Crichinella spiralis detection kit based on CRISPR / Cas12a system and click chemistry as well as use method and application of trichinella spiralis detection kit

Abstract

The invention discloses a trichina detection kit based on a CRISPR / Cas12a system and click chemistry as well as a use method and application thereof, and belongs to the technical field of immunological detection. The invention provides a trichina detection kit in order to solve the technical problems that a trichina detection method in the prior art is time-consuming and labor-consuming, low in sensitivity and easy to cause leak detection, a CRISPR / Cas12a system signal output probe and a manual click enzyme are complex in preparation process, and a nano material is not easy to store and transport. The kit comprises a CRISPR / Cas12a system, a click reaction system, a 96-well reaction plate, a magnetic bead probe, a colloidal gold probe, a scrubbing solution, a CuSO4 solution, an SA solution and a standard substance. The trichina detection kit provided by the invention has the advantages of high specificity, long-term stability, strong practicability, controllable quality, simplicity in operation, high sensitivity, easiness in storage and the like, can be applied to trichina detection, and has a good market prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunological detection, and specifically relates to a Trichinella spiralis detection kit based on CRISPR / Cas12a system and click chemistry, and a use method and application thereof. Background Art

[0002] Trichinellosis is a foodborne parasitic disease caused by Trichinella spiralis. It is widespread around the world and poses a serious threat to food safety and public health. In 2014, the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WOAH) ranked parasitic diseases according to nine criteria, and the results showed that the economic losses caused by trichinellosis ranked first among 24 common foodborne parasitic diseases. Trichinellosis is usually caused by eating raw meat containing Trichinella spiralis larvae, with pigs being the main source of infection; therefore, accurate and highly sensitive detection of meat contaminated by Trichinella spiralis is crucial to ensuring food safety.

[0003] The pooled sample digestion method recommended by the International Trichinella Commission (ICT) is the "gold standard" for detecting Trichinella. However, this method has the risk of infection with live larvae during sample processing, and the process is time-consuming and labor-intensive. It requires experienced technicians to perform microscopic examinations, which can easily lead to missed detections. Quantitative real-time PCR (qPCR) is also commonly used to detect Trichinella in meat products, but it still has limitations in detecting low-abundance targets, quantitative accuracy, and equipment costs. Therefore, there is an urgent need for a high-throughput and highly sensitive method for detecting Trichinella in meat samples to further ensure food safety.

[0004] Enzyme-linked immunosorbent assay (ELISA) has been widely used in the detection of foodborne parasites due to its high specificity and stability; however, ELISA has difficulty in detecting low-concentration pathogens, and improving its sensitivity remains a major challenge for technicians in the field. CRISPR / Cas system detection technology has atomic-level sensitivity, accuracy, and portability. Combined with biosensor modules such as aptamers, antigen-antibody interactions, and enzyme reactions, it can be applied to the detection of non-nucleic acid targets. Technicians in the field organically combine ELISA with the CRISPR / Cas12a system to perform high-sensitivity detection of pathogens; however, since the CRISPR / Cas12a system mainly relies on FQ probes to output signals, the above-mentioned probes are expensive to use and are easily affected by oxidation, which limits the practical application of the above-mentioned method in biochemical analysis.

[0005] Click chemistry is a highly sensitive signal output method with the advantages of fast reaction rate, mild conditions and high selectivity. The copper (I)-catalyzed azide-alkyne addition (CuAAC) reaction is a revolutionary click chemistry reaction; however, the preparation process of copper-based nanocatalysts (such as copper nanoflowers, copper nanozymes and copper MOFs) currently used for CuAAC reactions is complicated and time-consuming, and the storage and transportation conditions of nanomaterials are relatively harsh, which affects the practical application of click chemistry in biochemical analysis. Summary of the invention

[0006] The present invention aims to solve the technical problems that the prior art methods for detecting Trichinella are time-consuming and labor-intensive, have low sensitivity, are prone to missed detection, and the signal output probes of the CRISPR / Cas12a system and the complex preparation process of artificial click enzymes, as well as the difficulty in storage and transportation of nanomaterials. A detection kit for Trichinella based on the CRISPR / Cas12a system and click chemistry, as well as a method for using and application of the kit are provided.

[0007] One of the objects of the present invention is to provide a Trichinella detection kit based on CRISPR / Cas12a system and click chemistry, the kit comprising: CRISPR / Cas12a system, click reaction system, 96-well reaction plate, magnetic bead probe, colloidal gold probe, washing solution, CuSO4 solution, SA solution and standard.

