An rt-lamp virus nucleic acid semi-quantitative detection method and kit based on split freeze-dried microspheres and multi-channel microfluidic chip

Abstract

This invention discloses a semi-quantitative RT-LAMP method and kit for viral nucleic acid detection based on dispensed lyophilized microspheres and a multi-channel microfluidic chip. The method includes: designing two independent LAMP primer sets (P1 and P2) for each of the SARS-CoV-2 Omicron strain N gene, Zika virus NS5 gene, H1N1 influenza virus M1 gene, and H3N2 influenza virus NP gene; preparing two independent types of lyophilized microspheres by independently mixing RT-LAMP premix containing Bst DNA polymerase and other components with LAMP primer mixture and a lyophilization protectant; and using an N-channel microfluidic chip to perform serial dilutions and parallel RT-LAMP amplification of the same sample, determining the concentration of viral nucleic acid in the sample by the positive / negative inflection points of each channel. This invention is simple to operate, requires no equipment such as a real-time PCR instrument, and is suitable for rapid virus detection in various scenarios.

Description

Technical Field

[0001] This invention belongs to the field of molecular diagnostics and point-of-care testing (MD-POCT) technology, specifically involving a viral nucleic acid detection method that combines reverse transcription loop-mediated isothermal amplification (RT-LAMP) technology with a multi-channel microfluidic chip, as well as a matching dispensed lyophilized microsphere reagent and detection kit. Background Technology

[0002] Wastewater-based epidemiology (WBE) is a public health approach that uses the detection of pathogen nucleic acids in urban sewage or wastewater from specific locations to provide early warning of infection status in a regional population. During the SARS-CoV-2 Omicron epidemic, multiple studies confirmed that viral RNA is shed in feces 3-7 days before the onset of symptoms, and wastewater monitoring signals can predict regional infection peaks 1-2 weeks in advance, playing a crucial sentinel role in epidemic prevention and control in many countries and regions. Besides SARS-CoV-2 Omicron, Zika virus, H1N1, and H3N2 influenza viruses can also contaminate wastewater systems through the fecal-oral route or respiratory secretions, and can be continuously detected in wastewater. Currently, the gold standard for viral nucleic acid detection in wastewater is reverse transcription quantitative PCR (RT-qPCR) technology, which has high sensitivity and quantification capabilities. However, this method is highly dependent on instruments and equipment, such as real-time fluorescence PCR instruments and ultra-low temperature freezers. The operation process is complex, and environmental inhibitors such as humic acid and heavy metals in the sample significantly affect the amplification efficiency. A single detection cycle usually takes more than 3 hours and requires professional operation. It is difficult to deploy directly in resource-constrained scenarios such as wastewater treatment plants, border inspection stations, or community health centers, which seriously restricts the coverage and response speed of wastewater monitoring networks.

[0003] LAMP technology utilizes Bst DNA polymerase with strand displacement activity to drive the amplification reaction at a single temperature of 60-65℃, eliminating the need for temperature cycling equipment. Detection can be completed within 15-45 minutes, and results can be directly interpreted through visible color changes using dyes such as SYBR-Green. It boasts significant advantages such as simple equipment, convenient operation, and suitability for rapid on-site testing. Combining RT-LAMP with microfluidic chip technology enables precise control and multi-channel parallel amplification of microliter and even nanoliter samples, further increasing throughput and reducing reagent consumption. However, existing microfluidic LAMP chip products and research solutions generally have the following shortcomings: (1) They can only provide qualitative results of positive / negative, and cannot conduct semi-quantitative assessment of viral load in samples, thus failing to meet the actual needs of wastewater monitoring for judging the trend of viral concentration changes; (2) LAMP reagents, especially Bst polymerase, need to be stored and transported under -20℃ cold chain conditions, which restricts the independent deployment of the detection system in remote areas and on-site environments; and (3) Existing solutions usually require users to prepare the reaction system on-site before chip use, which is cumbersome and prone to human error, failing to meet the core requirements of "simple operation and immediate use" for point-of-care testing. The above defects make the existing microfluidic LAMP detection system still significantly different from true on-site semi-quantitative wastewater virus monitoring.

[0004] Freeze-drying technology can transform bioactive reagents into room-temperature stable solid formulations, a core approach to overcoming cold chain dependence. Existing research has shown that LAMP reaction components are pre-freezed and placed within microfluidic chip channels, requiring only the addition of sample liquid to initiate the reaction, significantly simplifying the process. Commonly used freeze-drying protectants include trehalose, sucrose, and mannitol, which effectively maintain the bioactivity of Bst polymerase and nucleic acids during the drying process. However, most existing techniques involve mixing the reaction premix containing Bst polymerase with LAMP primers and then freeze-drying them together. This approach has a fundamental, yet-to-be-fully-understood, flaw: during freeze-drying and reconstitution, the primers and polymerase are in prolonged contact. Furthermore, some components of the freeze-drying protectant (such as oligosaccharides) may affect the onset temperature of non-specific amplification, easily triggering template-independent primer self-amplification. This can lead to false-positive amplification signals in the absence of target nucleic acids, severely interfering with the interpretation of detection results, particularly in the detection of complex background matrices such as wastewater. Currently, there is no mature technical solution to systematically solve the above-mentioned false positive problem of mixed freeze-drying through freeze-drying process design, nor is there an integrated microfluidic detection platform designed for wastewater virus monitoring scenarios that combines room temperature storage stability and semi-quantitative detection capabilities. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies for viral nucleic acid detection in wastewater and environmental samples, such as cold chain dependence, qualitative interpretation only, false positives caused by mixed freeze-drying, and complex operation. This invention provides a semi-quantitative RT-LAMP method for viral nucleic acid detection based on dispensed freeze-dried microspheres and a multi-channel microfluidic chip, along with a matching reagent kit. This method enables stable room temperature storage of multiple viral targets, on-site use, and parallel semi-quantitative detection, meeting the practical needs of wastewater epidemiological monitoring, port border inspection, and rapid viral nucleic acid detection in resource-constrained scenarios.

[0006] Technical solution 1. Primer composition This invention provides a primer composition for RT-LAMP detection of multiple viral nucleic acids, comprising LAMP primer sets designed for at least two of the following viral target genes. Each primer set contains six oligonucleotides: F3, B3, FIP, BIP, LF, and LB. Two independent primer sets, P1 and P2, are provided for each target gene to improve detection coverage and specificity. (1) Primers for the N gene of the SARS-CoV-2 Omicron mutant strain: OmiN P1 group: F3 primer as shown in 1, B3 primer as shown in 2, FIP primer as shown in 3, BIP primer as shown in 4, LF primer as shown in 5, LB primer as shown in 6; OmiN P2 group: F3 primer as shown in 7, B3 primer as shown in 8, FIP primer as shown in 9, BIP primer as shown in 10, LF primer as shown in 11, LB primer as shown in 12; (2) Zika virus NS5 gene primers: Zika-NS5 P1 group: F3 primer as shown in 13, B3 primer as shown in 14, FIP primer as shown in 15, BIP primer as shown in 16, LF primer as shown in 17, LB primer as shown in 18; Zika-NS5 P2 group: F3 primer as shown in 19, B3 primer as shown in 20, FIP primer as shown in 21, BIP primer as shown in 22, LF primer as shown in 23, LB primer as shown in 24; (3) Primers for the M1 gene of H1N1 influenza virus: H1N1 M1 P1 group: F3 primer as shown in 25, B3 primer as shown in 26, FIP primer as shown in 27, BIP primer as shown in 28, LF primer as shown in 29, LB primer as shown in 30; H1N1 M1 P2 group: F3 primer as shown in 31, B3 primer as shown in 32, FIP primer as shown in 33, BIP primer as shown in 34, LF primer as shown in 35, LB primer as shown in 36; (4) Primers for the NP gene of H3N2 influenza virus: H3N2 NP P1 group: F3 primer as shown in 37, B3 primer as shown in 38, FIP primer as shown in 39, BIP primer as shown in 40, LF primer as shown in 41, LB primer as shown in 42; H3N2 NP P2 group: F3 primer as shown in 43, B3 primer as shown in 44, FIP primer as shown in 45, BIP primer as shown in 46, LF primer as shown in 47, LB primer as shown in 48.

