A pet helicobacter pylori detection kit based on cas12a protein

Abstract

The application provides a pet Helicobacter pylori detection kit based on a Cas12a protein, and belongs to the technical field of biological detection. The kit comprises a primer pair for a MIRA reaction and crRNA, wherein the primer pair for the MIRA reaction comprises primers MIRA F1 and MIRA R1, and the sequences of the MIRA F1, MIRA R1 and crRNA are shown in SEQ ID NO: 1-3 in sequence. The pet Helicobacter pylori is detected by using the kit, and the accuracy, specificity and sensitivity are high. Pet gastric mucosa biopsy samples or fresh feces can be used as detection objects, no false positive results are generated, no expensive professional instruments need to be purchased, on-site rapid detection can be realized, and the technical requirements for operators are low.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology detection, specifically relating to a detection kit for Helicobacter pylori in pets based on the Cas12a protein. Background Technology

[0002] Helicobacter pylori (Hp) is a common zoonotic disease. It was first isolated in 1983. According to incomplete statistics, approximately 50% of the global population is infected with Hp. Hp typically colonizes the stomach and is the only known bacterium that can survive in the highly acidic environment of the stomach. It is a common gastrointestinal pathogen and also an opportunistic pathogen. Early infection may cause halitosis, indigestion, and diarrhea; in severe cases, it can lead to chronic gastritis, gastric ulcers, duodenal ulcers, and MALT (mucosa-associated lymphoid tissue lymphoma). Long-term infection has a high potential to develop into gastric cancer. Because Hp is a major cause of gastric cancer, it was classified as a Group 1 carcinogen for human gastric cancer by the WHO (World Health Organization) in 1994. 89% of non-cardiac cancers are associated with Hp infection. As a zoonotic disease, *Helicobacter pylori* (Hp) can be naturally transmitted to humans and other animals besides humans. Common pets such as cats, dogs, and rodents are natural hosts. While macaques have also been reported as natural hosts, their role in human daily life is limited. Therefore, this invention focuses primarily on common domestic pets: cats and dogs. Studies have shown that cats and dogs can transmit Hp to their owners, with a cross-infection rate reaching up to 100%. However, the specific transmission routes of Hp are currently undetermined, although some literature suggests that oral-oral and fecal-oral routes are potential pathways for Hp infection. If a pet is infected with Hp, its owner is also highly likely to be infected simultaneously; therefore, Hp infection in pets poses a certain threat to human health. Due to the unclear transmission routes of Hp and the increasing drug resistance during Hp treatment, accurate early detection of the presence of Hp in the host is particularly important. Currently, in the human medical field, mature *Helicobacter pylori* (Hp) detection methods mainly include the C13 / C14 breath test and SAT (fecal antigen detection colloidal gold test strips). However, considering the difficulty for animals to cooperate during the testing process, the C13 / C14 breath test is difficult to perform on pets and cannot be well applied to pet clinical testing. Market research shows that the only product currently specifically used for *Hp* detection in pets is the fecal antigen test strip. However, the fecal antigen test is prone to false positives, and the product instructions clearly state that it can only be used as an auxiliary means to determine whether *Hp* is infected, and should not be used as the final judgment. Some veterinary hospitals choose to use molecular biology methods such as PCR and qPCR to detect *Hp* in samples, but these require expensive professional laboratory equipment and highly trained personnel. Other veterinary hospitals choose to use bacterial culture methods, but this method is time-consuming, costly, and has low sensitivity. Summary of the Invention

[0003] The purpose of this invention is to provide a Cas12a protein-based Helicobacter pylori detection kit for pets, which has high accuracy, specificity, and sensitivity. It can use pet gastric mucosal biopsy samples or fresh feces as the detection target, does not produce false positives, does not require the purchase of expensive professional instruments, can achieve rapid on-site testing, and has low technical requirements for operators.

[0004] The objective of this invention is achieved through the following technical solution:

[0005] A Cas12a protein-based detection kit for Helicobacter pylori in pets includes: a primer pair for the MIRA reaction and crRNA, wherein the primer pair for the MIRA reaction includes primers MIRAF1 and MIRAR1, and the sequences of MIRAF1, MIRAR1 and crRNA are shown in SEQ ID NO:1-3 in sequence.

