Primer probe composition and kit for detecting lower respiratory tract infection pathogens

Abstract

The invention belongs to the technical field of biology, and particularly relates to a primer probe composition and a kit for detecting lower respiratory tract infection pathogens. The primer probe composition is used for detecting mycobacterium tuberculosis, mycobacterium avium, mycobacterium abscessus, mycobacterium kansasii, pseudomonas aeruginosa, klebsiella pneumoniae, acinetobacter baumannii and haemophilus influenzae, and the primer probe composition is used for detecting mycobacterium tuberculosis, mycobacterium avium, mycobacterium abscessus, mycobacterium kansasii, pseudomonas aeruginosa, klebsiella pneumoniae, acinetobacter baumannii and haemophilus influenzae. The composition is one or more of a composition for detecting staphylococcus aureus, a composition for detecting streptococcus pneumoniae, a composition for detecting escherichia coli, a composition for detecting moraxella catarrhalis, a composition for detecting serratia marcescens, a composition for detecting mycoplasma pneumoniae, a composition for detecting candida albicans or a composition for detecting adenovirus; the RAA-RDB technology has the core advantages of rapidness, high sensitivity, low cost and easy operation, and is especially suitable for multiple pathogen screening and basic medical application.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a primer-probe combination and a kit for detecting pathogens of lower respiratory tract infection. Background Art

[0002] Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a major infectious disease prevalent worldwide. Mycobacteria are primarily divided into three major groups: the Mycobacterium tuberculosis complex (MTC), Mycobacterium leprae, and non-tuberculous mycobacteria (NTM). Lower respiratory tract infections (LRTIs) are common infectious diseases with high morbidity and mortality, with bacterial pneumonia accounting for approximately 80% of these cases. In recent years, with the emergence of new pathogens, advances in preventive measures, and the widespread use of antimicrobial drugs, the distribution of pathogens has shifted significantly, with NTM infections showing a clear upward trend. Notably, patients with underlying lung diseases are more susceptible to pathogens due to their compromised immune system. Therefore, early differential diagnosis of Mtb, NTM, and common respiratory pathogens such as Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus is crucial.

[0003] Currently, the following technologies are mainly used in clinical laboratory testing for respiratory pathogens: Traditional microbial culture methods rely on colony growth for identification by inoculating sputum or lavage fluid samples into culture media. This method is low-cost but time-consuming (2-7 days), has low sensitivity (especially in patients who have already used antibiotics), and some pathogens (such as Mtb and Mycoplasma) are difficult to culture; conventional PCR / real-time fluorescence quantitative PCR can rapidly amplify the specific gene sequence of a preset single pathogen (1-2 hours), with high sensitivity but cannot achieve high-throughput pathogen detection; immunological tests (such as colloidal gold method and ELISA) achieve rapid screening based on antigen-antibody reactions (15-30 minutes), but are easily affected by window periods and cross-reactions; metagenomic sequencing technology (mNGS) comprehensively detects microbial nucleic acids in samples through high-throughput sequencing, covering unknown pathogens, but is costly and complex to analyze, requiring specialized instruments and operators, making it difficult for primary medical institutions to implement; it is easily affected by environmental DNA contamination or interference from colonizing bacteria; mass spectrometry technology (such as MALDI-TOF While MS can rapidly identify the protein profile of pure cultured bacterial colonies, it requires prior culturing and expensive equipment, making it infeasible to directly test clinical samples. Each technology has its own advantages and disadvantages, and selection or combination must be tailored to clinical needs to balance speed, cost, and accuracy. Therefore, developing rapid, accurate, and cost-effective detection methods is a key challenge in the current field of respiratory pathogen diagnosis.

[0004] Recent research in nucleic acid amplification (NAA) technology focuses on isothermal reactions, which enable rapid amplification of target gene sequences under constant temperature conditions without the need for expensive laboratory equipment. Recombinase-mediated amplification (RAA), a novel isothermal NAA technique, exponentially amplifies target sequences at a constant temperature of 37°C–42°C, reaching detectable levels within 10 minutes. Compared with traditional PCR, RAA offers advantages such as high specificity, high sensitivity, ease of use, rapid reaction times, low cost, and excellent stability. It holds promise as a rapid and reliable method for bacterial identification at the bedside and in resource-limited settings. The combination of RAA with reverse dot blot (RDB) further enhances detection efficiency. In the RAA amplification system, biotinylated primers bind to recombinase to form a complex. With the assistance of single-stranded binding proteins, DNA polymerase initiates exponential amplification of the target sequence, generating biotinylated RAA products. These products are then hybridized with probes pre-immobilized on a nylon membrane, and the hybridization signal is visually interpreted using a colorimetric reaction.

[0005] Therefore, there is an urgent need for a primer-probe combination and a kit for combining RAA technology with RDB for the interspecies identification of mycobacteria and the identification of lower respiratory tract pathogens. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention aims to screen for highly conserved and species-specific gene targets in 16 common lower respiratory tract pathogens and develop a multiplex detection method combining RAA and RDB technologies to achieve rapid, visual differential diagnosis of Mtb and other common lower respiratory tract pathogens. This provides a highly sensitive, specific, low-cost, and easy-to-use pathogen diagnostic tool for clinical use.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: a primer-probe composition for detecting pathogens of lower respiratory tract infection, wherein the primer-probe composition is one or more of a composition for detecting Mycobacterium tuberculosis, a composition for detecting Mycobacterium avium-intracellulare, a composition for detecting Mycobacterium abscessus, a composition for detecting Mycobacterium kansasii, a composition for detecting Pseudomonas aeruginosa, a composition for detecting Klebsiella pneumoniae, a composition for detecting Acinetobacter baumannii, a composition for detecting Haemophilus influenzae, a composition for detecting Staphylococcus aureus, a composition for detecting Streptococcus pneumoniae, a composition for detecting Escherichia coli, a composition for detecting Moraxella catarrhalis, a composition for detecting Serratia marcescens, a composition for detecting Mycoplasma pneumoniae, a composition for detecting Candida albicans, or a composition for detecting adenovirus;

