Primer design method, compositions, kit, use thereof and method

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.

Description

Primer design methods, compositions, kits, uses and methods Technical Field

[0001] 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

[0002] 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.

[0003] Summary of the Invention

[0004] Based on this, this application provides a primer design method for microbial detection, including:

[0005] The gene sequence of the 16S rRNA of the microorganism was obtained;

[0006] Sequence alignment of the gene sequence was performed to obtain the conservation results of the gene sequence among species;

[0007] Based on the conservation results, a sequence of conservative regions is obtained;

[0008] 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.

[0009] On the other hand, this application also provides a composition comprising, obtained by the primer design method described herein:

[0010] 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

[0011] An upstream decoy oligonucleotide and a 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: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

[0012] 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.

[0013] 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.

[0014] In another aspect, this application also provides a kit comprising the compositions described herein.

[0015] On the other hand, this application also provides the use of the compositions or kits described herein in microbial detection.

[0016] On the other hand, this application also provides the use of the compositions or kits described herein in the detection of microorganisms by qPCR.

[0017] On the other hand, this application also provides a method for detecting microorganisms using qPCR, comprising:

[0018] 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.

[0019] 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.

[0020] 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 learned by practicing the application. Other advantages of this application can be realized and obtained by means of the embodiments described in the description and the accompanying drawings.

[0021] Overview of the attached figures

[0022] The accompanying drawings are used to provide an understanding of the technical solutions of this application and constitute a part of the specification. They are used together with the embodiments of this application to explain the technical solutions of this application and do not constitute a limitation on the technical solutions of this application.

[0023] Figure 1 is a schematic diagram illustrating the amplification effect of one embodiment of this application.

[0024] Figure 2 shows the NCBI MSA Viewer alignment results of the gene sequence of E. coli 16S rRNA (Gene Bank: J01859.1) in the example after BLAST, in which 5000 alignment sequences were selected.

[0025] Figure 3 shows the amplification detection results of the negative control (enzyme-free water) for each primer and probe combination in Example 2.

[0026] Figure 4 shows the amplification detection results of negative control (enzyme-free water) of six brands of Taq DNA polymerase in Example 3.

[0027] Figure 5 shows the amplification detection results of the negative control (enzyme-free water) for combinations 1-6 in Example 4.

[0028] Figure 6 shows the amplification detection results of the negative control (enzyme-free water) for combinations 7-12 in Example 4.

[0029] Figure 7 shows the amplification detection results of the negative control (enzyme-free water) of combinations 13-18 in Example 4.

[0030] Figure 8 shows the amplification detection results of the negative control (enzyme-free water) for combinations 19-23 in Example 4.

[0031] Figure 9 shows the amplification detection results of positive plasmids (E. coli 16S rRNA gene plasmids) in combinations 1-23 of Example 4.

[0032] Detailed Explanation

[0033] Unless otherwise stated, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this application pertains. When a quantity, concentration, or other value or parameter is expressed as a range, preferred range, or preferred upper and lower numerical limits, it should be understood that this is equivalent to specifically disclosing any range by combining any pair of upper or preferred values ​​with any lower or preferred value, regardless of whether the range is specifically disclosed. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within that range.

[0034] When used with a numerical variable, the terms "about" or "approximately" usually mean that the value of the variable and all values ​​of the variable are within the experimental error (e.g., within a 95% confidence interval for the mean) or within ±10% of the specified value, or a wider range.

[0035] The expression "comprising," or similar expressions such as "including," "containing," and "having," is open-ended and does not exclude additional unlisted elements, steps, or components. The expression "consisting of," excludes any unspecified elements, steps, or components. The expression "substantially consisting of," limits the scope to the specified elements, steps, or components, plus optional elements, steps, or components that do not materially affect the essential and novel features of the claimed subject matter. It should be understood that the expression "comprising" encompasses both the expressions "substantially consisting of" and "consisting of."

[0036] The expression "at least one" or "one or more" indicates 1, 2, 3, 4, 5, 6, 7, 8, 9 or more kinds.