[0006] In this invention, the kit further includes a single-stranded reporter gene and a Cas12a protein. The nucleotide sequence of the single-stranded reporter gene is shown in SEQ ID NO:4, with FAM labeled at the 5' end and Biotin labeled at the 3' end.

[0007] In this invention, the concentrations of primers MIRAF1 and MIRAR1 are 8-12 μM, the concentration of crRNA is 1.5-3.5 μM, the concentration of single-stranded reporter gene is 3-5 μM, and the concentration of Cas12a protein is 1.5-3.5 μM.

[0008] In this invention, the kit further includes a colloidal gold test strip, wherein a conjugate of gold particles and a FITC tag is incubated on the conjugate pad of the colloidal gold test strip, streptomycin and cytosine are incubated at the control line on the detection membrane, and goat anti-mouse secondary antibody is incubated at the detection line.

[0009] In this invention, the colloidal gold test strip includes a support pad and a detection membrane disposed on the support pad. The two ends of the detection membrane are respectively connected to a conjugate pad and an absorbent pad. The conjugate pad is connected to a sample pad. From the sample pad to the absorbent pad, the detection membrane is provided with a control line and a detection line in sequence.

[0010] This invention's kit is specifically designed for pets (cats and dogs). Two main methods are provided depending on the sampling site. The first method involves collecting a gastric mucosal biopsy sample from the pet on-site, incubating it with a nucleic acid release agent at 95°C for 3 minutes to obtain nucleic acid, performing a MIRA reaction, and then subjecting the amplified product to a CRISPR / Cas12a trans-cleavage reaction. Finally, the result is visualized using a colloidal gold test strip. The second method involves collecting a fresh fecal sample on-site, extracting nucleic acid from the fecal sample using a fecal sample genomic DNA extraction kit, and then following the same steps as the first method. Because fecal samples are high-salt and complex, the nucleic acid initially extracted using only a nucleic acid release agent is likely to contain other impurities. The subsequent MIRA reaction is a multi-enzyme reaction system, and impurities in the feces can cause enzyme shrinkage, degradation, or inactivation, significantly affecting amplification efficiency. Therefore, a fecal sample genomic DNA extraction kit is used to obtain nucleic acid from the fecal sample.

[0011] Beneficial Effects: Unlike conventional PCR and qPCR, which amplify target signals individually, this invention's kit utilizes isothermal amplification and CRISPR technology for dual amplification of the target signal. It does not rely on specialized experimental equipment and requires no technical expertise from operators. On-site sampling and testing are possible, offering high portability and a short processing time of approximately 25-45 minutes from sample collection to result interpretation. It exhibits excellent specificity; no false positives were observed in cross-sectional testing of several common gastrointestinal pathogens. High sensitivity is also demonstrated, with a detection limit (LOD) of 4 copies / reaction determined through sensitivity testing. Compared to conventional PCR and qPCR, it significantly reduces testing time and saves on instrument costs. Unlike SAT, this kit does not exhibit false positives or false negatives, demonstrating high accuracy. Furthermore, based on different sample types, we provide two testing methods for rapid on-site and rapid laboratory testing, meeting the testing needs of various situations. Attached Figure Description

[0012] Figure 1 This is a schematic diagram of the composition of a colloidal gold test strip: 1-support pad, 2-sample pad, 3-binding pad, 4-detection membrane, 4-1 is the control line (C line), 4-2 is the detection line (T line), and 5-absorbent pad.

[0013] Figure 2 The complete detection process of this kit is shown, including gastric mucosal biopsy / fecal sample collection, sample nucleic acid extraction, MIRA amplification, CRISPR / Cas12a reaction, and interpretation of colloidal gold test strip results.

[0014] Figure 3The diagram shows how to interpret the results using the test strips. If the T line is visible, the result is positive; if only the C line is visible, the result is negative; if neither the C line nor the T line is visible, the result is invalid.

[0015] Figure 4 This demonstrates the good specificity of the kit. From left to right, the test strips contain the following samples: Vibrio parahaemolyticus, Vibrio cholerae, Salmonella typhimurium, Escherichia coli, Yersinia enterocolitica, Enterococcus faecalis, Helicobacter pylori, positive control, and negative control.

[0016] Figure 5 This kit demonstrates good sensitivity; the concentrations of the Hp-positive standard plasmids from left to right are 10... 4 copies / reaction, 10 3 copies / reaction, 10 2 Copies / reaction, 10 copies / reaction, 4 copies / reaction, 2 copies / reaction, 1 copy / reaction, positive control, and negative control.