[0008] In a preferred embodiment of the present invention, the CRISPR / Cas12a system includes: 3.6 mL of 1X NEB 2.1 buffer, 5 µL of 20 µM gRNA, 10 µL of 10 µM Cas12a and 36 µL of a 100 µM DNA chain containing a 20 thymine region; the gRNA sequence is shown in SEQ ID NO.2.

[0009] In a preferred embodiment of the present invention, the click reaction system comprises: 0.25 mM 3-azido-7-hydroxycoumarin and 2000-fold diluted phenylacetylene.

[0010] In a preferred embodiment of the present invention, the magnetic bead probe is a probe for labeling a polyclonal antibody, and the preparation method of the magnetic bead probe is: S1: Wash 10 mg of COOH-modified magnetic beads three times with 0.05 M, pH 6 MES buffer; S2: 500 µL of 4 mg / mL EDC and 5.5 mg of sulfo-NHS were added to the magnetic beads obtained in S1 and incubated at room temperature for 15 min for activation; S3: Wash the activated magnetic beads in S2 three times with 500 µL of coupling solution; the coupling solution has a pH of 7.5 and is prepared from 0.1 M Na3PO4 and 0.15 M NaCl; S4: Mix 500 µL of coupling solution and 0.5 mg of polyclonal antibody, and shake the mixture at room temperature for 4 h. S5: Use the magnetic beads activated in S3 to magnetically separate the unbound polyclonal antibodies in S4, then add 500 µL of 0.1% BSA and shake at room temperature for 30 min to obtain the magnetic bead probe; S6: Wash the magnetic beads obtained in S5 three times with 10 mM PBS, pH = 7.2, resuspend in 1 mL PBS, and store at 4°C.

[0011] In a preferred embodiment of the present invention, the colloidal gold probe is a probe for labeling ssDNA and monoclonal antibodies, and the preparation method of the colloidal gold probe is: 1 mL of 4.2 nM AuNP was mixed with 4 µL of 100 mM K2CO3 and shaken at room temperature for 3 min. Then 10 µL of 0.1 mM labeled ssDNA and 6 µg of monoclonal antibody were added to obtain the probe. The probe was frozen at -20°C for 1 h. After thawing, the probe was centrifuged three times, each at 12800 g for 20 min, and the precipitate was resuspended in 10 mM PBS, pH=7.4.

[0012] In a preferred embodiment of the present invention, the monoclonal antibody is Ts-WN10-1H9, and the Ts-WN10-1H9 monoclonal antibody is prepared by hybridoma cell line WN10-1H9, and the deposit number of the hybridoma cell line WN10-1H9 is CGMCC No.18316.

[0013] In a preferred embodiment of the present invention, the sequence of the ssDNA is shown as SEQ ID NO.1.

[0014] In a preferred embodiment of the present invention, the washing solution is PBST, the standard uses PBS as a solvent, and contains crude Trichinella antigens at concentrations of 0, 3.125, 6.25, 12.5, 25, 50 and 100 ng / mL, respectively.

[0015] A second object of the present invention is to provide a method for using the above-mentioned kit for non-diagnostic purposes, wherein the method specifically comprises the following steps: S11: 15 µL of 10 mg / mL magnetic bead probe was mixed with 1 mL of the sample to be tested and the standard respectively, incubated at 37°C for 1 h, and then magnetically separated using a magnet, and washed three times with a washing solution to obtain a mixture 1; S22: Mix the mixture 1 obtained in S11 with 5 µL of 84 nM colloidal gold probe and 95 µL of PBS, incubate the mixture at 37°C for 1 h, perform magnetic separation using a magnet, and wash the mixture three times using a washing solution to obtain a mixture 2; S33: Add 10 µL of CRISPR / Cas12a system to the mixture 2 obtained in S22, and incubate at 37°C for 30 minutes; add 5 µL of 0.5 mM CuSO4 solution and 5 µL of 5 mM SA solution, and let stand for 2 minutes; add 80 µL of click reaction system, and incubate at 37°C for 10 minutes to obtain the processed sample to be tested; S44: Detect the fluorescence intensity of the sample obtained in S33, establish a standard curve with the concentration of the standard as the horizontal axis and the fluorescence intensity F460 value with an excitation wavelength of 350 nm and an emission wavelength of 460 nm as the vertical axis, and obtain the content of the crude antigen of Trichinella spiralis in the sample to be tested according to the standard curve.

[0016] The third object of the present invention is to provide the use of the above-mentioned kit in the detection of Trichinella spiralis.