[0007] The above primer combinations can be used for the detection of a single viral target, or two or more viral primer sets can be combined to achieve joint detection of multiple viruses.

[0008] 2. Lyophilized microsphere composition This invention provides a lyophilized microsphere composition for RT-LAMP detection, characterized by comprising two independently packaged types of lyophilized microspheres: a premixed lyophilized microsphere, prepared by mixing a thermostable DNA polymerase (Bst DNA polymerase or a variant thereof) containing reverse transcriptase activity, a mixture of deoxyribonucleoside triphosphates (dNTPs), a magnesium ion source, an amplification enhancer, and a visualization indicator dye with a lyophilization protectant, followed by liquid nitrogen rapid freezing and programmed lyophilization; the lyophilization protectant comprises a polyhydroxy oligosaccharide and a polymer, wherein the polyhydroxy oligosaccharide is selected from at least two of trehalose, sucrose, mannitol, and cyclodextrin, and the polymer is selected from polyethylene glycol (PEG) with a molecular weight range of 40 kDa-70 kDa; Primer lyophilized microspheres are prepared by mixing LAMP primer mixtures designed for specific viral target genes with a lyophilization protectant, followed by liquid nitrogen rapid freezing and programmed lyophilization. The premixed lyophilized microspheres and primer lyophilized microspheres exist independently in solid form throughout the preparation and storage process. They are placed in dry solid partitions within the channel and are only reconstituted and mixed together to initiate the reaction after the addition of liquid sample. This avoids long-term non-specific interactions between primers and polymerase under liquid coexistence conditions.

[0009] In a preferred embodiment, the volume ratio of the premixed solution to the lyophilized microspheres is 15-20:3; and the volume ratio of the primer mixture to the lyophilized microspheres is 2-5:1.

[0010] In a preferred embodiment, the freeze-drying protectant is composed of the following components in parts by weight: 3-5 parts trehalose, 3-5 parts sucrose, 0.2-0.6 parts mannitol, 0.2-0.6 parts cyclodextrin, 2-4 parts PEG (molecular weight 40k-70kDa), and 3-5 parts water.

[0011] 3. Preparation method of freeze-dried microspheres This invention provides a method for preparing the above-mentioned freeze-dried microspheres, comprising the following steps: (1) Preparation of protective agent: According to the formula, trehalose, sucrose, mannitol, cyclodextrin and polyethylene glycol are added to an appropriate amount of water for injection, stirred and dissolved at 40-60℃, filtered through a 0.22 μm sterile filter membrane, and stored at 4℃ for later use; (2) Preparation and mixing of premix: According to the component ratio of the RT-LAMP reaction system, add buffer, MgSO4, dNTPs, betaine amplification enhancer, SYBR-Green visualization dye and Bst DNA polymerase in sequence, and mix thoroughly to obtain the premix; mix the premix with the lyophilization protectant at a volume ratio of 15-20:3, and operate on ice. All operations are carried out in an environment free from nucleic acid contamination. (3) Preparation and mixing of primer mixture: Mix the six primers F3, B3, FIP, BIP, LF and LB according to the final concentration ratio to obtain primer mixture; mix the primer mixture with the freeze-drying protectant at a volume ratio of 2-5:1 until homogeneous; (4) Quick-freezing in liquid nitrogen: The premix containing the protectant and the primer mixture containing the protectant are respectively added dropwise to liquid nitrogen using a quantitative pipette, keeping the droplets spherical, and the quick-freezing time is not less than 60 seconds to form the premix freeze-dried microsphere precursor and the primer freeze-dried microsphere precursor. (5) Freeze-drying: Quickly transfer the quick-frozen spheres to a freeze dryer pre-cooled to -45°C and freeze-dry according to the following program: Pre-freezing stage: -45°C for 120 minutes; Freeze-sublimation stage: -45°C for 120 minutes, raise the temperature to -35°C for 300 minutes, raise the temperature to -25°C for 360 minutes, with a vacuum degree below 10 Pa; Desorption-sublimation stage: raise the temperature to 25°C for 60 minutes, raise the temperature to 30°C for 120 minutes; After removal from the freezer, repackage and seal in a dry environment and store at 15-30°C away from light. (6) Pre-place the premixed liquid lyophilized microspheres and primer lyophilized microspheres into the corresponding channels of the multi-channel microfluidic chip and seal them for preservation.

[0012] 4. Semi-quantitative detection method The core technical solution of this invention is a semi-quantitative detection method for viral nucleic acids using RT-LAMP based on a multi-channel microfluidic chip and lyophilized microspheres, comprising the following steps: (1) Sample pretreatment: Nucleic acid extraction is performed on the sample to be tested (wastewater concentrate, clinical sample or environmental water sample) to obtain nucleic acid extract containing viral RNA, which is used as the original nucleic acid solution (CO). (2) Gradual dilution: The original nucleic acid solution C0 is continuously diluted with dilution factor n (n is an integer from 2 to 10, preferably 10) to prepare at least 4 gradient concentrations of diluted samples, denoted as C0, C1, C2, C3...C_(k-1), for a total of k concentration gradients. The conversion relationship between the concentration of the diluted sample in each channel and the concentration of the original nucleic acid solution C0 is as follows: Where n is the dilution factor, j is the channel number (j=1,2,...,k); C1=C0 (undiluted), C2=C0 / n, .

[0013] (3) Add samples to the multi-channel microfluidic chip. Take a multi-channel microfluidic chip containing premixed lyophilized microspheres and primer lyophilized microspheres. The total number of chip channels N≥2. Add samples diluted at each gradient concentration to N-1 detection channels respectively. Add a negative control or positive control to the Nth channel. Seal the chip after adding the samples. (4) Isothermal amplification: The sealed microfluidic chip is placed in an isothermal heating device and reacted at 50-72℃ for 15-60 minutes to rehydrate the lyophilized microspheres in each channel and start the RT-LAMP amplification reaction. (5) Visual signal interpretation After the reaction is completed, the color change of each channel is read by visual observation or portable fluorescence detector. When SYBR-Green is used as indicator dye, the negative channel is yellow and the positive channel is yellow-green to green. When turbidity is used as the interpretation method, the positive channel is obviously white and turbid. Record the positive ("+") or negative ("-") result of each channel. (6) Determination of semi-quantitative concentration range Record the positive (+) or negative (-) results for each detection channel (arranged in ascending order of dilution factor) to determine the positive-to-negative inflection point; if the j-th detection channel (corresponding to dilution factor) The result was positive and the (j+1)th detection channel (corresponding to the dilution factor) was [missing information]. If the result is negative, then the concentration C0 of the target virus nucleic acid in the original nucleic acid solution is determined to meet the following conditions: C j+1 <C0≤ C j Where C j For the dilution concentration corresponding to the j-th channel, C (j+1) C0 represents the dilution concentration corresponding to the (j+1)th channel. If all detection channels are positive, C0 is higher than the detectable range of the highest dilution channel, and further dilution and retesting are required. If all detection channels are negative, C0 is lower than the detection limit.

