A kit and a detection method for detecting common pathogenic bacteria of otitis media based on multi-enzyme constant temperature rapid nucleic acid amplification technology

Abstract

The application provides a kit for detecting common pathogenic bacteria of otitis media based on a multi-enzyme constant temperature rapid nucleic acid amplification technology, which comprises a first primer pair for detecting Streptococcus pneumoniae, a second primer pair for detecting Haemophilus influenzae, a third primer pair for detecting Moraxella catarrhalis, a fluorescent probe combination and a colloidal gold probe combination; the nucleotide sequences of the first primer pair are SEQ ID NO: 2 and SEQ ID NO: 4 respectively, the nucleotide sequences of the second primer pair are SEQ ID NO: 9 and SEQ ID NO: 10 respectively, and the nucleotide sequences of the third primer pair are SEQ ID NO: 13 and SEQ ID NO: 18 respectively. The application provides a triple multi-enzyme constant temperature rapid synchronous detection kit and a detection method, which have high sensitivity, high specificity and convenient operation, and meet the requirements of rapid diagnosis on the spot in a clinic.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection and clinical diagnostic technology, specifically involving a triple detection kit and detection method based on multi-enzyme isothermal rapid amplification (MIRA) technology. It is particularly suitable for the simultaneous detection of common pathogens of otitis media—Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. It can achieve dual application of real-time fluorescence detection and colloidal gold lateral chromatography visualization detection. Background Technology

[0002] Otitis media is a common infectious disease in children worldwide, with bacterial otitis media accounting for over 60%. Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis are the three most prevalent pathogens, collectively causing approximately 85% of bacterial otitis media cases. If the causative agent is not identified promptly, it can easily lead to antibiotic overuse, delayed treatment, and consequently, serious complications such as tympanic membrane perforation, hearing loss, and even intracranial infection, causing irreversible damage to children's health.

[0003] Currently, commonly used clinical methods for detecting pathogens have significant limitations: traditional bacterial culture, as the gold standard for diagnosis, requires 24-48 hours to obtain results, and its positive detection rate is only 50%-60% due to factors such as sample collection quality and prior antibiotic use, failing to meet the needs of early diagnosis; while conventional PCR technology shortens the detection time, it relies on high-precision thermal cycling instruments, making it complex and costly, unsuitable for primary healthcare institutions; existing multiplex detection technologies are mostly designed for general respiratory infection scenarios and have not been specifically optimized for middle ear secretion samples, resulting in problems such as primer-probe cross-interference, weak resistance to sample inhibitors, and long detection times (≥40 minutes), making it difficult to meet the clinical needs for rapid diagnosis of otitis media. Furthermore, existing technologies often require cumbersome nucleic acid extraction steps, increasing operational difficulty and contamination risks, limiting the implementation of point-of-care testing.

[0004] Multienzyme isothermal rapid nucleic acid amplification (MIRA) is an isothermal amplification technology that has emerged in recent years. It relies on three core enzymes—DNA polymerase, single-stranded DNA-binding protein, and recombinase—to assist in DNA amplification. MIRA does not require complex temperature amplification equipment, has a short amplification reaction time, and enables rapid detection of amplified products by designing primers and probes with fluorescently modified groups, making it particularly suitable for rapid on-site detection.

[0005] Therefore, developing a MIRA detection kit that combines rapid isothermal function, triple synchronization, and dual detection modes (fluorescence / colloidal gold), and has undergone systematic primer screening and system optimization, to achieve full coverage of "precise detection in high-end institutions and rapid screening in grassroots institutions," has become an urgent technical problem to be solved in clinical practice. Summary of the Invention

[0006] To address the shortcomings of existing detection technologies, such as long processing time, complex operation, and insufficient specificity and anti-interference capabilities, this invention provides a triple multi-enzyme isothermal rapid synchronous detection kit and method, enabling one-step simultaneous detection of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. It combines high sensitivity, high specificity, and ease of operation, meeting the needs of rapid point-of-care diagnosis in clinical settings.

[0007] This invention provides a kit for detecting common pathogens of otitis media based on multi-enzyme isothermal rapid nucleic acid amplification technology, which includes a first primer pair for detecting Streptococcus pneumoniae, a second primer pair for detecting Haemophilus influenzae, a third primer pair for detecting Moraxella catarrhalis, a fluorescent probe combination, and a colloidal gold probe combination.

[0008] The nucleotide sequences of the first primer pair are SEQ ID NO:2 and SEQ ID NO:4, the nucleotide sequences of the second primer pair are SEQ ID NO:9 and SEQ ID NO:10, and the nucleotide sequences of the third primer pair are SEQ ID NO:13 and SEQ ID NO:18.