[0017] Beneficial effects of the present invention: The present invention provides a kit for detecting Trichinella spiralis, which is a Trichinella spiralis antigen detection platform constructed based on CRISPR / Cas12a system and click chemistry, and the kit comprises: CRISPR / Cas12a system, click reaction system, 96-well reaction plate, magnetic bead probe, colloidal gold probe, washing solution, CuSO4 solution, SA solution and standard.

[0018] The magnetic bead probe (labeled with polyclonal antibody) is used to promote antigen enrichment and reduce the reaction volume of the CRISPR / Cas12a system, thereby realizing the amplification of the first layer of signals; the colloidal gold probe labels ssDNA and monoclonal antibodies, wherein a large amount of activator ssDNA labeled on the colloidal gold is used to activate the CRISPR / Cas12a system and realize the amplification of the second layer of signals; in order to activate more CRISPR / Cas12a systems to cut the target T20 (a DNA chain composed of 20 consecutive thymine bases), the present invention designs a repeating sequence activator ssDNA (shown in SEQ ID NO.1) to realize the amplification of the third layer of signals; CuNP generated in situ using T20 as a template is used as a click enzyme to effectively catalyze the CuAAC reaction, improve the efficiency of the CuAAC reaction and generate a fluorescent signal, thereby realizing the amplification of the fourth layer of signals.

[0019] The present invention forms a sandwich structure by combining a magnetic bead probe labeled with a polyclonal antibody with a cysteine ​​protease inhibitor-like protein (CLP, an antigen component of Trichinella spiralis and excretory secretion products), and a colloidal gold probe (labeled with ssDNA and monoclonal antibodies); for the first time, the in situ generated click enzyme-mediated CuAAC reaction is introduced into the CRISPR / Cas12a system to achieve signal amplification and stable signal readout. Moreover, compared with the copper-based nanocatalyst used for CuAAC reaction in the prior art, the present invention solves the problem of harsh storage and transportation conditions of nanomaterials, and improves the practical application of click chemistry reactions in biochemical analysis.

[0020] The Trichinella detection kit provided by the present invention adopts a four-layer signal amplification strategy. Compared with the traditional FQ probe, the in situ generated click enzyme is more cost-effective and stable, and eliminates the challenges in pre-preparation and storage; compared with the traditional ELISA, the detection sensitivity is improved by three orders of magnitude.

[0021] The Trichinella detection kit provided by the present invention is based on CRISPR / Cas12a-mediated click chemistry immunoassay and does not require the careful preparation of artificial click enzymes or complex covalent fixation steps, while still being able to achieve the purpose of ultra-sensitive detection of Trichinella in meat samples. The kit has the advantages of controllable quality, simple operation, high sensitivity, etc., and does not cross-react with other parasites during detection, with high specificity; and has the characteristics of long-term stability, strong practicality, easy preservation, market development value and wide promotion space, and has good market prospects.

[0022] [Biological Deposit Information]: The hybridoma cell line is named WN10-1H9. The deposit number of the hybridoma cell line WN10-1H9 is CGMCC No.18316, and the classification name is hybridoma cell line. It is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, and the deposit date is August 15, 2019. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The schematic diagram of the Trichinella spiralis detection kit based on CRISPR / Cas12a system and click chemistry in Example 3; A is a schematic diagram of probe construction; B is a schematic diagram of kit design; MNPs are magnetic bead probes, AuNPs are colloidal gold probes, Trichinella spiralis CLP Ag is Trichinella spiralis CLP antigen, CLP is the antigen component of Trichinella spiralis and excretion and secretion products, CLP PcAb is a polyclonal antibody, CLP McAb is a monoclonal antibody, and ssDNA is single-stranded DNA; Wavelength is wavelength, and FL Intensity is fluorescence intensity; Figure 2 : It is a detection diagram related to the CuAAC reaction in Example 3; A is a schematic diagram of in situ generation of CuNP using T20 template; B is a fluorescence spectrum of in situ formation of CuNP using T20 as a template; C is a TEM image of CuNP, with the horizontal axis Diameter being the diameter and the vertical axis Frequency being the frequency; D is a schematic diagram of CuNP catalyzing the CuAAC reaction; E is a fluorescence spectrum of the CuAAC reaction catalyzed by CuNP; Wavelength is the wavelength; FL Intensity is the fluorescence intensity; T20 is a DNA chain containing 20 thymine regions, CuNP is CuNP click enzyme, Azide 1 is azide 1, and Alkyne 2 is alkyne 2; Figure 3 It is the relevant detection diagram of the CRISPR / Cas12a system in Example 3; A is the working principle diagram of the CRISPR / Cas12a system; B is the relevant detection diagram of the CRISPR / Cas12a system combined with the CuAAC reaction; Under LED blue light is the blue light detection under LED; C is the fluorescence spectrum of the CRISPR / Cas12a system combined with the CuAAC reaction; Contain all components means including all components; Figure 4 : This is a comparison diagram of the effects of different single-stranded DNA activators on the CRISPR / Cas12a system in Example 3: A is a schematic diagram of the structure of the M21-Ry activator; B is a diagram of the action of the M21-Ry activator; C is a schematic diagram of the structure of the Ry-M21 activator; D is a diagram of the action of the Ry-M21 activator; E is a schematic diagram of the structure of the repeat sequence activator; F is a diagram of the action of the repeat sequence activator; Figure 5 : is the performance evaluation diagram in Example 4; A is the linear relationship diagram between fluorescence intensity and crude protein concentration of Trichinella spiralis; B is the specificity detection diagram, Trichinella spiralis is Trichinella spiralis, Clonorchis sinensis is Clonorchis sinensis, Cryptosporidiumn Tyzzer is Cryptosporidium, and Leishmania spp is Leishmania; C is the sensitivity detection diagram, Larvae number is the number of larvae; D is the practicality detection diagram, Pig is pig, Dog is dog, Rat is rat, and Rabbit is rabbit. DETAILED DESCRIPTION

