Method for simultaneously and rapidly detecting multi-target moulds

Abstract

The invention discloses a method for simultaneously and rapidly detecting multi-target moulds. The method comprises the following steps: S1, preparing an integrated micro-fluidic chip; s2, injecting a sample through a sample inlet in the chip, enriching the sample, and intercepting mold spores; s3, injecting a spore lysis solution to carry out chemical-thermal synergistic wall breaking; step S4, introducing the LAMP reaction premixed liquid to carry out isothermal amplification; and S5, realizing detection through the color change of the HNB dye. According to the invention, multiple moulds can be detected at the same time.

Description

Technical Field

[0001] This invention relates to a method for the simultaneous and rapid detection of multiple target molds. Background Technology

[0002] Traditional methods for mold detection generally employ culture methods. For example, potato dextrose agar is used to culture the fungi for 5-7 days, followed by counting the total number of colonies to analyze mold contamination. While culture methods offer high accuracy, they are time-consuming and difficult to use for real-time monitoring of environmental mold contamination. Furthermore, approximately 70% of fungi cannot be cultured, leading to significant false negatives. Alternatively, direct microscopic examination can be used. Although this method is faster, it has low sensitivity and heavily relies on the operator's subjective experience, with an error margin of up to 35%, making it prone to false positives and false negatives. Summary of the Invention

[0003] The main objective of this invention is to provide a method for the rapid detection of multiple target molds simultaneously.

[0004] The technical solution adopted by this invention to solve its technical problem is:

[0005] A rapid method for simultaneous detection of multiple target fungi includes the following steps:

[0006] Step S1: Prepare an integrated microfluidic chip, comprising an upper layer and a lower layer, and a PDMS sponge located between the upper and lower layers; the upper layer has an upper inlet, and the number of outlets is progressively increased by dividing the upper inlet into two, and then into four, with each outlet connected to an upper chamber; the lower layer has a lower outlet, and the number of inlets is progressively increased by dividing the lower outlet into two, and then into four, with each inlet connected to a lower chamber; each upper chamber corresponds to a lower chamber, and the two are separated by the PDMS sponge; each upper chamber is pre-embedded with specific LAMP primers for the target mold;

[0007] Step S2: Inject the sample through the sample inlet on the chip to enrich the sample and extract mold spores;

[0008] Step S3: Inject spore lysis buffer for chemical-thermal synergistic cell disruption;

[0009] Step S4: Introduce LAMP reaction premix for isothermal amplification;

[0010] Step S5: Detection is achieved through the color change of HNB dye.

[0011] Further, in the chip of step S1, the upper layer has an upper-layer inlet, which is connected to one end of the upper-layer primary flow channel. The other end of the upper-layer primary flow channel is connected to the middle part of the upper-layer secondary flow channel. The two ends of the upper-layer secondary flow channel are respectively connected to the middle parts of two upper-layer tertiary flow channels. The two ends of one upper-layer tertiary flow channel are respectively connected to the upper-layer first chamber and the upper-layer second chamber, and the two ends of the other upper-layer tertiary flow channel are respectively connected to the upper-layer third chamber and the upper-layer fourth chamber. The two upper-layer tertiary flow channels are arranged in the same row, and the upper-layer tertiary flow channel is arranged in parallel with the upper-layer primary flow channel and the upper-layer secondary flow channel.

[0012] The lower layer has a lower layer outlet, which is connected to one end of the lower layer primary flow channel. The other end of the lower layer primary flow channel is connected to the middle section of the lower layer secondary flow channel. The two ends of the lower layer secondary flow channel are respectively connected to the middle sections of two lower layer tertiary flow channels. The two ends of one lower layer tertiary flow channel are respectively connected to the lower layer first chamber and the lower layer second chamber, and the two ends of the other lower layer tertiary flow channel are respectively connected to the lower layer third chamber and the lower layer fourth chamber. The two lower layer tertiary flow channels are arranged in parallel, and the lower layer tertiary flow channel, the lower layer primary flow channel, and the upper layer secondary flow channel are arranged in parallel.

[0013] The upper and lower layers are stacked together, with the upper first chamber and the lower first chamber positioned vertically, and a first PDMS sponge between them; the upper second chamber and the lower second chamber positioned vertically, and a second PDMS sponge between them; the upper third chamber and the lower third chamber positioned vertically, and a third PDMS sponge between them; and the upper fourth chamber and the lower fourth chamber positioned vertically, and a fourth PDMS sponge between them.