[0037] Due to the high sensitivity and specificity of qPCR technology, non-specific amplification is highly likely to occur during its development process due to primers, probes, or raw materials. Commercial Taq polymerases primarily use engineered strains (such as *E. coli*) for recombinant expression; therefore, a certain amount of host genomic DNA remains in the Taq polymerase. Currently, methods for removing bacterial DNA contamination from Taq DNA polymerases include physical radiation treatment, chemical treatment, physical radiation combined with chemical treatment, and biological enzyme treatment. Many commercially available DNA polymerases contain varying degrees of bacterial DNA contamination, especially when bacterial DNA fragments are digested to below 200 bp, making complete removal impossible. Existing qPCR methods typically use primers designed to amplify fragments of 80-150 bp for microbial detection. This can lead to the detection of residual host DNA below 200 bp in the Taq polymerase during amplification, thus affecting result interpretation and product development.

[0038] Patent CN107099518A discloses a method for removing host DNA contamination from Taq DNA polymerase using the broad-spectrum nuclease Binuclease. However, its verification method is conventional PCR + agarose gel electrophoresis, which has lower sensitivity compared to qPCR. If the process is not thorough, it cannot determine whether trace amounts of host DNA fragments exist in the Taq enzyme. To address this issue, this application uses the 16S rRNA gene of microorganisms as the detection target. Through primer design, the amplification region is enlarged, resulting in an amplification product length of 250-1000 bp. This prevents the amplification of residual host DNA fragments smaller than 200 bp in the Taq enzyme, thus ensuring that the detection results are not affected.

[0039] Based on this, this application provides a primer design method for microbial detection, including:

[0040] Obtain the gene sequence of the 16S rRNA of microorganisms;

[0041] Gene sequences are aligned to obtain the results of gene sequence conservation among species;

[0042] Based on the conservation results, obtain the sequence of conservative regions within the conservative regions;

[0043] Upstream and downstream primers were designed using conserved region sequences as templates, resulting in PCR products with lengths of 250-1000 bp obtained by amplifying the genomes of various microorganisms using upstream and downstream primers.

[0044] In all other respects disclosed herein, the primer may be referred to as a “bait oligonucleotide,” “oligonucleotide primer,” or “nucleic acid primer.” The primer may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The primer may contain one or more non-natural nucleotides. Non-natural nucleotides may be, for example, deoxyinosine. The primer may be a forward primer. The primer may be a reverse primer. The primer length may be from about 5 to about 50 nucleotides. The primer length may be at least 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 or more base pairs. The primer length may be at most 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, or 5 nucleotides. The primer length can be approximately 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50 base pairs.

[0045] A primer pair may consist of a forward primer (upstream primer) and a reverse primer (downstream primer). The forward primer may be configured to hybridize with a first region (e.g., the 3' end) of the nucleic acid sequence, while the reverse primer may be configured to hybridize with a second region (e.g., the 5' end) of the nucleic acid sequence, thereby being configured to amplify the nucleic acid sequence under conditions sufficient for nucleic acid amplification. Different primer pairs may be configured to amplify different target nucleic acid sequences.

[0046] In some implementations, primers can be designed for different microorganisms, including but not limited to bacteria, fungi, archaea, mycoplasma, chlamydia, and rickettsiae. The designed primers can detect the corresponding microorganisms. In some implementations, detectable microorganisms include Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Streptococcus pyogenes, Micrococcus sp., Candida albicans, Aspergillus niger, Clostridium sporogenes, or Propionibacterium acnes.

