A signal amplification composition for quadruple detection of respiratory pathogens, and a detection kit and application thereof

Abstract

The application discloses a signal amplification composition for quadruple detection of respiratory pathogens, a detection kit thereof and application, and belongs to the technical field of biology. The signal amplification composition is composed of a fluorescently-labeled AuNS@MoS2-Probe and an amino-modified silica colloidal crystal SiO2 PC-NH2, and realizes double amplification of signals. For low-concentration clinical samples, the application can advance the Ct value, so that samples that need to be rechecked can be directly determined as positive, effectively solves the clinical problem that the signal of a low-concentration sample is weak and is easily submerged by background noise, and reduces the risk of missed detection. The kit is a freeze-dried preparation, has good normal-temperature stability, can simultaneously detect influenza virus A, influenza virus B, a novel coronavirus and mycoplasma pneumoniae, has the advantages of high sensitivity, strong specificity and low missed detection rate, and is suitable for clinical popularization and application.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a signal amplification composition for the four-in-one detection of respiratory pathogens, its detection kit, and its application. Background Technology

[0002] Acute respiratory infections caused by respiratory pathogens are characterized by high infectivity, rapid spread, short incubation period, and acute onset, exhibiting high morbidity and mortality rates in children, the elderly, and vulnerable populations with weakened immune systems. Common pathogenic respiratory pathogens include influenza A virus, influenza B virus, SARS-CoV-2, and Mycoplasma pneumoniae. Developing detection technologies capable of simultaneously detecting and accurately quantifying multiple pathogens is crucial for early diagnosis, disease assessment, and treatment monitoring.

[0003] Reverse transcription quantitative polymerase chain reaction (RT-qPCR) technology has become the "gold standard" for nucleic acid detection of respiratory pathogens due to its high sensitivity, strong specificity, and accurate quantification. Traditional liquid RT-qPCR reagents must be stored and transported at -20°C or even lower temperatures, and require repeated freeze-thaw cycles or on-site preparation before use, making them prone to performance degradation due to temperature fluctuations. Therefore, researchers have introduced vacuum freeze-drying technology into the preparation of RT-qPCR reagents. Existing freeze-dried RT-qPCR reagents have achieved breakthroughs in room temperature storage and transportation. However, in actual clinical applications, when the concentration of pathogen nucleic acid in the sample is below 50 copies / μL, the exponential growth phase of the amplification curve is significantly delayed (Ct value > 35), and the fluorescence signal intensity is extremely weak. These weak signals are often drowned out by background noise generated by various factors such as sample autofluorescence, non-specific probe adsorption, and impurities in the reaction system, making it difficult for detection instruments to effectively distinguish between the true signal and background interference, easily leading to false negatives or weak positives during clinical interpretation. Furthermore, in multi-target detection systems, it is difficult to completely balance the amplification efficiency of different targets and the luminescence intensity of fluorescent probes. The dominant amplification of high-abundance targets often inhibits the signal acquisition of low-abundance targets, further exacerbating the risk of missed detection of low-concentration targets. Therefore, how to effectively improve the detection capability of lyophilized reagents for low-concentration samples while maintaining their convenience, and clearly distinguish weak signals from background noise, has become a pressing technical challenge in this field.

[0004] To improve the signal-to-noise ratio of fluorescence detection, various signal amplification strategies have been explored by those skilled in the art. One approach involves adding chemical enhancers, such as glycerol, betaine, trehalose, or BSA, to the reaction system to improve amplification efficiency by enhancing enzyme activity and the reaction environment. Another approach is to develop novel high-brightness fluorescent materials, such as quantum dots, time-resolved fluorescent microspheres, or upconversion nanoparticles, to replace traditional organic fluorescent groups. However, these methods all have significant limitations in practical applications: chemical enhancers offer limited sensitivity enhancement (usually no more than 2-3 times), and some enhancers (such as high-concentration DMSO or glycerol) can affect the morphology and reconstitution properties of lyophilized formulations. While novel nanomaterials possess excellent optical properties, issues such as dispersibility in complex multi-component lyophilization systems, compatibility with lyophilization processes, and long-term storage stability have not been effectively resolved, making direct application in mature RT-qPCR lyophilized reagent systems difficult. Summary of the Invention

[0005] To address the deficiencies and shortcomings of the existing technologies, this invention provides a signal amplification composition for the quadruple detection of respiratory pathogens, along with its detection kit and applications. This application constructs a signal amplification system that combines excellent lyophilization compatibility and long-term storage stability by combining fluorescently linked probe-labeled AuNS@MoS2-Probe with amino-modified silica silicocrystalline SiO2PC-NH2. This effectively solves the technical challenge of low-concentration nucleic acid detection sensitivity and provides a novel technical solution for the early, rapid, and accurate detection of respiratory pathogens.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a signal amplification composition for a quadruple detection of respiratory pathogens, the signal amplification composition being composed of fluorescently linked probe-labeled AuNS@MoS2-Probe and amino-modified silica volumetric crystals SiO2PC-NH2 in a mass ratio of 1: (1-5). The signal amplification composition has at least one of the properties described in (1)-(3): (1) Reduce the number of cycles Ct required for amplification to reach the threshold; (2) Improve the signal-to-noise ratio of the detection signal; (3) Reduce the risk of missed detection of low-concentration samples and / or weakly positive samples; The low-concentration sample is a sample with a pathogen nucleic acid concentration ≤50 copies / μL, and the weakly positive sample is a sample with a Ct value between 35 and 40 during RT-qPCR detection; The respiratory pathogens mentioned are influenza A virus, influenza B virus, novel coronavirus, and Mycoplasma pneumoniae.

[0007] In clinical testing of respiratory pathogens, the concentration of pathogen nucleic acid in samples is not constant but dynamically changes with the stage of infection, patient age, and immune status. Especially in the early stages of infection (window period), in special populations with weakened immune systems (such as the elderly, infants, and organ transplant patients), and in occult or mixed infections, pathogens are often in a low-level replication state, with nucleic acid concentrations as low as below 50 copies / μL. For such low-concentration samples, conventional testing methods often fail due to weak signals and a significantly delayed exponential growth phase of the amplification curve (Ct value > 35), making them easily drowned out by background noise. This leads to difficulties in interpreting results, often requiring retesting, and can even result in false negatives. This not only increases the workload of laboratories and patient waiting times, but more importantly, it makes it difficult to identify and intervene early in these "silent" sources of infection or potentially high-risk groups, thus missing the optimal window for prevention and treatment. This invention provides a solution to the sensitivity issue of low-concentration nucleic acid detection, enabling clinical diagnosis to be made before patients develop typical symptoms or become sources of transmission. This buys valuable time for early isolation and treatment, and is of great significance for blocking community transmission and nosocomial infections of respiratory infectious diseases.