[0014] Furthermore, the LAMP reaction premix contains hydroxynaphthol blue (HNB) dye, and quantitative detection is achieved by analyzing the RGB grayscale values ​​of the color spots after the reaction.

[0015] Furthermore, in step S3, the spore lysis solution contains 1-2 mg / mL chitinase.

[0016] Furthermore, enzymatic hydrolysis at a constant temperature of 35-45℃ for 10-20 minutes achieves synergistic chemical-thermal cell disruption.

[0017] Furthermore, in step S4, the LAMP amplification conditions are a constant temperature reaction at 65°C for 30 minutes, and the results are determined by the color change of HNB dye: positive results show sky blue, while negative results remain violet.

[0018] Further, step S5 involves quantitative detection, which is achieved by establishing a standard curve using a 2×LAMP Master Mix system containing HNB dye, with reaction conditions of 63-67℃ for 25-35 minutes.

[0019] Further, in step S1, the method for preparing the PDMS sponge includes the following steps:

[0020] Step 10: Take water-soluble salt, grind it to a size of less than 1 micrometer using a ball mill, then put it into a container, compact it, and then introduce uncured PDMS. By applying forward pressure or reverse vacuuming, the PDMS is impregnated with water-soluble salt. Then, heat it to cure the PDMS, and then take it out for later use.

[0021] Furthermore, in step S10, the shape of the container is adapted to the shape of the lower chamber.

[0022] Furthermore, the PDMS sponge is used to prepare a film, which includes step S20: taking a PDMS sponge and cutting it to a thickness of 0.3-1.0 mm to become a PDMS sponge slice; placing the PDMS sponge slice in a mold, the mold including upper and lower layers with a gap between the upper and lower layers and a cavity for placing the PDMS sponge slice; after installing the mold, filling uncured PDMS through the inlet and evacuating the outlet to accelerate the flow of PDMS; after the PDMS fills the mold cavity, heating to cure the PDMS and then removing it, placing it in hot water to dissolve water-soluble salts, and drying it to obtain a PDMS sponge film rich in mesopores.

[0023] Furthermore, the number of cavities used to place PDMS sponge slices is the same as the number of lower or upper chambers.

[0024] Compared with the prior art, this technical solution has the following advantages:

[0025] 1. The design of the upper and lower chamber structure of the chip in this invention enables the use of PDMS sponge to trap fungal spores in a closed environment, which is safer. On the other hand, the flow channel of the multi-target collaborative detection inlet is divided into two and then into four to ensure that the sample can be evenly distributed in the four reaction chambers.

[0026] 2. This invention employs an integrated microfluidic chip for enrichment and detection, enabling simultaneous enrichment, lysis, and detection of four types of molds. The chip's four-chamber parallel design allows for simultaneous detection of all four molds in a single injection, improving detection efficiency, reducing batch-to-batch variability, and enhancing the reliability of results. The enriched spores are lysed using a chemical-thermal lysis scheme involving chitinase (1-2 mg / mL) and 40°C heat treatment, releasing complete nucleic acids within 15 minutes without subsequent purification, simplifying the process and laying the foundation for automation. Subsequently, the LAMP method is used to expand the specific target genes of the four molds. Each chamber of the chip is relatively independent, with each chamber pre-embedded with a specific LAMP primer for one mold, ensuring high target specificity. Simultaneously, the HNB dye colorimetric method is used to indicate the LAMP reaction results. Analysis of the RGB grayscale values ​​of the spots in each reaction chamber after photographing enables simultaneous, rapid, and quantitative detection of multiple molds.

[0027] 3. This invention uses a specially designed PDMS sponge to capture spores, achieving a fungal capture efficiency of over 95%, while the efficiency of ordinary commercial filter membranes is only around 50%. Furthermore, the PDMS sponge has a higher flux due to its significantly higher porosity compared to ordinary filter membranes. Attached Figure Description

[0028] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0029] Figure 1 This is a schematic diagram of the upper structure of the chip for simultaneous rapid detection of multiple target molds according to the present invention.