[0017] Figure 6 The different CRISPR cleavage efficiencies guided by crRNA and crRNA2 in the kit of this invention are shown. The horizontal axis represents reaction time, and the vertical axis represents fluorescence value. RNase-free water was used as a negative control (N).

[0018] Figure 7 The effect of products amplified with different MIRA primers on the efficiency of CRISPR cleavage is shown. The horizontal axis represents time, and the vertical axis represents fluorescence intensity. Primer 1 indicates that MIRAF1 and MIRAR1 are MIRA amplification primers; Primer 2 indicates that MIRAF2 and MIRAR2 are MIRA amplification primers. Using Hp-positive standard plasmid (10... 10 A sample of RNase-free water (copies / μL) was used as a positive control (P), and RNase-free water was used as a negative control (N).

[0019] Figure 8 The results of the test using the kit of the present invention on 34 collected gastric mucosal biopsy samples are shown, of which 21 were positive and 13 were negative.

[0020] Figure 9 The results of testing 34 fecal samples collected using the kit of the present invention are shown, of which 5 were positive and 29 were negative.

[0021] Figure 10The results of qPCR testing of 34 gastric mucosal biopsy samples are shown, of which 21 were positive and 13 were negative.

[0022] Figure 11 The results of qPCR testing of 34 collected fecal samples are shown, of which 5 were positive and 29 were negative. Detailed Implementation

[0023] Example 1: Composition of a Cas12a protein-based Helicobacter pylori detection kit for pets

[0024] A Cas12a protein-based Helicobacter pylori detection kit for pets (hereinafter referred to as the kit of this invention) includes: a primer pair for the MIRA reaction, crRNA, a Reporter (single-stranded reporter gene), Cas12a protein, and a colloidal gold test strip.

[0025] (1) Primer pairs for the MIRA reaction

[0026] The primer pair for the MIRA reaction includes the upstream primer MIRAF1 and the downstream primer MIRAR1. The sequence of MIRA F1 (SEQ ID NO:1) is as follows: 5'-CTCTTTTAAAACCTGATGATGTGATGGATGG-3'; the sequence of MIRAR1 (SEQ ID NO:2) is as follows: 5'-TTTTTCTCTGTCAAAGTCTAAGCATCTATTC-3'.

[0027] The concentration of MIRAF1 was 10 μM, and the concentration of MIRAR1 was 10 μM.

[0028] The upstream primer MIRAF1 and the downstream primer MIRAR1 were synthesized by Tsingke Biotech Co, Ltd. (Beijing, China).

[0029] (2) crRNA

[0030] The sequence of crRNA (SEQ ID NO:3) is as follows: 5'-AGAACAACUCACCAGGAACUAAU-3'.

[0031] crRNA was synthesized by Tsingke Biotech Co, Ltd. (Beijing, China).

[0032] The concentration of crRNA was 2.5 μM.

[0033] (3) Reporter

[0034] The nucleotide sequence (SEQ ID NO:4) of the Reporter gene is as follows: 5'-TTTTTTTATTTTTTT-3', with FAM (carboxyfluorescein) labeled at the 5' end and Biotin labeled at the 3' end.

[0035] The concentration of the Reporter was 4 μM.

[0036] The Reporter was synthesized by Tsingke Biotech Co., Ltd. (Beijing, China).

[0037] (4) Cas12a protein

[0038] Cas12a protein, at a concentration of 2.5 μM, was purchased from New England Biolabs (Ipswich, Massachusetts, USA), product number #M0653T.

[0039] (5) Colloidal gold test strips

[0040] Figure 1 This is a schematic diagram of the composition of a colloidal gold test strip. The colloidal gold test strip includes a support pad 1 and a detection membrane 4 disposed on the support pad. The two ends of the detection membrane are respectively connected to a conjugate pad 3 and an absorbent pad 5. The conjugate pad is connected to a sample pad 2. From the sample pad to the absorbent pad, the detection membrane is provided with a control line (C line) 4-1 and a detection line (T line) 4-2 in sequence.