[0014] 5. Ready-to-use detection unit encapsulated with lyophilized microspheres The present invention also provides a ready-to-use detection unit, comprising: a multi-channel microfluidic chip having N independent reaction channels (N≥2, preferably N=6), each channel pre-filled with premixed liquid lyophilized microspheres and primer lyophilized microspheres, the two types of microspheres being placed in separate zones within the channel; the chip is sealed with a sealing film for the sample application port, and a waterproof and breathable film covers the venting port, and the entire unit is encapsulated in a moisture-proof aluminum foil bag containing a desiccant; the detection unit has a shelf life of not less than 7 days, preferably not less than 30 days, when stored at 15-30℃.

[0015] 6. Test kit This invention provides a kit for semi-quantitative detection of nucleic acids of various viruses, comprising: (1) at least one ready-to-use multichannel microfluidic chip detection unit encapsulated with dispensed lyophilized microspheres; (2) a gradient dilution solution for continuous gradient dilution of sample nucleic acid extract; (3) a positive control containing a known concentration of synthetic target gene RNA or inactivated virus particles; (4) a negative control containing a blank matrix solution without target gene nucleic acid; (5) an instruction manual containing the operation procedure, interpretation rules and concentration range conversion table; and (6) optional accessories: a portable constant temperature heating device and a visual fluorescence reading module.

[0016] Beneficial effects (1) Aliquoting and lyophilization fundamentally eliminates false positives and ensures the reliability of detection. In this invention, the premixed solution containing Bst DNA polymerase and LAMP primers are lyophilized separately into microspheres. The two types of microspheres are completely isolated before reconstitution, avoiding prolonged contact between the polymerase and primers during lyophilization, storage, and reconstitution. This fundamentally eliminates the conditions for template-independent primer self-amplification. Comparative experiments have shown that the false positive rate of the aliquoting and lyophilization method is significantly lower than that of the mixed lyophilization method, which is particularly prominent in the detection of complex matrix samples such as wastewater. This effectively ensures the specificity and reliability of the detection results and solves the core pain point that has long existed in the existing lyophilized LAMP reagent technology.

[0017] (2) Stable storage and transportation at room temperature, completely eliminating reliance on cold chain. Under the protection of an optimized freeze-drying protectant formulation (trehalose, sucrose, mannitol, cyclodextrin, and high molecular weight PEG composite system), the bioactivity of Bst DNA polymerase and primers can be stably maintained for at least 7 days at 15-30℃, and the stability of the optimized formulation can reach more than 30 days. The entire detection system does not require cold chain storage and transportation or refrigeration equipment, which greatly reduces logistics costs and the threshold for on-site deployment. This allows the detection system to operate independently in scenarios where cold chain resources are scarce, such as border inspection stations, sewage treatment plants, field monitoring points, and grassroots community health institutions, greatly expanding the geographical scope of application for viral nucleic acid detection.

[0018] (3) Semi-quantitative detection capability breaks through the technical bottleneck of existing LAMP systems, which can only provide qualitative interpretation. This invention achieves semi-quantitative detection of RT-LAMP reaction by performing gradient dilution and parallel amplification of the same sample on a multi-channel microfluidic chip, and directly calculating the range of nucleic acid concentration of the sample by utilizing the positive and negative inflection points of each channel. This overcomes the fundamental limitation of existing microfluidic LAMP systems, which can only provide qualitative results. This semi-quantitative capability has special value for wastewater virus monitoring, as it can dynamically track the changing trend of viral load in sewage and provide key information on concentration dimension for public health early warning. No existing commercial microfluidic LAMP product has this function.

[0019] (4) Joint detection of multiple viral targets, obtaining information on multiple pathogens in a single operation. This invention targets 8 target gene regions of 4 viruses, including the N gene of SARS-CoV-2 Omicron strain, the NS5 gene of Zika virus, the M1 gene of H1N1 influenza virus, and the NP gene of H3N2 influenza virus. Two independent LAMP primer sets, P1 and P2 (a total of 48 primers, 1-48), were designed for each virus. Each primer set has undergone rigorous specificity verification and has a low cross-reactivity rate. With the parallel amplification capability of the multi-channel microfluidic chip, multiple viral targets can be detected simultaneously in the same detection unit. This is especially suitable for complex epidemiological monitoring scenarios where multiple pathogens coexist in wastewater, significantly improving detection throughput and saving sample volume.

[0020] (5) The operation is extremely simplified, fully meeting the requirements of point-of-care testing (POCT). This invention encapsulates all the reagents (enzymes, substrates, primers, dyes) required for the RT-LAMP reaction in the form of pre-prepared lyophilized microspheres within the chip channel. Users only need to complete four steps: "sample addition - sealing - temperature control - interpretation". The entire process does not require on-site preparation of the reaction system, and the operation time is shortened from several hours in traditional methods to within 30-60 minutes. Moreover, only a simple portable temperature control device is required, and the results can be visually interpreted without the need for professional instruments such as quantitative PCR instruments. The above characteristics make this invention fully meet the core requirements of point-of-care testing for "simplicity, speed, low cost, and on-site operability", and can be independently completed by non-professionals with simple training.

[0021] (6) Dual primer set design improves detection coverage and enhances resistance to mutation. Two independent LAMP primer sets, P1 and P2, are designed simultaneously for each viral target gene. The two sets of primers target different segments of the same gene. When the performance of one set of primers deteriorates due to changes in the primer binding site caused by viral mutation, the other set of primers can still maintain effective amplification, thereby significantly improving the robustness of the detection system against viral variants. This design strategy is particularly important for viruses with high mutation rates, such as SARS-CoV-2 Omicron, as it can effectively extend the lifespan of the primer set and reduce the risk of missed detection due to viral mutation.

[0022] (7) The microfluidic chip packaging design ensures operational safety and reduces the risk of aerosol contamination. The entire amplification reaction is carried out in the closed microfluidic chip channel. After the reaction, there is no need to open the cover to read the product. The product can be directly visualized through the outer wall of the chip or detected by fluorescence through the transparent cover. This avoids aerosol diffusion and cross-contamination in the laboratory caused by opening the tube of amplification product, ensures the biosafety of the operator, and effectively prevents false positive interference of amplification product in subsequent detection. Attached Figure Description

[0023] Figure 1 Top view of a multi-channel microfluidic chip.

[0024] Figure 2 This invention presents a schematic diagram of the entire process for preparing lyophilized microspheres. It sequentially illustrates the steps of premix preparation, primer mixture preparation, mixing of each with a lyophilization protectant, rapid freezing into microspheres in liquid nitrogen, programmed lyophilization (including temperature-time curves for three stages: pre-freezing, freeze-sublimation, and desorption-sublimation), and final packaging and sealing of the finished microspheres.

[0025] Figure 3 : Schematic diagram of operation process and result interpretation. Figure 3 The color change diagram of the semi-quantitative detection result interpretation channel shows the positive and negative transition patterns corresponding to different concentrations of four viruses (from left to right: SARS-CoV-2 Omicron, Zika, H1N1, H3N2).

[0026] Figure 4: Figure 4A , 4B Figures 4C and 4D show the RT-LAMP real-time fluorescence amplification curves for each primer set (P1 and P2) of four viruses (SARS-CoV-2 Omicron, Zika, H1N1, and H3N2). Cycle number is plotted on the horizontal axis, and fluorescence value (RFU) is plotted on the vertical axis. This displays the typical amplification curve of the positive template corresponding to the target gene for each primer set, as well as the flat baseline of the negative control (no template control, NTC). It visually presents the amplification efficiency (Tp value) of each primer set and its difference from NTC, validating the effectiveness of the primer design.

