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CN-121992083-A - Rapid sterile detection method and kit based on 16S/18S rRNA-cDNA digital PCR technology

CN121992083ACN 121992083 ACN121992083 ACN 121992083ACN-121992083-A

Abstract

The invention discloses a rapid sterile detection method and a kit based on a 16S/18S rRNA-cDNA digital PCR technology, comprising the steps of S1, extracting total RNA in a sample to be detected to obtain bacterial 16S rRNA and fungal 18S rRNA, S2, carrying out reverse transcription on the bacterial 16S rRNA and the fungal 18S rRNA to obtain a stable cDNA segment, and S3, carrying out digital PCR amplification by taking the cDNA segment as a template, and judging whether thallus pollution exists according to an amplification result to realize rapid sterile detection. Aiming at the characteristics of high conservation and high copy of bacterial 16S rRNA/fungal 18S rRNA, the invention verifies 30 sets of universal primer probe combinations, selects two optimal primer probe combinations, can cover >99.99 percent of clinically and pharmaceutically common polluted bacteria/fungi, and has no cross amplification with human, hamster, cow and other mammal mitochondria rRNA.

Inventors

  • LIU YIZHEN

Assignees

  • 杭州沁瀚医药科技有限公司
  • 金华沁浩医药科技有限公司

Dates

Publication Date
20260508
Application Date
20260214

Claims (10)

  1. 1. A rapid sterile detection method based on a 16S/18S rRNA-cDNA digital PCR technology is characterized by comprising the following steps: S1, extracting total RNA in a sample to be detected to obtain bacterial 16S rRNA and fungal 18S rRNA; S2, carrying out reverse transcription on bacterial 16S rRNA and fungal 18S rRNA to obtain a stable cDNA fragment; and S3, carrying out digital PCR amplification by taking the cDNA fragment as a template, and judging whether the bacteria pollution exists according to the amplification result so as to realize rapid sterile detection.
  2. 2. The rapid sterile detection method based on the 16S/18S rRNA-cDNA digital PCR technique according to claim 1, wherein in step S1, total RNA in the sample to be detected is extracted to obtain bacterial 16S rRNA and fungal 18S rRNA, specifically comprising: s1.1, firstly adding lysozyme buffer solution and muramidase buffer solution into a sample to be detected; s1.2, adding guanidine isothiocyanate lysate containing beta-mercaptoethanol for cracking; S1.3, after the lysis, the bacterial 16S rRNA and the fungal 18S rRNA are obtained by centrifugation and elution through a silica gel membrane centrifugal column.
  3. 3. The rapid sterile detection method based on the 16S/18S rRNA-cDNA digital PCR technique according to claim 1, wherein in step S2, stable cDNA fragments are obtained by reverse transcription of bacterial 16S rRNA and fungal 18S rRNA lines, specifically comprising: S2.1, performing pre-denaturation on the extracted RNA by using a 16S/18S universal primer; s2.2, cDNA is synthesized by ProtoScript II reverse transcriptase after pre-denaturation; s2.3, obtaining stable cDNA fragments after enzyme heat inactivation.
  4. 4. The rapid sterile detection method based on the 16S/18S rRNA-cDNA digital PCR technology according to claim 3, wherein in the step S2.1, the 16S/18S universal primer is a universal primer designed by a bacterial 16S rRNA gene and a fungus 18S rRNA gene conserved region.
  5. 5. The rapid sterility test method based on the 16S/18S rRNA-cDNA digital PCR technique as set forth in claim 3, wherein in step S2.1, the pre-denaturation is performed at 55-75℃for 4-10 min.
  6. 6. The rapid sterile detection method based on the 16S/18S rRNA-cDNA digital PCR technology according to claim 3, wherein in the step S2.2, cDNA is synthesized at 50-60 ℃ for 30-70 min.
  7. 7. The rapid sterile detection method based on the 16S/18S rRNA-cDNA digital PCR technique according to claim 1, wherein in step S3, digital PCR amplification is performed using cDNA fragments as templates, specifically comprising: adding cDNA fragments, 16S/18S universal primers, bacterial probes, fungal probes and amplification premix into a sample plate of a digital PCR instrument, and then placing the sample plate into the digital PCR instrument for digital PCR amplification; The reaction conditions of the digital PCR amplification are that the initial denaturation is carried out for 1-4 min at 90-100 ℃, and then 30-50 cycles of denaturation at 90-100 ℃ for 15-60 s and annealing/extension at 50-70 ℃ for 15-120 s.
  8. 8. The rapid sterile detection method based on the 16S/18S rRNA-cDNA digital PCR technique according to claim 7, wherein in the step S3, the bacterial probe is a universal probe designed with a conserved region of bacterial 16S rRNA gene; The fungus probe is a universal probe designed by a fungus 18S rRNA gene conservation region.
  9. 9. The rapid sterile detection method based on the 16S/18S rRNA-cDNA digital PCR technology according to claim 1, wherein in the step S3, the presence or absence of contamination by the bacteria is judged according to the amplification result, specifically comprising: when the copy number concentration of the negative control is less than 2 copies/mu L and the corrected copy number concentration of the sample is more than or equal to 1 copy/mu L, judging that the sample is polluted by bacteria, wherein the corrected copy number concentration of the sample is the copy number concentration actually measured by the sample-the copy number concentration of the negative control; the sterility was judged to be contaminated when the copy number concentration of the negative control was < 2 copies/. Mu.L and the corrected copy number concentration of the sample was <1 copy/. Mu.L; And when the copy number concentration of the negative control is more than or equal to 2 copies/. Mu.L, judging that the experimental environment has bacterial contamination.
  10. 10. A kit for implementing the rapid sterility test method based on the 16S/18S rRNA-cDNA digital PCR technique as set forth in any one of claims 1 to 9.