[0008] Preferably, the method for preparing the signal amplification composition includes the following steps: (1) Synthesize gold nanostars, disperse the gold nanostars in MoS2 solution to obtain MoS2-coated gold nanostars AuNS@MoS2, perform surface carboxylation modification on AuNS@MoS2 to obtain carboxylation-modified AuNS@MoS2-COOH, and couple an amino-modified fluorescent linker probe to the surface of AuNS@MoS2-COOH to obtain fluorescent linker probe-labeled AuNS@MoS2-Probe; (2) Synthesize silica nanoparticles, emulsify-evaporate the silica nanoparticles to form silica gel crystals, and modify the silica gel crystals with amino to obtain amino-modified silica gel crystals SiO2PC-NH2. (3) The fluorescently linked probe-labeled AuNS@MoS2-Probe was resuspended in MES buffer at pH=5, and amino-modified silica gel crystals SiO2PC-NH2 were added. The signal amplification composition PC-AuNS@MoS2-Probe was obtained by electrostatic adsorption assembly.

[0009] This invention achieves synergistic signal amplification through multiple physical mechanisms by constructing a composite system of fluorescently linked probe-labeled AuNS@MoS2-Probe and amino-modified SiO2PC-NH2. On one hand, the localized surface plasmon resonance (LSPR) effect of gold nanostars enhances the excitation efficiency of the fluorescent probe; on the other hand, the photonic bandgap effect of silica bulk crystals optimizes the directional collection of the fluorescence signal. As shown in Table 4, the combination of the two produces a significantly better dual-effect than that of a single material (synergistic enhancement coefficient 1.68-1.70).

[0010] This invention introduces MoS2 as a spacer layer on the surface of gold nanostars. As a two-dimensional semiconductor material, MoS2 acts as a precise physical spacer, solving the problem of non-radiative energy transfer (i.e., fluorescence quenching) that easily occurs when metal nanomaterials are in direct contact with fluorescent molecules. The MoS2 introduced in this invention, as a two-dimensional semiconductor spacer layer, effectively suppresses energy loss induced by the metal surface, allowing the intrinsic fluorescence of the probes to be fully released. Table 4 shows experimental data that strongly supports this mechanism: Comparative Example 1 (without MoS2) achieved dual physical enhancement, but due to energy loss from some probes being too close, the signal only increased by 3.1-3.4 times; while Example 4 (with MoS2) resulted in a further signal increase of approximately 1.9 times compared to Comparative Example 1, effectively solving the clinical problem of weak signals in low-concentration samples that are easily drowned out by background noise.

[0011] In step (1), the nucleotide sequences of the fluorescent ligation probes are shown in SEQ ID NO:3, 6, 9, 12, and 15, respectively, and the 3′ ends of the fluorescent ligation probes are labeled with FAM, ROX, HEX, CY5, and CY7 fluorescent groups, respectively.

[0012] In a second aspect, the present invention provides the use of the above-described signal amplification composition for the quadruple detection of respiratory pathogens in any one of the following (1)-(3): (1) Prepare a detection product that reduces the number of cycles required to reach the amplification threshold; (2) Prepare detection products that improve the signal-to-noise ratio of the detection signal; (3) Prepare detection products that reduce the risk of missed detection of low concentration samples and / or weak positive samples.

[0013] In a third aspect, the present invention provides a detection kit for a four-in-one detection of respiratory pathogens. The kit is a lyophilized preparation and comprises the above-mentioned signal amplification composition, specific primers, free fluorescent probes, reverse transcriptase, Taq DNA polymerase, buffer system, and lyophilization protectant.

[0014] Preferably, the kit contains a free fluorescent probe and a coupled fluorescent probe; the free fluorescent probe is free in the reaction system to ensure the kinetic efficiency of the amplification reaction; the coupled fluorescent probe is coupled to the surface of the signal amplification composition to generate a fluorescent signal synergistically enhanced by plasmon resonance and photonic crystal effect; the free fluorescent probe and the coupled fluorescent probe have the same nucleotide sequence and the same fluorescent group.

[0015] In the embodiments of this invention, the detection effect of retaining only the ligation probe without using the free probe is not ideal. This is because conventional free probes diffuse freely in solution, and when Taq enzyme extends along the DNA template to the probe binding site, it can efficiently collide with and cleave the probe, releasing fluorescent groups to generate a signal. However, the ligation probe is covalently coupled to the AuNS@MoS2-COOH surface via the 5′ amino group and further assembled onto SiO2PC-NH2 colloidal crystal microspheres, forming a large complex structure. This immobilized state creates significant steric hindrance for the probe: during amplification, Taq enzyme has difficulty accessing and effectively cleaving these ligation probes "attached" to the surface of the nanomaterial. Even if target DNA is generated, the fluorescence signal generation efficiency is extremely low, resulting in a severely lagging Ct value (38.5±1.2) and a low detection rate (37.5%).

[0016] Based on the detection results of this invention, in actual RT-qPCR detection, improper design (lack of free probe coordination) can actually interfere with the RT-qPCR reaction. In this invention, the free probe diffuses freely in solution, allowing it to bind to the template immediately and be efficiently cleaved by Taq enzyme, ensuring a large number of target DNA copies are generated in the system after successful amplification. These copies then bind to the ligation probe immobilized on the signal amplification composition. At this point, the ligation probe no longer primarily relies on Taq enzyme cleavage to generate a signal (although it may be partially cleaved), but instead acts as a signal receiver. This invention's dual-probe design cleverly utilizes the free probe to solve the amplification efficiency problem and the ligation probe to solve the signal intensity problem, achieving true functional complementarity and synergistic enhancement.

[0017] It should be noted that the probe in this invention is designed with a 5′ end coupled and a 3′ end free and labeled with a fluorescent group, without any traditional quenching group. Therefore, the probe has fluorescence background in its initial state. However, this design does not interfere with the detection of real-time quantitative PCR. The core detection principle of real-time quantitative PCR technology is to monitor the relative increase of fluorescence signal. The fluorescence background in the initial stage of the reaction is automatically calibrated by the instrument and used as a baseline for subtraction, and will not affect the subsequent identification of specific signal growth.

[0018] The specific primers include: upstream oligonucleotide primer FluA-F and downstream primer FluA-R with sequences as shown in SEQ ID NO: 1-SEQ ID NO: 2 for detecting the M1 gene of influenza A virus; The sequences shown in SEQ ID NO: 4-SEQ ID NO: 5 are the upstream and downstream oligonucleotide primers FluB-F and FluB-R used to detect the NP gene of influenza B virus; The sequences shown in SEQ ID NO: 7-SEQ ID NO: 8 are the upstream and downstream oligonucleotide primers nCoV-F and nCoV-R used to detect the N gene of the novel coronavirus. The sequences shown in SEQ ID NO: 10-SEQ ID NO: 11 are the upstream and downstream oligonucleotide primers MP-F and MP-R used to detect the Mycoplasma pneumoniae 16S rRNA gene.