[0030] Figure 2 This is a schematic diagram of the lower layer structure of the chip for simultaneous rapid detection of multiple target molds according to the present invention.

[0031] Figure 3 A schematic diagram of the upper mold structure for preparing PDMS sponge.

[0032] Figure 4 A schematic diagram of the lower mold structure for preparing PDMS sponge.

[0033] Figure 5 This is a schematic diagram of the upper and lower mold assembly structure.

[0034] Figure 6 This is a schematic diagram of the structure of a PDMS thin film.

[0035] Figure 7 This is a schematic diagram of the overall structure of the chip for the simultaneous rapid detection of multiple targets of mold using commercial filter membranes, as described in this invention.

[0036] In the picture:

[0037] 100 - Upper layer 110 - Upper layer inlet 120 - Upper layer primary flow channel 130 - Upper layer secondary flow channel 140 - Upper layer tertiary flow channel

[0038] 150 - Upper three-stage flow channel; 160 - Upper first chamber; 170 - Upper second chamber; 180 - Upper third chamber; 190 - Upper fourth chamber

[0039] 200 - Lower Level 201 - Indicator Marker 210 - Lower Level Outlet 220 - Lower Level Primary Flow Channel 230 - Lower Level Secondary Flow Channel 240 - Lower Level Tertiary Flow Channel 250 - Lower Level Tertiary Flow Channel 260 - Lower Level First Chamber 270 - Lower Level Second Chamber 280 - Lower Level Third Chamber 290 - Lower Level Fourth Chamber

[0040] 300-PDMS film, 310-PDMS sponge chips

[0041] 400-Upper mold 410-Inlet 420-Outlet 430-Cavity

[0042] 500-lower mold 530-round cavity

[0043] 600-Commercial Filter Membrane

[0044] Figure 8 This is a schematic diagram of the structure of a PDMS sponge slice.

[0045] Figure 9 These are micrographs of fluorescent microparticles trapped in a sponge within a chip. Among them, A is a bright-field image of fluorescent microspheres trapped in a PDMS sponge, B is a fluorescence-field image of fluorescent microspheres trapped in a PDMS sponge, C is a bright-field image of fluorescent microspheres trapped in a commercial filter membrane, and D is a fluorescence-field image of fluorescent microspheres trapped in a commercial filter membrane.

[0046] Figure 10 This is a standard curve for detecting Aspergillus terreus within the chip. Detailed Implementation

[0047] Example 1 Chip

[0048] See Figures 1 to 5 The present invention provides a chip for simultaneous rapid detection of multiple target molds, comprising an upper layer 100, a lower layer 200 and a filter membrane 300 located between the upper and lower layers.

[0049] The upper layer 100 has an upper inlet 110, which is connected to one end of the upper primary flow channel 120. The other end of the upper primary flow channel 120 is connected to the middle part of the upper secondary flow channel 130. The two ends of the upper secondary flow channel 130 are respectively connected to the middle parts of the upper tertiary flow channels 140 and 150. The two ends of the upper tertiary flow channel 140 are respectively connected to the upper first chamber 160 and the upper second chamber 170, and the two ends of the upper tertiary flow channel 150 are respectively connected to the upper third chamber 180 and the upper fourth chamber 190. The upper tertiary flow channels 140 and 150 are arranged in the same row, and the upper tertiary flow channels (140 and 150) are arranged parallel to the upper primary flow channel 120 and the upper secondary flow channel 130.

[0050] The lower layer 200 has a lower layer outlet 210, which is connected to one end of the lower primary flow channel 220. The other end of the lower primary flow channel 220 is connected to the middle portion of the lower secondary flow channel 230. The two ends of the lower secondary flow channel 230 are respectively connected to the middle portions of the lower tertiary flow channel 240 and the lower tertiary flow channel 250. The two ends of the lower tertiary flow channel 240 are respectively connected to the lower first chamber 260 and the lower second chamber 270, and the two ends of the lower tertiary flow channel 250 are respectively connected to the lower third chamber 280 and the lower fourth chamber 290. The lower tertiary flow channels 240 and 250 are arranged in the same row, and the lower tertiary flow channels (240 and 250) are arranged parallel to the lower primary flow channel 220 and the upper secondary flow channel 230.