[0009] In one embodiment of the present invention, the fluorescent probe assembly comprises three probe sequences adapted to a first primer pair, a second primer pair, and a third primer pair, respectively. Each probe sequence does not overlap with the recognition sites of the first primer pair, the second primer pair, and the third primer pair. The probe sequence is 46-52 nt in length and does not contain palindromic sequences, internal secondary structures, or continuous repeating bases. Each probe sequence contains four modification sites: a dSpacer (tetrahydrofuran, THF) is marked at a position ≥35 nt from the 5' end; a fluorescent group is marked on the T base upstream of the THF site in each probe sequence, and a quencher group corresponding to the fluorescent group is marked on the T base downstream of the T base, with a spacing of 2-4 nt between the two groups; the THF site is ≥15 nt from the 3' end, and a C3-Spacer modification group is marked at the 3' end; the fluorescent group and the quencher group of each probe sequence are non-repeating.

[0010] In one embodiment of the invention, the fluorescent probe assembly comprises nucleic acid molecules with nucleotide sequences SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively.

[0011] In one embodiment of the present invention, the colloidal gold probe assembly comprises three colloidal gold probe sequences adapted to the first primer pair, the second primer pair, and the third primer pair, respectively. Each colloidal gold probe sequence is 46-52 nt in length. Each colloidal gold probe sequence is a nucleotide sequence complementary to the target fragment amplified by the first primer pair, the second primer pair, and the third primer pair, respectively. Each colloidal gold probe sequence is modified with an antigen label at its 5' end, a dSpacer is labeled at a sequence position approximately 30 nt from the 5' end, and each colloidal gold probe sequence is labeled with a modification group at its 3' end.

[0012] In one embodiment of the invention, the colloidal gold probe assembly comprises nucleic acid molecules with nucleotide sequences SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, respectively.

[0013] In one embodiment of the present invention, the kit further includes a dissolution buffer, lyophilized enzyme powder, magnesium acetate solution, and buffer.

[0014] The dissolution buffer comprises 50 mM Tris buffer and 150 mM potassium acetate; the lyophilized enzyme powder comprises 500 ng / μL recombinase, 400 ng / μL recombinase cofactor, 900 ng / μL single-stranded DNA binding protein, 200 ng / μL DNA polymerase, 100 ng / μL reverse transcriptase, 3 mM ATP, 100 mM creatine phosphate, 300 ng / μL creatine kinase, 500 μM dNTPs, 10% w / v polyethylene glycol 20000, and 5 mM dithiothreitol.

[0015] This invention also provides a nucleic acid composition for detecting common pathogens of otitis media based on multi-enzyme isothermal rapid nucleic acid amplification technology, which includes a first primer pair for detecting Streptococcus pneumoniae, a second primer pair for detecting Haemophilus influenzae, a third primer pair for detecting Moraxella catarrhalis, a fluorescent probe combination, and a colloidal gold probe combination.

[0016] The nucleotide sequences of the first primer pair are SEQ ID NO:2 and SEQ ID NO:4, the nucleotide sequences of the second primer pair are SEQ ID NO:9 and SEQ ID NO:10, and the nucleotide sequences of the third primer pair are SEQ ID NO:13 and SEQ ID NO:18.

[0017] And / or, the fluorescent probe assembly comprises nucleic acid molecules with nucleotide sequences SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21, respectively;

[0018] And / or, the colloidal gold probe assembly comprises nucleic acid molecules with nucleotide sequences SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, respectively.

[0019] In another aspect, the present invention provides the use of the above-described kit or nucleic acid composition in the preparation of diagnostic reagents for detecting pathogens associated with otitis media infection.

[0020] Another aspect of the present invention provides a detection method based on multi-enzyme isothermal rapid nucleic acid amplification technology, comprising:

[0021] 1) Using the above-mentioned kit or nucleic acid composition, the sample is subjected to MIRA amplification to obtain amplification products; preferably, the sample is from a patient with otitis media;

[0022] 2) Analyze the fluorescence detection results after amplification; preferably, the fluorescence detector is set to the following fluorescence detection program: constant temperature 39ºC; acquire the fluorescence value of each channel (the selection of the signal acquisition channel is consistent with the design of the fluorescence probe) every 30 seconds; reaction time 20 min;

[0023] 3) Detect the amplification product using colloidal gold; preferably, after diluting the amplification product 20 times, take 80 microliters and apply it to the colloidal gold detection reagent strip, and let it stand at room temperature for 5 minutes.

[0024] In one embodiment of the present invention, the MIRA amplification reaction system comprises: 29.4 μL of dissolution buffer, 0.6 μL each of 10 μM Streptococcus pneumoniae upstream and downstream primers, 0.18 μL of 10 μM Streptococcus pneumoniae probe, 0.6 μL each of 10 μM Haemophilus influenzae upstream and downstream primers, 0.8 μL of 10 μM Haemophilus influenzae probe, 0.8 μL each of 10 μM Moraxella catarrhalis upstream and downstream primers, 0.24 μL of 10 μM Moraxella catarrhalis probe, 5 μL of template DNA to be tested, 8.5 μL of sterile double-distilled water, 50 mg of MIRA lyophilized enzyme powder, and 2.5 μL of 280 mM magnesium acetate.