[0019] The sequences shown in SEQ ID NO: 13-SEQ ID NO: 14 are the upstream primer β2M-F and the downstream primer β2M-R used to detect the human internal reference gene β2M.

[0020] The free fluorescent probes include: FluA-P, a fluorescent probe for detecting influenza A virus as shown in SEQ ID NO: 3; FluB-P, a fluorescent probe for detecting influenza B virus as shown in SEQ ID NO: 6; nCoV-P, a fluorescent probe for detecting novel coronavirus as shown in SEQ ID NO: 9; MP-P, a fluorescent probe for detecting Mycoplasma pneumoniae as shown in SEQ ID NO: 12; and β2M-P, a fluorescent probe for detecting human internal reference gene β2M as shown in SEQ ID NO: 15.

[0021] In a fourth aspect, the present invention provides a method for performing a quadruple detection of respiratory pathogens in ex vivo samples using the above-mentioned detection kit, comprising the following steps: (1) Extracting nucleic acid from in vitro samples; (2) The extracted nucleic acid was amplified by reverse transcription fluorescence quantitative PCR using a detection kit. The amplification program included: reverse transcription at 55℃ for 5 min; pre-denaturation at 95℃ for 5 s; denaturation at 95℃ for 5 s; annealing and extension at 58℃ for 15 s and fluorescence collection, for a total of 40 cycles. (3) Determine whether the sample contains influenza A virus, influenza B virus, novel coronavirus and / or mycoplasma pneumoniae based on the fluorescence signal detection results.

[0022] In step (3), the criterion for determination is: ① Positive determination: When the control validity determination is valid, the corresponding fluorescence channel shows an increase in fluorescence index and the Ct value is ≤35, indicating that the sample has detected influenza A virus or / and influenza B virus or / and novel coronavirus N gene or / and Mycoplasma pneumoniae with replication function; ② Negative determination: When the control validity determination is established, there is no increase in fluorescence index in the corresponding fluorescence channel and the Ct value is ≥40, indicating that the sample does not detect influenza A virus or / and influenza B virus or / and novel coronavirus N gene or / and Mycoplasma pneumoniae with replication function; ③ Critical value retest: If the fluorescence index increases in the channel and the Ct value is between 35 and 40 when the control validity is established, the test should be repeated once. If the fluorescence index still increases after the retest and the Ct value is between 35 and 40, it is judged as positive; otherwise, it is judged as negative.

[0023] The criteria for determining the validity of the control group are as follows: The positive control showed an increase in fluorescence index in all fluorescence channels, with a Ct value ≤ 35; the negative control showed no increase in fluorescence index in all fluorescence channels, with a Ct value ≥ 40. If both of the above conditions are met, the control validity is established and the experiment is valid; otherwise, the control validity is not established, the experiment is invalid, and retesting is required.

[0024] The beneficial effects of this invention are: 1. This invention achieves dual amplification of detection signals by combining AuNS@MoS2-Probe labeled with a fluorescent linker probe and amino-modified silica gel crystalline SiO2PC-NH2. Experiments show that the signal enhancement effect of this composition is significantly higher than that of the single material (synergistic enhancement coefficient 1.68-1.70 times), which can improve the fluorescence signal intensity of low-concentration samples (50 copies / μL), advance the Ct value, and achieve detection sensitivity at the single-copy level, which is significantly better than conventional lyophilized reagents.

[0025] 2. The detection kit provided by this invention contains both free fluorescent probes and linked fluorescent probes. The former ensures rapid response and kinetic efficiency of PCR amplification, while the latter generates a synergistically enhanced high-intensity signal. Both probes have identical sequences and fluorescent groups, ensuring both specificity for multiplex detection and effective signal amplification. For weakly positive clinical samples with Ct values ​​in the critical range of 35-40, the kit provided by this invention can advance the Ct value by an average of 3.2 cycles, improving the signal-to-noise ratio to 4.4 times that of the control group. This allows samples that would otherwise require retesting to be directly identified as positive, demonstrating good stability in repeated testing.

[0026] 3. This invention introduces a MoS2 spacer layer on the surface of gold nanostars, effectively suppressing nonradiative energy transfer between the metal surface and the fluorophore, thus preventing fluorescence quenching. This further enhances the signal amplification effect by approximately 1.9 times, and advances the Ct value by 1.3-1.5 cycles. Attached Figure Description

[0027] Figure 1 This is a transmission electron microscope (TEM) image of a gold nanostar.

[0028] Figure 2 This is the UV-Vis absorption spectrum of gold nanostars.

[0029] Figure 3 Transmission electron microscope (TEM) image of gold nanostars AuNS@MoS2 coated with MoS2.

[0030] Figure 4 This is a scanning electron microscope image of silica gel crystals.

[0031] Figure 5 Transmission electron microscope (TEM) image of the signal amplification composition PC-AuNS@MoS2-Probe.

[0032] Figure 6 The image shows the results of quantitative fluorescence amplification of respiratory pathogens using the kit prepared in Example 4, where A represents Mycoplasma pneumoniae, B represents influenza B virus, C represents novel coronavirus, and D represents influenza A virus. Detailed Implementation

[0033] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0034] The specific embodiments of the present invention will be described in further detail below with reference to examples. The following detailed descriptions are illustrative and intended to provide further explanation of this application, rather than limiting the scope of the invention.

[0035] The gold nanostars and silica gel crystalline materials described in this invention are both known materials in the art and can be obtained commercially or prepared according to the methods described in the embodiments of this specification and other known techniques in the art. The specific sequence information of the primers and probes used in this invention is shown in Table 1: Table 1 Primer and probe sequences for influenza A virus, influenza B virus, novel coronavirus, Mycoplasma pneumoniae, and β2M gene. Note: R:A / G Example 1: Preparation of fluorescently labeled AuNS@MoS2-Probe 1. Synthesis of gold nanostars (1) Take 0.5 mL of 1% (w / v) chloroauric acid solution and add it to 50 mL of ultrapure water. Heat to boiling and quickly add 1.5 mL of 1% (w / v) trisodium citrate solution. Continue boiling for 15 min. The solution color changes from light yellow to wine red to obtain the gold seed solution. Cool to room temperature for later use.