[0008] The composition for detecting Mycobacterium tuberculosis comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 1; a downstream primer having a nucleotide sequence as shown in SEQ NO: 2; and a probe having a nucleotide sequence as shown in SEQ NO: 3;

[0009] The composition for detecting Mycobacterium avium intracellulare comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 4; a downstream primer having a nucleotide sequence as shown in SEQ NO: 5; and a probe having a nucleotide sequence as shown in SEQ NO: 6;

[0010] The composition for detecting Mycobacterium abscessus comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 7; a downstream primer having a nucleotide sequence as shown in SEQ NO: 8; and a probe having a nucleotide sequence as shown in SEQ NO: 9;

[0011] The composition for detecting Mycobacterium kansasii comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 10; a downstream primer having a nucleotide sequence as shown in SEQ NO: 11; and a probe having a nucleotide sequence as shown in SEQ NO: 12;

[0012] The composition for detecting Pseudomonas aeruginosa comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 13; a downstream primer having a nucleotide sequence as shown in SEQ NO: 14; and a probe having a nucleotide sequence as shown in SEQ NO: 15;

[0013] The composition for detecting Klebsiella pneumoniae comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 16; a downstream primer having a nucleotide sequence as shown in SEQ NO: 17; and a probe having a nucleotide sequence as shown in SEQ NO: 18;

[0014] The composition for detecting Acinetobacter baumannii comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 19; a downstream primer having a nucleotide sequence as shown in SEQ NO: 20; and a probe having a nucleotide sequence as shown in SEQ NO: 21;

[0015] The composition for detecting Haemophilus influenzae comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 22; a downstream primer having a nucleotide sequence as shown in SEQ NO: 23; and a probe having a nucleotide sequence as shown in SEQ NO: 24;

[0016] The composition for detecting Staphylococcus aureus comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 25; a downstream primer having a nucleotide sequence as shown in SEQ NO: 26; and a probe having a nucleotide sequence as shown in SEQ NO: 27;

[0017] The composition for detecting Streptococcus pneumoniae comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 28; a downstream primer having a nucleotide sequence as shown in SEQ NO: 29; and a probe having a nucleotide sequence as shown in SEQ NO: 30;

[0018] The composition for detecting Escherichia coli comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 31; a downstream primer having a nucleotide sequence as shown in SEQ NO: 32; and a probe having a nucleotide sequence as shown in SEQ NO: 33;

[0019] The composition for detecting Moraxella catarrhalis comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 34; a downstream primer having a nucleotide sequence as shown in SEQ NO: 35; and a probe having a nucleotide sequence as shown in SEQ NO: 36;

[0020] The composition for detecting Serratia marcescens comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 37; a downstream primer having a nucleotide sequence as shown in SEQ NO: 38; and a probe having a nucleotide sequence as shown in SEQ NO: 39;

[0021] The composition for detecting Mycoplasma pneumoniae comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 40; a downstream primer having a nucleotide sequence as shown in SEQ NO: 41; and a probe having a nucleotide sequence as shown in SEQ NO: 42;

[0022] The composition for detecting Candida albicans comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO:43; a downstream primer having a nucleotide sequence as shown in SEQ NO:44; and a probe having a nucleotide sequence as shown in SEQ NO:45;

[0023] The composition for detecting adenovirus includes: an upstream primer having a nucleotide sequence as shown in SEQ NO: 46; a downstream primer having a nucleotide sequence as shown in SEQ NO: 47; and a probe having a nucleotide sequence as shown in SEQ NO: 48.

[0024] Furthermore, the primer-probe combination is a composition for detecting Mycobacterium tuberculosis, a composition for detecting Mycobacterium avium-intracellulare, a composition for detecting Mycobacterium abscessus, and a composition for detecting Mycobacterium kansasii.

[0025] Furthermore, the composition for detecting Pseudomonas aeruginosa, the composition for detecting Klebsiella pneumoniae, the composition for detecting Acinetobacter baumannii and the composition for detecting Haemophilus influenzae.

[0026] Furthermore, the composition for detecting Staphylococcus aureus, the composition for detecting Streptococcus pneumoniae, the composition for detecting Escherichia coli and the composition for detecting Moraxella catarrhalis.

[0027] Furthermore, the composition for detecting Serratia marcescens, the composition for detecting Mycoplasma pneumoniae, the composition for detecting Candida albicans and the composition for detecting adenovirus.

[0028] A kit for detecting pathogens of lower respiratory tract infection containing the above primer-probe combination, comprising RAA detection reagent and RDB detection membrane;

[0029] The RAA detection reagent includes a buffer, ddH2O, an upstream primer, and a downstream primer;

[0030] A probe is fixed on the RDB detection membrane.

[0031] Furthermore, the reaction system of the RAA detection reagent includes: 25 μL of A Buffer, 4 μL of mixed upstream and downstream primers, 13.5 μL of ddH2O, 2.5 μL of B Buffer, and 5 μL of the DNA sample to be detected.

[0032] Furthermore, the preparation method of the RDB detection membrane includes:

[0033] Soak the nylon membrane in pure water for 5 minutes, then in 20×SSC for 5 minutes, take 0.5 μL of the probe and spot it on the membrane, and fix the probe to the membrane by UV cross-linking.

[0034] The benefits of the present invention mainly lie in:

[0035] The RAA-RDB technology of the present invention has the core advantages of rapidity, high sensitivity, low cost and easy operation, and is particularly suitable for multiple pathogen screening and primary medical applications. Efficient, rapid, and highly sensitive, RAA amplification only takes 20 minutes, much faster than the 1.5-2 hours of traditional PCR or several days of culture methods, making it particularly suitable for rapid screening of emergency or nosocomial infections; it can achieve single-copy sensitivity, which is superior to antigen detection and some antibody detection; it eliminates non-target amplification through specific probes, making it more reliable than ordinary PCR; high-throughput detection: RDB hybridization can simultaneously detect multiple targets, with a higher throughput than single-plex qPCR or antigen detection; low equipment and energy requirements: constant temperature amplification (37°C-42°C), no expensive PCR instrument is required, reducing equipment investment (RAA only requires a constant temperature water bath); results are interpreted by the naked eye, eliminating the need for a fluorescent quantifier or sequencer, making it suitable for scenarios with limited resources; easy operation and visual results: "one-step" hybridization and pre-fixed probes avoid complex steps such as electrophoresis and sequencing; color development reactions can be directly observed without the need for specialized equipment, making it suitable for primary hospitals; strong adaptability and flexible expansion: it can tolerate complex samples such as sputum and BALF, and is superior to some isothermal amplification technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a result diagram of a multiple RAA system according to an embodiment of the present invention;