[0025] The beneficial effects of the above-described technical solution of the present invention are as follows:

[0026] 1. Rapid and Efficient: The triple multi-enzyme isothermal amplification system simultaneously detects and differentiates Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in a single-tube reaction system. The entire process (sample processing + amplification + detection) takes only 25-30 minutes, reducing the time by more than 90% compared to traditional culture techniques and more than 50% compared to conventional multiplex PCR techniques, achieving "sampling and testing, testing and results in a flash."

[0027] 2. High specificity: Primers and probes are designed based on the specific conserved genes of pathogenic bacteria, the probe structure is optimized and THF modification is introduced, which effectively avoids cross-reaction between the three sets of primers and probes. Experimental verification shows that there are no false positive results for 10 common non-target bacteria such as Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, and Klebsiella pneumoniae, which has good specificity.

[0028] 3. High sensitivity: The composition of this invention exhibits excellent detection sensitivity. Its triple MIRA fluorescence method achieves detection limits of 3×10² CFU / mL for Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis, respectively. 2 CFU / mL, 2×10 2 The detection sensitivity is comparable to that of PCR methods, with a concentration of CFU / mL. Its triple MIRA colloidal gold method achieves detection limits of 3 × 10² CFU / mL, 5 × 10² CFU / mL, and 5 × 10² CFU / mL for Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis, respectively. 2 CFU / mL, 2×10 3 CFU / mL.

[0029] 4. Simple operation: Sample processing does not require professional technicians; amplification does not require precision thermal cycling instruments, and ordinary constant temperature metal baths are sufficient; results can be intuitively interpreted through fluorescence curves, which can quickly distinguish between single infection and mixed infection, making it suitable for primary healthcare and point-of-care testing scenarios.

[0030] 5. Simultaneous detection: A single-tube reaction can simultaneously detect the three core pathogens, clarify the infection type, provide a direct basis for the precise use of antibiotics in clinical practice, and avoid drug resistance problems caused by blind drug use. Attached Figure Description

[0031] Figure 1 Image showing the results of primer screening for Streptococcus pneumoniae;

[0032] Figure 2 Image showing the results of primer screening for Haemophilus influenzae;

[0033] Figure 3 Image showing the primer screening results for Moraxella catarrhalis;

[0034] Figure 4 This is a graph showing the results of a specific fluorescence assay for Streptococcus pneumoniae.

[0035] Figure 5 The image shows the results of the specific-colloidal gold method for detecting Streptococcus pneumoniae.

[0036] Figure 6 This is a graph showing the results of the Haemophilus influenzae specific-fluorescence assay.

[0037] Figure 7 The image shows the results of the specific-colloidal gold method for detecting Haemophilus influenzae.

[0038] Figure 8 This is a graph showing the results of the Moraxella catarrhalis specific-fluorescence assay.

[0039] Figure 9 The image shows the results of the Moraxella catarrhalis specific-colloidal gold method detection.

[0040] Figure 10 This is a graph showing the results of the single-sensitivity fluorescence assay for detecting Streptococcus pneumoniae.

[0041] Figure 11 The image shows the results of the single-sensitivity colloidal gold method for detecting Streptococcus pneumoniae.

[0042] Figure 12 The image shows the results of the single-sensitivity fluorescence assay for detecting Haemophilus influenzae.

[0043] Figure 13 The image shows the results of the single-sensitivity colloidal gold method for detecting Haemophilus influenzae.

[0044] Figure 14 The image shows the results of the single-sensitivity fluorescence method for detecting Moraxella catarrhalis.

[0045] Figure 15 The image shows the results of the single-sensitivity colloidal gold method for detecting Moraxella catarrhalis.

[0046] Figure 16 Figure showing the results of the triple system optimization and fluorescence detection method;

[0047] Figure 17 Optimization of the triple system - Detection results using colloidal gold method;

[0048] Figure 18 The graph shows the sensitivity detection results of the triple fluorescence method for three bacteria.

[0049] Figure 19 The graph shows the sensitivity test results of triple colloidal gold against three bacteria. Detailed Implementation

[0050] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0051] Example 1 Primer Design

[0052] Based on the isolation of pathogens causing otitis media reported in the literature, this invention selected *Streptococcus pneumoniae* lytA gene, *Haemophilus influenzae* hpd gene, and *Moraxella catarrhalis* copB gene for primer design. Three forward primers (F1, F2, F3) and three reverse primers (R1, R2, R3) were designed for each pathogen. Primers of 30–35 bp were designed to avoid hairpin structures and dimers, controlling the amplified fragment to 150–300 bp (≤500 bp). The primers had a GC content of 30%–70%, random bases, and no secondary structure in the amplified fragment, ensuring amplification efficiency and detection sensitivity. The primer sequences of this invention are shown in Table 1.