[0047] In some implementations, detectable microorganisms include *Acetobacter aurantius*, *Acinetobacter baumannii*, *Actinomyces Israelii*, *Agrobacterium radiobacter*, *Agrobacterium tumefaciens*, *Azorhizobium caulinodans*, *Azotobacter vinelandii*, *Anaplasma phagocytophilum*, *Anaplasma marginale*, *Bacillus anthracis*, *Bacillus brevis*, *Bacillus cereus*, *Bacillus fusiformis*, *Bacillus licheniformis*, and *Bacillus megaterium*. Bacillus mecoides, Bacillus stearothermophilus, Bacillus subtilis, Bacteroides fragilis, Bacteroides gingivalis, Bacteroides melaminogenica (Prevotella melaminogenicus), Bartonella henselae, Bartonella quintana, Bordetella bronchiseptica, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella melitensis, Brucella swine Burkholderia mallei (suis), Burkholderia mallei (mercanthosis suis), Burkholderia mallei (mercanthosis suis)pseudomallei), Burkholderia cepacia complex, Burkholderia cenocepacia, Calymmatobacterium granulomatis, Campylobacter coli, Campylobacter fetus, Campylobacter jejuni, Campylobacter pylori, Chlamydia trachomatis, Chlamydophila (such as Chlamydia pneumoniae, Chlamydia psittaci), Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetanus Corynebacterium diphtheriae, Corynebacterium fusiforme, Coxiella bumetii, Ehrlichia chaffeensis, Enterobacter cloacae, Enterococcus avium, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus galllinarum, Enterococcus maloratus, Escherichia coli, Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis Haemophilus ducreyi, Haemophilus influenzae, Haemophilus parainfluenzaeHaemophilus parainfluenzae, Haemophilus pertussis, Haemophilus vaginalis, Helicobacter pylori, Klebsiella pneumoniae, Lactobacillus acidophilus, Lactobacillus casei, Lactococcus lactis, Legionella pneumophila, Listeria monocytogenes, Methanobacterium extroquens, Bacteroides polymorpha, Micrococcus luteus, Moraxella catarrhalis, Mycobacterium avium, Mycobacterium bovis, Mycobacterium diphtheriae, Mycobacterium intracellulare, Mycobacterium leprae Mycobacterium leprae, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycoplasma fermentans, Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma penetrans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Pasteurella tularensis, Peptostreptococcus, Porphyromonas gingivalis *Gingivalis*, *Pseudomonas aeruginosa*, and *Rhizobium*The following bacteria are listed: *Rhizobacter*, *Rickettsia prowazekii*, *Rickettsia psittaci*, *Rickettsia quintana*, *Rickettsia rickettsii*, *Rickettsia trachomae*, *Rochalimaea henselae*, *Rochalimaea quintana*, *Rothia dentocariosa*, *Salmonella enteritidis*, *Salmonella typhi*, *Salmonella typhimurium*, *Serratia marcescens*, *Shigella dysenteriae*, and *Staphylococcus aureus*. Streptococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus avium, Streptococcus bovis, Streptococcus cricetus, Streptococcus faceium, Streptococcus faecalis, Streptococcus ferus, Streptococcus gallinarum, Streptococcus lactis, Streptococcus mitior, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus pneumoniae Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus rattus, Streptococcus salivarius*Streptococcus salivarius*, *Streptococcus sanguis*, *Streptococcus sobrinus*, *Treponema pallidum*, *Treponema denticola*, *Vibrio cholerae*, *Vibrio comma*, *Vibrio parahaemolyticus*, *Vibrio vulnificus*, *Wolbachia*, *Yersinia enterocolitica*, *Yersinia pestis*, and *Yersinia pseudotuberculosis*.

[0048] In some implementations, after obtaining the 16S rRNA gene sequence of the microorganism, sequence analysis is performed on the gene sequence. For example, a BLAST scan on NCBI is used to find multiple sequences (e.g., 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000 or more) that are similar to the gene sequence. All of these are compared to identify conserved regions. Different conserved regions can be selected depending on the type of microorganism to be detected. Primers are then designed based on the selected conserved regions. Microorganisms containing the selected conserved regions can then be amplified and detected.

[0049] In some implementations, the upstream and downstream primers are located in different conserved sequences. This results in PCR products with lengths between 250 and 1000 bp amplified using these primers. With this design, only the target microbial species containing conserved sequences in the sample can be amplified, while remnants of host DNA shorter than 200 bp in the Taq enzyme cannot be amplified. This significantly reduces the occurrence of false positives.

[0050] In some implementations, the upstream and downstream primers 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.

[0051] In some embodiments, this application provides a method for designing primers for microbial detection, the method further comprising:

[0052] Probe sequences were designed using conserved region sequences as templates, so that the probe sequences were complementary to the sequences located between the upstream and downstream primers in the PCR product.

[0053] In some embodiments, 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 and downstream primers. Preferably, the Tm value of the probe sequence is 68-72℃. In some embodiments, the probe length can be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30 or more nucleotides.

[0054] In this document, the terms "detection probe" and "probe" are used interchangeably. A probe can be a nucleic acid (e.g., DNA, RNA, etc.). A probe may contain a region complementary to a region of a target nucleic acid. The concentration of the probe may be such that it is in excess relative to other components in the detection mixture. A probe may be a signal-generating nucleic acid probe. A signal-generating nucleic acid probe may contain the characteristics of a probe as described herein, and the characteristics of generating a signal upon application of a stimulus, modification of the probe, or hydrolysis of the probe. For example, a probe may be hydrolyzed by the exonuclease activity of a polymerase to generate a signal. A signal-generating nucleic acid probe may generate any signal as described elsewhere herein. For example, the signal-generating probe may generate a fluorescent signal. A signal-generating probe may contain a signal tag. A signal tag may generate a signal. A signal tag may generate a signal in response to a reaction or stimulus. For example, a signal tag may generate a signal upon degradation by exonuclease activity.