[0036] (2) Take 50 mL of 0.25 mM chloroauric acid solution, add 50 μL of 1 M HCl to adjust the pH to acidic, then add 100 μL of the gold seed solution obtained in step (1), quickly add 200 μL of 3 mM silver nitrate solution and 250 μL of 100 mM ascorbic acid solution, gently mix, and let stand at room temperature for 30 min until the solution gradually turns blue-green. Centrifuge at 8000 rpm for 10 min, discard the supernatant, resuspend the precipitate in ultrapure water, wash twice, and finally resuspend in 10 mL of ultrapure water to obtain the gold nanostar solution, which is stored at 4℃ for later use. Transmission electron microscopy shows a star-shaped multi-branched structure ( Figure 1 Using UV-Vis absorption spectroscopy, a characteristic localized surface plasmon resonance absorption peak appeared at 700 nm. Figure 2 ).

[0037] 2. Preparation of MoS2-coated gold nanostars Take 10 mL of the above gold nanostar solution, disperse it in a DMF solution containing 0.1 mg / mL molybdenum disulfide (MoS2), sonicate for 30 min, centrifuge and wash to remove unbound MoS2, and obtain MoS2-coated gold nanostars AuNS@MoS2. Figure 3 ).

[0038] 3. Carboxylation modification 10 mg AuNS@MoS2 was resuspended in 10 mL MES buffer (0.1 M, pH 6.0), and 1 mL of 1 mMHS-PEG-COOH was added. The mixture was stirred at room temperature for 4 h. After the reaction was complete, the mixture was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the precipitate was washed twice with ultrapure water to obtain carboxylated AuNS@MoS2-COOH, which was then resuspended in PBS buffer (0.01 M, pH 7.4) for later use.

[0039] 4. Coupling of fluorescent linker probes Amino-modified fluorescent ligation probes (sequences shown in SEQ ID NO:3, 6, 9, 12, 15, with FAM, ROX, HEX, CY5, CY7 labeled at the 3′ end and amino-modified at the 5′ end, respectively) were dissolved in PBS buffer (0.01 M, pH 7.4) to prepare 100 μM stock solutions.

[0040] Take 1 mL of carboxyl-modified AuNS@MoS2-COOH solution, add 20 μL EDC (10 mg / mL) and 20 μL NHS (10 mg / mL), and activate at room temperature for 30 min. Immediately after activation, centrifuge at 10000 rpm for 5 min to remove excess activator, and resuspend the precipitate in PBS buffer. Then add different fluorescent probe stock solutions and react at room temperature for 2 h. After the reaction, centrifuge and wash 3 times to remove unbound probes, obtaining five different fluorescently labeled AuNS@MoS2-Probes (denoted as AuNS@MoS2-FluA-P, AuNS@MoS2-FluB-P, AuNS@MoS2-nCoV-P, AuNS@MoS2-MP-P, and AuNS@MoS2-β2M-P), resuspend in PBS, and store at 4℃ protected from light.

[0041] Example 2: Preparation of Aminated Silica Gel Crystals (1) Mix 10 mL of tetraethyl orthosilicate (TEOS) with 100 mL of anhydrous ethanol, then quickly add 10 mL of ammonia (28%) and 10 mL of water, and stir at room temperature for 12 h. After the reaction is complete, centrifuge to collect the precipitate, wash it three times with ethanol to obtain monodisperse silica nanoparticles.

[0042] (2) Monodisperse silica nanoparticles were dispersed in ethanol to prepare a 10 mg / mL suspension. 10 mL of this suspension was mixed with 40 mL of paraffin oil containing 2% (w / v) Span 80, and emulsified at 10000 rpm for 2 min to form a W / O emulsion. The emulsion was transferred to a 60℃ oven and allowed to stand for 6 h to allow the ethanol to evaporate slowly. The precipitate was collected by centrifugation, washed three times with ethanol and cyclohexane to remove the paraffin oil, and dried at 60℃ to obtain silica crystalline solids. Figure 4 ).

[0043] (3) Disperse 0.1 g of silica gel crystals in 20 mL of anhydrous ethanol, add 0.2 mL of 3-aminopropyltriethoxysilane (APTES), and reflux at 60 °C for 4 h. After the reaction is complete, centrifuge, wash three times with ethanol, and dry at 60 °C to obtain amino-modified silica gel crystals SiO2PC-NH2.

[0044] Example 3: Assembly and stability evaluation of the signal amplification composition (1) Assembly of the signal amplification composition Five fluorescent probe-labeled AuNS@MoS2-Probes prepared in Example 1 were mixed in equal volume ratios. 2 mg of the mixed AuNS@MoS2-Probes were resuspended in 1 mL of MES buffer (pH 5.0), and then 10 mg of SiO2PC-NH2 was added. The mixture was incubated at room temperature with shaking for 2 h. This allowed the negatively charged AuNS@MoS2-Probes to bind to the positively charged SiO2PC-NH2 surface via electrostatic adsorption. After assembly, the mixture was centrifuged at 3000 rpm for 5 min to remove unbound AuNS@MoS2-Probes from the supernatant. The precipitate was washed twice with ultrapure water to obtain the signal amplification composition PC-AuNS@MoS2–Probe (…). Figure 5 ).

[0045] (2) Stability assessment of the signal amplification composition The obtained signal amplification composition was dispersed in PBS buffer containing 0.05% sodium azide and stored at 4°C in the dark. Fluorescence intensity was measured on days 0, 7, 14, and 30, and the results showed that the fluorescence intensity retention rate was >95% within 30 days. After vacuum freeze-drying and reconstitution, the fluorescence intensity retention rate was >90%, indicating that the composite probe has good freeze-drying compatibility and storage stability.

[0046] Example 4: Preparation of a lyophilized RT-qPCR kit containing a signal amplification composition (1) Preparation of frozen intervention mixture The lyophilized RT-qPCR kit of the present invention comprises a lyophilized reaction powder, a positive control, and a negative control. The formulation of the lyophilized reaction powder is shown in Table 2.

[0047] Table 2: Formulation of lyophilized RT-qPCR reaction solution Note: In this embodiment, the fluorescent probe exists in two forms: (1) Free probe: with a concentration of 0.1 μM, used as a conventional RT-qPCR probe to ensure the rapid response and kinetic efficiency of the amplification reaction; (2) Ligation probe: that is, a fluorescent probe coupled to the surface of the signal amplification composition, whose nucleotide sequence is exactly the same as the corresponding free probe, as shown in SEQ ID NO:3, 6, 9, 12, 15.

[0048] The ligation probe is coupled to the surface of the gold nanostar via its 5′ amino group, and its 3′ end is labeled with a fluorescent group identical to that of the free probe. Both probes have the same sequence and fluorescent group, but exist in different forms: the free probe is free in solution, ensuring a rapid response in the amplification reaction; the ligation probe is located on the surface of the signal amplification composition, generating a synergistically enhanced high-intensity signal.