[0027] Figure 5: Freeze-dried microspheres before being placed at room temperature for one week ( Figure 5A )back( Figure 5B Morphological comparison chart.

[0028] Figure 6: Visualization results of isothermal amplification of SARS-CoV-2 Omicron RNA at different dilution gradients after rehydration of lyophilized microspheres (Figure 6: Visualization results of isothermal amplification of SARS-CoV-2 Omicron RNA at different dilution gradients) Figure 6A ) and fluorescence change curve ( Figure 6B ). Detailed Implementation

[0029] The following examples are used to illustrate the technical solutions of the present invention, but do not constitute a limitation on the scope of protection of the present invention. Unless otherwise specified, the reagents and instruments used in the examples are all commercially available conventional products. All experimental data are expressed as mean ± standard deviation (Mean ± SD), and the number of replicates n ≥ 3.

[0030] Example 1: Using the N gene sequence of the SARS-CoV-2 Omicron variant (representative strains BA.2 / BA.4 / BA.5), the NS5 gene sequence of the Zika virus Asian lineage, the M1 gene sequence of the H1N1 influenza virus (pdm09 strain), and the NP gene sequence of the H3N2 influenza virus (strains circulating in the last three years) from the NCBI database as templates, LAMP primers were automatically designed using PrimerExplorer V5 software. Conserved regions were screened as primer binding sites using multiple sequence alignment (MUSCLE algorithm). Two independent primer sets, P1 and P2 (a total of 48 primers, 1-48), were designed for each target gene. Primer synthesis was performed by a third-party synthesis company, and the primers were purified at the HPLC level.

[0031] The RT-LAMP reaction system (total volume 25 μL) included: 2.5 μL of 10×ThermoPol buffer, MgSO4 (final concentration 8 mmol / L), dNTPs mixture (final concentration of each dNTP 1.2 mmol / L), FIP / BIP primers (final concentration of each 1.6 μmol / L), F3 / B3 primers (final concentration of each 0.4 μmol / L), LF / LB primers (final concentration of each 0.8 μmol / L), Bst 2.0 WarmStart DNA polymerase (8 U / μL, final concentration 0.32 U / μL), SYBR-Green I (final concentration 180 nmol / L), betaine (final concentration 0.8 mol / L), RNase-free water added to 25 μL, and finally 2 μL of RNA template added.

[0032] Positive template: SARS-CoV-2 Omicron N gene in vitro transcribed RNA (concentration gradient: 10) 6 10 5 10 4 10 3 10 2 10 1 copies / μL); Zika virus NS5 gene synthesized RNA (same gradient as above); H1N1 M1 gene synthesized RNA (same gradient as above); H3N2 NP gene synthesized RNA (same gradient as above). Negative control: template-free control (NTC, with RNase-free water instead of template).

[0033] Reaction conditions: Amplification at 63℃, real-time fluorescence detection mode, fluorescence values ​​read every 30 seconds, for a total of 90 reads (45 minutes). Amplification efficiency was characterized by the time to peak amplification (Tp value, i.e., the time corresponding to the maximum slope of the fluorescence curve); a smaller Tp value indicates faster amplification. Instrument: Bio-Rad CFX96 real-time quantitative PCR instrument (isothermal mode).

[0034] The results are shown in the table below. The results indicate that the eight LAMP primer sets (1-48) designed in this invention all exhibited highly efficient and specific amplification performance for their respective target genes, with a detection limit of 10. 1 -10 2 copies / μL, NTC all negative. Each primer set was then compared with positive RNAs from three other viruses (10 copies / μL). 6 Cross-reaction tests were performed using primers (copies / μL), and the results showed that there was no cross-amplification in any primer set, with a specificity of 100% (n=3), meeting the requirements for multi-virus joint detection.

[0035] Table 1-1: Amplification performance of SARS-CoV-2 Omicron OmiN P1 and P2 groups (n=3, Mean±SD)

[0036] Table 1-2: Amplification performance of Zika NS5 P1 and P2 groups (n=3, Mean±SD)

[0037] Table 1-3: Amplification performance of each primer set for H1N1 M1 and H3N2 NP (n=3, Mean±SD, calculated as 10⁻⁶). 4 (Copies / μL represent the concentration)

[0038] Example 2:

[0039] Preparation of freeze-drying protectant: Weigh 4.0g of trehalose, 4.0g of sucrose, 0.4g of mannitol, 0.4g of β-cyclodextrin, and 3.0g of PEG (molecular weight 40kDa). Add 4.0mL of water for injection and stir in a 50℃ water bath until completely dissolved. Filter through a 0.22 μm sterile filter membrane and dispense into sterile EP tubes. Store at 4℃ for later use.

[0040] Premix preparation: Under ice bath conditions, add the following to a sterile EP tube sequentially: 10×ThermoPol buffer, MgSO4 solution (final concentration 8 mmol / L), dNTPs mixture (1.2 mmol / L each), betaine (0.8 mol / L), SYBR-GreenI (180 nmol / L), and Bst 2.0 WarmStart DNA polymerase (8 U / μL). Mix gently, avoiding the formation of air bubbles. Combine the premix with the lyophilization protectant at a volume ratio of 18:3, gently invert to mix 5 times, and operate on ice.

[0041] Primer mixture preparation: Mix F3 / B3 (final concentration 0.4 μmol / L each), FIP / BIP (1.6 μmol / L each), and LF / LB (0.8 μmol / L each) in the specified proportions to obtain the primer mixture; mix the primer mixture with the lyophilization protectant at a volume ratio of 3:1.

[0042] Liquid nitrogen flash freezing: Above a Dewar flask filled with liquid nitrogen, use a 10 μL pipette to drop the premixed solution containing the cryoprotectant drop by drop (approximately 10 μL per drop) onto the liquid nitrogen surface. After the droplets have completely frozen into regular spherical shapes (approximately 90 seconds), transfer them to a pre-cooled centrifuge tube using pre-cooled tweezers. Process the primer mixture in the same way. The two liquid handling procedures must be strictly separated to avoid cross-contamination.

[0043] Freeze-drying program: Place centrifuge tubes containing spheres (without caps) on the sample rack of the freeze dryer and run the following program: ① Pre-freeze: -45℃, 120 minutes; ② First drying (freeze-sublimation): -45℃ / 120 minutes → -35℃ / 300 minutes → -25℃ / 360 minutes, maintain vacuum ≤8Pa; ③ Second drying (desorption-sublimation): 25℃ / 60 minutes → 30℃ / 120 minutes; ④ After the program is completed, purge with dry nitrogen to break the vacuum, remove the sample in a clean workbench with relative humidity <20%, immediately aliquot into aluminum foil sealed bags, add desiccant, heat seal, and store at room temperature (22±2℃).

[0044] Activity assay: Take a portion each of the premixed solution / primer mixture before lyophilization (control group) and the lyophilized microspheres (experimental group), add the same volume of RNase-free water to reconstitute the lyophilized microspheres, and add the remaining missing components according to the system described in Example 1 (no addition is required for the control group before lyophilization). Add SARS-CoV-2 Omicron N gene RNA (10 μL) uniformly. 4 Using copies / μL as templates, react at 63℃ for 45 minutes and record the Tp value.

[0045] The experimental results are as follows: Table 2-1: Comparison of RT-LAMP amplification activities before and after freeze-drying (n=3, Mean±SD)

[0046] The difference in Tp values ​​before and after freeze-drying was 0.9 minutes (<1 minute), and the difference was not statistically significant after paired t-test (p=0.21>0.05). The retention rate of fluorescence intensity at the endpoint was 95.9%±3.4%, indicating that the freeze-drying process had no significant effect on the LAMP reaction activity.

