Visual paper-based cancer miRNA marker detection kit

Abstract

The invention discloses a visual paper-based cancer miRNA marker detection kit, the kit comprises an integrally formed foldable T-shaped filter paper, and a DNA probe TEprobe, urease and a color developing agent dropwise added on the filter paper, and the principle of the kit is as follows: the TEprobe is combined with a cancer nucleic acid marker (miRNA) to release Ag < + > to inhibit the activity of the urease; the composite signal response of the pH response probe phenol red and fluorescein is triggered, and the detection sensitivity is as low as 10-12 M. The kit provided by the invention can significantly shorten the detection time and reduce the sample demand, and realizes zero false positive rate and 97.10% specificity by combining with machine learning algorithm classification during result analysis. The performance is successfully verified in real samples of prostatic cancer, liver cancer and colorectal cancer, and a cheap and convenient means is expected to be provided for family self-inspection and cancer screening and diagnosis in resource-deficient areas.

Description

Technical Field

[0001] This invention relates to the fields of biomaterials science and detection technology, specifically to a visual paper-based cancer miRNA biomarker detection kit. Background Technology

[0002] Today, prevalent malignant tumors pose a significant challenge to global public health, such as prostate cancer, colorectal cancer, and liver cancer. These cancers often present with subtle symptoms in their early stages. For example, prostate cancer grows slowly and often develops in young men, but is frequently not detected until the age of 40-50. Therefore, early cancer screening is crucial.

[0003] Currently, conventional cancer screening methods vary, but all have limitations. For example, prostate cancer screening mainly relies on serum prostate-specific antigen (PSA) testing and digital rectal examination, colorectal cancer screening commonly uses colonoscopy and fecal occult blood testing, while liver cancer screening focuses on ultrasound and alpha-fetoprotein (AFP) testing. However, these methods all have varying degrees of deficiencies in sensitivity, specificity, accuracy, invasiveness, cost, and / or accessibility. Therefore, exploring highly specific biomarkers for different cancer types and their corresponding, better detection methods is of great significance for improving the early screening and diagnosis capabilities for various cancers.

[0004] Taking prostate cancer as an example, the long non-coding RNA PCA3, discovered in 1999, has been established as a prostate cancer-specific biomarker due to its extremely high expression level in cancerous tissue (10-100 times that of adjacent benign tissue) and its detectability in urinary sediments. Commercially available diagnostic kits based on PCA3 (such as Progensa PCA3), combined with other clinical parameters, have shown the potential to reduce unnecessary invasive procedures (such as biopsies). Similarly, in fields such as colorectal cancer and liver cancer, new specific molecular markers are constantly being explored and validated (such as certain gene mutations or methylation markers in colorectal cancer, and abnormal prothrombin / PIVKA-II in liver cancer). However, many current advanced molecular detection methods (including PCA3-based detections) often face bottlenecks such as time consumption, high cost, and reliance on sophisticated instruments and professional personnel, which greatly limits their promotion and popularization in grassroots or resource-scarce areas.

[0005] Against this backdrop, developing a simple, rapid, inexpensive, and easy-to-interpret detection platform suitable for various cancer screenings is particularly important. Colorimetric methods, due to their ease of operation, lack of complex equipment, and ability to achieve rapid visual readings, are considered one of the ideal candidate technologies for on-site testing. However, traditional colorimetric methods also have significant drawbacks such as weak anti-interference ability, insufficient specificity, and poor reagent stability requiring repeated preparation, which hinder their widespread application. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a visual paper-based cancer miRNA biomarker detection kit. The detection results of this kit are identifiable to the naked eye. It is easy to operate, has rapid color development, high sensitivity, and rich color variations. It can realize the construction and immediate detection of multiple cancer miRNA biomarkers, and has broad application prospects in disease diagnosis and pre-detection.

[0007] The specific technical solution adopted in this invention is as follows:

[0008] This invention provides a visual paper-based cancer miRNA biomarker detection kit. The kit includes a hydrophobically treated filter paper in the shape of a T, with a central blank folded area. Three edges of the blank folded area are respectively provided with a probe addition area, a urease addition area, and a chromogenic reagent addition area. After folding, the probe addition area, urease addition area, and chromogenic reagent addition area overlap with the blank folded area.