[0051] Among them, after the upper layer 100 and the lower layer 200 are stacked, the upper first chamber 160 and the lower first chamber 260 are vertically aligned and have a first filter membrane between them; the upper second chamber 170 and the lower second chamber 270 are vertically aligned and have a second filter membrane between them; the upper third chamber 180 and the lower third chamber 280 are vertically aligned and have a third filter membrane between them; the upper fourth chamber 190 and the lower fourth chamber 290 are vertically aligned and have a fourth filter membrane between them.

[0052] The "filter membrane" mentioned in this invention includes a self-made PDMS sponge membrane or a commercial filter membrane.

[0053] Preparation method of PDMS (polydimethylsiloxane) sponge slices: Sodium chloride is ground to a size of less than 1 micrometer using a ball mill, then placed into a stainless steel tube with an inner diameter of 3 mm, compacted, and then uncured PDMS is introduced. The PDMS is impregnated with sodium chloride by applying forward pressure or reverse vacuuming, followed by heating at 90 degrees Celsius for 30 minutes. After the PDMS has cured, it is removed for later use. The PDMS sponge is then cut to a thickness of 0.3 mm to obtain PDMS sponge slices 310.

[0054] PDMS sponge film preparation method: see Figures 3 to 6 A specially designed mold, comprising an upper mold 400 and a lower mold 500, is used. The upper mold has an inlet 410, an outlet 420, and four circular cavities 430. The positions of these four circular cavities 430 correspond to the four chamber positions of the chip. The lower mold 500 has four circular cavities 530 corresponding to the circular cavities 430. Four PDMS sponge slices 310 are placed in the circular cavities of the mold. After the upper and lower molds are installed, uncured PDMS is filled through the inlet 410, and a vacuum is drawn through the outlet 420 to accelerate the flow of PDMS. After the PDMS fills the mold cavity, it is heated at 90 degrees Celsius for 30 minutes. After the PDMS has cured, it is removed and placed in hot water to dissolve the sodium chloride in the PDMS slices 310 (an ultrasonic cleaner can be used to accelerate the dissolution process). The remaining PDMS is then dried to obtain a PDMS sponge film 300 rich in mesoporous material. The PDMS sponge slices 310 have the following morphology: Figure 8 As shown. After the film is formed, four PDMS sponge slices 310 are connected together by the PDMS film.

[0055] Chip bonding: The chip consists of a lower layer 200, a middle layer (PDMS sponge film 300) and an upper layer 100. During bonding, the PDMS sponge of the middle layer needs to be aligned with the reaction chamber of the lower layer, and the chamber of the upper layer needs to be aligned with the middle PDMS sponge and the chamber of the lower layer to seal the reaction chamber.

[0056] Detection

[0057] The upper first chamber 160, upper second chamber 170, upper third chamber 180, and upper fourth chamber 190 are pre-embedded with specific LAMP primers for the target molds (aspergillus flavus target gene aflR, aspergillus fumigatus target gene anxC4, aspergillus niger target gene GOD, and aspergillus terreus target gene benA; primer sequences are shown in the table below), with each chamber targeting one target mold.

[0058] The primer sequences are as follows:

[0059]

[0060] Each primer component was prepared into a primer premix (10×) at the following concentrations: FIP and BIP at 16 µM, F3 and B3 at 2 µM, and LF and LB at 4 µM. The filter membranes or sponges in each chamber were immersed in the premixes of primers for different fungal target genes, freeze-dried, and then used for the fabrication of the detection chip.

[0061] Air samples were collected using a vacuum pump and introduced into the chip through inlet 110. During track etching of the filter membrane, mold spores were efficiently trapped on the membrane. After gas collection, a spore lysis buffer containing chitinase was injected into the chamber through inlet 110, and chemo-thermal lysis was performed at a constant temperature of 40°C for 15 minutes. After lysis, LAMP reaction reagents [containing HNB (hydroxynaphthol blue, 1 mM), MgSO4 (100 mM), four dNTPs (all at 10 mM), nuclease-free water, and Bst DNA polymerase (400 U / mL)] were introduced. Subsequently, the target genes of the four molds were amplified at a constant temperature of 65°C. Qualitative and semi-quantitative detection of the four molds was performed based on HNB color development and RGB grayscale analysis, achieving integrated operation of collection, lysis, DNA amplification, and detection of four molds on a single chip.