[0055] In some implementations, a signal can be generated simultaneously with hybridization of the probe to a nucleic acid region. For example, a probe (e.g., a molecular beacon) can generate a signal (e.g., a fluorescent signal) after hybridization with a nucleic acid. In some cases, a signal can be generated after the probe has hybridized with a nucleic acid region, or after the probe has been degraded by a nuclease. Where the probe contains a signal tag, the probe can be degraded when it binds to a region of a primer, thereby generating a signal. For example, a probe (e.g., a probe) can generate a signal after hybridization with a nucleic acid and subsequent degradation by a polymerase (e.g., during amplification such as PCR amplification). The probe can be degraded by the exonuclease activity of a nuclease.

[0056] In some implementations, the probe sequence is modified with fluorescent reporter groups and corresponding fluorescent quencher groups at both ends. The probe, modified with both fluorescent reporter and quencher groups, is used in qPCR to indicate the relative amount of synthesized DNA product. In the absence of amplification, the fluorescent reporter and quencher groups at both ends of the probe sequence are attached together; the fluorescence of the fluorescent reporter group is quenched by the quencher group, and no fluorescence is emitted. When PCR synthesis begins, the probe sequence complementaryly pairs with the target fragment in the genomic DNA and anneals together. If the target microorganism (i.e., the target fragment) is present in the sample, the probe sequence can bind to the target fragment. Once the PCR reaction begins, if the PCR reaction occurs, the Taq enzyme dissociates the probe sequence bound to the target fragment, thereby separating the fluorescent reporter group from the quencher group, at which point the fluorescence emitted by the fluorescent reporter group can be detected. As the PCR reaction proceeds, the more DNA amplification product is synthesized, the more fluorescent reporter groups are dissociated. By detecting the fluorescence intensity, the amount of DNA amplification product can be accurately quantified.

[0057] In some embodiments, the fluorescent reporter group and the fluorescent quencher group can be selected from various fluorescent reporter groups and corresponding fluorescent quencher groups commonly used by those skilled in the art in qPCR. In some embodiments, the fluorescent reporter group can be selected from FAM, VIC, HEX, ROX or CY5, and correspondingly, the fluorescent quencher group can be selected from BHQ1, BHQ2, BHQ3 or MGB.

[0058] On the other hand, this application also provides a composition comprising, obtained by the primer design method described herein:

[0059] 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

[0060] 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

[0061] 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.

[0062] In some embodiments, the upstream decoy oligonucleotide is derived from at least 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 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:2; or

[0063] Upstream and downstream decoy oligonucleotides, wherein the upstream decoy oligonucleotide is derived from at least 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 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:3; or

[0064] The upstream decoy oligonucleotide and the downstream decoy oligonucleotide, wherein the upstream decoy oligonucleotide is derived from at least 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 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:5.

[0065] In some embodiments, the upstream decoy oligonucleotide is derived from at least 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 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:2; or

[0066] Upstream and downstream decoy oligonucleotides, wherein the upstream decoy oligonucleotide is derived from at least 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 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:3; or

[0067] The upstream decoy oligonucleotide and the downstream decoy oligonucleotide, wherein the upstream decoy oligonucleotide is derived from at least 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 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:5.

[0068] In some embodiments, 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

[0069] 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

[0070] 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.

[0071] In some embodiments, 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. In some embodiments, the probe sequence is derived from at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in any one of SEQ ID NO: 6-7. In some embodiments, the probe sequence is derived from at least 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in any one of SEQ ID NO: 6-7.

[0072] In some implementations, the probe sequence is any one of the sequences shown in SEQ ID NO:6-7.

[0073] In some embodiments, the probe sequence is modified at both ends with a fluorescent reporter group and a corresponding fluorescent quencher group; the fluorescent reporter group is FAM, VIC, HEX, ROX, or CY5, and the fluorescent quencher group is BHQ1, BHQ2, BHQ3, or MGB. In some embodiments, the probe sequence is modified at both ends with FAM and BHQ1. In some embodiments, the 5' end of the probe sequence is modified with FAM and the 3' end of the probe sequence is modified with BHQ1.