[0053] Table 1 Primer sequences

[0054]

[0055] Example 2: Design of Fluorescent Probes

[0056] After determining the primers, probes for each strain were designed according to the MIRA fluorescent probe design principles. The design principles are as follows: the probe sequence does not overlap with the specific primer recognition site, the length is 46-52 nt, and the sequence avoids palindromic sequences, internal secondary structures, and continuous repeating bases. The probe has four modification sites: a dSpacer (tetrahydrofuran, THF) is labeled at the midpoint ≥35 nt from the 5' end as the exonuclease recognition site; a fluorescent group is labeled upstream of the THF site, and a quenching group is labeled downstream, with a spacing of 2-4 nt between the two groups; the THF site is ≥15 nt from the 3' end, and a C3-Spacer modification group is labeled at the 3' end. Specifically, the fluorescent group for the *Streptococcus pneumoniae* probe is FAM; the fluorescent group for the *Haemophilus influenzae* probe is ROX; the fluorescent group for the *Moraxella catarrhalis* probe is CY5; the quenching group for the *Streptococcus pneumoniae* probe is BHQ1; the quenching group for the *Haemophilus influenzae* probe is BHQ2; and the quenching group for the *Moraxella catarrhalis* probe is BHQ3. The fluorescent probe sequences of the present invention are shown in Table 2.

[0057] Table 2 Fluorescent probe sequences

[0058]

[0059] Example 3: Design of a colloidal gold probe

[0060] After determining the primers, colloidal gold probes for each strain were designed according to the MIRA colloidal gold probe design principles. The design principles are as follows: A 46-52 nt complementary sequence to the target fragment was designed between the upstream and downstream primers as the colloidal gold probe; the probe sequence should not overlap with the specific primer recognition site, and its length should be 46-52 nt. Palindromic sequences, internal secondary structures, and consecutive repetitive bases should be avoided. The probe has three modification sites: an antigen marker (typical FAM) is modified at the 5' end; a dSpacer (tetrahydrofuran, THF) is labeled approximately 30 nt from the 5' end as the nfo recognition site; and the THF is approximately 15 nt from the 3' end, with a C3-spacer modification group labeled at the 3' end. In specific implementations, the 5' end of the Streptococcus pneumoniae probe is modified with an antigen label named FAM; the 5' end of the Haemophilus influenzae probe is modified with an antigen label named TAMRA; and the 5' end of the Moraxella catarrhalis probe is modified with an antigen label named Digoxin. The FAM, TAMRA, and DIG groups labeled at the 5' end can bind to the specific antibodies coated on the colloidal gold test strip, respectively, to achieve triple visualization detection. The colloidal gold probe sequences of this invention are shown in Table 3.

[0061] Table 3 Colloidal gold probe sequences

[0062]

[0063] Example 4: Design of colloidal gold reverse primers

[0064] Colloidal gold primers should be 30-35 bp in length. Primers that are too long or too short will affect the amplification speed and detection sensitivity (in special cases where it is difficult to design longer primer sequences, the primer length can be shortened to 25 bp, but it is best not to be less than 25 bp). Primer sequence requirements: 1. High randomness of base arrangement, GC content between 30% and 70%; 2. Avoid the formation of secondary structures in the amplification fragment, which will affect amplification; 3. The length of the amplification fragment is recommended to be 150-300 bp, usually not exceeding 500 bp; 4. A modification group (biotin is commonly used) is labeled at the 5' end of the downstream primer.

[0065] Table 4. Colloidal gold reverse primer sequences

[0066]

[0067] Example 5 Primer Screening

[0068] Primers were synthesized by Beijing Liuhe BGI Genomics Co., Ltd.

[0069] Single-phase MIRA amplification was performed using candidate primer combinations (F1 / R1, F1 / R2, F1 / R3, F2 / R1, F2 / R2, F2 / R3, F3 / R1, F3 / R2, F3 / R3) for Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. Using standard strain nucleic acid as a template, fluorescence values ​​were collected every 30 seconds (the signal acquisition channel selection was consistent with the fluorescent probe design); the reaction time was 20 minutes. The amplification effect was detected using a fluorescent probe method.

[0070] Primer screening for Streptococcus pneumoniae can be found in [link to documentation]. Figure 1 In the figure, F2R2 is the optimal primer pair, and N represents the negative control;

[0071] Primer screening for Haemophilus influenzae can be found in [link to relevant documentation]. Figure 2 In the figure, F3R1 is the optimal primer pair, and N represents the negative control;

[0072] Primer screening for Moraxella catarrhalis can be found in [link to primer screening]. Figure 3 In the figure, F1R3 is the optimal primer pair, and N represents the negative control.

[0073] The results showed that the candidate combinations of Streptococcus pneumoniae F2 / R2, Haemophilus influenzae F3 / R1, and Moraxella catarrhalis F1 / R3 exhibited the best singleton amplification efficiency.