[0062] See Figure 7 The PDMS sponge membrane 300 can also be replaced by a commercial filter membrane 600. Furthermore, the commercial filter membrane 600 is a track-etched filter membrane with a thickness of 100 μm and a pore diameter of 0.4 μm.

[0063] To evaluate the retention efficiencies of track-etched filter membranes and PDMS sponges for mold spores, fluorescent microspheres with a particle size of 1.2 μm were used to simulate mold spores in retention experiments. 100 μL of microsphere suspension (concentration 0.1 mg / mL) was introduced into the chip (using track-etched filter membranes or PDMS sponges as retention components) at a constant flow rate (100 μL / h). The fluorescence intensity in the microsphere solution before loading and in the waste liquid after loading was measured. The retention efficiencies of the commercial filter membrane and PDMS sponge for fluorescent microspheres were calculated using the differential method. The results showed that the retention efficiency of the commercial filter membrane was ~47%, and the retention efficiency of the PDMS sponge was ~95%. Bright-field and fluorescence microscopic images of the chamber region were also acquired. The results are shown below. Figure 9 As shown.

[0064] In this invention, using *Tetrandrobium* and PDMS sponge film as examples, a quantitative detection standard curve was plotted. A 20 μL LAMP reaction system with different concentrations of spores was constructed, with the specific component configuration as follows:

[0065]

[0066] After the system is prepared, gently mix it by pipetting and briefly centrifuge at room temperature to ensure the reaction solution collects at the bottom of the tube. Then place the reaction tube in a PCR instrument and run the following program: amplify at 65 ℃ for 30 minutes; maintain 4 ℃ continuously; set the lid temperature to 105 ℃ to prevent condensation. After 30 minutes, remove the reaction tube and let it stand at room temperature for 2 minutes. Observe the color change against a pure white background. A positive reaction will appear sky blue, while a negative reaction will remain violet.

[0067] Quantitative detection standard curve as follows Figure 10 As shown, the detection limit or sensitivity of this method is 10 CFU / mL.

[0068] As can be seen from the examples above:

[0069] 1. Traditional culture methods require 3-7 days or even longer to obtain results. While molecular biology methods such as PCR shorten the identification time, they still require lengthy sample pretreatment (enrichment, DNA extraction, purification). This invention seamlessly integrates four key steps—air sampling, spore enrichment, cell disruption, and nucleic acid amplification and detection—onto a single microchip, achieving a "sample in, result out" detection model. Time costs are significantly reduced: the entire process, from collecting air samples to obtaining qualitative and semi-quantitative results for four common pathogenic molds, can be completed within tens of minutes. This speed is unattainable with traditional methods, enabling real-time environmental quality assessment and risk warning.

[0070] 2. Significantly faster decision-making response: In places with strict requirements for microbial concentration, such as hospital ICUs, operating rooms, and aseptic food workshops, once the number of airborne colonies exceeds the standard, this invention can provide near real-time data to help managers quickly locate the source of contamination, assess the effectiveness of disinfection, and take intervention measures before a hazard occurs, thus avoiding large-scale contamination or infection events caused by delayed detection.

[0071] 3. High Detection Sensitivity and Accuracy: Precise Control from "Million-Level" to "Micro-Level". The core advantage of microfluidic technology lies in the precise manipulation of fluids and reactions at the microscopic scale. Low Sample Consumption and High Enrichment Efficiency: The chip-integrated PDMS sponge has high retention efficiency for 1-5μm particles (corresponding to the size of mold spores), ensuring that target spores in limited air samples are captured and enriched to the maximum extent in the reaction chamber without significant loss. This provides a more robust sensitivity foundation compared to traditional impaction or centrifugation methods, which may result in spore bounce, death, or dispersion.

[0072] 4. Avoid cross-contamination and ensure specificity: Each reaction chamber is physically isolated and pre-embedded with different specific primers. This allows for simultaneous parallel amplification of nucleic acids targeting different targets in four independent "micro-factories" after a sample enters. This design fundamentally eliminates problems such as primer interference and uneven amplification efficiency that may exist in multiplex PCR, ensuring the specificity and accuracy of multi-target detection. The detection results are clear and unambiguous, eliminating the need for complex melting curves or sequencing to distinguish different molds, thus simplifying the interpretation process.