[0037] Figure 2 Specific electrophoresis diagram of the multiple RAA-RDB detection method according to an embodiment of the present invention;

[0038] Figure 3 Specific hybridization diagram of the multiple RAA-RDB detection method according to an embodiment of the present invention;

[0039] Figure 4 is the sensitivity of the multiple RAA-RDB detection method according to an embodiment of the present invention;

[0040] Figure 5 The repeatability of the multiple RAA-RDB detection method of the embodiment of the present invention;

[0041] Figure 6 This is a distribution diagram of the hybridized membrane of an embodiment of the present invention. DETAILED DESCRIPTION

[0042] The specific implementation is further described below with reference to the accompanying drawings.

[0043] The reagents and instruments in the following examples are all conventional experimental reagents and instruments.

[0044] Example 1:

[0045] A primer-probe composition for detecting pathogens of lower respiratory tract infections, wherein the primer-probe composition is one or more of a composition for detecting Mycobacterium tuberculosis, a composition for detecting Mycobacterium avium-intracellulare, a composition for detecting Mycobacterium abscessus, a composition for detecting Mycobacterium kansasii, a composition for detecting Pseudomonas aeruginosa, a composition for detecting Klebsiella pneumoniae, a composition for detecting Acinetobacter baumannii, a composition for detecting Haemophilus influenzae, a composition for detecting Staphylococcus aureus, a composition for detecting Streptococcus pneumoniae, a composition for detecting Escherichia coli, a composition for detecting Moraxella catarrhalis, a composition for detecting Serratia marcescens, a composition for detecting Mycoplasma pneumoniae, a composition for detecting Candida albicans, or a composition for detecting adenovirus;

[0046] By consulting relevant literature, the target genes were preliminarily determined to be the IS6110 sequence of Mycobacterium tuberculosis, the DT1 gene of Mycobacterium avium-intracellulare, the A3O00_RS07180 gene of Mycobacterium abscessus, the MKAN_RS06095 gene of Mycobacterium kansasii, the oprl gene of Pseudomonas aeruginosa, the phoE gene of Klebsiella pneumoniae, the ITS1 gene of Candida albicans, the lytA gene of Streptococcus pneumoniae, the omp6 gene of Haemophilus influenzae, the pgaD gene of Acinetobacter baumannii, the cap5F gene of Staphylococcus aureus, the 23sRNA sequence of Stenotrophomonas maltophilia, the uspA gene of Moraxella catarrhalis, the LuxS gene of Serratia marcescens, the ybbw gene of Escherichia coli, and the P1 gene of Mycoplasma pneumoniae. Multiple gene sequences of pathogens were retrieved and downloaded from the National Center for Biotechnology Information (NCBI). Multiple sequences of the same gene were aligned using sequence alignment software such as MEGA11. The alignment results were analyzed to determine the highly conserved and species-specific sequence range in the target gene.

[0047] Gene sequences and related information were confirmed using the GenBank database. RAA primers for each gene were designed using Oligo7 software. The primer design requirements for RAA nucleic acid amplification differ somewhat from those for conventional PCR. A primer pair consists of two oligonucleotides, each specifically recognizing the upstream and downstream nucleotide sequences of a nucleic acid target. Primers should be between 30 and 35 bp in length and contain no palindromes, consecutive single-base repeats, or internal secondary structures. Primer Tm values ​​are not a primary consideration during design. The optimal primer pair is determined through experimental optimization and screening, requiring a single amplification product with no nonspecific amplification or significant primer dimers. Primer sequences should avoid repeated bases, and the amplified band size should not exceed 500 bp, as this directly affects detection sensitivity and speed. Furthermore, the GC content of the target fragment should be between 40% and 60%, and repetitive sequences should be avoided. Primer sequence specificity and various parameters were verified and selected using the Primer-BLAST module on the NCBI website and Oligo7 software. Biotin was modified at the 5' end of the forward primer or the backward primer, respectively, and synthesized after secondary screening and purified by high performance liquid chromatography. The specific primers are shown in Table 1:

[0048] Table 1

[0049]

[0050]

[0051]

[0052] The complete gene sequence of each bacterium was downloaded from the NCBI database. Multiple sequence alignments were performed using MegAlign in DNASTAR software to analyze homology and identify highly variable regions of the gene. Probes were designed from the highly variable regions identified in the multiple sequence alignments, and the probes were evaluated for self-dimerization and hairpin structure. The principles for oligonucleotide probe design are as follows:

[0053] The optimal probe length is 18-30 bases.

[0054] b Base composition: 40%-60% (G+C content).

[0055] c Avoid the repetition of a single base (no more than four), such as -GGGG-.

[0056] d. Avoid complementary regions inside the probe as much as possible to reduce the presence of "hairpin" structures.

[0057] e. The probe sequence has no more than 70% homology with the non-target region, or has 8 or more consecutive bases of homology.

[0058] f. Adjust the probe lengths to ensure that the melting temperatures of all probes are within 6°C so that they can be processed under the same hybridization and washing conditions.

[0059] The specificity of the probe sequence was verified using Primer-BLAST on the NCBI website and the best probe sequence was selected. 30 T tails were added to the 3' end of the probe to synthesize the probe. The specific probes are shown in Table 2:

[0060] Table 2

[0061]

[0062] A kit for detecting pathogens of lower respiratory tract infection using a primer-probe combination.

[0063] Includes RAA detection reagent and RDB detection membrane;

[0064] The RAA detection reagent includes a buffer, ddH2O, an upstream primer, and a downstream primer;

[0065] A probe is fixed on the RDB detection membrane.

