Search

WO-2026091032-A1 - PRIMER DESIGN METHOD, COMPOSITIONS, KIT, USE THEREOF AND METHOD

WO2026091032A1WO 2026091032 A1WO2026091032 A1WO 2026091032A1WO-2026091032-A1

Abstract

The present invention relates to a primer design method, compositions, a kit, the use thereof and a method. A 16s rRNA gene of a microorganism is used as a detection target, and an amplification region is enlarged by means of primer and probe design, that is, the length of an amplification product is 250-1000 bp, such that a host DNA residual fragment in Taq enzyme of 200 bp or less cannot be amplified, thereby not affecting the interpretation of a detection result, and reducing false positives in the detection result while not affecting the amplification efficiency.

Inventors

  • ZHAN, Mingming
  • ZHANG, YAQI

Assignees

  • 京东方科技集团股份有限公司
  • 北京京东方技术开发有限公司

Dates

Publication Date
20260507
Application Date
20241031

Claims (16)

  1. A method for designing primers for microbial detection, comprising: The gene sequence of the 16S rRNA of the microorganism was obtained; Sequence alignment of the gene sequence was performed to obtain the conservation results of the gene sequence among species; Based on the conservation results, a sequence of conservative regions is obtained; Upstream and downstream primers were designed using the conserved region sequence as a template, such that the length of the PCR products obtained by amplifying the genomes of various microorganisms using the upstream and downstream primers was 250-1000 bp.
  2. According to the method of claim 1, the upstream primer and the downstream primer are located in different conserved region sequences.
  3. According to the method of claim 1, the upstream primer and the downstream primer are 15-30 bp in length; the GC content is 40-60%; ΔG < 58.61 KJ/mol; and the Tm value is 58-62 °C.
  4. The method according to claim 1, wherein the method further comprises: A probe sequence is designed using the conserved region sequence as a template, such that the probe sequence is complementary to the sequence located between the upstream primer and the downstream primer in the PCR product.
  5. According to the method of claim 4, the probe sequence is 15-30 bp in length and its 5' end is not G; the Tm value of the probe sequence is higher than that of the upstream primer and the downstream primer, preferably, the Tm value of the probe sequence is 68-72℃.
  6. According to the method of claim 5, the probe sequence is modified with a fluorescent reporter group and a fluorescent quencher group corresponding to the fluorescent reporter group at both ends; the fluorescent reporter group is FAM, VIC, HEX, ROX or CY5, and the fluorescent quencher group is BHQ1, BHQ2, BHQ3 or MGB.
  7. According to any one of claims 1-6, the microorganism is Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Streptococcus pyogenes, Micrococcus sp., Candida albicans, Aspergillus niger, Clostridium sporogenes, or Propionibacterium acnes.
  8. A composition comprising: obtained by the method of any one of claims 1-7: Upstream and downstream decoy oligonucleotides, wherein the upstream decoy oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:1, and the downstream decoy oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:2; or Upstream and downstream decoy oligonucleotides, wherein the upstream decoy oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:1, and the downstream decoy oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:3; or The upstream decoy oligonucleotide and the downstream decoy oligonucleotide, wherein the upstream decoy oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:4, and the downstream decoy oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:5.
  9. The composition according to claim 8, wherein the upstream decoy oligonucleotide is the sequence shown in SEQ ID NO:1, and the downstream decoy oligonucleotide is the sequence shown in SEQ ID NO:2; or The upstream decoy oligonucleotide is the sequence shown in SEQ ID NO:1, and the downstream decoy oligonucleotide is the sequence shown in SEQ ID NO:3; or The upstream decoy oligonucleotide is the sequence shown in SEQ ID NO:4, and the downstream decoy oligonucleotide is the sequence shown in SEQ ID NO:5.
  10. The composition according to claim 8 or 9, wherein the composition further comprises a probe sequence derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in any one of SEQ ID NO: 6-7.
  11. The composition according to claim 10, wherein the probe sequence is the sequence shown in any one of SEQ ID NO: 6-7.
  12. The composition according to claim 10 or 11, wherein the probe sequence is modified at both ends with a fluorescent reporter group and a fluorescent quencher group corresponding to the fluorescent reporter group; the fluorescent reporter group is FAM, VIC, HEX, ROX or CY5, and the fluorescent quencher group is BHQ1, BHQ2, BHQ3 or MGB.
  13. A kit comprising the composition of any one of claims 8-12.
  14. Use of the composition of any one of claims 8-12 or the kit of claim 13 in microbial detection.
  15. Use of the composition of any one of claims 8-12 or the kit of claim 13 in the detection of microorganisms by qPCR.
  16. A method for detecting microorganisms using qPCR, comprising: The composition of any one of claims 8-12 or the kit of claim 13 is used to perform qPCR on the sample to be tested on a qPCR instrument, and the presence and/or species of microorganisms in the sample are determined by the qPCR results.