[0009] The blank folding region is used to add the sample to be tested; the probe adding region is used to add a detection probe mixture, which includes a detection probe, AgNO3 solution, and NaNO3 solution, and the detection probe includes a Rec chain and a Block chain; the urease adding region is used to add urease; and the chromogenic reagent adding region is used to add a chromogenic reagent, which includes a mixture of urea, phenol red, and water.

[0010] Preferably, the detection probes are TEprobes 1, TEprobes 2, or TEprobes 3, with the following sequences:

[0011] TEprobes 1:

[0012] Rec chain: 5'-ACGATAGGCAGTCAGCAGCCTTTCTTATTTC-3';

[0013] Block chain: 5'-AAAGGCTGCTGACTCCCTATCGT-3';

[0014] TEprobes 2:

[0015] Rec chain: 5'-ACGATAGGCTCAACATCAGTCTGATAAGCTA-3';

[0016] Block chain: 5'-CAGACTGATGTTGACCCTATCGT-3';

[0017] TEprobes 3:

[0018] Rec chain: 5'-ACGATAGGCACAGGCCGGGACAAGTGCAATA-3';

[0019] Block chain: 5'-TTGTCCCGGCCTGTCCCTATCGT-3'.

[0020] Preferably, the detection probe mixture is prepared by the following method:

[0021] Two single-stranded DNA strands, Rec and Block, constituting the detection probe were synthesized. The Rec strand, Block strand, AgNO3 solution, and NaNO3 solution were mixed thoroughly and incubated at a constant temperature to form a double-helix structure, then frozen for storage. The volume ratio of Rec strand, Block strand, AgNO3 solution, and NaNO3 solution was 1:1:1:3–7, and the concentration ratio was 1:1:1–2:10. 8 ~10 9 .

[0022] Preferably, the incubation temperature is 20~30 ℃ and the incubation time is 10~30 min.

[0023] Preferably, the volume ratio of the detection probe, urease, and chromogenic agent is 1:1:1~3, the concentration ratio of the detection probe to urease is 1~3:1, the volume ratio of urea, phenol red, and H2O is 1:1:1~3, the concentration of urea is 1~4 M, and the concentration of phenol red is 0.5~2 mM.

[0024] Preferably, the detection method of the kit includes the following steps:

[0025] The detection probe mixture, urease, and chromogenic reagent are added to the probe addition area, urease addition area, and chromogenic reagent addition area, respectively.

[0026] Add the sample to be tested to the blank folded area;

[0027] By folding the blank folded area to coincide with the probe drop-in area, the sample to be tested and the test probe mixture come into contact, and then the mixture is incubated at a constant temperature.

[0028] By folding the urease addition zone to align with the blank folding zone, and then incubating at a constant temperature to await the release of Ag. + Fully bind with urease;

[0029] The colorimetric reagent addition area is folded to coincide with the blank folding area, and then kept at a constant temperature to wait for the colorimetric results. The colorimetric results are observed and photographed, and the results are analyzed to obtain the detection results.

[0030] Preferably, the volume of the sample to be tested is 2~10 μL.

[0031] Preferably, the temperature for constant temperature settling is 20~30 ℃, and the settling time is 5~20 min.

[0032] As a preferred approach, the results analysis can be performed using a machine learning model.

[0033] The principle of this invention is as follows: TEproobes consist of a Rec chain and a Block chain, forming a cytosine-cytosine (CC) mismatch. This mismatch can interact with Ag. + Selective binding occurs, forming metal-mediated C-Ag bonds through base-metal cation interactions. + -C artificial base pairs. Due to the thermodynamic energy penalty caused by single-base pair mismatch, this structure possesses a positive free energy. When the target miRNA is present, the corresponding base sequence fragment of the target miRNA competitively binds to the Rec strand of TEproobes, releasing Ag. + Ag + As an inhibitor of urease, its content is sufficient to inhibit the urease cleavage of urea to produce NH4. + The amplification effect plays a regulatory role. + The amount of the product alters the pH of the environment, triggering the color development of phenol red and fluorescent yellow. Therefore, by using pH indicators, cancer miRNA biomarkers can be identified and visualized through visual observation or using a smartphone.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] (1) This invention utilizes nucleic acid chain shift reaction, thermodynamic energy penalty related to single base pair mismatch, and Ag + The controlled urease cleavage effectively amplifies the recognition of target miRNAs, enabling the efficient detection of low-abundance target biomarkers in early-stage cancer patient samples, thus improving the accuracy and sensitivity of the detection kit. Furthermore, integrating this detection system into an origami kit fully leverages the synergistic effect of filter paper to simplify sample pretreatment steps, significantly reduce sample dosage, and accelerate detection time, showing broad application prospects in the field of point-of-care testing for cancer screening and diagnosis.