[0073] 5. Traditional airborne microbial detection primarily provides quantitative data on colony-forming units (CFU / m³), but culture methods cannot quantify specific species. This invention achieves semi-quantitative analysis of specific molds through ingenious dye design. The HNB dye, acting as a real-time indicator for the LAMP reaction, exhibits a color change (violet → sky blue) directly correlated with the amount of magnesium pyrophosphate precipitate (i.e., DNA amplification). This color change is not a simple "on / off" signal, but rather its intensity exhibits a linear relationship with the amount of initial template within a certain range.

[0074] Objective RGB grayscale analysis: By capturing images of the color spots after the reaction using a smartphone camera or a simple scanning device, and then extracting their RGB values ​​(especially the blue channel, B value) using image processing software for grayscale analysis, subjective color judgments can be transformed into objective, quantifiable digital signals. Using a pre-established standard curve, the relative content of specific mold spores in the sample can be calculated, achieving a cognitive upgrade from "whether it exists" to "approximately how much." This is crucial for assessing the degree of contamination, tracing the source of contamination, and monitoring the effectiveness of disinfection.

[0075] 6. This invention compresses and solidifies a complex process requiring expensive equipment, multiple specialized laboratories, and professional operators onto a chip the size of a business card. The operational process is extremely simplified: a "one-click" operation reduces human error; users only need to perform three simple steps: connect the chip to the vacuum pump for sampling -> sequentially inject lysis buffer and reaction premix through the inlet -> incubate at a constant temperature and observe / photograph the color change. The entire process eliminates the need for complex nucleic acid extraction and purification, cumbersome reagent preparation, expensive real-time fluorescence PCR instruments, and opening the chip for electrophoresis detection. This significantly reduces the technical background and professional training requirements for operators, enabling community health workers, factory quality inspectors, and even home users with minimal training to reliably complete the tests, greatly improving the accessibility and widespread adoption of the technology.

[0076] 7. Extremely Low Cost per Detection: Low-cost chip materials: PDMS is a common and inexpensive polymer material, ideally suited for large-scale mass production via soft lithography, molding, and other processes, thus keeping the cost per chip extremely low. Minimal reagent consumption: The micro-scale reaction system means that the amount of expensive reagents such as primers, enzymes, and dNTPs used is reduced by several times or even an order of magnitude compared to traditional methods. The reagent cost per detection is significantly reduced. Low equipment investment and maintenance costs: As mentioned earlier, the absence of expensive large instruments also means no corresponding maintenance, calibration costs, or huge power consumption.

[0077] 8. High-throughput parallel detection capability: Four parallel reaction chambers are integrated on a single chip, enabling simultaneous processing of four different target molds in a single air sample. This not only improves detection efficiency but also further reduces the detection cost per target. This architecture has good scalability; by designing more chambers or using a multi-layer chip structure, it can achieve simultaneous detection of more types of microorganisms or environmental indicators (such as endotoxins and allergens). For example, if needed, this invention can be divided into eight parts to detect even more types of molds.

[0078] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for simultaneous and rapid detection of multiple target fungi, characterized in that, Includes the following steps: Step S1: Prepare an integrated microfluidic chip, comprising an upper layer and a lower layer, and a polydimethylsiloxane sponge film located between the upper and lower layers; the upper layer has an upper inlet, and the number of outlets is progressively increased by dividing the upper inlet into two, and then into four, with each outlet connected to an upper chamber; the lower layer has a lower outlet, and the number of inlets is progressively increased by dividing the lower outlet into two, and then into four, with each inlet connected to a lower chamber; each upper chamber corresponds to a lower chamber, and the two are separated by PDMS sponge; each upper chamber is pre-embedded with specific LAMP primers for the target mold; Step S2: Inject the sample through the sample inlet on the chip to enrich the sample and extract mold spores; Step S3: Inject spore lysis buffer for chemical-thermal synergistic cell disruption; Step S4: Introduce LAMP reaction premix for isothermal amplification; Step S5: Detection is achieved through the color change of HNB dye.

