc tttgttgcca gcggtccggccgggaactca 1141 aaggaactg ccagtgataa actggaggaa ggtggggatg acgtcaagtcatcatggccc 1201 ttacgaccag ggctacacac gtgctacaat ggcgcataca aagagaagcgacctcgcgag 1261 together cctcataaag tgcgtcgtag tccggattgg agtctcgactccat 1321 gagtcgga tcgctagtaa tcgtggatca gaatgccacg gtgaatacgttcccggggcct 1381 tgtacacacc gcccgtcaca ccatggggagt gggttgcaaa agaagtaggtagcttaactt 1441 cggggaggggcg 18S rRNA synthesis: Candida albicans CBS 562 18S rRNA gene 1 tatctggttg atcctgccag tagtcatatg cttgtctcaa agattaagcc atgcatgtct 61 aagtataagc aattataca gtgaaactgc gaatggctca ttaaatcagt tatcgtttat 121 ttgatagtac cttactactt ggataaccgt ggtaattcta gagctaatac atgcttaaaa 181 tcccgactgt ttggaagga tgtatttatt agataaaaaa tcaatgcctt cgggctcttt 241 gatgattcat aataactttt cgaatcgcat ggccttgtgc tggcgatggt tcattcaaat 301 ttctgcccta tcaactttcg atggtaggat agtggcctac catggtttca acgggtacg 361 gggataagg gttcgattcc gggagggag cctgagaaac ggctaccaca tccaaggaag 421 gcagcaggcg cgcaaattac ccaatcccga cacggggagg tagtgacaat aaataacgat 481 acagggccct tttgggtctt gtaattggaa tgagtacaat gtaaatacct taacgaggaa 541 caattggagg gcaagtctgg tgccagcagc cgcggtaatt ccagctccaa aagcgtatat 601 taaagttgtt gcagttaaaa agctcgtagt tgaaccttgg gcttggctgg ccggtccatc 661 tttttgatgc gtactggacc cagccgagcc tttccttctg ggtagccatt tatggcgaac 721 caggactttt actttgaaaa aattagagtg ttcaaagcag gcctttgctc gaatatatta 781 gcatggaata atagaatagg acgttatggt tctattttgt tggtttctag gaccatcgta 841 atgattaata gggacggtcg ggggtatcag tattcagttg tcagaggtga aattcttgga 901 tttactgaag actaactact gcgaaagcat ttaccaagga cgttttcatt aatcaagaac 961 gaaagttagg ggatcgaaga tgatcagata ccgtcgtagt cttaaccata aactatgccg 1021 actagggatc ggttgttgtt cttttattga cgcaatcggc accttacgagaaatcaaagt 1081 ctttgggttc tggggggagt atggtcgcaa ggctgaaact taaaggaattgacggaaggg 1141 caccaccagg agtggagcct gcggcttaat ttgactcaac acggggaaactcaccaggtc 1201 cagacacaat aaggattgac agattgagag ctctttcttg attttgtgggtggtggtgca 1261 tggccgttct tagttggtgg agtgatttgt ctgcttaatt gcgataacgaacgagacctt 1321 aacctactaa atagtgctgc tagcatttgc tggtatagtc acttcttagagggactatcg 1381 acttcaagtc gatggaagtt tgaggcaata acaggtctgt gatgcccttagacgttctgg 1441 gccgcacgcg cgctacactg acggagccag cgagtataag ccttggccgagaggtctggg 1501 aaatcttgtg aaactccgtc gtgctggggga tagagcattg taattgttgctcttcaacga 1561 ggaattccta gtaagcgcaa gtcatcagct tgcgttgatt acgtccctgccctttgtaca 1621 caccgcccgt cgctactacc gattgaatgg cttagtgagg cctccggattggtttaggaa 1681 aggggcaac ctcattctgg aaccgagaag ctggtcaaac ttggtcatttagaggaagta 1741 aaagtcgtaa caaggtttcc gtaggtgaac ctgcggaagg atcatta 。

Claims

1. A rapid aseptic detection method based on 16S / 18S rRNA-cDNA digital PCR technology, characterized in that, Includes the following steps: S1: Extract total RNA from the sample to be tested to obtain bacterial 16S rRNA and fungal 18S rRNA; S2: Stable cDNA fragments were obtained by reverse transcription of bacterial 16S rRNA and fungal 18S rRNA. S3: Digital PCR amplification is performed using cDNA fragments as templates, and the presence or absence of bacterial contamination is determined based on the amplification results, enabling rapid aseptic detection.