[0074] In some embodiments, this application provides a composition comprising:

[0075] The upstream decoy oligonucleotide, the downstream decoy oligonucleotide, and the probe sequence, 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; 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; and the probe sequence 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:6; or

[0076] The upstream decoy oligonucleotide, the downstream decoy oligonucleotide, and the probe sequence, 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; 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; and the probe sequence 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:6; or

[0077] The upstream decoy oligonucleotide, the downstream decoy oligonucleotide, and the probe sequence, 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; 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; and the probe sequence 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:7.

[0078] In some embodiments, this application provides a composition comprising:

[0079] The upstream bait oligonucleotide, the downstream bait oligonucleotide, and the probe sequence, wherein the upstream bait oligonucleotide is derived from at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:1; the downstream bait oligonucleotide is derived from at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:2; and the probe sequence is derived from at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:6; or

[0080] The upstream bait oligonucleotide, the downstream bait oligonucleotide, and the probe sequence, wherein the upstream bait oligonucleotide is derived from at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:1; the downstream bait oligonucleotide is derived from at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:3; and the probe sequence is derived from at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:6; or

[0081] The upstream decoy oligonucleotide, the downstream decoy oligonucleotide, and the probe sequence, wherein the upstream decoy oligonucleotide is derived from at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:4, the downstream decoy oligonucleotide is derived from at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:5, and the probe sequence is derived from at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:7.

[0082] In some embodiments, this application provides a composition comprising:

[0083] An upstream bait oligonucleotide, a downstream bait oligonucleotide, and a probe sequence, wherein the upstream bait oligonucleotide is derived from at least 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:1; the downstream bait oligonucleotide is derived from at least 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:2; and the probe sequence is derived from at least 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:6; or

[0084] An upstream bait oligonucleotide, a downstream bait oligonucleotide, and a probe sequence, wherein the upstream bait oligonucleotide is derived from at least 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:1; the downstream bait oligonucleotide is derived from at least 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:3; and the probe sequence is derived from at least 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:6; or

[0085] The upstream decoy oligonucleotide, the downstream decoy oligonucleotide, and the probe sequence, wherein the upstream decoy oligonucleotide is derived from at least 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:4, the downstream decoy oligonucleotide is derived from at least 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:5, and the probe sequence is derived from at least 95%, 96%, 97%, 98%, 99%, or 100% of the sequence shown in SEQ ID NO:7.

[0086] In some embodiments, this application provides a composition comprising:

[0087] The upstream decoy oligonucleotide, downstream decoy oligonucleotide, and probe sequence are specified. The upstream decoy oligonucleotide is the sequence shown in SEQ ID NO:1, the downstream decoy oligonucleotide is the sequence shown in SEQ ID NO:2, and the probe sequence is the sequence shown in SEQ ID NO:6; or

[0088] The upstream decoy oligonucleotide, downstream decoy oligonucleotide, and probe sequence are specified. The upstream decoy oligonucleotide is the sequence shown in SEQ ID NO:1, the downstream decoy oligonucleotide is the sequence shown in SEQ ID NO:3, and the probe sequence is the sequence shown in SEQ ID NO:6; or

[0089] The upstream decoy oligonucleotide, the downstream decoy oligonucleotide, and the probe sequence are: the upstream decoy oligonucleotide is the sequence shown in SEQ ID NO:4, the downstream decoy oligonucleotide is the sequence shown in SEQ ID NO:5, and the probe sequence is the sequence shown in SEQ ID NO:7.

[0090] In some implementations, this application provides primer and probe combinations as shown in the table below:

[0091] In another aspect, this application also provides a kit comprising the composition described herein.

[0092] In some implementations, the kit may contain one or more sets of primers (or “decoy oligonucleotides”) as described herein. A set of primers may contain paired primers. Paired primers may contain a forward primer and a reverse primer. A set of primers may be configured to amplify a nucleic acid sequence corresponding to a specific target. For example, a forward primer may be configured to hybridize with a first region (e.g., the 3' end) of the nucleic acid sequence, while a reverse primer may be configured to hybridize with a second region (e.g., the 5' end) of the nucleic acid sequence, thereby being configured to amplify the nucleic acid sequence. Different sets of primers may be configured to amplify nucleic acid sequences. In one example, a first set of primers may be configured to amplify a first nucleic acid sequence, and a second set of primers may be configured to amplify a second nucleic acid sequence. Primers configured to amplify nucleic acid molecules may be used in the disclosed methods. In some cases, all primers in the kit are lyophilized.