[0041] Preparation of colloidal gold test strips: Gold particles were prepared using the sodium citrate reduction method. A 0.01% aqueous solution of chloroauric acid hydrate was heated to boiling, and then 1 mL of a 1% sodium citrate aqueous solution was rapidly added while stirring, turning the solution wine-red. The solution was then boiled for another 15 minutes and cooled to room temperature to obtain the gold particle solution. 1 mL of the above gold particle solution was coupled with 4 μL of 1 mg / mL FITC (fluorescein isothiocyanate) and incubated at the conjugate pad. The C-line of the detection membrane was incubated with 1 mg / mL streptomycin (SA), and the T-line was incubated with 1 mg / mL goat anti-mouse secondary antibody.

[0042] The goat anti-mouse secondary antibody was ProteinFind Goat Anti-Mouse IgG (H+L), FITCConjugate, purchased from TransGen Biotech, Beijing, catalog number HS211-01.

[0043] Example 2: Method for detecting samples using the kit of the present invention

[0044] The entire detection process for testing samples using the kit of this invention is shown in the following flowchart. Figure 2 As shown, it includes the following steps:

[0045] (1) Sample collection and nucleic acid extraction

[0046] Samples include gastric mucosal biopsies or feces from pets (cats, dogs, etc.).

[0047] ① Collection and nucleic acid extraction of gastric mucosal biopsy samples: A sesame seed-sized (approximately 15 mg) gastric mucosal biopsy sample was cut using gastroscopy forceps, added to a 1.5 mL centrifuge tube, and 150 μL of nucleic acid release agent (purchased from Suzhou Xianda Gene Technology Co., Ltd., product number NR201) was added. The mixture was shaken and incubated in a 95℃ water bath for 3 minutes to obtain the nucleic acid extract of the gastric mucosal biopsy sample.

[0048] ② Collection of fecal samples and nucleic acid extraction: Collect 180-220 mg of fresh fecal sample from the pet into a clean 2 mL centrifuge tube and place the centrifuge tube on ice. Use a fecal genomic DNA extraction kit (#DP328, purchased from Tiangen Biotech (Beijing) Co., Ltd.) to rapidly extract nucleic acids from the fecal sample to obtain the fecal nucleic acid extract.

[0049] (2) MIRA reaction

[0050] The MIRA kit (purchased from AMP-Future Biotech Co., Ltd. (Shandong, China), product number WLN8201KIT) was used to amplify target DNA using nucleic acid extracts from gastric mucosal biopsy samples or fecal samples as templates.

[0051] MIRA reaction system: 2 μL nucleic acid extract, 2 μL upstream primer MIRAF1 (10 μM), 2 μL downstream primer MIRAR1 (10 μM), 29.4 μL A Buffer, 2.5 μL B Buffer, 12.1 μL ddH2O. The total reaction volume was 50 μL. The MIRA reaction system was incubated at 37 °C for 15 min to obtain the MIRA amplification product.

[0052] A Buffer and B Buffer are reagents in the MIRA kit.

[0053] (3) CRISPR / Cas12a trans cleavage reaction

[0054] The CRISPR / Cas12a reaction system consisted of 2 μL of MIRA amplification product and 10 μL of 10×NE buffer. TM2.1 (Purchased from New England Biolabs (Ipswich, Massachusetts, USA), product number #B7202S), 1 μL crRNA (2.5 μM), 1 μL Cas12a protein (2.5 μM), 1 μL Reporter (4 μM), 85 μL RNase-free water. The total reaction volume was 100 μL. The above system was subjected to a CRISPR / Cas12a trans-cleavage reaction at 37 °C for 10 min.

[0055] (4) Interpretation results of colloidal gold test strips

[0056] Insert the sample pad of the test strip directly into the liquid after the reaction in step (3), and interpret the results within 10 minutes. A positive result indicates the presence of Helicobacter pylori in the sample if only a T line appears; a negative result indicates the absence of Helicobacter pylori in the sample if only a C line appears; and an invalid result indicates no C or T line. Result interpretation is as follows: Figure 3 As shown.

