CN-122012770-A - Simultaneous rapid detection method for multi-target mould

CN122012770ACN 122012770 ACN122012770 ACN 122012770ACN-122012770-A

Abstract

The invention discloses a simultaneous rapid detection method of multi-target mould, which comprises the following steps of S1, preparing an integrated microfluidic chip, S2, injecting a sample through a sample inlet on the chip for sample enrichment, intercepting mould spores, S3, injecting spore lysate for chemical-thermal synergistic wall breaking, S4, introducing LAMP reaction premix for isothermal amplification, and S5, and detecting through HNB dye color change. The invention can realize the simultaneous detection of a plurality of moulds.

Inventors

CHEN XIAOFENG
DU JINGJING
XU SHUNQING
ZHU LIN

Assignees

海南大学

Dates

Publication Date: 20260512
Application Date: 20260126
Priority Date: 20251104

Claims (10)

1. The simultaneous and rapid detection method of the multi-target mould is characterized by comprising the following steps of: Step S1, preparing an integrated microfluidic chip, which comprises an upper layer, a lower layer and a polydimethylsiloxane sponge film positioned between the upper layer and the lower layer, wherein the upper layer is provided with an upper layer inlet, the upper layer inlet is divided into two and four to amplify the number of outlets step by step, each outlet is connected with an upper layer chamber, the lower layer is provided with a lower layer outlet, the lower layer outlet is divided into two and four to amplify the number of inlets step by step, each inlet is connected with a lower layer chamber, each upper layer chamber corresponds to a lower layer chamber, and the upper layer chamber and the lower layer chamber are separated by PDMS sponge, and each upper layer chamber is respectively embedded with a specific LAMP primer aiming at target mould; S2, injecting a sample through a sample inlet on the chip, enriching the sample, and intercepting mould spores; s3, injecting spore lysate to perform chemical-thermal synergistic wall breaking; S4, introducing LAMP reaction premix to perform isothermal amplification; And S5, detecting through HNB dye color change.
2. The method for simultaneous and rapid detection of multiple-target mold according to claim 1, wherein in the chip of step S1, the upper layer has an upper inlet, and the upper inlet is communicated with one end of the upper primary flow channel. The two ends of the upper layer three-level runner are respectively connected with the upper layer first chamber and the upper layer second chamber, and the two ends of the other upper layer three-level runner are respectively connected with the upper layer third chamber and the upper layer fourth chamber; The lower layer is provided with a lower layer outlet which is communicated with one end of a lower layer primary runner, the other end of the lower layer primary runner is connected with the middle part of a lower layer secondary runner, the two ends of the lower layer secondary runner are respectively connected with the middle parts of two lower layer tertiary runners, the two ends of one lower layer tertiary runner are respectively connected with a lower layer first chamber and a lower layer second chamber, the two ends of the other lower layer tertiary runner are respectively connected with a lower layer third chamber and a lower layer fourth chamber, the two lower layer tertiary runners are arranged in the same row, and the lower layer tertiary runner is parallel to the lower layer primary runner and the lower layer secondary runner; After the upper layer and the lower layer are laminated, the positions of the upper layer first chamber and the lower layer first chamber are vertically corresponding, a first PDMS sponge is arranged between the upper layer first chamber and the lower layer first chamber, the positions of the upper layer second chamber and the lower layer second chamber are vertically corresponding, a second PDMS sponge is arranged between the upper layer second chamber and the lower layer second chamber, the positions of the upper layer third chamber and the lower layer third chamber are vertically corresponding, a third PDMS sponge is arranged between the upper layer third chamber and the lower layer third chamber, the positions of the upper layer fourth chamber and the lower layer fourth chamber are vertically corresponding, and a fourth PDMS sponge is arranged between the upper layer fourth chamber and the lower layer fourth chamber.
3. The microfluidic chip of claim 1, wherein the LAMP reaction premix contains hydroxynaphthol blue dye, and quantitative detection is realized by color spot RGB gray scale analysis after reaction.
4. The method for simultaneous and rapid detection of multiple-target mold according to claim 1, wherein in the step S3, the spore lysate contains 1-2mg/mL chitinase.
5. The simultaneous rapid detection method of multi-target mould according to claim 4, wherein in the step S3, chemical-thermal synergistic wall breaking is realized by enzymolysis for 10-20 minutes at the constant temperature of 35-45 ℃.
6. The simultaneous rapid detection method of multi-target mould according to claim 1, wherein in the step S4, the LAMP amplification conditions are 63-67 ℃ and the isothermal reaction is carried out for 25-35 minutes, and the color change of HNB dye judges that the color of the dye is positive, the color of the dye is sky blue, and the color of the dye is negative, and the violet is kept.
7. The simultaneous and rapid detection method of multi-target mold according to claim 1, wherein the quantitative detection is performed in the step S5, the quantitative detection is realized by establishing a standard curve, a 2X LAMP MASTER Mix system containing HNB dye is used, and the reaction condition is 63-67 ℃ for 25-35 minutes.
8. The method for simultaneous and rapid detection of multi-target mold according to any one of claims 1 to 6, wherein in step S1, the preparation method of the PDMS sponge comprises the following steps: And 10, taking a water-soluble salt, grinding the water-soluble salt to a size below 1 micron by a ball mill, then filling the water-soluble salt into a container, tamping, introducing uncured PDMS, soaking the PDMS into the water-soluble salt by means of forward pressure or reverse vacuumizing, heating to cure the PDMS, and taking out the PDMS for later use.
9. The method for simultaneous and rapid detection of multiple-target mold according to claim 8, wherein the shape of the container and the shape of the lower chamber are adapted in step S10.
10. The method for simultaneously and rapidly detecting the multi-target mold according to claim 8, wherein the method is characterized in that the PDMS sponge is prepared into a film for use, and the method comprises the steps of S20, cutting the PDMS sponge to a thickness of 0.3-1.0mm to obtain a PDMS sponge slice, placing the PDMS sponge slice into a mold, wherein the mold comprises an upper layer and a lower layer, a gap is reserved between the upper layer and the lower layer, a cavity for placing the PDMS sponge slice is reserved between the upper layer and the lower layer, filling uncured PDMS from an inlet, vacuumizing from an outlet to accelerate the flow of the PDMS after the mold cavity is filled with the PDMS, heating to solidify the PDMS, then taking out the PDMS, dissolving water soluble salt in hot water, and drying to obtain the PDMS sponge film with partial areas rich in mesopores.