2. The method for simultaneous rapid detection of multiple target molds according to claim 1, characterized in that, In the chip of step S1, the upper layer has an upper-layer inlet, which is connected to one end of the upper-layer primary flow channel. The other end of the upper-layer primary flow channel is connected to the middle part of the upper-layer secondary flow channel; the two ends of the upper-layer secondary flow channel are respectively connected to the middle parts of two upper-layer tertiary flow channels; the two ends of one upper-layer tertiary flow channel are respectively connected to the upper-layer first chamber and the upper-layer second chamber, and the two ends of the other upper-layer tertiary flow channel are respectively connected to the upper-layer third chamber and the upper-layer fourth chamber; the two upper-layer tertiary flow channels are arranged in the same row, and the upper-layer tertiary flow channel is arranged in parallel with the upper-layer primary flow channel and the upper-layer secondary flow channel. The lower layer has a lower layer outlet, which is connected to one end of the lower layer primary flow channel; the other end of the lower layer primary flow channel is connected to the middle part of the lower layer secondary flow channel; the two ends of the lower layer secondary flow channel are respectively connected to the middle parts of two lower layer tertiary flow channels; the two ends of one lower layer tertiary flow channel are respectively connected to the lower layer first chamber and the lower layer second chamber, and the two ends of the other lower layer tertiary flow channel are respectively connected to the lower layer third chamber and the lower layer fourth chamber; the two lower layer tertiary flow channels are arranged in the same row, and the lower layer tertiary flow channel is arranged in parallel with the lower layer primary flow channel and the lower layer secondary flow channel; The upper and lower layers are stacked together, with the upper first chamber and the lower first chamber positioned vertically, and a first PDMS sponge between them; the upper second chamber and the lower second chamber positioned vertically, and a second PDMS sponge between them; the upper third chamber and the lower third chamber positioned vertically, and a third PDMS sponge between them; and the upper fourth chamber and the lower fourth chamber positioned vertically, and a fourth PDMS sponge between them.

3. The microfluidic chip according to claim 1, characterized in that: The LAMP reaction premix contains hydroxynaphthol blue dye, and quantitative detection is achieved by analyzing the RGB grayscale values ​​of the color spots after the reaction.

4. The method for simultaneous rapid detection of multiple target fungi according to claim 1, characterized in that: In step S3, the spore lysis buffer contains 1-2 mg / mL chitinase.

5. The method for simultaneous rapid detection of multiple target fungi according to claim 4, characterized in that: In step S3, enzymatic hydrolysis at a constant temperature of 35-45℃ for 10-20 minutes achieves chemical-thermal synergistic cell wall disruption.

6. The method for simultaneous rapid detection of multiple target fungi according to claim 1, characterized in that: In step S4, the LAMP amplification conditions are a constant temperature reaction at 63-67℃ for 25-35 minutes, and the results are determined by the color change of HNB dye: positive results show sky blue, while negative results remain violet.

7. The method for simultaneous rapid detection of multiple target fungi according to claim 1, characterized in that: Step S5 involves quantitative detection, which is achieved by establishing a standard curve using a 2×LAMP Master Mix system containing HNB dye, with reaction conditions of 63-67℃ for 25-35 minutes.

8. A method for simultaneous rapid detection of multiple target fungi according to any one of claims 1 to 6, characterized in that: In step S1, the preparation method of the PDMS sponge includes the following steps: Step 10: Take water-soluble salt, grind it to a size of less than 1 micrometer using a ball mill, then put it into a container, compact it, and then introduce uncured PDMS. By applying forward pressure or reverse vacuuming, the PDMS is impregnated with water-soluble salt. Then, heat it to cure the PDMS, and then take it out for later use.

9. The method for simultaneous rapid detection of multiple target fungi according to claim 8, characterized in that: In step S10, the shape of the container is adapted to the shape of the lower chamber.

10. The method for simultaneous rapid detection of multiple target fungi according to claim 8, characterized in that: The PDMS sponge is prepared into a film for use, which includes step S20: taking PDMS sponge and cutting it to a thickness of 0.3-1.0 mm to become PDMS sponge slices; placing the PDMS sponge slices in a mold, the mold including upper and lower layers with a gap between the upper and lower layers and a cavity for placing the PDMS sponge slices; after installing the mold, filling uncured PDMS from the inlet and evacuating the outlet to accelerate the flow of PDMS; after the PDMS fills the mold cavity, heating to cure the PDMS and then taking it out, placing it in hot water to dissolve water-soluble salts, and drying it to obtain a PDMS sponge film with mesoporous areas.

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