[0074] Example 6: Specific screening for Streptococcus pneumoniae

[0075] For specificity testing, ten common non-detection pathogenic bacteria were selected as standard strains as samples: *Pseudomonas aeruginosa* ATCC27853, *Enterococcus faecalis* ATCC29212, *Escherichia coli* ATCC25922, *Staphylococcus saprophyticus* BAA750, *Stenotrophomonas maltophilia* ATCC17666, *Enterococcus pyogenes* ATCC700327, *Enterobacter cloacae* ATCC700323, *Klebsiella pneumoniae* ATCC700603, *Staphylococcus epidermidis* ATCC12228, and *Acinetobacter baumannii* ATCC19606. All standard strains were purchased from the American Type Culture Collection (ATCC). MIRA fluorescence and colloidal gold reactions were performed using the DNA of these ten non-detection bacteria as templates. The fluorescence results are shown in the figure below. Figure 4 As shown, only Streptococcus pneumoniae was positive; the other 10 non-detected bacteria were negative. N represents the negative control. The colloidal gold results are as follows: Figure 5 As shown, only Streptococcus pneumoniae was positive, while the other 10 non-detected bacteria were negative. NC represents the negative control.

[0076] The results showed that all 10 non-tested bacteria were negative, and no cross-reaction occurred with the tested bacteria.

[0077] Example 7 Specific detection of Haemophilus influenzae

[0078] For specificity testing, ten common non-detection pathogenic bacteria were selected as standard strains as samples: *Pseudomonas aeruginosa* ATCC27853, *Enterococcus faecalis* ATCC29212, *Escherichia coli* ATCC25922, *Staphylococcus saprophyticus* BAA750, *Stenotrophomonas maltophilia* ATCC17666, *Enterococcus pyogenes* ATCC700327, *Enterobacter cloacae* ATCC700323, *Klebsiella pneumoniae* ATCC700603, *Staphylococcus epidermidis* ATCC12228, and *Acinetobacter baumannii* ATCC19606. MIRA fluorescence and colloidal gold reactions were performed using the DNA of these ten non-detection bacteria as templates. Both the MIRA fluorescent reagent and the colloidal gold reagent were purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd., and the fluorescence results are shown in the figure below. Figure 6 As shown, only Haemophilus influenzae was positive; the other 10 non-detected bacteria were negative. N represents the negative control. The colloidal gold results are as follows: Figure 7 As shown, only Haemophilus influenzae was positive, while the other 10 non-detected bacteria were negative. NC represents the negative control.

[0079] The results showed that all 10 non-tested bacteria were negative, and no cross-reaction occurred with the tested bacteria.

[0080] Example 8 Specific detection of Moraxella catarrhalis

[0081] For the specificity test, ten common non-detection pathogenic bacteria were selected as standard strains as samples: *Pseudomonas aeruginosa* ATCC27853, *Enterococcus faecalis* ATCC29212, *Escherichia coli* ATCC25922, *Staphylococcus saprophyticus* BAA750, *Stenotrophomonas maltophilia* ATCC17666, *Enterococcus pyogenes* ATCC700327, *Enterobacter cloacae* ATCC700323, *Klebsiella pneumoniae* ATCC700603, *Staphylococcus epidermidis* ATCC12228, and *Acinetobacter baumannii* ATCC19606. MIRA fluorescence and colloidal gold reactions were performed using the DNA of these ten non-detection bacteria as templates. The fluorescence results are shown in the figure below. Figure 8 As shown, only Moraxella catarrhalis was positive, while the other 10 non-detected bacteria were negative. N represents the negative control. The colloidal gold results are as follows: Figure 9 As shown, only Moraxella catarrhalis was positive, while the 10 non-detected bacteria were all negative. NC represents the negative control.

[0082] The results showed that all 10 non-tested bacteria were negative, and no cross-reaction occurred with the tested bacteria.

[0083] Example 9: Sensitivity of MIRA fluorescence method for detecting Streptococcus pneumoniae in a single reaction

[0084] (1) Preparation of 3 CFU / mL ~ 3 × 106 Samples containing the ATCC6305 Streptococcus pneumoniae standard strain CFU / mL were used to extract total DNA from the samples using a DNA extraction kit purchased from Tiangen Biotech (Beijing) Co., Ltd.

[0085] (2) Using the extracted total DNA as the template to be tested, the MIRA amplification reaction system includes: 29.4 μL of dissolving buffer, 2 μL each of 10 μM upstream and downstream primers, 0.6 μL of 10 μM fluorescent probe, 5.0 μL of template DNA to be tested, 8.5 μL of sterile double-distilled water, 50 mg of MIRA lyophilized enzyme powder, and 2.5 μL of 280 mM magnesium acetate.

[0086] (3) Place the above system in an ice box and immediately place it on a PCR fluorescence amplification instrument for amplification. The program is set to constant temperature 39℃; collect the fluorescence value of the channel every 30s (the selection of the signal acquisition channel is consistent with the design of the fluorescence probe); reaction time 20min.