[0047] The freeze-drying process described in this invention (liquid nitrogen quick-freezing + programmed freeze-drying + composite protective agent formulation) can effectively protect the biological activity of Bst DNA polymerase and primers. After freeze-drying, the amplification performance of the microspheres after reconstitution is comparable to that of the freshly prepared reagents. No false positives occurred during the freeze-drying process, which verifies the reliability of the preparation process.

[0048] Example 3:

[0049] The lyophilized microspheres prepared according to Example 2 (premixed microspheres and primer microspheres were aliquoted into aluminum foil sealed bags containing desiccant) were placed at room temperature (22±2℃, relative humidity 40-60%, protected from light) for 0, 3, 7, 14, and 30 days, respectively. Samples were taken at each time point, reconstituted according to the method described in Example 2, and the SARS-CoV-2 Omicron N gene RNA (10T) was detected. 4 The amplification activity of NTC samples (copies / μL) was measured, with Tp value as the primary evaluation indicator. The false positive rate (number of positive results in the NTC group / total number of tests × 100%) was also recorded. Each time point had n=3.

[0050] The experimental results are as follows: Table 3-1: Room temperature stability data of dispensed freeze-dried microspheres (n=3, Mean±SD)

[0051] Using a Tp delay of no more than 5 minutes and an endpoint fluorescence retention rate of ≥80% as the criteria for activity qualification, the lyophilized microspheres still met the qualification criteria after 30 days of storage at room temperature (Tp delay 5.4 minutes, fluorescence retention rate 78.3%, close to the qualification threshold); they fully met the qualification criteria within 7 days (Tp delay ≤1.1 minutes, fluorescence retention rate ≥95%); and they met the strict qualification criteria within 14 days (fluorescence retention rate 89.7%). The overall NTC false positive rate was 0%.

[0052] The freeze-dried microspheres prepared by this invention can be stably stored at room temperature (22±2℃) for no less than 14 days (preferably within 7 days with basically no performance loss), with no false positives occurring throughout the process. This fully verifies the feasibility of storage outside the cold chain and meets the actual needs of on-site testing for room temperature storage and transportation.

[0053] Example 4: Validation of the semi-quantitative detection method The detection chip is a 6-channel microfluidic chip made of PDMS material. Each channel has an effective volume of about 25 μL. Each channel is pre-filled with one premixed lyophilized microsphere (containing Bst polymerase, dNTPs, etc.) and one lyophilized microsphere of SARS-CoV-2 Omicron OmiN P1 primer. The sample well is sealed with a sealing membrane and the vent is covered with a waterproof and breathable membrane.

[0054] Using SARS-CoV-2 Omicron N gene in vitro transcribed RNA standard (concentration C0=10) 5 The original nucleic acid solution (copies / μL) was serially diluted 10-fold with RNase-free water to prepare CO(10) solutions. 5 C1 (10) 4 C2 (10) 3 C3 (10) 2 C4 (10) 1 Five concentration gradients were used. After removing the sealing film, 2 μL of C0 to C4 were added sequentially from channel 1 to channel 5, and 2 μL of negative control (RNase-free water, RNase-free) was added to channel 6. The sample wells were resealed, and the microspheres were gently centrifuged (500×g, 30 seconds) to ensure sufficient contact between the sample and the lyophilized microspheres. The chip was placed on a flatbed thermostat and incubated at 63℃ for 35 minutes. After the reaction, the color of each channel was visually observed under a standard white LED light box. SYBR-Green was used as the indicator dye; positive channels appeared yellow-green, and negative channels appeared yellow. The results were recorded using a portable 470nm excitation light source. The positive and negative results for each channel were recorded, and the inflection point from positive to negative (i.e., the last positive channel) was identified. The original sample concentration range was determined as [last positive channel concentration × 10, last positive channel concentration × 100]. The experiment was repeated three times (n=3).

[0055] The experimental results are as follows: Table 4-1: Semi-quantitative detection results of 6-channel chip (n=3, results of each channel in each experiment)

[0056] Note: "+" indicates positive (yellow-green), "-" indicates negative (yellow).

[0057] The turning point for the first and second tests is located in channel 5 (10). 1 Between copies / μL) and channel 6 (NTC), according to the judgment rule C0, C satisfies C j+1 <C0≤ C j Channel 5 (C) j = 10 1 (copies / μL) positive and channel 6 (C) (j+1)= NTC (meaning below the detection limit) negative, indicating that the detection limit of this detection system under these experimental conditions is approximately 10. 1 copies / μL, original sample C0 concentration ≥10 5 The determination of copies / μL is consistent with the actual concentration. The turning point of the third detection is located in channel 4 (10 2 copies / μL) and channel 5 (10 1 Between copies / μL), according to the judgment rules, channel 4 (C) j = 10 2 (copies / μL) positive and channel 5 (C) (j+1) =10 1 If the test result is negative (copies / μL), the detection limit is 10. 1 Up to 10 2 The range between copies / μL is within the normal detection fluctuation at this concentration gradient point (10). 1 (Copies / μL are at the edge of detection sensitivity), but the judgment result still matches the actual concentration.

[0058] This technique can accurately determine the positive or negative results of each dilution gradient channel, and the results from three experiments show good internal consistency (the detection limit for two / three experiments is 10). 1 -10 2 copies / μL, 1 / 3 of the time is 10 1 ~10 2 (copies / μL boundary), semi-quantitative detection dynamic range covers 10 1 Up to 10 5 The results, with copies / μL (spanning four orders of magnitude) and no false positives throughout the process (no negative controls), fully validated the accuracy and stability of this semi-quantitative method. The Tp value showed a good linear relationship with increasing dilution factor (Rp). 2 =0.992), confirming the quantitative correlation between the detection signal and the template concentration.

[0059] The semi-quantitative detection method described in this invention can accurately determine the concentration range of viral nucleic acid in the sample by using a gradient dilution and parallel amplification strategy on a 6-channel microfluidic chip, with a detection dynamic range covering 10. 1 Up to 10 5 The results of three independent experiments showed good consistency across copies / μL (spanning four orders of magnitude), and the negative control throughout the process showed no false positives, validating the accuracy and repeatability of the semi-quantitative detection method.

[0060] Example 5:

[0061] A 6-channel microfluidic chip made of PDMS material is used, with an effective volume of approximately 25 μL for each channel. Each channel is pre-filled with one premixed lyophilized microsphere (containing Bst DNA polymerase, dNTPs, MgSO4, betaine, and SYBR-Green I) and one lyophilized microsphere of Zika-NS5 P1 primers. The sample well is sealed with a sealing membrane, and the vent is covered with a waterproof and breathable membrane.

[0062] RNA standards were synthesized using the Zika NS5 gene (initial concentration C0 = 10). 4 Starting with copies / μL, the solution was prepared by serially diluting RNase-free water at 10-fold gradients using RNase-free water: Channel 1 (10 copies / μL) 4 copies / μL), Channel 2 (10 copies / μL), Channel 2 (10 3 copies / μL), channel 3 (10 copies / μL), channel 3 (10 2 copies / μL), channel 4 (10 copies / μL), channel 4 (10 1 Four concentration gradients were used (copies / μL), with channel 5 serving as a negative control (NTC, RNase-free water) and channel 6 as a blank control, for a total of five detection channels, including one NTC channel. The sealing film was removed, and 2 μL of the corresponding concentration diluted sample was added sequentially to channels 1 through 4. 2 μL of RNase-free water was added to channel 5. The sample wells were resealed, and the samples were gently centrifuged (500×g, 30 seconds). The chip was placed on a flatbed thermostat and incubated at 63℃ for 35 minutes. After the reaction, the samples were visually observed under a standard white LED light box, and photographed using a portable 470 nm excitation light source. The SYBR-Green positive channel appeared yellow-green, and the negative channel appeared yellow. The experiment was independently repeated three times (n=3).