2. The rapid aseptic detection method based on 16S / 18S rRNA-cDNA digital PCR technology according to claim 1, characterized in that, In step S1, total RNA is extracted from the sample to be tested to obtain bacterial 16S rRNA and fungal 18S rRNA, specifically including: S1.1: First, add lysozyme buffer and lysozyme buffer to the sample to be tested; S1.2: Then add... β The guanidine isothiocyanate cleavage buffer of β-mercaptoethanol was used for cleavage. S1.3: After lysis, bacterial 16S rRNA and fungal 18S rRNA were obtained by centrifugation and elution using a silica membrane centrifuge column.

3. The rapid aseptic detection method based on 16S / 18S rRNA-cDNA digital PCR technology according to claim 1, characterized in that, In step S2, stable cDNA fragments are obtained by reverse transcription of bacterial 16S rRNA and fungal 18S rRNA, specifically including: S2.1: Pre-denaturate the extracted RNA using 16S / 18S universal primers; S2.2: cDNA was synthesized using ProtoScript II reverse transcriptase after pre-denaturation; S2.3: Stable cDNA fragments are obtained after enzyme heat inactivation.

4. The rapid aseptic detection method based on 16S / 18S rRNA-cDNA digital PCR technology according to claim 3, characterized in that, In step S2.1, the 16S / 18S universal primers are universal primers designed based on the conserved regions of bacterial 16S rRNA genes and fungal 18S rRNA genes.

5. The rapid aseptic detection method based on 16S / 18S rRNA-cDNA digital PCR technology according to claim 3, characterized in that, In step S2.1, pre-denaturation is performed at 55~75 ℃ for 4~10 min.

6. The rapid aseptic detection method based on 16S / 18S rRNA-cDNA digital PCR technology according to claim 3, characterized in that, In step S2.2, cDNA is synthesized at 50-60 ℃ for 30-70 min.

7. The rapid aseptic detection method based on 16S / 18S rRNA-cDNA digital PCR technology according to claim 1, characterized in that, In step S3, digital PCR amplification is performed using the cDNA fragment as a template, specifically including: Add the cDNA fragment, 16S / 18S universal primers, bacterial probe, fungal probe, and amplification premix to the sample plate of the digital PCR instrument, and then place the sample plate into the digital PCR instrument for digital PCR amplification. The reaction conditions for digital PCR amplification are as follows: initial denaturation at 90-100 ℃ for 1-4 min; followed by 30-50 cycles of denaturation at 90-100 ℃ for 15-60 s and annealing / extension at 50-70 ℃ for 15-120 s.

8. The rapid aseptic detection method based on 16S / 18S rRNA-cDNA digital PCR technology according to claim 7, characterized in that, In step S3, the bacterial probe is a universal probe designed with the conserved region of the bacterial 16S rRNA gene. The fungal probe described is a universal probe designed with the conserved region of the fungal 18S rRNA gene.

9. The rapid aseptic detection method based on 16S / 18S rRNA-cDNA digital PCR technology according to claim 1, characterized in that, In step S3, the presence or absence of bacterial contamination is determined based on the amplification results, specifically including: When the copy number concentration of the negative control is < 2 copies / μL and the corrected copy number concentration of the sample is ≥ 1 copy / μL, it is determined to be contaminated with bacteria. The corrected copy number concentration of the sample is the measured copy number concentration of the sample minus the copy number concentration of the negative control. When the copy number concentration of the negative control is < 2 copies / μL and the corrected copy number concentration of the sample is < 1 copy / μL, it is considered to be sterile. When the copy number concentration of the negative control is ≥ 2 copies / μL, it is determined that the experimental environment is contaminated with bacteria.

10. A kit for implementing the rapid sterile detection method based on 16S / 18S rRNA-cDNA digital PCR technology as described in any one of claims 1 to 9.

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