Description

Simultaneous rapid detection method for multi-target mould Technical Field The invention relates to a simultaneous rapid detection method of multi-target mould. Background In the traditional method, a culture method is generally adopted for mould detection. For example, potato dextrose agar is used for 5-7 days, and then the total number of colonies is counted to analyze the mold contamination. The culture method has higher accuracy, but takes too long time, and is difficult to monitor the mould pollution condition in the environment in real time. And about 70% of the fungi cannot be cultivated, resulting in serious missed detection errors in the cultivation method. In addition, a direct microscopic examination method can be adopted, and the method has low detection sensitivity although consuming shorter time, and the method is seriously dependent on subjective experience of operators, and the detection error can reach 35 percent, so that the situations of false detection, missing detection and the like are easy to occur. Disclosure of Invention The invention mainly aims at providing a simultaneous rapid detection method for multi-target mould. The technical scheme adopted for solving the technical problems is as follows: A simultaneous rapid detection method of multi-target mould comprises the following steps: Step S1, preparing an integrated microfluidic chip, which comprises an upper layer, a lower layer and PDMS sponge positioned between the upper layer and the lower layer, wherein the upper layer is provided with an upper layer inlet, the number of outlets is gradually amplified in a mode of dividing the upper layer inlet into two parts and four parts, and each outlet is connected with an upper layer cavity; S2, injecting a sample through a sample inlet on the chip, enriching the sample, and intercepting mould spores; s3, injecting spore lysate to perform chemical-thermal synergistic wall breaking; S4, introducing LAMP reaction premix to perform isothermal amplification; And S5, detecting through HNB dye color change. Further, in the chip in step S1, the upper layer has an upper layer inlet, and the upper layer inlet is communicated with one end of the upper layer primary flow channel. The other end of the upper layer primary runner is connected with the middle part of the upper layer secondary runner. Two ends of the upper layer secondary flow channel are respectively connected with the middle parts of the two upper layer tertiary flow channels. Two ends of one upper layer three-level runner are respectively connected with the upper layer first chamber and the upper layer second chamber, and two ends of the other upper layer three-level runner are respectively connected with the upper layer third chamber and the upper layer fourth chamber. The two upper three-level flow channels are arranged in the same row, and the upper three-level flow channel, the upper first-level flow channel and the upper second-level flow channel are arranged in parallel. The lower layer is provided with a lower layer outlet which is communicated with one end of the lower layer primary runner. The other end of the lower-layer primary runner is connected with the middle part of the lower-layer secondary runner. Two ends of the lower-layer secondary flow channel are respectively connected with the middle parts of the two lower-layer tertiary flow channels. Two ends of one lower-layer three-level runner are respectively connected with the lower-layer first chamber and the lower-layer second chamber, and two ends of the other lower-layer three-level runner are respectively connected with the lower-layer third chamber and the lower-layer fourth chamber. The two lower three-level flow channels are arranged in the same row, and the lower three-level flow channels, the lower first-level flow channels and the upper second-level flow channels are arranged in parallel. After the upper layer and the lower layer are laminated, the positions of the upper layer first chamber and the lower layer first chamber are vertically corresponding, a first PDMS sponge is arranged between the upper layer first chamber and the lower layer first chamber, the positions of the upper layer second chamber and the lower layer second chamber are vertically corresponding, a second PDMS sponge is arranged between the upper layer second chamber and the lower layer second chamber, the positions of the upper layer third chamber and the lower layer third chamber are vertically corresponding, a third PDMS sponge is arranged between the upper layer third chamber and the lower layer third chamber, the positions of the upper layer fourth chamber and the lower layer fourth chamber are vertically corresponding, and a fourth PDMS sponge is arranged between the upper layer fourth chamber and the lower layer fourth chamber. Further, the LAMP reaction premix contains hydroxynaphthol blue (HNB) dye, and quantitative detection is realized through color spot RGB gray scale value