[0066] The preparation method of the RDB detection film comprises:

[0067] 1. Establishment of multiple RAA detection methods

[0068] Based on the biological characteristics and clinical relevance of pathogens, and considering the competitive inhibition between multiple primers in a reaction system, respiratory pathogens were divided into four different groups through primer screening and verification, and four sets of quadruple RAA reaction detection systems were established:

[0069] ①Mtb, Mycobacterium intracellulare, Mycobacterium abscessus, Mycobacterium kansasii

[0070] ② Pseudomonas aeruginosa, Haemophilus influenzae, Mycoplasma pneumoniae, Acinetobacter baumannii

[0071] ③ Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli

[0072] ④ Candida albicans, Moraxella catarrhalis, Serratia marcescens, adenovirus

[0073] Add 25 μL of ABuffer, 2 μL of forward primer mix, 2 μL of backward primer mix, 13.5 μL of purified water, and 5 μL of each mixed template to the reaction tube. Add 2.5 μL of B Buffer to the detection unit tube cap, cover the tube, shake it upside down and mix thoroughly 5-6 times, and centrifuge at low speed for 10 seconds. Place the detection unit tube in a 37°C constant temperature metal bath (or constant temperature water bath) and incubate for 30 minutes. After the reaction is completed, add 50 μL of phenol: chloroform: isoamyl alcohol (25:24:1) extraction solution to the detection unit tube, mix thoroughly, and centrifuge at 12000 rpm for 5 minutes. Take the supernatant for electrophoresis detection ( Figure 1 A). Figure 1 In A, M: Marker 1: Pseudomonas aeruginosa, Acinetobacter baumannii, Haemophilus influenzae, Mycoplasma pneumoniae Group 2: Moraxella catarrhalis, Candida albicans, Serratia marcescens, adenovirus Group 3: Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae Group 4: Mycobacterium tuberculosis, Mycobacterium kansasii, Mycobacterium abscessus, Mycobacterium intracellulare Group;

[0074] The reaction system is shown in Table 3:

[0075] Table 3

[0076]

[0077] The steps for extracting DNA samples are as follows:

[0078] Use an inoculating loop to pick up a loopful of bacteria and resuspend them in an EP tube containing 1 mL of normal saline. Inactivate the cells in a metal bath at 80°C for 30 minutes. After 30 minutes of inactivation, slowly cool the tube, taking care not to crack it. Centrifuge at 12,000 rpm for 5 minutes. Discard the supernatant and add 200 μL of normal saline and 25 μL of 10 mg / mL lysozyme. Vortex to mix thoroughly and incubate at 37°C for 16 hours. Add 5 μL of RNase A and invert several times to mix thoroughly. Let the tube rest for 10 minutes. Add 35 μL of 10% SDS and 3 μL of 20 mg / mL Proteinase K, invert to mix thoroughly, and incubate at 65°C for 10 minutes. Add 50 μL of 5 mol / L NaCl and 50 μL of CTAB / NaCl and vortex to mix thoroughly. A milky white flocculent precipitate will appear in the tube. Incubate at 65°C for 10 minutes. Add 750 μL of chloroform, mix by inversion, and centrifuge at 12,000 rpm for 5 minutes. The liquid in the tube should now separate into three layers. Carefully aspirate the upper layer and transfer it to a new EP tube. Add 300 μL of pre-chilled isopropanol and incubate at -20°C for 30 minutes. Centrifuge at 12,000 rpm for 15 minutes. Discard the supernatant, wash the precipitate with 1 mL of ice-cold 70% ethanol, and centrifuge at 12,000 rpm for 5 minutes. Dry at 37°C for 10 minutes. Dissolve the precipitate in 20 μL of ddH2O and store at -20°C for up to 1 month.

[0079] 2. Construction of RDB system

[0080] Soak the nylon membrane in pure water for 5 minutes, then in 20× SSC for 5 minutes. Spot 0.5 μL of the probe onto the membrane and fix the probe to the membrane via UV crosslinking (0.30 J). Equilibrate the hybridization detection reagent to room temperature, and preheat the hybridization solution to 45°C before use.

[0081] Preparation before hybridization:

[0082] 1) Turn on the power of the hybridizer and install the waste liquid tank at the waste liquid outlet at the back of the hybridizer.

[0083] 2) Follow the control panel instructions, select [Manual Mode], press [Enter] to enter the temperature input interface, enter the temperature as 45°C, and press [Enter] to confirm the temperature increase.

[0084] 3) Fill the reaction chamber with distilled water, place the metal porous plate and turn on the water pump. After the water on the porous plate is removed, turn off the water pump.

[0085] 4) Place a plastic film with the number of holes corresponding to the number of experimental samples on a porous metal plate. Use tweezers to place the hybridization membrane over the corresponding holes in the plastic film. If there are any extra holes, cover them with Parafilm. Ensure that the hybridization membrane is moist and free of bubbles. Place the silicone seal and divider on the hybridization membrane and secure the snap-on cover.

[0086] 5) Turn on the pump to remove the water droplets remaining on the membrane and then turn off the pump.

[0087] RAA product hybridization process

[0088] 1) Heat all RAA products at 95°C for 5 minutes, then immediately place in an ice-water bath for 5-10 minutes.

[0089] 2) Perform hybridization experiments at 45°C under the conditions prepared before hybridization.

[0090] 3) Add 1 mL of hybridization solution preheated to 45°C to the hybridization well, cover the well, incubate for at least 2 minutes, turn on the pump to discharge the pre-hybridization solution, and then turn off the pump.

[0091] 4) Add 0.5 mL of hybridization solution preheated to 45°C to the denatured DNA sample solution prepared in step 1), mix well, and then add it to the hybridization membrane. Cover the membrane with the cover plate and incubate for 20 minutes before turning on the pump for flow-through hybridization.

[0092] 5) Rinse the membrane three times with 0.8 mL of hybridization solution preheated to 45°C at 45°C, turning off the water pump.

[0093] Note: The hybridization solution temperature should be kept at 45°C during the above operations.

[0094] 6) Press [ESC] to enter the temperature modification screen. Set the hybridization instrument temperature to 25°C and press [Enter] to confirm. At 25°C (±3°C), add 0.5 mL of enzyme-labeled solution. After 5 minutes of enzymatic catalysis, pump out all the solution and set the temperature to 36°C.