[0049] (2) Freeze-drying process Aliquot the prepared premixed solution into 0.2 mL PCR eight-tube strips, 25 μL per tube. Place the eight-tube strips in a freeze dryer and freeze-dry according to the following procedure: Pre-freezing: Keep at -45℃ for 2 hours; First sublimation: Heat to -30°C (hold for 2 h) while evacuating to a pressure below 1 mbar; then heat to -15°C (hold for 2 h). Secondary sublimation: Heat to 10℃ (hold for 2 hours); Finally, raise the temperature to 25°C (and maintain for 2 hours).

[0050] After lyophilization, dry nitrogen gas is introduced to atmospheric pressure, and the container is quickly capped and sealed to obtain lyophilized RT-qPCR reagent containing the signal amplification composition. The reagent is then stored at room temperature in the dark.

[0051] (3) Kit composition The kit prepared in this embodiment includes: (1) Reaction lyophilized powder; (2) Positive control: Five plasmids containing the M1 gene of influenza A virus, the NP gene of influenza B virus, the N gene of novel coronavirus, the 16S rRNA gene of Mycoplasma pneumoniae, and the human β2M gene fragment (each with a concentration of 1×10⁻⁶) were used. 8 Mix equal volumes of the stock solution (copies / μL). Then, dilute the stock solution sequentially with diluent to a concentration of 10. 5 10 4 10 3 10 2 10 1 10 0 Concentration gradient of copies / μL.

[0052] (3) Negative control: Nuclease-free water; (4) Reconstituted solution: Nuclease-free water.

[0053] Example 5: Method for quadruple detection of respiratory pathogens in ex vivo samples using a detection kit This embodiment uses the lyophilized RT-qPCR kit containing the signal amplification composition prepared in Example 4 as an example to provide a specific operating method for the quadruple detection of influenza A virus, influenza B virus, novel coronavirus and mycoplasma pneumoniae in in vitro samples.

[0054] 1. Sample collection and nucleic acid extraction (1) Sample collection: Use a special sampling swab to collect pharyngeal or nasopharyngeal swab samples from the person to be tested. Place the collected swab head into a sampling tube containing virus preservation solution, seal it and send it for testing as soon as possible.

[0055] (2) Nucleic acid extraction: Take 200 μL of the sample to be tested and use a commercial viral RNA extraction kit according to the instructions. The specific steps include: lysing the virus, adsorbing nucleic acid onto the adsorption column, removing impurities, washing and eluting, and finally obtaining 50-100 μL of nucleic acid eluent, which can be used directly for amplification detection or stored at -80℃ for later use.

[0056] 2. Kit preparation and sample loading (1) Reconstitution: Take out the eight-tube pack of lyophilized reaction powder from the kit stored at room temperature and protected from light, add 25 μL of nuclease-free water (reconstitution solution) to each tube, gently blow or vortex to completely dissolve the lyophilized powder, and then centrifuge briefly before use.

[0057] (2) Sample addition: Add 5 μL of extracted nucleic acid of the sample to be tested to the reconstituted PCR reaction tube. Simultaneously set up positive and negative controls: Add 5 μL of positive control sample (a mixed plasmid containing fragments of influenza A virus M1 gene, influenza B virus NP gene, novel coronavirus N gene, Mycoplasma pneumoniae 16S rRNA gene, and human β2M gene, all at a concentration of 1×10⁻⁶) to the positive control tube. 5 (Copies / μL); Add 5 μL of nuclease-free water to the negative control tube. The total volume of each tube is 30 μL.

[0058] 3. Quantitative Real-Time PCR Amplification Place the PCR eight-tube strips containing the samples into a real-time PCR instrument and perform amplification according to the following procedure: Table 3: Reaction Procedure The fluorescence channel settings for each target are as follows: Influenza A virus (FluA): FAM channel Influenza B virus (FluB): ROX channel Novel Coronavirus (nCoV): HEX Channel Mycoplasma pneumoniae (MP): CY5 channel Human internal reference gene β2M: CY7 channel 4. Result Judgment After the amplification is completed, the results are judged according to the fluorescence signals. The judgment criteria are as follows: (1)Judgment of control validity: Positive control: There is an increase in fluorescence index in all FAM, ROX, HEX, CY5, and CY7 channels, and the Ct value ≤ 35; Negative control: There is no increase in fluorescence index in all FAM, ROX, HEX, CY5, and CY7 channels, and the Ct value ≥ 40.

[0059] When both of the above two conditions are met, this experiment is valid; otherwise, the experiment is invalid and needs to be retested.

[0060] (2)Judgment of sample results: Positive: There is an increase in fluorescence index in the corresponding fluorescence channel, and the Ct value ≤ 35, indicating that the corresponding pathogen (influenza A virus, influenza B virus, novel coronavirus, and / or Mycoplasma pneumoniae) is detected in the sample.

[0061] Negative: There is no increase in fluorescence index in the corresponding fluorescence channel, and the Ct value ≥ 40, indicating that the corresponding pathogen is not detected in the sample.

[0062] Critical value retest: If there is an increase in fluorescence index in the corresponding fluorescence channel, and 35 < Ct value < 40, it should be retested once. If there is still an increase in fluorescence index after retesting, and the Ct value is between 35 - 40, it is judged as positive; otherwise, it is judged as negative.

[0063] Comparative Example 1: Preparation of a Composite Probe without a MoS2 Spacer Layer and Its Kit 1. Preparation of the probe Prepare gold nanospheres (AuNS) and perform carboxylation modification according to the method of Example 1 to obtain AuNS-PEG-COOH.

[0064] The difference from Example 1 is that: MoS2 coating is not carried out. The carboxylated gold nanospheres are directly dispersed in MES buffer, and the fluorescent linker probe is coupled according to the method of step 4 of Example 1 to obtain AuNS-Probe.

[0065] 2. Assembly of the signal amplification composition According to the method of Example 3, electrostatic adsorption assembly is performed on the above AuNS-Probe and the amino-functionalized silica colloidal crystal (SiO2PC-NH2) prepared in Example 2 to obtain PC-AuNS-Probe (i.e., a composite probe without MoS2).

[0066] 3. Preparation of the kit The signal amplification composition (PC-AuNS@MoS2-Probe) in Example 4 was replaced with PC-AuNS-Probe prepared in Comparative Example 1. The remaining components, lyophilization process and detection method were the same as in Example 4, resulting in a comparative kit.

[0067] Comparative Example 2: Kit containing only AuNS@MoS2-Probe (without silica gel crystals) 1. Probe preparation AuNS@MoS2-Probe (MoS2-coated, coupled fluorescent probe) was completely prepared according to the method in Example 1. Electrostatic adsorption assembly with silica bulk crystals was not performed. That is, AuNS@MoS2-Probe was directly used as a signal amplification material.