[0063] The experimental results are shown in the table below: Table 5-1 Comparison of semi-quantitative detection results for Zika samples (n=3)

[0064] Note: "+" indicates a positive result (yellow-green), and "-" indicates a negative result (yellow). Tp values ​​show a significant linear increasing trend with increasing dilution factor (Rp). 2 =0.995, n=3), compared with the Zika primer data in Table 1-2 of Example 1 (Zika P1 group: 10 4 →11.8±0.5min, 10 3 →17.9±0.8 min, 10 2 →27.1±1.3 min, 10 1 →35.8±2.4 min) The high internal consistency verified the equivalence between the freeze-dried microspheres and the liquid-phase reaction system.

[0065] The first and second tests were positive in channels 1-4, and negative in channel 5 (NTC). The turning point was in channel 4 (10). 1 Between channels 1 and 2 (copies / μL) and channel 5 (NTC), the inflection point was in channel 4 (C4=10). 1 Between copies / μL) and channel 5 (NTC), according to C j+1 <C0≤ C j , judge 10 1 <C0≤ 10 4 copies / μL, compared to the actual spiked concentration of 10 4 Copies / μL match. Third test, channel 4 (10...) 1 The result was negative for copies / μL, with the inflection point in channel 3 (10 copies / μL). 2 copies / μL) and channel 4 (10 1 The detection limit is determined to be between (copies / μL) and (10 copies / μL). 1 ~10 2 The copies / μL boundary is within the normal range of detection fluctuations.

[0066] The semi-quantitative detection method using lyophilized microspheres and multi-channel microfluidic chips described in this invention is also applicable to the semi-quantitative detection of the Zika virus NS5 gene. The lyophilized microspheres containing Zika-NS5 P1 primers exhibited a stable gradient amplification pattern in a 6-channel chip, with a significant linear relationship between the Tp value and the logarithm of the template concentration (Rp). 2 =0.995), dynamic range coverage 10 1 Up to 10 4 The results showed no false positives for NTC throughout the entire process, validating the universality and semi-quantitative detection capability of this method on a variety of viral targets.

[0067] Example 6:

[0068] Secondary effluent from a municipal wastewater treatment plant (taken from the effluent outlet after the aerated grit chamber and before the membrane bioreactor) was collected on-site. Large particulate impurities were removed by coarse filtration through a 0.22 μm membrane. The samples were then aliquoted and stored at 4℃. The experiment was completed within 24 hours. RT-qPCR and NTC validation using this method confirmed that the SARS-CoV-2 Omicron nucleic acid test result in the original wastewater sample was negative (CT value > 40, considered negative).

[0069] SARS-CoV-2 Omicron N gene in vitro transcribed RNA standard was artificially added to wastewater samples to prepare a final concentration of 10. 3Spiked positive samples of wastewater were collected at 1000 copies / μL; wastewater without added standards was retained as a negative control. Both samples underwent the following pretreatment: heat inactivation at 95℃ for 5 minutes, followed by cooling on ice, and nucleic acid extraction using a magnetic bead nucleic acid extraction kit (ZR Viral RNA Kit). The elution volume was 50 μL (approximately 10-fold concentrated), and the extracted nucleic acid solution was used as the original nucleic acid solution. Concentration conversion path: final concentration of wastewater spiked 10... 3 Copies / μL × Magnetic bead extraction concentration factor 10 × (50 μL wastewater → 5 μL elution buffer) = Nucleic acid extraction buffer C0 = 10 4 copies / μL; After a 10-fold serial dilution of C0, the concentrations of each channel were: Channel 1 (10 copies / μL); 4 ), Channel 2 (10) 3 ), Channel 3 (10) 2 ), Channel 4 (10) 1 (copies / μL).

[0070] A 6-channel microfluidic chip containing lyophilized microspheres of SARS-CoV-2 Omicron OmiN P1 primers was used to perform 10-fold serial dilutions of nucleic acid extraction solution spiked in wastewater (10... 4 →10 3 →10 2 →10 1 The chip has four detection channels (copies / μL), one NTC channel, and one chip blank channel. The reaction is carried out at 63℃ for 35 minutes. A pure water matrix control group (RNase-free water with the same spiked sample concentration and identical pretreatment) is also included, and samples are detected on a separate chip under the same conditions. Each group is independently replicated three times (n=3).

[0071] The results are as follows: Table 6-1: Comparison of semi-quantitative detection results of spiked samples with wastewater matrix and pure water matrix (n=3)

[0072] Table 6-2: Tp values ​​for each positive channel (wastewater matrix, n=3, Mean±SD)

[0073] Positive samples of wastewater matrix spiked in channel 1 (10) 4 ), Channel 2 (10) 3 ), Channel 3 (10) 2 All were positive (3 / 3), channel 4 (10) 1 Both channel 3 and channel 4 (NTC) were negative (0 / 3), with the inflection point located between channel 3 and channel 4. The semi-quantitative determination concentration range was 10.2 ≤ C0<10 3 copies / μL, compared to the actual spiked concentration (approximately 10 copies / μL in the nucleic acid extract). 4 The values ​​(copies / μL, corresponding ranges after dilution in 4 channels) were consistent. All channels of the wastewater negative sample (unspecified) were negative, indicating 100% specificity. Compared to the pure water matrix control group: channels 1-4 of the pure water control were positive, with channel 4 (10 μL) showing the highest positive result. 1 The presence of copies / μL also indicates that the wastewater matrix reduces the detection sensitivity by approximately one order of magnitude (the lowest detection value for the wastewater group is 10). 2 copies / μL vs. pure water group: minimum detection 10 1 The Tp value was delayed by about 2 to 3 minutes at the same concentration, indicating that environmental inhibitors such as humic acid and heavy metals in the wastewater have a certain matrix effect on the RT-LAMP reaction.

[0074] The semi-quantitative detection method using lyophilized microspheres and multi-channel microfluidic chips described in this invention can still achieve semi-quantitative detection of SARS-CoV-2 Omicron nucleic acid in real municipal wastewater matrix. The wastewater matrix reduces the detection sensitivity by approximately one order of magnitude compared to pure water matrix (due to matrix effects such as humic acid and heavy metals), but it can still achieve 10... 2 The semi-quantitative concentration range determination at the copy / μL level meets the practical needs of wastewater epidemiological monitoring for judging viral load trends; the false positive rate of wastewater negative samples is 0%, and the aliquoting and freeze-drying strategy is also effective in eliminating non-specific amplification in complex wastewater matrices. This experiment verifies the feasibility and practical value of this invention in real wastewater monitoring applications.

[0075] Comparative Example 1: Mixed lyophilized group (control group): The LAMP premix (containing Bst polymerase, dNTPs, MgSO4, SYBR-Green, and betaine) was directly mixed with the SARS-CoV-2 Omicron OmiN P1 primer mixture, and then a lyophilization protectant (same formula as in Example 2, with the volume ratio calculated according to the equivalence principle) was added. The mixture was then rapidly frozen in liquid nitrogen and lyophilized according to the same lyophilization procedure as in Example 2 to obtain mixed lyophilized microspheres.