[0095] 7) Wash the membrane thoroughly with solution A three times, 0.8 mL each time; and wash the membrane three times with solution B, 0.8 mL each time.

[0096] 8) Add 0.5 mL of color developing solution, cover the plate and allow the solution to develop for at least 10 minutes, then pump out the solution.

[0097] 9) Wash the membrane three times with 1 mL of solution B each time, then rinse with 2 mL of distilled water. Turn off the pump, open the snap cap, remove the compartment, remove the hybridization membrane with tweezers and place it on absorbent paper. Analyze the results within 1 hour. Figure 1 B), Figure 1 In B, a2: positive control; a3: negative control; b1-b4: Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Streptococcus pneumoniae, respectively; c1-c4: Pseudomonas aeruginosa, Haemophilus influenzae, Mycoplasma pneumoniae, and Acinetobacter baumannii, respectively; d1-d4: adenovirus, Serratia marcescens, Moraxella catarrhalis, and Candida albicans, respectively; e1-e4: Mtb, Mycobacterium kansasii, Mycobacterium abscessus, and Mycobacterium intracellulare, respectively.

[0098] The test results are determined as follows: If clear blue-purple spots appear in both the positive control and the test area on the membrane, while the negative control shows no color, the result is considered positive, indicating the presence of the target substance in the test sample. If blue-purple spots appear in the positive control, but neither the negative control nor the test area show any color, the result is considered negative, indicating that the target substance was not detected in the test sample. Furthermore, if the positive control shows no color, this indicates that the membrane strip or amplification reagent may be ineffective or incorrectly operated, and retesting is necessary to ensure the validity of the experiment.

[0099] 3. Multiple RAA-RDB system specificity detection

[0100] The genomic DNA of common clinical pathogens was extracted using the CTAB method. In the experimental design, enzyme-free sterile water was used as a blank control to ensure the accuracy and reliability of the experimental results. After the optimized multiple RAA amplification technology was used to prepare the target fragments labeled with biotin, the products were subjected to reverse dot hybridization with nylon membranes fixed with probes to specifically detect the above bacterial DNA templates. The results of the multiple RAA-RDB detection method showed that ( Figure 2-3 ), Figure 2A is the Kabaisha gland group, including 1, Kabaisha gland group mixed template 2, Baozhi Tongliu group mixed template 3, intestinal Jinkelian group mixed template 4, nodular pus group mixed template 5, negative control; Figure 2 B is the Baozhi Tongliu group, including 1, Baozhi Tongliu group mixed template 2, Kabaisha gland group mixed template 3, Baozhi Tongliu group mixed template 4, cytosolic pus group mixed template 5, negative control; Figure 2 C is the intestinal Jinke chain group, including 1, mixed template 2 of the intestinal Jinke chain group, mixed template 3 of the Kabaisha gland group, mixed template 4 of the Baozhi Tongliu group, mixed template 5 of the cystic pus group, and negative control; Figure 2 D is the cystic pus group, including 1, cystic pus group mixed template 2, Kabaisha gland group mixed template 3, Baozhi Tongliu group mixed template 4, intestinal Jinke chain group mixed template 5, Gordon, scrofula, Surga, Mal mixed template 6, negative control

[0101] Figure 3 a2: positive control; a3: negative control; b1-b4 are Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Streptococcus pneumoniae, respectively; c1-c4 are Pseudomonas aeruginosa, Haemophilus influenzae, Mycoplasma pneumoniae, and Acinetobacter baumannii, respectively; d1-d4 are adenovirus, Serratia marcescens, Moraxella catarrhalis, and Candida albicans, respectively; e1-e4 are Mtb, Mycobacterium kansasii, Mycobacterium abscessus, and Mycobacterium intracellulare, respectively.

[0102] In the same detection system, blue-purple spots appeared on the nylon membrane only when the corresponding target pathogen DNA template was added. The results of the multiple RAA agarose gel electrophoresis test were basically consistent with the RDB results, further verifying the good specificity among the four groups of target strains, the lack of nonspecific amplification, and the absence of cross-reactions. This result demonstrates that the multiple RAA-RDB detection method constructed in this study has good specificity and can effectively distinguish different pathogens.

[0103] 4. Multiple RAA-RDB system sensitivity detection

[0104] The CTAB method was used to extract the genomic DNA of common clinical pathogens, ensuring aseptic operation during the extraction process to avoid contamination. After the extraction was completed, the concentration and purity of each DNA sample were measured using an ultraviolet spectrophotometer, and the A260 / A280 ratio was recorded. Subsequently, the DNA template was diluted 10 times in a gradient manner using ddH2O, and DNA solutions of different concentrations were prepared in turn, with enzyme-free sterile water as a no-template control. The hybridization results showed that the minimum detection limit of the Cabaisha gland group was 0.916fg / μL, the minimum detection limit of the intestinal gold chain group was 9.71fg / μL, the minimum detection limit of the Baozhi copper flow group was 0.8fg / μL, and the minimum detection limit of the cystic pus group was 5.5fg / μL ( Figure 4 ) Figure 4 AF represents 10% of DNA template -1 to 10 -7 Multiple dilution series; a2: positive control; a3: negative control; b1-b4: Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Streptococcus pneumoniae, respectively; c1-c4: Pseudomonas aeruginosa, Haemophilus influenzae, Mycoplasma pneumoniae, and Acinetobacter baumannii, respectively; d1-d4: adenovirus, Serratia marcescens, Moraxella catarrhalis, and Candida albicans, respectively; e1-e4: Mtb, Mycobacterium kansasii, Mycobacterium abscessus, and Mycobacterium intracellulare, respectively.