[0068] 2. Preparation and performance verification of the reagent kit AuNS@MoS2-Probe (in the form of free nanostars) prepared in Comparative Example 2 was added to the premixed solution in the same amount as in Example 4. The remaining components, lyophilization process and detection method were the same as in Example 4 to obtain the comparative kit.

[0069] Comparative Example 3: Kit containing only SiO2PC-NH2 (without gold nanostars) 1. Material preparation Aminated silica gel crystalline solids (SiO2PC-NH2) were prepared according to the method in Example 2. No gold nanostars or fluorescent linker probes were coupled.

[0070] 2. Preparation and performance verification of the reagent kit The SiO2PC-NH2 prepared in Comparative Example 3 was added to the premixed solution in the same mass as the signal amplification composition in Example 4. In this comparative example, the fluorescence signal originated only from the 0.1 μM free probe in the system. The remaining components, lyophilization process, and detection method were the same as in Example 4, resulting in a comparative kit.

[0071] Comparative Example 4: A standard lyophilized RT-qPCR kit without any nanomaterials 1. Preparation of the reagent kit The kit was prepared according to the formulation and lyophilization process of Example 4. No signal amplification composition was added to the formulation (i.e., PC-AuNS@MoS2-Probe was not included); signal detection relied solely on a conventional free probe (0.1 μM). All other components and the lyophilization process were the same as in Example 4, resulting in a conventional lyophilized RT-qPCR kit.

[0072] Experimental Example 1: Performance Verification of Lyophilized Reagents Containing Signal Amplification Compositions 1. Detection sensitivity Samples were tested using the lyophilized reagent containing the signal amplification composition prepared in Example 4, with conventional lyophilized reagent without the signal amplification composition serving as a control. Each concentration in each group was tested eight times, amplified according to the following procedure: reverse transcription at 55°C for 5 min; pre-denaturation at 95°C for 5 s; 95°C for 5 s, 58°C for 15 s (fluorescence acquisition), for 40 cycles. The Ct values ​​for each channel were recorded.

[0073] Table 4: Comparison of detection rates for samples with different concentrations (n=8) Note: 10 0 At a concentration of copies / μL, the Ct values ​​of the experimental group were all between 35 and 37 in 8 tests, which is within the "critical value retest" range of the judgment criteria of this invention. After retesting, all 8 cases were judged to be positive.

[0074] The results showed that the lyophilized reagent containing the signal amplification composition was effective at 10... 0 It can still detect 100% even at copies / μL, and the detection sensitivity is about 100 times higher than that of conventional lyophilized reagents.

[0075] To verify the synergistic enhancement effect of the signal amplification composition, the detection kits prepared in Example 4 and Comparative Examples 1-4 were used to test samples. Using a mixed standard of four pathogen plasmids at a concentration of 50 copies / μL as a template, each group was tested 8 times. The endpoint fluorescence intensity (RFU) and Ct value of the four fluorescence channels (FAM, ROX, HEX, CY5) were recorded and the average values ​​were compared.

[0076] Table 5: Comparison of signal intensity among different enhancement groups (four detection targets) Note: Signal enhancement factor = RFU of each group / RFU of comparison group 4 (no enhancement group), rounded down.

[0077] Table 6: Summary of Synergistic Effects of Each Target Note: Synergy enhancement coefficient = RFU of complete synergy group / (RFU of single A group + RFU of single B group).

[0078] As can be seen from Table 5 and Table 6, for the four different detection targets (Influenza A, Influenza B, COVID-19, Mycoplasma pneumoniae), the end fluorescence intensity of the complete synergy group was significantly higher than that of other groups, and showed a highly consistent increasing trend. The RFU of the complete synergy group was significantly higher than the sum of the RFU of the single A group and the single B group under the same target (synergy enhancement coefficient 1.68 - 1.70). This result fully proves that the combination of AuNS@MoS2-Probe and SiO2PC-NH2 is not a simple functional superposition, but produces a synergistic enhancement effect of "1 + 1 > 2", and this effect is generally present in four different fluorescence channels, independent of the target type and fluorophore.

[0079] It is worth noting that the Ct values of the non-enhancement group (Comparative Example 4) were distributed between 34.8 - 35.6. Among them, the Ct values of the Influenza A, Influenza B, and Mycoplasma pneumoniae targets (35.2, 35.5, 35.6 respectively) exactly fell into the "critical value recheck" interval (35 < Ct < 40) defined by the judgment standard of this invention, and even approached the lower limit of the critical value. This data precisely reveals that when conventional lyophilized reagents detect low-concentration samples, their signal intensity can only reach the critical detectable level, and they are extremely vulnerable to background noise interference, resulting in unstable detection results and a high risk of missed detection. In contrast, the Ct values of the complete synergy group were all stable between 29.5 - 30.3, far lower than the positive judgment threshold of 35, showing a clear and definite strong positive signal. This result fully indicates that the signal amplification composition constructed in this invention can effectively amplify the originally weak signal in the critical state to a strong positive level that can be clearly interpreted, significantly improving the detection reliability of low-concentration samples.

[0080] (2)Verification of the role of the MoS2 spacer layer Results of the complete synergy group and the group without MoS2: The RFU of the complete synergy group was about 1.9 times that of the group without MoS2 (average of four targets); The Ct value of the complete synergy group was 1.3 - 1.5 cycles earlier than that of the group without MoS2.

[0081] This result shows that the MoS2 spacer layer effectively inhibits the fluorescence quenching caused by non-radiative energy transfer between the metal surface and the fluorophore. When the probe is directly connected to the surface of gold nanorods (group without MoS2), part of the fluorescence energy is dissipated by the metal, and the signal enhancement effect is limited; after being coated with MoS2 (complete synergy group), quenching is avoided, and more fluorophores are in the effective enhancement region, further enhancing the signal amplification effect.

[0082] (3)Simultaneous improvement of Ct values Based on Ct values, the complete synergistic group showed Ct values ​​4.9-5.4 cycles earlier than the non-enhanced group, 2.5-2.9 cycles earlier than the single A group, and 3.1-3.5 cycles earlier than the single B group. This significant advancement in Ct values ​​means that, with the same number of cycles, the reagent of this invention can detect the target's presence earlier, which has important clinical significance for rapid screening of low-concentration samples.

[0083] The above results demonstrate that the signal amplification composition PC-AuNS@MoS2-Probe constructed in this invention exhibits a significant synergistic enhancement effect in the detection of four respiratory pathogens. It increases the fluorescence signal intensity of low-concentration samples (50 copies / μL) by approximately 6 times and advances the Ct value by 5 cycles. This effect is universal in the four fluorescence channels (FAM, ROX, HEX, and CY5), fully demonstrating that the signal amplification composition of this invention is suitable for quadruple detection systems and can simultaneously improve the detection sensitivity of four targets.