[0093] The kit may contain one or more nucleases. The nuclease may be a nucleic acid polymerase. The nucleic acid polymerase may be a deoxyribonucleic acid polymerase (DNase). The DNase may be Taq polymerase or a variant thereof. The nuclease may be a ribonucleic acid polymerase (RNase). The RNase may be RNase III. The nuclease may be an endonuclease. The endonuclease may be endonuclease I. Endonuclease I may be T7 endonuclease I. The nuclease may be able to degrade nucleic acids containing non-natural nucleotides. The nuclease may be endonuclease V, such as E. coli endonuclease V. The nuclease may be a polymerase (e.g., DNA polymerase). The polymerase may be Taq polymerase or a variant thereof. Non-restricted examples of DNA polymerases include Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, VENT polymerase, DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase, Expand polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mth polymerase, Pho polymerase, ES4 polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tih polymerase, Tfi polymerase, Platinum Taq polymerase, Hi-Fi polymerase, Tbr polymerase, Tfl polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenow fragments, and their variants, modified products, and derivatives. For a given hot-start polymerase, a denaturation step of 2 to 10 minutes at 94–95°C may be required, which may vary the thermal profile depending on the polymerase. Nucleases can degrade probes under appropriate conditions. For example, a nuclease can be a polymerase and has exonuclease activity, degrading the probe to generate a detectable signal. Under appropriate conditions, the nuclease may be able to release a quencher from the probe. The kit may include instructions on the use of any of the aforementioned substances described herein.

[0094] On the other hand, this application also provides the use of the compositions or kits described herein in microbial detection.

[0095] On the other hand, this application also provides the use of the compositions or kits described herein in the detection of microorganisms by qPCR.

[0096] On the other hand, this application also provides a method for detecting microorganisms using qPCR, comprising:

[0097] 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.

[0098] In some embodiments, the sample may be a biological sample. The sample may be derived from a biological sample. Biological samples may be, for example, blood, plasma, serum, urine, saliva, mucosal secretions, sputum, feces, or tears. Biological samples may be fluid samples. Fluid samples may be blood or plasma. Biological samples may contain cell-free nucleic acids (e.g., cell-free RNA, cell-free DNA, etc.). In some embodiments, the sample may be a cell product to be tested.

[0099] Samples can be processed simultaneously with, before, or after the methods disclosed herein. Samples can be processed to purify or enrich nucleic acids (e.g., purifying nucleic acids from plasma samples). Samples containing nucleic acids can be processed to purify or enrich the target nucleic acid.

[0100] This application uses the 16S rRNA gene of microorganisms as a template to design primers and probes with amplification fragments ranging from 250 to 1000 bp in length. This addresses the false positive problem caused by the detection of host DNA fragments that cannot be completely removed from Taq DNA polymerase, while also ensuring that there are no missed detections. Using the qPCR method provided in this application to detect microorganisms in samples significantly improves the efficiency of microbial detection in cell products, reduces release time, and increases product production efficiency.

[0101] This application describes several embodiments, but these descriptions are exemplary and not restrictive, and it will be apparent to those skilled in the art that many more embodiments and implementations are possible within the scope of the embodiments described herein. Although many possible combinations of features are shown in the accompanying drawings and discussed in the detailed description, many other combinations of the disclosed features are also possible. Unless specifically limited, any feature of any embodiment may be used in combination with any other feature in any other embodiment, or may substitute for any other feature in any other embodiment.

[0102] This application includes and contemplates combinations of features known to those skilled in the art. The embodiments and features disclosed in this application can also be combined with any conventional features to form a unique inventive scheme as defined by the claims. Any feature of any embodiment can also be combined with features from other inventive schemes to form another unique inventive scheme as defined by the claims. Therefore, it should be understood that any feature shown and / or discussed in this application can be implemented individually or in any suitable combination. Therefore, the embodiments are not limited except by the limitations imposed by the appended claims and their equivalents. Furthermore, various modifications and changes can be made within the scope of the appended claims.