[0087] (4) After the reaction was completed, the results were analyzed based on the fluorescence curve. The results showed that the singlet sensitivity of Streptococcus pneumoniae was 3 × 10⁻⁶. 2 CFU / mL. The result curve is as follows: Figure 10 As shown, its sensitivity is 3×10 2 CFU / mL, N represents the negative control.

[0088] Example 10: Sensitivity of MIRA Colloidal Gold Detection of Streptococcus pneumoniae in a Single Reaction

[0089] (1) Preparation of 3 CFU / mL ~ 3 × 10 6 Total DNA was extracted from samples containing the ATCC6305 Streptococcus pneumoniae standard strain at CFU / mL using a DNA extraction kit.

[0090] (2) Using the extracted total DNA as the template to be tested, the MIRA amplification reaction system includes: 29.4 μL of dissolving buffer, 2 μL each of 10 μM upstream and downstream primers, 0.6 μL of 10 μM colloidal gold probe, 5.0 μL of template DNA to be tested, 8.5 μL of sterile double-distilled water, 50 mg of MIRA lyophilized enzyme powder, and 2.5 μL of 280 mM magnesium acetate.

[0091] (3) Place the above system in an ice box and immediately place it on a PCR fluorescence amplification instrument for amplification. The program is set to constant temperature 39℃ and reaction time 20min.

[0092] (4) After the reaction, add 10 μL to a centrifuge tube containing 190 μL ddH2O, mix well, insert the sample end of the colloidal gold test strip into the centrifuge tube to equilibrate, and observe the results of the control line and the detection line within 5 mins. The colloidal gold test strip was purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd. The results showed that the single-strike sensitivity for Streptococcus pneumoniae was 3 × 10⁻⁶. 2 CFU / mL. The result curve is as follows: Figure 11 As shown, its sensitivity is 3×10 2 CFU / mL, NC indicates negative control.

[0093] Example 11 Sensitivity of MIRA fluorescence method for detecting Haemophilus influenzae in a single reaction

[0094] (1) Preparation of 5 CFU / mL ~ 5 × 10 6 Total DNA was extracted from samples containing the ATCC19418 Haemophilus influenzae standard strain at CFU / mL using a DNA extraction kit.

[0095] (2) The subsequent steps were the same as in Example 8, and the results showed that the singlet sensitivity of Haemophilus influenzae was 5 × 10⁻⁶. 2 CFU / mL, the result curve is as follows Figure 12 As shown, its sensitivity is 5×10 2 CFU / mL, N represents the negative control.

[0096] Example 12 Sensitivity of MIRA Colloidal Gold Detection of Haemophilus influenzae Single Reaction

[0097] (1) Preparation of 5 CFU / mL ~ 5 × 10 6 Total DNA was extracted from samples containing the ATCC19418 Haemophilus influenzae standard strain at CFU / mL using a DNA extraction kit.

[0098] (2) The subsequent steps were the same as in Example 9, and the results showed that the singlet sensitivity of Haemophilus influenzae was 5 × 10⁻⁶. 2 CFU / mL, the result curve is as follows Figure 13 As shown, its sensitivity is 5×10 2 CFU / mL, NC indicates negative control.

[0099] Example 13 Sensitivity of MIRA fluorescence method for detecting Moraxella catarrhalis single reaction

[0100] (1) Preparation of 2 CFU / mL ~ 2 × 10 6 Total DNA was extracted from samples containing the ATCC25238 Moraxella catarrhalis standard strain at CFU / mL using a DNA extraction kit.

[0101] (2) The subsequent steps were the same as in Example 8, and the results showed that the singlet sensitivity of Haemophilus influenzae was 2 × 10⁻⁶. 2 CFU / mL, the result curve is as follows Figure 14 As shown, its sensitivity is 2×10 2 CFU / mL, N represents the negative control.

[0102] Example 14: Sensitivity of MIRA colloidal gold detection for Moraxella catarrhalis single reaction

[0103] (1) Preparation of 2 CFU / mL ~ 2×10 6 Total DNA was extracted from samples containing the ATCC25238 Moraxella catarrhalis standard strain at CFU / mL using a DNA extraction kit.

[0104] (2) The subsequent steps were the same as in Example 9, and the results showed that the singlet sensitivity of Haemophilus influenzae was 2 × 10⁻⁶. 3 CFU / mL, the result curve is as follows Figure 15 As shown, its sensitivity is 2×10 3 CFU / mL, NC indicates negative control.

[0105] Example 15 Construction and Optimization of Fluorescence-Based Triple Reaction System

[0106] (1) To achieve simultaneous detection of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in a single tube, it is proposed to mix the primers and probes of the three bacteria in one tube and find the optimal triplet reaction system by screening different concentrations of primers and probes. The primers and probes are added according to the following 8 systems, and each system is further supplemented with mixed nucleic acid of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis as template DNA. The optimal system is then selected based on the fluorescence curve. The specific optimized system loading is shown in Table 5.