[0105] 5. Repeatability experiment of multiple RAA-RDB systems

[0106] The original sample was diluted to 0 times (i.e. the original sample), 10 -1 times and 10 -2 Each dilution gradient was independently divided into 3 parallel samples, for a total of 9 samples to be tested. Samples with the same dilution factor were tested every three days. The optimized multiple RAA-RDB method was used for testing. In this experiment, the appearance of blue-purple spots on the membrane was considered positive, and no quantitative analysis was involved. Experimental results ( Figure 5 )show, Figure 5 In, AC: template 0, 10 -1 , 10 -2 1-fold serial dilution; a2: positive control; a3: negative control; b1-b4: Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Streptococcus pneumoniae, respectively; c1-c4: Pseudomonas aeruginosa, Haemophilus influenzae, Mycoplasma pneumoniae, and Acinetobacter baumannii, respectively; d1-d4: adenovirus, Serratia marcescens, Moraxella catarrhalis, and Candida albicans, respectively; e1-e4: Mtb, Mycobacterium kansasii, Mycobacterium abscessus, and Mycobacterium intracellulare, respectively. Under the three dilution conditions, spot coloration was observed in three repeated tests, indicating that the experimental operation and analytical method are highly repeatable and robust, and are suitable for subsequent batch testing or research.

[0107] 6. Evaluation of the application value of clinical samples

[0108] (1) Sample inclusion and exclusion criteria

[0109] Patients with respiratory infections who visited a hospital between 2022 and 2024 were selected for the study, with a total of 300 samples included, including 75 samples of Mtb infection and 225 samples of other bacterial infections. In addition, samples from 60 patients suspected of bacterial infection were collected for testing. Basic information such as gender, age, and hospitalization number of the participants was collected. All participants signed informed consent forms. All experiments complied with ethical requirements, and all samples underwent rigorous clinical diagnosis and laboratory confirmation. The specimens involved in this study included bronchoalveolar lavage fluid specimens and sputum specimens. The study aimed to analyze the pathogenic characteristics and effectiveness of detection methods by comparing samples of different infection types, providing a scientific basis for clinical diagnosis and treatment. After liquefaction of the patient's sputum or bronchoalveolar lavage fluid with 4% NaOH, 1 mL was taken and inactivated at 80°C for 30 minutes and stored at -80°C. This study excluded patients with HIV, HBV, severe autoimmune diseases, or severe underlying diseases or complications. The inclusion criteria were as follows: (1) TB patients included the following main clinical manifestations: long-term cough, sputum, chest pain, hemoptysis, weight loss, and fatigue; positive sputum culture and acid-fast staining; chest X-ray and CT showing lung lesions; and no HIV, syphilis, or other diseases. (2) Patients with microbial infection included the following: positive bacterial culture results; clear drug sensitivity test results; and clinical symptoms consistent with infection.

[0110] (2) DNA extraction from clinical sputum specimens

[0111] The sputum sample was mixed with 4 times the volume of 4% NaOH solution, thoroughly mixed using a vortex oscillator, and allowed to stand at room temperature for about 30 minutes to fully liquefy the sputum; 0.5 mL of the sample was transferred to an EP tube and allowed to stand at room temperature for 10 minutes to fully balance the sample. The EP tube was placed in a high-speed centrifuge and centrifuged at 13,000 rpm for 5 minutes to ensure that the particulate matter in the sample was fully precipitated; after the centrifugation, the supernatant was carefully discarded (avoid touching the precipitate), 1 mL of sterile saline was added to the precipitate, and the vortex was mixed to ensure that the precipitate was completely resuspended, and the precipitate was centrifuged at 13,000 rpm for 5 minutes; the above washing steps were repeated once to remove impurities. After the last centrifugation, most of the supernatant was discarded, leaving only about 100 μL of liquid and precipitate, which was shaken and mixed to make the precipitate evenly dispersed in the liquid; 100 μL of liquid sample was aspirated, an equal volume of DNA concentrate was added, the vortex was mixed for 5 seconds, and the 10,000 g centrifuge was used for 10 minutes to concentrate the DNA precipitate; the supernatant was discarded (avoid touching the precipitate), and 50 μL of Vortex the DNA extract for 5-10 seconds, centrifuge for a few seconds, and place the EP tube in 100°C boiling water for 10 minutes to fully release the DNA. Centrifuge at 10,000g for 5 minutes. The supernatant is the purified DNA template. Transfer the DNA template to a sterile EP tube, seal it, and store it in a -20°C refrigerator for subsequent experiments.

[0112] (3) Multiple RAA-RDB method for sample detection

[0113] The extracted sputum sample DNA was subjected to multiple RAA amplification using the optimized reaction system according to the above method, and the amplified products were hybridized according to the above method.

[0114] (4) Comparative evaluation of diagnostic methods

[0115] Statistical analysis was performed using SPSS 26.0 software. Using clinical diagnostic results as the reference standard, the detection rates of the four Mtb detection methods were compared using the McNemar test and paired chi-square test, with an α level of 0.05. Kappa tests were used for consistency analysis. A Kappa value ≥ 0.8 indicates strong consistency, 0.4 ≤ Kappa < 0.6 indicates good consistency, and a Kappa value < 0.4 indicates poor consistency. A P value < 0.05 indicated statistical significance. Detailed results are shown in Tables 4-8 in the accompanying figures.

[0116] Table 4 Comparison of the detection efficiency of four methods for diagnosing mycobacteria

[0117]

[0118] Table 5 Comparison of the multiple RAA-RDB method with the other three methods for the diagnosis of mycobacteria

[0119]

[0120]

[0121] Table 6 Preliminary clinical diagnostic value of the multiple RAA-RDB method

[0122]

[0123] Table 7 Efficacy evaluation of the multiple RAA-RDB method for detecting suspected samples

[0124]

[0125] Table 8 Diagnostic efficacy of the multiple RAA-RDB method for detecting respiratory pathogens

[0126]

[0127]

[0128] This application integrates RAA technology and RDB detection technology into the same system for the first time. RAA technology achieves efficient nucleic acid amplification through constant temperature reaction; RDB technology achieves high-throughput detection through membrane strip hybridization. The two work together to break through the traditional PCR's dependence on precision equipment and establish a new integrated "amplification-detection" technology path. The first detection kit that can simultaneously identify 4 types of mycobacteria and 12 types of respiratory pathogens has been successfully developed. By optimizing the primer probe combination and hybridization conditions, a single test can cover more than 90% of common respiratory pathogens, achieving efficient diagnosis with one test and multiple screening. The design of the "single tube 4 types × four tubes" combination supports the selection of single-tube independent detection or multi-tube combined detection mode on demand, and new pathogens can be included by replacing the specific probe area to adapt to different detection scenarios and needs.