[0076] Freeze-dried group (experimental group): Following the method described in Example 2, the premixed solution and primer mixture were freeze-dried separately into microspheres, and then combined into the same reaction system upon reconstitution.

[0077] False positive rate determination: For each group, lyophilized microspheres (stored at room temperature for 0 days and 7 days) were reconstituted and RNase-free water was added as a "template" (targetless nucleic acid). The mixture was reacted at 63℃ for 45 minutes, and the number of positive results (Tp value < 45 minutes or endpoint color turning green) was counted. n=6 per time point for each group.

[0078] Meanwhile, the fluorescence baseline at 0 days of storage of the lyophilized microspheres was used as the negative determination threshold to assess the occurrence of false positives in both groups.

[0079] The experimental results are shown in Table 7 below. Table 7-1: Comparison of false positive rates between mixed lyophilization and repackaged lyophilization (n=6, Mean±SD)

[0080] With prolonged storage time, the template-independent amplification (TIA) in the mixed lyophilized group continued to accumulate in the mixed lyophilized group, and the false positive rate gradually increased from 16.7% at day 0 to 50.0% at day 7, 66.7% at day 14, and reached as high as 83.3% (5 / 6) at day 30. Furthermore, the false positive Tp value significantly decreased with prolonged storage time (from 41.2 min to 28.1 ± 2.9 min), suggesting that the primer-polymerase nonspecific complex continued to accumulate over time under solid-state coexistence conditions. The aliquot lyophilized group maintained a stable false positive rate of 0% at all four time points (days 0, 7, 14, and 30), showing a highly significant difference from the mixed lyophilized group (Fisher's exact test p < 0.001 at each time point). These data fully demonstrate that the aliquot lyophilization strategy is the fundamental technical means to eliminate false positives in lyophilized LAMP reagents, and its protective effect remains effective throughout a 30-day room temperature storage period, providing solid experimental evidence for the long-term reliability of the lyophilized microspheres of this invention under field deployment conditions.

[0081] Table 7-2: Comparison of specificity of two freeze-drying methods in wastewater matrix samples (n=6, Mean±SD)

[0082] In wastewater matrix negative samples containing inhibitors such as humic acid and heavy metals (verified to be free of viral nucleic acid), the false positive rate of the mixed freeze-drying group was as high as 33.3%, while the false positive rate of the repackaged freeze-drying group was 0%. The detection rate of positive samples in both groups was 100%, proving that repackaged freeze-drying does not affect the detection sensitivity, but only significantly improves the specificity.

[0083] The mixed lyophilization method results in the long-term coexistence of Bst polymerase and primers during lyophilization and storage. With prolonged storage, template-independent amplification significantly increases, and the false positive rate rises from 16.7% to 50.0%, particularly severe in complex wastewater matrix samples. The aliquot lyophilization method described in this invention completely eliminates the aforementioned false positive risk by isolating and lyophilizing the two components separately and then reconstituted and combining them before use. The false positive rate is consistently maintained at 0%, fully validating the technical necessity and innovative value of the aliquot lyophilization strategy.

[0084] Tables 8-1 to 8-4 list the DNA sequences 1 to 48 included in this invention, and the sequence listing is an integral part of this application specification.

[0085] Table 8-1 Primers for the SARS-CoV-2 Omicron variant strain

[0086] Table 8-2 Zika virus NS5 gene primers

[0087] Table 8-3 Primers for the M1 gene of H1N1 influenza virus

[0088] Table 8-4 Primers for the NP gene of H3N2 influenza virus

Claims

1. A semi-quantitative detection method for viral nucleic acids based on RT-LAMP using dispensed lyophilized microspheres and a multi-channel microfluidic chip, characterized in that, Includes the following steps: Step (1) Sample nucleic acid extraction: Nucleic acid is extracted from the sample to be tested to obtain a nucleic acid extract containing viral RNA, which is used as the original nucleic acid solution; Step (2) Gradient dilution: The original nucleic acid solution is serially diluted with a dilution factor n, where the dilution factor n is an integer from 2 to 10, to prepare at least 4 gradient concentrations of diluted samples; Step (3) Sample addition: Take a multi-channel microfluidic chip with N independent reaction channels, where N≥2, and each channel is pre-filled with mutually independent premixed liquid lyophilized microspheres and primer lyophilized microspheres; add the samples diluted at each gradient concentration in step (2) to N-1 detection channels respectively, add a negative control or positive control to the Nth channel, and seal the chip; Step (4) Isothermal amplification: Place the sealed chip in an isothermal device and react at 50-72℃ for 15-60 minutes to reconstitute the premixed lyophilized microspheres and primer lyophilized microspheres in each channel and start the RT-LAMP amplification reaction. Step (5) Signal detection: After the reaction is completed, the amplification signal of each channel is detected to determine the positive or negative result of each channel; Step (6) Semi-quantitative concentration range determination: Record the positive (+) or negative (-) results of each detection channel (arranged from low to high dilution factor) to determine the positive-negative inflection point; If the j-th detection channel (corresponding to the dilution factor) The result was positive and the (j+1)th detection channel (corresponding to the dilution factor) was [missing information]. If the result is negative, then the concentration C0 of the target virus nucleic acid in the original nucleic acid solution is determined to meet the C... j+1 < C0 ≤ C j C j For the dilution concentration corresponding to the j-th channel, C (j+1) The dilution concentration corresponding to the (j+1)th channel is: if all detection channels are positive, then C0 is higher than the detection range of the highest dilution channel and further dilution and retesting are required; if all detection channels are negative, then C0 is lower than the detection limit; wherein the premixed liquid lyophilized microspheres and primer lyophilized microspheres are prepared independently and the two types of microspheres do not come into contact before sample addition in step (3).

2. The method according to claim 1, characterized in that: In step (2), the dilution factor n is 10, and 5-6 gradient concentrations of diluted samples are prepared; in step (3), the total number of channels N of the multi-channel microfluidic chip is 6, of which 5 channels are used to add gradient diluted samples and 1 channel is used to add negative control; in step (4), the isothermal amplification temperature is 60-65℃ and the reaction time is 30-45 minutes.

3. The method according to claim 1, characterized in that: The signal detection method described in step (5) is at least one of the following: visually interpreting the color change using SYBR-Green I as an indicator dye, with the negative channel appearing yellow and the positive channel appearing yellow-green to green; detecting the fluorescence intensity of each channel using a portable fluorescence reader at an excitation wavelength of 450-490nm; or visually or using a turbidimeter to detect the degree of turbidity caused by magnesium pyrophosphate precipitation in each channel; the sample to be tested is at least one of the following: wastewater concentrate, urban sewage, environmental water sample, nasopharyngeal swab eluent, oropharyngeal swab eluent, fecal suspension, serum or plasma.

4. A composition of dispensed lyophilized microspheres for RT-LAMP viral nucleic acid detection, characterized in that, It contains independently packaged premixed lyophilized microspheres and primer lyophilized microspheres. The premixed lyophilized microspheres are prepared by mixing a premix containing a thermostable chain-displacement DNA polymerase, a mixture of deoxyribonucleoside triphosphates, a magnesium ion source, an amplification enhancer, and a visualization indicator dye with a lyophilization protectant, followed by liquid nitrogen rapid freezing and programmed lyophilization. The primer lyophilized microspheres are prepared by mixing a primer mixture containing LAMP primers with a lyophilization protectant, followed by liquid nitrogen rapid freezing and programmed lyophilization. The lyophilization protectant contains polyhydroxy oligosaccharides and a high molecular weight polymer. The polyhydroxy oligosaccharides are selected from at least two of trehalose, sucrose, mannitol, and cyclodextrin, and the high molecular weight polymer is polyethylene glycol with a molecular weight of 40 kDa-70 kDa. The premixed lyophilized microspheres and primer lyophilized microspheres exist independently in solid form throughout the preparation and storage process. They are placed in dry solid partitions within the channel and are only reconstituted and mixed together after the addition of liquid samples to initiate the reaction, thereby avoiding long-term non-specific interactions between primers and polymerase under liquid coexistence conditions.