[0107] Table 5 System Optimization and Sampling Table

[0108]

[0109] (2) The MIRA fluorescence curve results are as follows Figure 16 As shown, based on the experimental results, system 2 was ultimately selected as the optimal system for the triple reaction and used in subsequent experiments. The MIRA colloidal gold results are as follows... Figure 17 As shown, system 2 was ultimately selected as the optimal reaction system.

[0110] (3) The final triple reaction system for detecting Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis is shown in Table 6.

[0111] Table 6. Preparation of Single-Tube Triple Reaction System

[0112]

[0113] Example 16 Sensitivity of Triple Reaction System

[0114] (1) Preparation of 3×10 6 Total DNA was extracted from samples containing the ATCC6305 Streptococcus pneumoniae standard strain at CFU / mL using a DNA extraction kit.

[0115] (2) Prepare 5×10 6 Total DNA was extracted from samples containing the ATCC19418 Haemophilus influenzae standard strain at CFU / mL using a DNA extraction kit.

[0116] (3) Prepare 2×10 6 Total DNA was extracted from samples containing the ATCC25238 Moraxella catarrhalis standard strain at CFU / mL using a DNA extraction kit.

[0117] (4) Mix the nucleic acid samples of the above three bacteria in equal proportions, and then dilute them with sterile double-distilled water at a ratio of 10 times to form template DNA to form nucleic acid mixtures of different concentrations for triple sensitivity testing.

[0118] (5) The MIRA amplification reaction system includes: 29.4 μL of dissolving buffer, 0.6 μL each of 10 μM Streptococcus pneumoniae upstream and downstream primers, 0.18 μL of 10 μM Streptococcus pneumoniae probe, 0.6 μL each of 10 μM Haemophilus influenzae upstream and downstream primers, 0.8 μL of 10 μM Haemophilus influenzae probe, 0.8 μL each of 10 μM Moraxella catarrhalis upstream and downstream primers, 0.24 μL of 10 μM Moraxella catarrhalis probe, 5 μL of template DNA to be tested, 8.5 μL of sterile double-distilled water, 50 mg of MIRA lyophilized enzyme powder, and 2.5 μL of 280 mM magnesium acetate.

[0119] (6) Place the above system in an ice box and immediately place it on a PCR fluorescence amplification instrument for amplification. The program is set to constant temperature 39℃; collect the fluorescence value of the channel every 30s (the selection of the signal acquisition channel is consistent with the design of the fluorescence probe); reaction time 20min.

[0120] (7) After the amplification reaction is completed, take 10 μL of the amplification product and add it to a centrifuge tube containing 190 μL of ddH2O. After mixing evenly, insert the sample end of the colloidal gold test strip into the centrifuge tube to balance. Observe the reading results of the control line and the detection line within 5 mins.

[0121] (8) Analysis of its fluorescence detection results showed that the detection limits for Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis were 3 × 10⁻⁶. 2 CFU / mL, 5×10 2 CFU / mL, 2×102 CFU / mL; the detection limits for Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis using the colloidal gold method were 3 × 10⁻⁶ CFU / mL, respectively. 2 CFU / mL, 5×10 2 CFU / mL, 2×10 3 CFU / mL. The fluorescence result curve is shown below. Figure 18 As shown, the sensitivities for Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis were 3 × 10⁻⁶. 2 CFU / mL, 5×10 2 CFU / mL, 2×10 2 CFU / mL; colloidal gold detection results are as follows: Figure 19 As shown, the sensitivities for Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis were 3 × 10⁻⁶. 2 CFU / mL, 5×10 2 CFU / mL, 2×10 3 CFU / mL. The composition of this invention exhibits excellent detection sensitivity; its triple MIRA fluorescence method achieves detection limits of 3 × 10⁻⁶ for Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. 2 CFU / mL, 5×10 2 CFU / mL, 2×10 2 The detection sensitivity is comparable to that of PCR methods, with a concentration of CFU / mL. Its triple MIRA colloidal gold method achieves detection limits of 3 × 10⁻⁶ for Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. 2 CFU / mL, 5×10 2 CFU / mL, 2×10 3 CFU / mL.

[0122] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A kit for detecting common pathogens of otitis media based on multi-enzyme isothermal rapid nucleic acid amplification technology, comprising a first primer pair for detecting Streptococcus pneumoniae, a second primer pair for detecting Haemophilus influenzae, a third primer pair for detecting Moraxella catarrhalis, a fluorescent probe combination, and a colloidal gold probe combination; The nucleotide sequences of the first primer pair are SEQ ID NO:2 and SEQ ID NO:4, the nucleotide sequences of the second primer pair are SEQ ID NO:9 and SEQ ID NO:10, and the nucleotide sequences of the third primer pair are SEQ ID NO:13 and SEQ ID NO:

18.