[0036] (2) The method of the present invention is applicable to a variety of cancer biomarkers and has the advantages of not requiring instruments, visually perceptible results, simple operation, high sensitivity, and rich color changes, thus minimizing operation steps and manual intervention. At the same time, the image recognition performance is further enhanced by combining machine learning models, providing accurate and reliable information support for precise screening of multiple cancers. Attached Figure Description

[0037] Figure 1This is a flowchart illustrating the principle of the present invention, where A represents the principle of nucleic acid visualization detection, and B represents signal acquisition and machine learning-assisted signal recognition to improve accuracy.

[0038] Figure 2 The images shown are gel electrophoresis images of the DNA and RNA chains in Example 1.

[0039] Figure 3 This is a linear relationship graph for detecting the prostate cancer marker PCA3 in Example 1.

[0040] Figure 4 This is a bar chart showing the maximum absorbance of miRNA 21, a liver cancer biomarker, in Example 2.

[0041] Figure 5 The bar chart shows the maximum absorbance of miRNA 92a, a colorectal cancer biomarker, as used in Example 3.

[0042] Figure 6 The image shows a colorimetric photograph of a real sample of urine from a normal person, obtained using the paper-based sensor in Example 1.

[0043] Figure 7 A colorimetric photograph of a real urine sample from a prostate cancer patient, obtained using the paper-based sensor in Example 1.

[0044] Figure 8 A comparative bar chart showing how machine learning improves signal recognition accuracy in Example 1. Detailed Implementation

[0045] The present invention will be further illustrated below with reference to the accompanying drawings and embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0046] like Figure 1 As shown, this invention provides a visual paper-based cancer miRNA biomarker detection kit. The kit includes a hydrophobically treated filter paper in the shape of a T. The central area of ​​the filter paper is a blank folded area (purple area, hereinafter referred to as page 1 in this embodiment) for adding the sample to be tested. At the three edges of the blank folded area are respectively provided a probe addition area (green area, hereinafter referred to as page 2 in this embodiment), a urease addition area (pink area, hereinafter referred to as page 3 in this embodiment), and a chromogenic reagent addition area (blue area, hereinafter referred to as page 4 in this embodiment). The probe addition area, urease addition area, and chromogenic reagent addition area overlap with the blank folded area after folding.

[0047] Specifically, the preparation and use of this test kit includes the following steps:

[0048] Step (1): Two single-stranded DNA strands (Rec strand and Block strand) that make up TEprobes are synthesized. The Rec strand, Block strand, AgNO3 solution and NaNO3 solution are mixed evenly and incubated at a constant temperature to form a double helix structure, and then frozen for storage; wherein the volume ratio of Rec strand solution, Block strand solution, AgNO3 solution and NaNO3 solution is 1:1:1:4, and the concentration ratio is 1:1:1~2:10. 9 The incubation temperature is 25℃, and the incubation time is 10~30 min.

[0049] In step (1), TEproobes consist of a Rec strand and a Block strand. The Rec strand contains both forward and reverse fulcrums, while the Block strand contains only the reverse fulcrum. The two strands are linked together by a complementary base segment (designed according to the target miRNA) to form a double helix structure. The reverse fulcrum contains a set of cytosine-cytosine mismatches, which can interact with Ag... + Selective binding occurs, forming metal-mediated C-Ag bonds through base-metal cation interactions. + -C artificial base pairs.

[0050] Step (2): Print the pattern onto filter paper using a wax-spray printer. Then, heat the paper in a metal bath to melt the wax ink and form a hydrophobic barrier. The heating temperature is 100 ℃, and the heating time is 10~30 s. Next, add a mixture of TEprobes probe, urease, and chromogenic agent (urea + phenol red + H2O) to pages 2, 3, and 4 of the origami paper, respectively. The volume ratio of TEprobes probe, urease, and chromogenic agent mixture is 1:1:1~3, the concentration ratio of TEprobes probe to urease is 1~3:1, the volume ratio of urea, phenol red, and H2O is 1:1:1~3, the concentration of urea is 1~4 M, and the concentration of phenol red is 0.5~2 mM. Store the prepared origami paper at -4 ℃.