[0129] Example 2:

[0130] A detection method for a kit for detecting lower respiratory tract infection pathogens using a primer-probe combination,

[0131] 1. Sample collection

[0132] Sputum samples were collected from patients suspected of Mtb, NTM, or respiratory pathogen infection in a hospital. Samples included but were not limited to sputum, bronchoalveolar lavage fluid, and pleural and ascites fluids.

[0133] 2. Genome Extraction

[0134] The DNA template of the sample to be tested was obtained by high-temperature lysis using a commercial kit. The reagents were purchased from Guangzhou Daan Gene Co., Ltd. The extraction operation was carried out strictly according to the instructions. The specific steps are as follows:

[0135] (1) Add 4 times the volume of 4% NaOH to the sputum, shake well, and place at room temperature for about 30 minutes to liquefy. Take 0.5 mL into an EP tube, add 0.5 mL of 4% NaOH, and place at room temperature for 10 minutes.

[0136] (2) Discard the supernatant, add 1 mL of sterile saline to the precipitate and mix thoroughly. Centrifuge at 13,000 rpm for 5 min. Repeat the washing process once more, remove most of the supernatant, retain about 100 μL of liquid precipitate, and oscillate to mix thoroughly.

[0137] (3) Discard the supernatant and add 100 μL of DNA concentrate. Resuspend the precipitate by shaking on an oscillator and centrifuge at 10,000 rpm for 10 min.

[0138] (4) Remove the supernatant and add 50 μL of DNA extraction solution to the precipitate. Mix vigorously on an oscillator for 5-10 seconds, centrifuge briefly for a few seconds, and heat at 100°C for 10 minutes. Centrifuge at 10,000 g for 5 minutes and remove the supernatant for later use.

[0139] 3. RAA Amplification Reaction

[0140] The required primer and probe sequences and reagents are as described in the invention protocol, and the RAA amplification reaction is performed: ABuffer 25 μL; upstream primer (10 μmol / L) 2 μL; downstream primer (10 μmol / L) 2 μL; ddH2O 13.5 μL; DNA sample 5 μL; BBuffer 2.5 μL.

[0141] (1) According to the reaction quantity, a mixture containing water, ABuffer, upstream primer (10 μmol / L), and downstream primer (10 μmol / L) was prepared according to the reaction system. After mixing evenly, it was added to the detection unit tube containing the reaction dry powder.

[0142] (2) Add 5 μL of the DNA sample to be tested into the detection unit tube.

[0143] (3) Add 2.5 μL of B Buffer to the detection unit tube cap, close the tube cap, invert to mix, and centrifuge briefly.

[0144] (4) The RAA amplification reaction temperature was 37°C and the reaction time was 30 min.

[0145] 4.RDB reaction

[0146] The required reagents are as described in the invention scheme, and the RDB flow-through hybridization reaction is carried out: 0.2 mL of enzyme label solution; 100 mL of color development solution; 100 mL of solution A; 100 mL of solution B

[0147] (1) Heat all RAA products at 95°C for 5 min, then immediately place in an ice-water bath for 5-10 min.

[0148] (2) Add 1 mL of hybridization solution preheated to 45°C to the hybridization well, cover the well with the cover, incubate for 2 minutes, turn on the pump to discharge the pre-hybridization solution, and turn off the pump.

[0149] (3) Add 0.5 mL of hybridization solution preheated to 45°C to the denatured DNA sample solution, mix well, add it to the hybridization membrane, cover it with a cover plate, incubate for 20 minutes, and then turn on the pump for flow-through hybridization.

[0150] (4) Rinse the membrane three times with 0.8 mL of hybridization solution preheated to 45°C at 45°C, and turn off the pump.

[0151] (5) When the hybridization instrument temperature is set to 25°C (±3°C), add 0.5 mL of enzyme-labeled solution (1:2000 dilution). After 5 minutes of enzymatic incubation, turn on the pump to pump out all the solution and set the temperature to 36°C.

[0152] (6) Wash the membrane three times with solution A, 0.8 mL each time; and wash the membrane three times with solution B, 0.8 mL each time.

[0153] (7) Add 0.5 mL of color developing solution, cover the plate and allow the solution to develop for at least 10 minutes, then pump out the solution.

[0154] (8) Wash the membrane with solution B three times, 1 mL each time, then rinse with 2 mL of distilled water and turn off the pump.

[0155] (9) Open the snap-on cover, remove the compartment, remove the hybridization membrane with tweezers and place it on absorbent paper. Analyze the results within 1 hour.

[0156] 5. Interpretation of results

[0157] Nylon membrane point design:

[0158] Hybridization membrane Figure 6 As shown:

[0159] PC: positive control NC: negative control 1: Staphylococcus aureus 2: Escherichia coli 3: Klebsiella pneumoniae 4: Streptococcus pneumoniae 5: Pseudomonas aeruginosa 6: Haemophilus influenzae 7: Mycoplasma pneumoniae 8: Acinetobacter baumannii 9: Adenovirus 10: Serratia marcescens 11: Moraxella catarrhalis 12: Candida albicans 13: Mycobacterium tuberculosis 14: Mycobacterium kansasii 15: Mycobacterium abscessus 16: Mycobacterium intracellulare.

[0160] The test results are determined as follows: If clear blue-purple spots appear in both the positive control and the test area on the membrane, while the negative control shows no color, the result is considered positive, indicating the presence of the target substance in the test sample. If blue-purple spots appear in the positive control, but neither the negative control nor the test area show any color, the result is considered negative, indicating that the target substance was not detected in the test sample. Furthermore, if the positive control shows no color, this indicates that the membrane strip or amplification reagent may be ineffective or incorrectly operated, and retesting is necessary to ensure the validity of the experiment.

[0161] The above embodiments are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made based on the essence of the present invention are intended to be covered by the scope of protection of the present invention.