[0084] 2. Verification of the necessity of the free probe To verify the necessity of free probes, the following three sets of comparisons were set up: Group A: Contains only the binding probe (no free probe), and the concentration of the composite probe is the same as in Example 2; Group B: Contains only free probe (no linked probe), free probe concentration 0.1 μM; Group C: Includes a connection probe and a free probe (same as in Example 2).

[0085] Using a mixed standard of 50 copies / μL as a template, each group was tested 8 times.

[0086] Table 7: Results of Verification of the Necessity of Free Probes The results showed that when only the ligation probe was used, the amplification efficiency decreased significantly, and the false negative rate was high for low-concentration samples. When only the free probe was used, amplification efficiency was maintained, but the signal intensity was limited. When both probes coexisted, both amplification efficiency and signal amplification were achieved, resulting in the highest detection sensitivity. These results demonstrate that the free probe is crucial for ensuring PCR amplification efficiency and forms a complementary synergistic design with the ligation probe.

[0087] 3. Stability study of lyophilized reagents The lyophilized reagent prepared in Example 4 was stored in the dark at 4°C, 25°C, and 37°C, respectively. It was removed at 0, 1, 2, 3, and 6 months, reconstituted, and tested. 3 Record the Ct value for a mixed standard of copies / μL.

[0088] Table 8: Results of stability study of lyophilized reagents (Ct values) The results showed that the Ct value changed by less than 1.5 after being stored at 25℃ for 6 months and by less than 1 after being stored at 37℃ for 1 month, indicating that the reagent has good storage stability at room temperature, is suitable for freeze-drying process, and is stable for a long time.

[0089] 4. Specificity verification of multiplex detection Using the kit prepared in Example 4, positive samples containing only a single target (e.g., containing only influenza A virus plasmid, only SARS-CoV-2 plasmid, etc.) and mixed samples containing four targets were detected. The results showed no cross-interference between channels. The FAM, ROX, HEX, and CY5 channels specifically detected their respective targets, while the CY7 channel detected the internal reference gene β2M. No non-specific amplification was observed. This indicates that the introduction of the signal amplification composition did not affect the specificity of multiplex PCR.

[0090] Experimental Example 2: Clinical Sample Testing 1. Accuracy of clinical sample testing A total of 60 pharyngeal swab samples were collected from clinically diagnosed patients with respiratory infections, including 15 positive for influenza A, 12 positive for influenza B, 15 positive for COVID-19, 12 positive for Mycoplasma pneumoniae, 3 mixed infections, and 3 negative. Parallel testing was performed using the lyophilized reagent containing the signal amplification composition prepared in Example 4 (experimental group) and a commercially available liquid RT-qPCR reagent with a medical device registration certificate (control group).

[0091] Table 9: Comparison of Clinical Sample Detection Results The results showed that the concordance rate between the experimental group and the control group was 100%.

[0092] 2. Detection accuracy of weakly positive samples To fully verify the advantages of the reagent of the present invention in the detection of weak positive samples, an in-depth analysis was conducted on the weak positive samples with low viral load among the above 60 clinical samples.

[0093] (1) Screening criteria for weak positive samples Based on the test results of the control group (commercially available liquid RT-qPCR reagent), samples with Ct values ​​in the range of 35-40 (i.e., the "borderline retesting zone" in the conventional judgment criteria) were defined as weakly positive samples. These samples cannot be directly judged as positive according to the standard reagent instructions and require retesting or a comprehensive judgment based on clinical symptoms. This presents issues of result uncertainty and extended reporting time due to the retesting process.

[0094] According to statistics, 8 out of 60 clinical samples met the criteria, specifically: 3 weakly positive for influenza A, 2 weakly positive for influenza B, 2 weakly positive for COVID-19, and 1 weakly positive for Mycoplasma pneumoniae. These samples represent low viral load scenarios such as the infection window period, occult infection, or immunosuppressed patients, which are sample types that are prone to interpretation difficulties in clinical testing.

[0095] (2) Comparison of detection sensitivity The lyophilized reagent containing the signal amplification composition prepared in Example 4 of this invention (experimental group) and commercially available liquid RT-qPCR reagent (control group) were used to perform parallel detection on the above 8 weakly positive samples. Each sample was tested three times, and the average Ct value, endpoint fluorescence intensity and signal-to-noise ratio of each sample were recorded.

[0096] Table 10: Comparison of Detection Results for Low-Concentration Samples Note: RFU is relative fluorescence unit, and the average value of 3 repeated tests is taken; fluorescence enhancement factor = experimental group RFU / control group RFU.

[0097] The data in the table shows that the Ct values ​​of the control group (commercially available reagents) for these 8 weakly positive samples ranged from 35.2 to 37.0, all falling within the "critical retest" range (35 < Ct < 40) in the conventional judgment criteria. This means that, according to the instructions of the conventional reagents, these samples cannot be directly judged as positive and a retesting procedure needs to be initiated, extending the reporting period. Furthermore, there is a risk of result fluctuations during the retesting process.

[0098] The Ct values ​​of the experimental group (using the reagents of this invention) were significantly reduced to 31.8-33.9, all below the positive threshold of 35, and could be directly judged as positive. The Ct values ​​were reduced by an average of 3.2 cycles, which is equivalent to the signal amplification effect improving the detection sensitivity by about an order of magnitude. This change has significant clinical implications: it transforms samples that were originally in the borderline range and required retesting into samples with a single positive result that can be clearly determined with a single test, thereby shortening the reporting time, reducing the reagent and labor costs caused by repeated testing, and reducing the waiting period for clinical decision-making.

[0099] (3) Signal-to-noise ratio analysis Signal-to-noise ratio (SNR) is a key indicator for measuring the ability of a detection system to distinguish between real signals and background noise. The calculation formula is: SNR = (mean value of sample fluorescence signal - mean value of negative control fluorescence signal) / standard deviation of negative control fluorescence signal.

[0100] The signal-to-noise ratio of 8 weakly positive samples was calculated, and the results showed: The average signal-to-noise ratio (SNR) of the control group was 4.2 ± 1.1 (SNR > 3 is generally considered a detectable signal, but it is close to the critical value). The average signal-to-noise ratio of the experimental group was 18.6 ± 3.5, which was 4.4 times that of the control group.

[0101] A high signal-to-noise ratio means that weak signals can be clearly separated from background noise, significantly improving the reliability and repeatability of detection results.

[0102] (4) Stability analysis of repeated tests To assess the stability of the test results for weakly positive samples, eight weakly positive samples were tested eight times (nucleic acid was extracted and amplified independently for each test), and the detection rate and standard deviation of Ct values ​​for each sample were calculated.