[0103] Furthermore, in describing representative embodiments, the specification may have presented methods and / or processes as a specific sequence of steps. However, the method or process should not be limited to the specific order of steps described herein, to the extent that it does not depend on such a specific order. As will be understood by those skilled in the art, other sequences of steps are also possible. Therefore, the specific order of steps set forth in the specification should not be construed as a limitation of the claims. Moreover, the claims concerning the method and / or process should not be limited to the steps performed in the written order, and those skilled in the art will readily understand that these orders can be varied and still remain within the spirit and scope of the embodiments of this application.

[0104] Experimental methods in the following embodiments without specific conditions are generally determined according to national standards. Experimental materials in the following embodiments without specified sources are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, then generally accepted international standards, conventional conditions, or conditions recommended by the manufacturer are followed. Unless otherwise defined or stated, all technical and scientific terms used in this application have the same meaning as those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to the methods of this application. Example

[0105] Example 1. Design of primer-probe combination

[0106] The gene sequence of *E. coli* 16S rRNA was found on NCBI (Gene Bank: J01859.1), as shown in SEQ ID NO:8. Subsequently, the Run Blast function on the NCBI system was run, and 5000 sequences were compared. The obtained sequences included, but were not limited to, the 16S rRNA gene sequences of *Staphylococcus aureus*, *Escherichia coli*, *Pseudomonas aeruginosa*, *Bacillus subtilis*, *Streptococcus pyogenes*, *Micrococcus sp.*, *Candida albicans*, *Aspergillus niger*, *Clostridium sporogenes*, and *Propionibacterium acnes*. The comparison results were then graphically analyzed using NCBI MSA Viewer, as shown in Figure 2.

[0107] Select the conservative regions from the alignment results to obtain the conservative sequence of the conservative regions.

[0108] During the alignment process, it was found that some conserved sequences sometimes contained bases that differed from the main conserved sequence in the original 16S rRNA. To ensure normal detection, degenerate bases (i.e., bases containing two or more different bases at the same site) were designed. The use of degenerate bases is as follows:

[0109] Primers and probes were designed using conserved sequences as templates. Specific results are shown in Table 1 below:

[0110] Table 1. Sequence information of primers and probes

[0111] By freely combining these different primers and probes, amplification fragments of different lengths can be generated.

[0112] Example 2. Validation-level screening of conventional primer and probe combinations

[0113] 1. Selection of primers and probes

[0114] The screening continued according to the conventional qPCR primer and probe design and combination methods, with all amplified fragments being <200bp in length. The specific combinations are shown in Table 2 below:

[0115] Table 2

[0116] 2. Validation with different primer combinations

[0117] Remove each reagent component and place it on the table. After equilibration to room temperature, gently shake to mix and then briefly centrifuge. Prepare the corresponding amplification reagents using the primers and probes described above, as shown in Table 3 below:

[0118] Table 3

[0119] Note: The specific quantity prepared is N+2, where "N" is the theoretical verification quantity. The actual quantity prepared should be based on the actual test count. The quantity used in this experiment was 32.

[0120] Mix the prepared amplification mix by shaking, dispense 25 μL into eight-tube strips, cap the tubes (check for tightness), and centrifuge briefly.

[0121] 3. Amplification detection

[0122] The specific steps are shown in Table 4 below:

[0123] Table 4

[0124] Place the above eight tubes into a real-time quantitative PCR machine and set the amplification channel according to the following parameters: Select the FAM channel.

[0125] 4. Results Analysis

[0126] The results are shown in Figure 3. These results indicate that the existing system, when using various primer and probe combinations for amplification, showed non-specific amplification in the negative control detection results, which could not meet the requirements for final detection.

[0127] Example 3. Screening of Taq DNA polymerase

[0128] Six brands of Taq DNA polymerase (Tiangen, Yisheng Bio, Novozymes, Thermofisher, Baorui Bio, and Feipeng Bio, corresponding to brands 1-6 respectively) were selected and combined with combination 10 from Example 2 (combination 10 showed the best amplification efficiency and minimal non-specific amplification). The corresponding amplification systems were prepared according to the instructions of the respective Taq DNA polymerases, and the negative control (enzyme-free water) was tested to observe its non-specific amplification. The results are shown in Figure 4. The results show that for brands 1-4, non-specific amplification was observed in the negative control at Ct values ​​between 30 and 35. Brands 5-6 showed relatively ideal results; although Ct values ​​were above 38, amplification signals were still present, but the detection requirements were not met.