[0057] Detection Principle: When *Helicobacter pylori* is present in the sample, target DNA is amplified via MIRA reaction. In the subsequent CRISPR reaction, the target DNA serves as the template. When a specific crRNA fragment is present in the system, the Cas12a protein, guided by the crRNA, binds to the target DNA. At this point, the trans-cleavage activity of the Cas12a protein is activated, allowing it to effectively cleave single-stranded DNA in the system without selection. The Reporter is a specially designed single-stranded DNA fragment. The 3' biotin of the Reporter can bind to the SA at the C-line and remain there. If the Reporter is cleaved by Cas12a, the FAM at the 5' end will be in a free state. Since FAM does not bind to SA, the free FAM will bind to the goat anti-mouse secondary antibody at the T-line and develop color. Therefore, the color development at the C-line and T-line can be used to determine whether the sample contains *Helicobacter pylori*.

[0058] Example 3: Specificity of the kit of the present invention

[0059] Construction of Hp-positive standard plasmid: After successfully ligating the pclone007 vector (purchased from Qingke Company) with the pet Hp-specific gene: ureA (Genebank accession number: AB032429.1), it was transformed into competent DH5α cells (purchased from Qingke Company, product number TSC-C14) and cultured overnight. The next day, single clones were picked and cultured in LB liquid medium containing ampicillin for 2 hours. They were then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The correctly sequenced bacterial culture was inoculated into 10 mL of LB liquid medium containing ampicillin at a volume ratio of 1:100. After overnight culture, the plasmid was extracted using Plasmid Mini Kit I (purchased from OMEGA Company, product number D6943-02), named Hp-positive standard plasmid, and the initial plasmid concentration was measured.

[0060] Using the kit of this invention, six common pet gastrointestinal bacteria, including Helicobacter pylori, Vibrio parahaemolyticus, Vibrio cholerae, Salmonella typhimurium, Escherichia coli, Yersinia enterocolitica, and Enterococcus faecalis isolated and cultured from pets, were detected according to the method in Example 2. A 10Hp positive standard plasmid was set up. 10 Copies per reaction were used as a positive control, and sterile water as a negative control. Results are as follows: Figure 4 As shown, only *H. pylori* and the positive control showed positive results, while the other six bacteria tested negative. Therefore, the kit of this invention has good specificity.

[0061] Example 4: Sensitivity of the reagent kit of the present invention

[0062] The sensitivity of the kit of the present invention was investigated by the following steps: Hp-positive standard plasmids were obtained according to the method in Example 3, and the initial plasmid concentration was measured. The Hp-positive standard plasmids were diluted with sterile water to a concentration of 10. 10 After being diluted to copies / μL, the Hp-positive standard plasmids were serially diluted 10-fold, and the results were analyzed using the method described in Example 2 to determine the limit of detection (LOD) of the kit in Example 1. The results are as follows: Figure 5 As shown, the LOD is 4 copies / reaction.

[0063] Example 5: Comparison of the detection method of the present invention's reagent kit with the control method

[0064] 1. Comparison of the detection method of the reagent kit of the present invention with control method 1

[0065] The kit of this invention was used to detect Helicobacter pylori samples isolated and cultured from pets. Simultaneously, control method 1 was used to detect the same Helicobacter pylori samples. The only difference between control method 1 and the kit of this invention is that the crRNA in the kit of this invention was replaced with crRNA 2, and the Reporter was replaced with Reporter II. The sequence of crRNA 2 is: 5'-GAUUUAACGCAUUGGUUGAUAG-3', and the sequence of Reporter II is: 5'-FAM-TTTTTTTTTTTT-BHQ1-3'. The kit of this invention was used with RNase-free water as a negative control. After the CRISPR / Cas12a trans-cleavage reaction in step (3), the CRISPR / Cas12a reaction products obtained using the kit of this invention and control method 1 were placed in a multifunctional microplate reader (purchased from Tecan Trading Co., Ltd. (Pudong, China)) to detect the fluorescence values ​​at 485nm-535nm. The results are as follows. Figure 6 As shown, it can be observed that the CRISPR cleavage efficiency guided by crRNA in the kit of this invention (complete reaction time of 28 minutes) is much greater than that guided by crRNA2 (complete reaction time of 48 minutes).