[0103] Control group: Among the 8 samples, 2 cases had no amplification in 1 out of 8 repeated tests (detection rate 87.5%), another case was detected in all 8 tests but the standard deviation of the Ct value was 1.6 (large fluctuation), and the remaining 5 cases had a detection rate of 100% and a standard deviation of Ct value <1.0.

[0104] Experimental group: All 8 samples were detected in 8 repeated tests (detection rate 100%), and the standard deviation of Ct values ​​for all samples was <0.8, indicating good repeatability.

[0105] The results show that conventional reagents exhibit a certain degree of repeatability fluctuations and potential false negatives in the detection of critical concentration samples; while the reagent of this invention significantly enhances the amplification signal through the synergistic enhancement effect of the signal amplification composition, thereby achieving higher detection stability and result reliability.

[0106] The above results indicate that for weakly positive samples with extremely low viral load (control reagent Ct value in the critical range of 35-40), the lyophilized reagent containing the signal amplification composition of this invention has the following advantages: (1) The Ct value is advanced by an average of 3.2 cycles, which allows samples that originally needed to be retested to be directly judged as positive, significantly shortening the reporting cycle; (2) The signal-to-noise ratio was increased to 4.4 times that of the control group, the distinction between weak signals and background noise was significantly improved, and the results were more reliable; (3) The detection rate of repeated testing is 100%, and the risk of missed detection and the volatility of results are significantly reduced.

[0107] The results fully demonstrate that the signal amplification composition prepared in this invention can effectively solve the technical problems of weak signals, easy falling into the critical range, and uncertain results in low-concentration nucleic acid detection, and has outstanding technical advantages in clinical low viral load sample detection.

[0108] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A signal amplification composition for respiratory pathogen quadruple detection, characterized in that, The signal amplification composition is composed of AuNS@MoS2-Probe labeled with a fluorescent linker probe and amino-modified silica bulk crystals SiO2 PC-NH2 in a mass ratio of 1: (1-5). The method for preparing the signal amplification composition includes the following steps: (1) Synthesize gold nanostars, disperse the gold nanostars in MoS2 solution to obtain MoS2-coated gold nanostars AuNS@MoS2, perform surface carboxylation modification on AuNS@MoS2 to obtain carboxylation-modified AuNS@MoS2-COOH, and couple an amino-modified fluorescent linker probe to the surface of AuNS@MoS2-COOH to obtain fluorescent linker probe-labeled AuNS@MoS2-Probe; (2) Synthesize silica nanoparticles, emulsify-evaporate the silica nanoparticles to form silica gel crystals, and modify the silica gel crystals with amino to obtain amino-modified silica gel crystals SiO2 PC-NH2. (3) The fluorescently linked probe-labeled AuNS@MoS2-Probe was resuspended in MES buffer at pH=5, and amino-modified silica gel crystals SiO2 PC-NH2 were added. The signal amplification composition PC-AuNS@MoS2-Probe was obtained by electrostatic adsorption assembly. The nucleotide sequences of the fluorescent linker probes are shown in SEQ ID NO:3, 6, 9, 12, and 15, respectively; the 3' end of the fluorescent linker probe shown in SEQ ID NO:3 is labeled with the FAM fluorescent group, the 3' end of the fluorescent linker probe shown in SEQ ID NO:6 is labeled with the ROX fluorescent group, the 3' end of the fluorescent linker probe shown in SEQ ID NO:9 is labeled with the HEX fluorescent group, the 3' end of the fluorescent linker probe shown in SEQ ID NO:12 is labeled with the CY5 fluorescent group, and the 3' end of the fluorescent linker probe shown in SEQ ID NO:15 is labeled with the CY7 fluorescent group. The signal amplification composition has at least one of the following properties (1)-(3): (1) Reduce the number of cycles Ct required for amplification to reach the threshold; (2) Improve the signal-to-noise ratio of the detection signal; (3) Reduce the risk of missed detection of low-concentration samples and / or weakly positive samples; The low-concentration sample is a sample with a pathogen nucleic acid concentration ≤50 copies / μL, and the weakly positive sample is a sample with a Ct value between 35 and 40 during RT-qPCR detection; The respiratory pathogens mentioned are influenza A virus, influenza B virus, novel coronavirus, and Mycoplasma pneumoniae.

2. The use of the signal amplification composition for respiratory pathogen quadruple detection according to claim 1 in any one of (1) to (3), characterized in that, include: (1) Prepare a detection product that reduces the number of cycles required to reach the amplification threshold; (2) Prepare detection products that improve the signal-to-noise ratio of the detection signal; (3) Prepare detection products that reduce the risk of missed detection of low-concentration samples and / or weakly positive samples; The detection products are designed to detect respiratory pathogens, including influenza A virus, influenza B virus, novel coronavirus, and Mycoplasma pneumoniae.

3. A test kit for respiratory pathogen quad test, characterized by, The kit is a lyophilized formulation, comprising the signal amplification composition of claim 1, specific primers, free fluorescent probes, reverse transcriptase, Taq DNA polymerase, buffer system, and lyophilization protectant; The detection kit contains a free fluorescent probe and a coupled fluorescent probe; the free fluorescent probe is free in the reaction system, and the coupled fluorescent probe is coupled to the surface of the signal amplification composition; the free fluorescent probe and the coupled fluorescent probe have the same nucleotide sequence and the same fluorescent group; The specific primers include: oligonucleotide upstream primer FluA-F and downstream primer FluA-R for detecting the M1 gene of influenza A virus, as shown in SEQ ID NO: 1-SEQ ID NO: 2; oligonucleotide upstream primer FluB-F and downstream primer FluB-R for detecting the NP gene of influenza B virus, as shown in SEQ ID NO: 4-SEQ ID NO: 5; oligonucleotide upstream primer nCoV-F and downstream primer nCoV-R for detecting the N gene of novel coronavirus, as shown in SEQ ID NO: 7-SEQ ID NO: 8; oligonucleotide upstream primer MP-F and downstream primer MP-R for detecting the 16S rRNA gene of Mycoplasma pneumoniae, as shown in SEQ ID NO: 10-SEQ ID NO: 11; and oligonucleotide upstream primer β2M-F and downstream primer β2M-R for detecting the human internal reference gene β2M, as shown in SEQ ID NO: 13-SEQ ID NO:

14. The free fluorescent probes include: FluA-P, a fluorescent probe for detecting influenza A virus as shown in SEQ ID NO: 3; FluB-P, a fluorescent probe for detecting influenza B virus as shown in SEQ ID NO: 6; nCoV-P, a fluorescent probe for detecting novel coronavirus as shown in SEQ ID NO: 9; MP-P, a fluorescent probe for detecting Mycoplasma pneumoniae as shown in SEQ ID NO: 12; and β2M-P, a fluorescent probe for detecting human internal reference gene β2M as shown in SEQ ID NO: 15.

Images