[0051] In step (2), the paper-based sensor constructs a hydrophobic barrier through inkjet printing and heating to limit the flow range of the solution sample. Different pages are functionally designed for specific regions, and the folding sequence guides the liquid flow and enables a stepwise reaction.

[0052] Step (3): Add the standard solution or body fluid sample containing the target miRNA to the first page of the origami paper, then fold the paper to the second page (containing the TEprobes probe), so that the RNA sample comes into contact with the TEprobes, and incubate at a constant temperature. The volume of the standard solution or body fluid sample is 2~10 μL, the incubation temperature is 20~30 ℃, and the incubation time is 10 min.

[0053] In step (3), the target miRNA undergoes a strand shift reaction with the TEprobes probe, releasing Ag. + .

[0054] Step (4): Fold page 2 onto page 3 (containing urease), incubate at 20-30°C for 5-20 minutes, and wait for the release of Ag. + It binds fully with urease.

[0055] In step (4), the released Ag + It is a typical "soft acid" that tends to bind with urease (soft base). The specific mechanism involves Ag... + It specifically binds to key cysteine ​​residues and structurally critical cysteine ​​residues (-SH) near the active site of urease, disrupting the function of the active site or altering the protein conformation, leading to a decrease or even loss of urease activity.

[0056] Step (5): Fold page 3 to page 4 (containing the color developer mixture), let stand at 20-30 ℃ for 5-20 minutes, and wait for the color development results. Take photos using a smartphone and a 3D printed camera (providing a stable light source). Input the photos into the application, and the test results will be output through a machine learning model.

[0057] In step (5), the bisNi of the urease active site 2+ The synergistic effect allows urea to be broken down into ammonia and carbamic acid. Ammonia increases the alkalinity of the environment, triggering a response from the pH probe phenol red and fluorescence, resulting in a variety of color changes. The machine learning model can calculate the content of the target miRNA based on the area of ​​the chromogenic region, thus enabling its use in screening for various cancers. The machine learning model can employ conventional image data processing models, such as convolutional neural networks (CNNs). This invention does not limit this approach; subsequent embodiments use CNNs only as examples.

[0058] The present invention will be further described below.

[0059] Example 1

[0060] (1) Preparation of TEprobes 1 probe

[0061] Probe chain designed based on prostate cancer marker PCA3:

[0062] Rec chain: 5'-ACGATAGGCAGTCAGCAGCCTTTCTTATTTC-3' (SEQ ID NO. 1);

[0063] Block chain: 5'-AAAGGCTGCTGACTCCCTATCGT-3' (SEQ ID NO. 2).

[0064] Mix 5 μL of 300 nM Rec chain, 5 μL of 300 nM Block chain, 5 μL of 300 nM AgNO3 solution, 15 μL of 0.6 M NaNO3 solution and 20 μL of H2O thoroughly, incubate at room temperature for 20 min and then store at 4 ℃.

[0065] (2) Preparation of a paper-based nucleic acid visualization kit

[0066] The pattern was printed onto filter paper using a Xerox ColorQube 8580 / 8880N color printer. The paper was then heated in a 100°C metal bath for 30 seconds to melt the wax and form a hydrophobic barrier. The dropper wells in each dropper area were not treated with hydrophobic material; the hydrophobic barrier in the remaining areas ensured that the liquid in the dropper wells did not leak out when the kit was folded. On pages 2, 3, and 4 of the folded paper, 5 μL of 3 nM TEprobes, 5 μL of 1 nM urease, and 5 μL of a chromogenic reagent mixture (1 μL 2.5 M urea + 1 μL 1.25 mM phenol red + 3 μL H2O) were added, respectively. The prepared folded paper was stored at 0–4°C.

[0067] (3) Add 5 μL of PCA3 standard solution to the first page of the origami paper, then fold the paper to the second page (containing TEprobes probes) to bring the RNA sample into contact with TEprobes 1. After incubating at room temperature for 10 min, fold the second page to the third page (containing urease), wait 10 min, and release Ag. + Fully bind with urease. Finally, fold page 3 over page 4 (containing the colorimetric reagent mixture) and incubate for 10 minutes. Take photos using a smartphone and a 3D-printed camera (providing a stable light source).