Claims

1. A primer-probe combination for detecting pathogens of lower respiratory tract infection, characterized by: The primer-probe composition is one or more of a composition for detecting Mycobacterium tuberculosis, a composition for detecting Mycobacterium avium-intracellulare, a composition for detecting Mycobacterium abscessus, a composition for detecting Mycobacterium kansasii, a composition for detecting Pseudomonas aeruginosa, a composition for detecting Klebsiella pneumoniae, a composition for detecting Acinetobacter baumannii, a composition for detecting Haemophilus influenzae, a composition for detecting Staphylococcus aureus, a composition for detecting Streptococcus pneumoniae, a composition for detecting Escherichia coli, a composition for detecting Moraxella catarrhalis, a composition for detecting Serratia marcescens, a composition for detecting Mycoplasma pneumoniae, a composition for detecting Candida albicans, or a composition for detecting adenovirus; The composition for detecting Mycobacterium tuberculosis comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 1; a downstream primer having a nucleotide sequence as shown in SEQ NO: 2; and a probe having a nucleotide sequence as shown in SEQ NO: 3; The composition for detecting Mycobacterium avium intracellulare comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 4; a downstream primer having a nucleotide sequence as shown in SEQ NO: 5; and a probe having a nucleotide sequence as shown in SEQ NO: 6; The composition for detecting Mycobacterium abscessus comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 7; a downstream primer having a nucleotide sequence as shown in SEQ NO: 8; and a probe having a nucleotide sequence as shown in SEQ NO: 9; The composition for detecting Mycobacterium kansasii comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 10; a downstream primer having a nucleotide sequence as shown in SEQ NO: 11; and a probe having a nucleotide sequence as shown in SEQ NO: 12; The composition for detecting Pseudomonas aeruginosa comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 13; a downstream primer having a nucleotide sequence as shown in SEQ NO: 14; and a probe having a nucleotide sequence as shown in SEQ NO: 15; The composition for detecting Klebsiella pneumoniae comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 16; a downstream primer having a nucleotide sequence as shown in SEQ NO: 17; and a probe having a nucleotide sequence as shown in SEQ NO: 18; The composition for detecting Acinetobacter baumannii comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 19; a downstream primer having a nucleotide sequence as shown in SEQ NO: 20; and a probe having a nucleotide sequence as shown in SEQ NO: 21; The composition for detecting Haemophilus influenzae comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 22; a downstream primer having a nucleotide sequence as shown in SEQ NO: 23; and a probe having a nucleotide sequence as shown in SEQ NO: 24; The composition for detecting Staphylococcus aureus comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 25; a downstream primer having a nucleotide sequence as shown in SEQ NO: 26; and a probe having a nucleotide sequence as shown in SEQ NO: 27; The composition for detecting Streptococcus pneumoniae comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 28; a downstream primer having a nucleotide sequence as shown in SEQ NO: 29; and a probe having a nucleotide sequence as shown in SEQ NO: 30; The composition for detecting Escherichia coli comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 31; a downstream primer having a nucleotide sequence as shown in SEQ NO: 32; and a probe having a nucleotide sequence as shown in SEQ NO: 33; The composition for detecting Moraxella catarrhalis comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 34; a downstream primer having a nucleotide sequence as shown in SEQ NO: 35; and a probe having a nucleotide sequence as shown in SEQ NO: 36; The composition for detecting Serratia marcescens comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 37; a downstream primer having a nucleotide sequence as shown in SEQ NO: 38; and a probe having a nucleotide sequence as shown in SEQ NO: 39; The composition for detecting Mycoplasma pneumoniae comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO: 40; a downstream primer having a nucleotide sequence as shown in SEQ NO: 41; and a probe having a nucleotide sequence as shown in SEQ NO: 42; The composition for detecting Candida albicans comprises: an upstream primer having a nucleotide sequence as shown in SEQ NO:43; a downstream primer having a nucleotide sequence as shown in SEQ NO:44; and a probe having a nucleotide sequence as shown in SEQ NO:45; The composition for detecting adenovirus includes: an upstream primer having a nucleotide sequence as shown in SEQ NO: 46; a downstream primer having a nucleotide sequence as shown in SEQ NO: 47; and a probe having a nucleotide sequence as shown in SEQ NO:

48.

2. The primer-probe combination for detecting pathogens of lower respiratory tract infection according to claim 1, characterized in that: The primer-probe composition is a composition for detecting Mycobacterium tuberculosis, a composition for detecting Mycobacterium avium-intracellulare, a composition for detecting Mycobacterium abscessus and a composition for detecting Mycobacterium kansasii.

3. The primer-probe combination for detecting pathogens of lower respiratory tract infection according to claim 1, characterized in that: The composition for detecting Pseudomonas aeruginosa, the composition for detecting Klebsiella pneumoniae, the composition for detecting Acinetobacter baumannii and the composition for detecting Haemophilus influenzae.

4. The primer-probe combination for detecting pathogens of lower respiratory tract infection according to claim 1, characterized in that: The composition for detecting Staphylococcus aureus, the composition for detecting Streptococcus pneumoniae, the composition for detecting Escherichia coli and the composition for detecting Moraxella catarrhalis.

5. The primer-probe combination for detecting pathogens of lower respiratory tract infection according to claim 1, characterized in that: The composition for detecting Serratia marcescens, the composition for detecting Mycoplasma pneumoniae, the composition for detecting Candida albicans and the composition for detecting adenovirus.

6. A kit for detecting pathogens of lower respiratory tract infection comprising the primer-probe combination according to any one of claims 1 to 5, characterized in that: Includes RAA detection reagent and RDB detection membrane; The RAA detection reagent includes a buffer, ddH2O, an upstream primer, and a downstream primer; A probe is fixed on the RDB detection membrane.

7. The kit for detecting pathogens of lower respiratory tract infection according to claim 6, characterized in that: The reaction system of the RAA detection reagent includes: 25 μL of A Buffer, 4 μL of mixed upstream and downstream primers, 13.5 μL of ddH2O, 2.5 μL of B Buffer, and 5 μL of the DNA sample to be detected.

8. The kit for detecting pathogens of lower respiratory tract infection according to claim 6, characterized in that: The preparation method of the RDB detection film comprises: Soak the nylon membrane in pure water for 5 minutes, then in 20×SSC for 5 minutes, take 0.5 μL of the probe and spot it on the membrane, and fix the probe to the membrane by UV cross-linking.

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