Description

Primer design methods, compositions, kits, uses and methods Technical Field This application relates generally to the field of biotechnology, and in particular to a method for designing primers, compositions, kits, uses thereof, and a method for detecting microorganisms using qPCR. Background Technology Cell and gene therapy products have become a research hotspot both domestically and internationally due to their significant efficacy in clinical studies of oncology. Therefore, the sterility testing and release of their intermediate products are particularly important. On October 26, 2021, the National Pharmacopoeia Commission published a draft of the "General Rules - Microbiological Examination Method for Cell Products," filling a gap in my country's rapid microbiological testing methods for cell products. Subsequently, the "Validation Technical Requirements for Rapid Sterility Testing Methods for Cell and Gene Therapy Products" was released on June 28, 2022, and implemented on July 28, 2022. This law further improved the regulations for the validation of rapid sterility testing methods in cell and gene therapy products in my country. Among these, rapid nucleic acid methods, represented by qPCR, will eventually make rapid sterility testing a possibility, bringing greater convenience to scientific research and drug production. Summary of the Invention Based on this, this application provides a primer design method for microbial detection, including: The gene sequence of the 16S rRNA of the microorganism was obtained; Sequence alignment of the gene sequence was performed to obtain the conservation results of the gene sequence among species; Based on the conservation results, a sequence of conservative regions is obtained; Upstream and downstream primers were designed using the conserved region sequence as a template, such that the length of the PCR products obtained by amplifying the genomes of various microorganisms using the upstream and downstream primers was 250-1000 bp. On the other hand, this application also provides a composition comprising, obtained by the primer design method described herein: Upstream and downstream decoy oligonucleotides, wherein the upstream decoy oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:1, and the downstream decoy oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:2; or Upstream and downstream decoy oligonucleotides, wherein the upstream decoy oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:1, and the downstream decoy oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:3; or The upstream decoy oligonucleotide and the downstream decoy oligonucleotide, wherein the upstream decoy oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:4, and the downstream decoy oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:5. In another aspect, this application also provides a composition comprising a probe sequence derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in any one of SEQ ID NO: 6-7. In another aspect, this application also provides a kit comprising the compositions described herein. On the other hand, this application also provides the use of the compositions or kits described herein in microbial detection. On the other hand, this application also provides the use of the compositions or kits described herein in the detection of microorganisms by qPCR. On the other hand, this application also provides a method for detecting microorganisms using qPCR, comprising: Perform qPCR on the sample to be tested using the composition or kit described herein on a qPCR instrument, and determine the presence and/or species of microorganisms in the sample based on the qPCR results. This application uses the 16S rRNA gene of microorganisms as the detection target. Through primer and probe design, the amplification region is enlarged, that is, the length of the amplification product is 250-1000bp, so that it cannot amplify the host DNA residual fragments in the Taq enzyme below 200bp, thus not affecting the interpretation of the detection results. This reduces false positives in the detection results while keeping the amplification efficiency unaffected. Other features and advantages of this application will be set forth in the following description, and will be apparent in part from the description, or may be lea