[0068] Gel electrophoresis images of each DNA / RNA strand are shown below. Figure 2 As shown.

[0069] The linear fit between the changes in absorbance signal of different concentrations of the prostate cancer marker PCA3 and the chromogenic reagent is as follows: Figure 3 As shown.

[0070] Example 2

[0071] (1) Preparation of TEprobes 2 probes

[0072] Probe strands were designed based on the liver cancer marker miRNA 21:

[0073] Rec chain: 5'-ACGATAGGCTCAACATCAGTCTGATAAGCTA-3' (SEQ ID NO.3);

[0074] Block chain: 5'-CAGACTGATGTTGACCCTATCGT-3' (SEQ ID NO. 4).

[0075] Other parameters and methods were the same as in Example 1, and TEprobes 2, a specific probe for liver cancer markers, was synthesized.

[0076] (2) Preparation of a paper-based nucleic acid visualization kit

[0077] The pattern was printed onto filter paper using a Xerox ColorQube 8580 / 8880N color printer. The paper was then heated in a 100°C metal bath for 30 seconds to melt the wax ink and form a hydrophobic barrier. On pages 2, 3, and 4 of the origami, 5 μL of a mixture of 3 nM TEprobes, 5 μL of 1 nM urease, and 5 μL of a chromogenic reagent (1 μL 2.5 M urea + 1 μL 1.25 mM phenol red + 3 μL H2O) was added, respectively. The prepared origami was stored at 0°C.

[0078] (3) Add 5 μL of miRNA 21 standard solution to the first page of the origami paper, then fold the paper to the second page (containing TEprobes probes) to bring the RNA sample into contact with TEprobes 2. After incubating at room temperature for 10 min, fold the second page to the third page (containing urease), wait 10 min, and release Ag. + Fully bind with urease. Finally, fold page 3 over page 4 (containing the colorimetric reagent mixture) and incubate for 10 minutes. Take photos using a smartphone and a 3D-printed camera (providing a stable light source).

[0079] The bar chart showing the relationship between different concentrations of the liver cancer marker miRNA 21 and the changes in absorbance signal of the chromogenic agent is shown below. Figure 4 As shown.

[0080] Example 3

[0081] (1) Preparation of TEprobes 3 probes

[0082] Probe strands were designed based on the colorectal cancer marker miRNA 92a:

[0083] Rec chain: 5'-ACGATAGGCACAGGCCGGGACAAGTGCAATA-3' (SEQ ID NO.5);

[0084] Block chain: 5'-TTGTCCCGGCCTGTCCCTATCGT-3' (SEQ ID NO. 6).

[0085] Other parameters and methods are the same as in Example 1, and TEprobes 3, a specific probe for colorectal cancer biomarkers, are synthesized.

[0086] (2) Preparation of a paper-based nucleic acid visualization kit

[0087] The pattern was printed onto filter paper using a Xerox ColorQube 8580 / 8880N color printer. The paper was then heated in a 100°C metal bath for 30 seconds to melt the wax ink and form a hydrophobic barrier. On pages 2, 3, and 4 of the origami, 5 μL of a mixture of 3 nM TEprobes, 5 μL of 1 nM urease, and 5 μL of a chromogenic reagent (1 μL 2.5 M urea + 1 μL 1.25 mM phenol red + 3 μL H2O) was added, respectively. The prepared origami was stored at 4°C.

[0088] (3) Add 5 μL of miRNA 92a standard solution to the first page of the origami paper, then fold the paper to the second page (containing TEprobes probes) to bring the RNA sample into contact with TEprobes 3. After incubating at room temperature for 10 min, fold the second page to the third page (containing urease), wait 10 min, and release Ag. + Fully bind with urease. Finally, fold page 3 over page 4 (containing the colorimetric reagent mixture) and incubate for 10 minutes. Take photos using a smartphone and a 3D-printed camera (providing a stable light source).

[0089] The bar graph showing the relationship between different concentrations of the colorectal cancer marker miRNA 92a and the changes in absorbance signal of the chromogenic agent is shown below. Figure 5 As shown.