5. The lyophilized microsphere composition according to claim 4, characterized in that: The lyophilization protectant is composed of the following components by weight: 3-5 parts trehalose, 3-5 parts sucrose, 0.2-0.6 parts mannitol, 0.2-0.6 parts cyclodextrin, 2-4 parts polyethylene glycol (molecular weight 40k-70kDa), and 3-5 parts water for injection; the volume ratio of the premix to the lyophilization protectant is 15-20:3, and the volume ratio of the primer mixture to the lyophilization protectant is 2-5:1; the programmed lyophilization includes the following stages: a pre-freezing stage at -40 to -50°C (preferably -45°C) for 90-150 minutes (preferably 120 minutes); and a freeze-sublimation stage at -40 to -50°C (preferably -45°C) for 90-150 minutes (preferably 120 minutes), followed by a freeze-sublimation stage at -30 to -40°C (preferably -35°C). The reagent is prepared at 240-360 minutes (preferably 300 minutes) and held at -20 to -30℃ (preferably -25℃) for 300-420 minutes (preferably 360 minutes) with a vacuum level below 10 Pa. During the desorption and sublimation stage, the temperature is raised to 20-30℃ (preferably 25℃) and held for 40-80 minutes (preferably 60 minutes), then raised to 25-35℃ (preferably 30℃) and held for 90-150 minutes (preferably 120 minutes). After lyophilization, the reagent is sealed and packaged in an environment with a relative humidity below 20% and stored at 15-30℃ in the dark. After 7 days of storage, the reconstitution amplification peak time (Tp) is delayed by no more than 2 minutes compared to the fresh reagent. After 30 days of storage, the Tp delay is no more than 6 minutes. The false positive rate is 0% throughout the process (independent determination of each batch, n≥6).

6. A primer composition for RT-LAMP detection of various viral nucleic acids, characterized in that, The primer set contains LAMP primers designed for at least two of the following viral target genes. Each primer set contains six oligonucleotides: F3, B3, FIP, BIP, LF, and LB. Each target gene also has two independent primer sets, P1 and P2: (1) Primers for the N gene of the SARS-CoV-2 Omicron mutant strain: The OmiN P1 group contains primers shown in 1-6, and the OmiN P2 group contains primers shown in 7-12; (2) Zika virus NS5 gene primers: Zika-NS5 P1 group contains primers shown in 13-18, and Zika-NS5 P2 group contains primers shown in 19-24. (3) Primers for the M1 gene of H1N1 influenza virus: H1N1 M1 P1 group contains primers shown in 25-30, and H1N1 M1 P2 group contains primers shown in 31-36. (4) Primers for the NP gene of H3N2 influenza virus: H3N2 NP P1 group contains primers shown in 37-42, and H3N2 NP P2 group contains primers shown in 43-48.

7. The primer composition according to claim 6, characterized in that: The primer composition comprises primer sets of all four viruses listed in items (1) to (4) above, for the joint detection of the nucleic acids of the four viruses in the same sample; or comprises primer sets of any two or three viruses listed in items (1) to (4) above, for the joint detection of the corresponding viruses; in the RT-LAMP reaction system, the final concentration range of the primers is 0.2-0.6 μmol / L for each of the F3 and B3 primers, 1.0-2.0 μmol / L for each of the FIP and BIP primers, and 0.4-1.2 μmol / L for each of the LF and LB primers.

8. A detection kit for semi-quantitative detection of nucleic acids from multiple viruses, characterized in that, Include: (1) At least one ready-to-use detection unit, the detection unit comprising a multi-channel microfluidic chip (N≥2) having N independent reaction channels, each channel pre-filled with the premixed liquid lyophilized microspheres and primer lyophilized microspheres as described in claim 4, and the two types of microspheres are placed in separate zones within each channel; (2) Gradient dilution buffer, used for continuous gradient dilution of nucleic acid extract of the sample to be tested; (3) Negative control, blank matrix solution containing no target gene nucleic acid; (4) Positive control, containing known concentrations of target gene synthetic RNA or inactivated virus particles; (5) Instruction manual, including operating procedures, result interpretation rules and concentration range conversion table.

9. The detection kit according to claim 8, characterized in that: The multichannel microfluidic chip is prepared from polydimethylsiloxane (PDMS) and includes 6 sample wells, 6 independent reaction channels, and 6 venting ports. The sample wells are sealed with a sealing membrane, and the venting ports are covered with a waterproof and breathable membrane. The effective reaction volume of each channel is 10-50 μL. The entire chip is packaged in a moisture-proof aluminum foil bag containing desiccant. The LAMP primers pre-placed in the primer lyophilized microspheres are at least one set of the primer composition described in claim 6, used to detect at least one of SARS-CoV-2 Omicron, Zika virus, H1N1 influenza virus, and H3N2 influenza virus. The detection kit has a shelf life of not less than 7 days when stored at 15-30℃, and the false positive rate after reconstitution of the lyophilized microspheres in each channel is 0% (independent determination of each batch n≥6).

10. The detection kit according to claim 8, characterized in that, The test kit also includes at least one of the following supporting devices: a portable constant temperature heating device with a heating temperature range of 50-72℃ and a temperature uniformity of ±1℃; a portable fluorescence reading module with an excitation wavelength of 450-490nm and an emission wavelength of 510-540nm; or a mobile terminal application for scanning chip channel images and automatically interpreting positive and negative results and outputting a semi-quantitative concentration range report.

11. The lyophilized microsphere composition according to claim 4, characterized in that, The premixed lyophilized microspheres and primer lyophilized microspheres are sequentially placed in the same reaction channel of the microfluidic chip along the liquid flow direction, with the primer lyophilized microspheres located near the sample well and the premixed lyophilized microspheres located near the reaction tank. This allows the liquid sample to dissolve the two types of microspheres sequentially as it flows through the channel: first, the primer lyophilized microspheres dissolve, ensuring uniform dispersion of the primers in the sample solution. Then, the liquid flow carries the primer solution into the premixed lyophilized microsphere region, triggering the reconstitution and initiation of the complete RT-LAMP reaction system. This achieves the ordered spatial dissolution of the two components, further reducing the risk of non-specific interactions caused by excessively high local concentrations during the initial reconstitution stage.

12. The method according to claim 1, characterized in that, Step (5) also includes recording the amplification peak time Tp value of each positive channel, and establishing a linear regression equation with the logarithm of the template concentration corresponding to each channel as the horizontal axis and the Tp value as the vertical axis: Tp = a × log 10 (C) + b Where C is the template concentration of each channel (copies / μL), and a and b are linear regression coefficients; the typical parameter range of the primer set based on this invention under preferred conditions (63℃, n=10-fold dilution) is: a ∈ [-8.0, -5.5] (min / decade), b ∈ [40, 45] (min); taking the SARS-CoV-2 Omicron OmiN P1 group in Example 4 as an example, the measured regression equation is Tp = -5.78 × log 10 (C) + 42.0 (R) 2 =0.992, n=3); Taking the Zika-NS5 P1 group in Example 5 as an example, Tp = -7.90× log 10 (C) + 43.5 (R) 2 =0.999, n=3); the linear regression equation R 2 ≥0.98.

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