[0129] Example 4. Screening and Validation of Primer and Probe Combinations for Long Amplified Fragments

[0130] 1. Primer and probe combinations were validated and screened based on amplified fragment lengths ranging from 250 to 1000 bp. Specific combinations are shown in Table 5 below:

[0131] Table 5

[0132] 2. Reagent preparation

[0133] Remove each reagent component and place it on the table to equilibrate to room temperature. Gently vortex to mix and then briefly centrifuge. Prepare the corresponding amplification reagents using the primers and probes described above. The polymerase used is the Hieff UCF.ME Hotstart Sensitive Taq DNA polymerase, which showed superior amplification performance as demonstrated in Example 3. The specific system preparation is shown in Table 6 below:

[0134] Table 6

[0135] Note: The specific quantity prepared is N+2, where "N" is the theoretical verification quantity. The actual quantity prepared should be based on the actual test count. The quantity in this experiment was 71.

[0136] The prepared amplification mix was shaken and mixed well, and 23 μL was dispensed into an eight-tube bundle. The dispensed mixture was then transferred to the sample preparation area for later use.

[0137] 3. Sample preparation

[0138] The plasmid containing the E. coli 16S rRNA gene was serially diluted with ultrapure water to obtain 1×10⁻⁶ g / mL of each plasmid. 9 Samples with a concentration of 1 copy / mL should be stored at -20℃ for later use.

[0139] Add 2 μL of 1×10 to the eight tubes containing the PCR mixture. 9 Mix a sample of E. coli 16S rRNA gene plasmid at a concentration of 1 copy / mL with enzyme-free water, cap the tube (to check for tightness), and centrifuge briefly.

[0140] 4. Amplification detection

[0141] The specific steps are shown in Table 7 below:

[0142] Table 7

[0143] Place the above eight tubes into a real-time quantitative PCR machine and set the amplification channel according to the following parameters: Select the FAM channel.

[0144] The results are shown in Figures 5-9. Overall, compared with the primer-probe combination with amplified fragments <200bp in Example 2, the primer combination with amplified fragments in the range of 250-1000bp showed higher specificity for detecting the negative control, i.e., a lower probability of false positive signals or a later Ct value.

[0145] Combination 1 served as the control group, with amplified fragment length <200bp, used to analyze the difference in amplification efficiency between primer combinations with long amplified fragments. Statistical analysis of the results showed that primer / probe combinations 3, 4, 6, 9, 12, 13, 14, 15, and 19 amplified no amplification signal in the negative control within 45 cycles. Further analysis of their amplification efficiency indicated that primer / probe combinations 3, 4, and 9 were unaffected.

[0146] Overall trend analysis shows that for primer-probe combinations with amplified fragments in the range of 250-1000bp, the false positive rate of the negative control decreases as the amplified fragments become larger, further verifying the primer-probe design and selection principles of this application.

[0147] Example 5. Sensitivity Verification

[0148] The sensitivity of primer-probe combinations 3, 4, and 9 selected in Example 4 was verified.

[0149] 1. Sample preparation

[0150] The E. coli 16S rRNA gene plasmid was serially diluted with enzyme-free water to obtain a concentration of 1×10⁻⁶. 3 Samples with concentrations of 100 copies / mL (S1), 500 copies / mL (S2), 200 copies / mL (S3), and 100 copies / mL (S4) were tested using the detection method described in Example 4. Primer-probe combinations 3, 4, and 9 were verified, and the amplification results are shown in Table 8 below.

[0151] Table 8

[0152] Note: "+" indicates the presence of an amplification signal, and "-" indicates the absence of an amplification signal.

[0153] 2. Results Analysis

[0154] Primer-probe combinations 3, 4, and 9 were validated, and all three combinations were able to detect samples with concentrations of 200 copies / mL or higher normally.

Claims

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. The method according to claim 1, wherein, The upstream primer and the downstream primer are located in different conserved regions of the sequence.

3. The method according to claim 1, wherein, The upstream and downstream primers are 15-30 bp in length; the GC content is 40-60%; ΔG < 58.61 KJ / mol; and the Tm value is 58-62℃.

4. The method according to claim 1, wherein, The method further includes: 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. The method according to claim 4, wherein, 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. The method according to claim 5, 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.

7. The method according to any one of claims 1-6, wherein, The microorganisms mentioned are 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: 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: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 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: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 any one of the sequences shown in 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.

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