[0090] Application Example 1

[0091] Urine samples were obtained from prostate cancer patients and healthy individuals from the hospital. Other parameters and methods were the same as in Example 1. After obtaining the final colorimetric results, the samples were photographed and observed, and then input into a CNN model for identification to provide more accurate diagnostic results.

[0092] Colorimetric images of real human urine samples detected using paper-based sensors, such as... Figure 6 As shown.

[0093] Colorized images of real urine samples from prostate cancer patients detected using paper-based sensors, such as... Figure 7 As shown.

[0094] A comparative histogram showing how machine learning models can help improve signal recognition accuracy. Figure 8 As shown, the specificity increased from 86% to 97%.

[0095] The embodiments and application examples described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A visual paper-based cancer miRNA marker detection kit, characterized in that, The kit comprises a hydrophobic treated filter paper in the shape of T, wherein a central region of the filter paper is a blank folding region, and three edges of the blank folding region are respectively provided with a probe dropping region, a urease dropping region and a color developing agent dropping region; the probe dropping region, the urease dropping region and the color developing agent dropping region are respectively overlapped with the blank folding region after folding. The blank folding region is used for dropping a sample to be detected; the probe dropping region is used for dropping a detection probe mixture, the detection probe mixture comprising a detection probe, an AgNO3 solution and a NaNO3 solution, the detection probe comprising a Rec chain and a Block chain; the urease dropping region is used for dropping urease; and the color developing agent dropping region is used for dropping a color developing agent, the color developing agent comprising a mixture of urea, phenol red and water.

2. The kit of claim 1, wherein The detection probe is TEprobes 1, TEprobes 2 or TEprobes 3, and the sequences thereof are respectively as follows: TEprobes 1: Rec chain: 5'-ACGATAGGCAGTCAGCAGCCTTTCTTATTTC-3'; Block chain: 5'-AAAGGCTGCTGACTCCCTATCGT-3'; TEprobes 2: Rec chain: 5'-ACGATAGGCTCAACATCAGTCTGATAAGCTA-3'; Block chain: 5'-CAGACTGATGTTGACCCTATCGT-3'; TEprobes 3: Rec chain: 5'-ACGATAGGCACAGGCCGGGACAAGTGCAATA-3'; Block chain: 5'-TTGTCCCGGCCTGTCCCTATCGT-3'.

3. The kit of claim 2, wherein The detection probe mixture is prepared by the following method: The two single-stranded DNAs Rec chain and Block chain constituting the detection probe are synthesized; the Rec chain, the Block chain, an AgNO3 solution and an NaNO3 solution are uniformly mixed, and incubated at a constant temperature to form a double-stranded helix structure, and are stored in a frozen state; wherein the volume ratio of the Rec chain, the Block chain, the AgNO3 solution and the NaNO3 solution is 1:1:1:3-7, and the concentration ratio is 1:1:1-2:10 8 ~10 9 .

4. The kit of claim 3, wherein The incubation temperature is 20-30 DEG C, and the incubation time is 10-30 min.

5. The kit of claim 1, wherein The volume ratio of the detection probe, the urease and the color developing agent is 1:1:1-3, and the concentration ratio of the detection probe to the urease is 1-3:

1.

6. The kit of claim 5, wherein The volume ratio of urea, phenol red and water is 1:1:1-3, the concentration of urea is 1-4 M, and the concentration of phenol red is 0.5-2 mM.

7. The kit of claim 1, wherein The detection method of the kit comprises the following steps: The detection probe mixture, the urease and the color developing agent are respectively dropped in the probe dropping region, the urease dropping region and the color developing agent dropping region; The sample to be detected is dropped in the blank folding region; The blank folding region is overlapped with the probe dropping region by folding, so that the sample to be detected and the detection probe mixture are contacted and incubated at constant temperature; By folding the urease drop zone coincides with the blank folding zone, constant temperature and wait for the release of Ag + With urease fully combined; The color developing agent dropping region is overlapped with the blank folding region by folding, and the color developing result is waited at constant temperature; the color developing result is observed and photographed, and the detection result is analyzed.

8. The kit of claim 7, wherein The volume of the sample to be detected is 2-10 μL.

9. The kit of claim 7, wherein The temperature for constant temperature standing is 20-30 DEG C, and the time for constant temperature standing is 5-20 min.

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