2. The kit according to claim 1, wherein, The fluorescent probe assembly comprises three probe sequences adapted to the first, second, and third primer pairs, respectively. Each probe sequence does not overlap with the recognition sites of the first, second, and third primer pairs. The probe sequence length is 46-52 nt, and the probe sequence does not contain palindromic sequences, internal secondary structures, or continuous repeating bases. Each probe sequence contains four modification sites: a dSpacer (tetrahydrofuran, THF) is marked at the midpoint ≥35 nt from the 5' end; a fluorescent group is marked on the T base upstream of the THF site in each probe sequence, and a quencher group corresponding to the fluorescent group is marked on the T base downstream of the T base, with a spacing of 2-4 nt between the two groups; the THF site is ≥15 nt from the 3' end, and a C3-Spacer modification group is marked at the 3' end; the fluorescent group and quencher group of each probe sequence are unique.

3. The kit according to claim 1 or 2, wherein, The fluorescent probe assembly comprises nucleic acid molecules with nucleotide sequences SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively.

4. The kit according to any one of claims 1-3, wherein, The colloidal gold probe assembly comprises three colloidal gold probe sequences adapted to the first primer pair, the second primer pair, and the third primer pair, respectively. Each colloidal gold probe sequence is 46-52 nt in length. Each colloidal gold probe sequence is complementary to the target fragment amplified by the first primer pair, the second primer pair, and the third primer pair, respectively. Each colloidal gold probe sequence is modified with an antigen label at the 5' end, a dSpacer is labeled at a sequence position approximately 30 nt from the 5' end, and a modification group is labeled at the 3' end of each colloidal gold probe sequence.

5. The kit according to any one of claims 1-4, wherein, The colloidal gold probe assembly comprises nucleic acid molecules with nucleotide sequences SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively.

6. The kit according to any one of claims 1-5, characterized in that, It also includes dissolution buffer, lyophilized enzyme powder, magnesium acetate solution, and buffer; The dissolution buffer comprises 50 mM Tris buffer and 150 mM potassium acetate; the lyophilized enzyme powder comprises 500 ng / μL recombinase, 400 ng / μL recombinase cofactor, 900 ng / μL single-stranded DNA binding protein, 200 ng / μL DNA polymerase, 100 ng / μL reverse transcriptase, 3 mM ATP, 100 mM creatine phosphate, 300 ng / μL creatine kinase, 500 μM dNTPs, 10% w / v polyethylene glycol 20000, and 5 mM dithiothreitol.

7. A nucleic acid composition for detecting common pathogens of otitis media based on multi-enzyme isothermal rapid nucleic acid amplification technology, comprising a first primer pair for detecting Streptococcus pneumoniae, a second primer pair for detecting Haemophilus influenzae, a third primer pair for detecting Moraxella catarrhalis, a fluorescent probe combination, and a colloidal gold probe combination; The nucleotide sequences of the first primer pair are SEQ ID NO:2 and SEQ ID NO:4, the nucleotide sequences of the second primer pair are SEQ ID NO:9 and SEQ ID NO:10, and the nucleotide sequences of the third primer pair are SEQ ID NO:13 and SEQ ID NO:

18. And / or, the fluorescent probe assembly comprises nucleic acid molecules with nucleotide sequences SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21, respectively; And / or, the colloidal gold probe assembly comprises nucleic acid molecules with nucleotide sequences SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, respectively.

8. Use of the kit according to any one of claims 1-6 or the nucleic acid composition according to claim 7 in the preparation of a diagnostic reagent for detecting pathogens associated with otitis media infection.

9. A detection method based on multi-enzyme isothermal rapid nucleic acid amplification technology, comprising: 1) Using the kit as described in any one of claims 1-6, or the nucleic acid composition as described in claim 7, the sample is subjected to MIRA amplification to obtain amplification products; preferably, the sample is from a patient with otitis media; 2) Analyze the fluorescence detection results after amplification; preferably, the fluorescence detector is set to the following fluorescence detection program: constant temperature 39℃; acquire the fluorescence value of each channel (the selection of the signal acquisition channel is consistent with the design of the fluorescence probe) every 30 seconds; reaction time 20 minutes; 3) Detect the amplification product using colloidal gold; preferably, after diluting the amplification product 20 times, take 80 microliters and apply it to the colloidal gold detection reagent strip, and let it stand at room temperature for 5 minutes.

10. The detection method as described in claim 9, wherein, The MIRA amplification reaction system includes: 29.4 μL of dissolution buffer, 0.6 μL each of 10 μM Streptococcus pneumoniae upstream and downstream primers, 0.18 μL of 10 μM Streptococcus pneumoniae probe, 0.6 μL each of 10 μM Haemophilus influenzae upstream and downstream primers, 0.8 μL of 10 μM Haemophilus influenzae probe, 0.8 μL each of 10 μM Moraxella catarrhalis upstream and downstream primers, 0.24 μL of 10 μM Moraxella catarrhalis probe, 5 μL of template DNA to be tested, 8.5 μL of sterile double-distilled water, 50 mg of MIRA lyophilized enzyme powder, and 2.5 μL of 280 mM magnesium acetate.

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