Description

Rapid sterile detection method and kit based on 16S/18S rRNA-cDNA digital PCR technology Technical Field The invention relates to a rapid sterile detection method and a kit based on a 16S/18S rRNA-cDNA digital PCR (dPCR) technology. The invention belongs to the technical field of microorganism detection. Background In quality control of sterile pharmaceutical products, biological agents, medical devices, and clinical specimens, rapid sterility testing (RAPID STERILITY TESTING, RST) is a key element to ensure product safety and effectiveness. The traditional pharmacopoeia method (such as the 2025 edition of Chinese pharmacopoeia, general rule 1101) depends on a 14-day culture period, and is difficult to meet the urgent requirements of modern biological pharmacy on quick release, real-time process control and closed-loop production. Particularly for short shelf life products such as CAR-T cell therapy, mRNA vaccines, gene therapy drugs, etc., release delays will not only lead to product rejection and supply chain interruption, but also create clinical medication risks. Although there are alternative methods such as growth monitoring, ATP bioluminescence, etc., they also require culture, have a long period and low sensitivity. At present, a method for rapidly distinguishing 0 living bacteria from 1 living bacteria is not available. Therefore, development of a broad-spectrum rapid sterility test method that can be completed within 5 hours and has the sensitivity to distinguish between 0 and 1 (absolute quantity, volume independent) bacteria has become an urgent need for the industry. The current mainstream rapid sterile detection scheme still takes 'genome DNA extraction-qPCR/loop-mediated isothermal amplification' as a core, and results can be obtained after a sample is cracked and amplified by using a 16S/18S rDNA universal primer for 3-4 hours. However, the high stability of DNA brings natural defects that ① dead bacteria DNA can still be stable and amplified and detected within a few days, the false positive rate is high, ② qPCR is easy to be inhibited by heme, heparin, polysaccharide and the like, the detection limit is still about 10 2 CFU/mL, an overnight bacteria increasing step is additionally required for low pollution of <10 CFU/mL, ③ single copy genome target signals are weak, ct drift in a complex matrix is large, the quantitative result deviation can reach an order of magnitude, and the accuracy requirement of process control cannot be met. Compared with the DNA extraction route, the RNA extraction route has the advantages in the field of rapid sterile detection that the total RNA of bacteria is firstly extracted, then the random primer or the 16S/18S specific primer is used for reverse transcription, and the high-abundance 16S/18S rRNA is converted into cDNA and then amplified. In gram-negative bacteria in the active growth phase, the 16S/18S rRNA copy number is 10 3~104 higher than its single-copy genome, the copy number of >10 2 is maintained even for gram-positive or dormant bacteria, and the 18S rRNA copy number is 10 4~105 higher than its single-copy genome at higher levels for fungi. The signal amplification effect enables the template quantity under the same pollution level to be improved by 100-1000 times, so that the bacteria increasing step is omitted. Meanwhile, RNA rapidly degrades within 5-10 min along with the leakage of ribonuclease after the death of the thalli, and residual signals of the dead thalli are shielded naturally, so that the detection has 'living bacteria selectivity'. The scheme omits chemical pretreatment of the chelating dead bacteria DNA such as PMA-photolysis, shortens the operation time by nearly 1 hour, and is beneficial to eliminating the potential pollution of phototoxic reagents to the environment and the defect of false positive caused by incomplete chelating of the dead bacteria DNA. The digital PCR (DIGITAL PCR, DPCR) is used as the third generation PCR, the amplification system is divided into thousands or even tens of thousands of independent nano-upgrading microdroplets through a microfluidic technology, each microdroplet contains 0 or 1 template molecule, positive microdroplets are directly counted after end-point amplification, single-molecule level absolute quantification is realized, a standard curve is not needed, and the false negative risk caused by matrix inhibition or amplification efficiency difference is remarkably reduced compared with qPCR. On the other hand, the tolerance of the dPCR to heme and cell lysate is higher than that of qPCR, and the dPCR is especially suitable for high-protein and high-nucleic acid background samples such as cell treatment end products. Secondly, the dPCR only judges whether the fluorescent signal exists or not in the reading stage, and does not depend on the Ct value, so that the situation that a low-copy sample is misjudged to be negative because the qPCR amplification curve does not enter the platform stage i