[0066] 2. Comparison of the kit of the present invention with control method 2

[0067] Helicobacter pylori samples isolated and cultured from pets were detected using the kit of this invention. Control method 2 was used to detect the same Helicobacter pylori samples, the only difference being that the primer pair (MIRA F1 and MIRAR1) for the MIRA reaction in the kit of this invention was replaced with primer pair 2 (primers MIRAF2 and MIRAR2). The sequence of primer MIRAF2 was 5'-AAGCAGTAGCTTTGATTAGTGCCCATATTA-3', and the sequence of primer MIRAR2 was 5'-CATTTTTAACTTTCACGCTTAACGGCTTTTT-3'. Simultaneously, step (3) of the kit of this invention was used to detect the Hp-positive standard plasmid (10... 10 A sample of RNase-free water was used as a positive control (P); the reagent kit of this invention was used as a negative control. After the reaction in step (3) of each method was completed, the fluorescence value at 485nm-535nm was detected in a multifunctional microplate reader, and the results are as follows. Figure 7As shown, it can be found that when MIRAF1 and MIRAR1 are used for amplification, the subsequent CRISPR cleavage reaction efficiency (complete reaction time of 28 minutes) is the highest, while the subsequent CRISPR cleavage reaction efficiency corresponding to primers MIRAF2 and MIRAR2 is lower (complete reaction time of 58 minutes).

[0068] Example 6: Accuracy of the reagent kit of the present invention

[0069] Thirty-four gastric mucosal biopsy samples and 34 fecal samples were collected from pet cats and dogs. These samples were tested using the kit and qPCR method of this invention (Schabereiter-Gurtner, C.; Hirschl, AM; Dragosics, B.; Hufnagl, P.; Puz, S.; Kovách, Z.; Rotter, M.; Makristathis, A., Novel real-time PCR assay for detection of Helicobacter pylori infection and simultaneous clarithromycin susceptibility testing of stool and biopsy specimens. J Clin Microbiol 2004, 42(10), 4512-8.), and the concordance rate was calculated. The concordance rate was 100%.

[0070] The detection results of the kit of this invention on gastric mucosal biopsy samples are as follows: Figure 8 Of the 21 positive samples, 13 were negative; the test results for the stool samples were as follows: Figure 9 As shown, there were 5 positive and 29 negative results.

[0071] Results of qPCR: Thirty-four gastric mucosal biopsy samples were tested using the qPCR method. The results are as follows: Figure 10 As shown, 21 samples were positive and 13 were negative; the stool samples were tested, and the results are as follows. Figure 11 As shown, there were 5 positive samples and 29 negative samples.

[0072] The comparison results of the two methods are shown in the table below:

[0073] The kit of this invention is used to detect gastric mucosal biopsy samples. 21 13 34 The kit of this invention is used to detect fecal samples. 5 29 34 qPCR method for detecting gastric mucosal biopsy samples 21 13 34 qPCR method for detecting fecal samples 5 29 34

[0074] The above results demonstrate that the detection results of 34 gastric mucosal biopsy samples and 34 fecal samples using the kit of this invention and the qPCR method were 100% consistent, indicating that the kit of this invention does not produce false positives and has high accuracy.

Claims

1. A Cas12a protein-based detection kit for Helicobacter pylori in pets, characterized in that, include: The kit includes a primer pair for the MIRA reaction and crRNA, wherein the primer pair for the MIRA reaction includes primers MIRA F1 and MIRA R1, and the sequences of MIRA F1, MIRA R1 and crRNA are shown in SEQ ID NO:1-3, respectively; the kit also includes a single-stranded reporter gene and Cas12a protein, wherein the nucleotide sequence of the single-stranded reporter gene is shown in SEQ ID NO:4, with FAM labeled at the 5' end and Biotin labeled at the 3' end.

2. The reagent kit according to claim 1, characterized in that... The concentrations of primers MIRA F1 and MIRA R1 were 8-12 μM, the concentration of crRNA was 1.5-3.5 μM, the concentration of single-stranded reporter gene was 3-5 μM, and the concentration of Cas12a protein was 1.5-3.5 μM.

3. The reagent kit according to claim 2, characterized in that... The kit also includes colloidal gold test strips, wherein gold particles and FITC-tagged conjugates are incubated on the conjugate pad of the colloidal gold test strips, streptomycin and cytosine are incubated at the control line on the detection membrane, and goat anti-mouse secondary antibody is incubated at the detection line.

4. The reagent kit according to claim 3, characterized in that... The colloidal gold test strip includes a support pad and a detection membrane disposed on the support pad. The two ends of the detection membrane are respectively connected to a conjugate pad and an absorbent pad. The conjugate pad is connected to a sample pad. From the sample pad to the absorbent pad, the detection membrane is provided with a control line and a detection line in sequence.

Images