[0024] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0025] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments and the accompanying drawings of the specification. The test methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained by those skilled in the art through commercial channels.

[0026] Example 1: A Trichinella detection kit based on CRISPR / Cas12a system and click chemistry biosensor The Trichinella detection kit described in this embodiment includes: CRISPR / Cas12a system, click reaction system, 96-well reaction plate, magnetic bead probe, colloidal gold probe, washing solution, CuSO4 solution, SA solution and standard; The CRISPR / Cas12a system includes: 3.6 mL of 1X NEB 2.1 buffer, 5 µL of 20 µM gRNA, 10 µL of 10 µM Cas12a, and 36 µL of 100 µM DNA chain (T20) containing 20 thymine regions; the sequence of the gRNA is shown in SEQ ID NO.2; The click reaction system includes: 0.25 mM 3-azido-7-hydroxycoumarin and 2000-fold diluted phenylacetylene; The magnetic bead probe is a probe for labeling polyclonal antibodies, and the preparation method of the magnetic bead probe is as follows: S1: Wash 10 mg of COOH-modified magnetic beads three times with 0.05 M, pH 6 MES buffer; S2: 500 µL of 4 mg / mL EDC and 5.5 mg of sulfo-NHS were added to the magnetic beads obtained in S1 and incubated at room temperature for 15 min for activation; S3: Wash the activated magnetic beads in S2 three times with 500 µL coupling solution (pH=7.5, 0.1 M Na3PO4, 0.15 M NaC); S4: Mix 500 µL of coupling solution and 0.5 mg of polyclonal antibody, and shake the mixture at room temperature for 4 h. S5: Use the magnetic beads activated in S3 to magnetically separate the unbound polyclonal antibodies in S4, then add 500 µL of 0.1% BSA and shake at room temperature for 30 min to obtain the magnetic bead probe; S6: Wash the magnetic beads obtained in S5 three times with 10 mM PBS, pH = 7.2, resuspend in 1 mL PBS, and store at 4°C; The colloidal gold probe is a probe for labeling ssDNA and monoclonal antibodies. The preparation method of the colloidal gold probe is as follows: 1 mL of 4.2 nM AuNP was mixed with 4 µL of 100 mM K2CO3 and shaken at room temperature for 3 min. Then 10 µL of 0.1 mM ssDNA and 6 µg of monoclonal antibody were added to obtain the probe, which was frozen at -20°C for 1 h. After thawing, the probe was centrifuged three times, each at 12800 g for 20 min, and the precipitate was resuspended in 10 mM PBS, pH=7.4. The monoclonal antibody is Ts-WN10-1H9, which is prepared by hybridoma cell line WN10-1H9, and the deposit number of the hybridoma cell line WN10-1H9 is CGMCC No.18316; The sequence of the ssDNA is shown in SEQ ID NO.1; The washing solution is PBST, and the standard uses PBS as a solvent, including crude Trichinella spiralis antigens with concentrations of 0, 3.125, 6.25, 12.5, 25, 50 and 100 ng / mL, respectively.

[0027] Example 2: Method for using the Trichinella spiralis detection kit S11: 15 µL of 10 mg / mL magnetic bead probe was mixed with 1 mL of the sample to be tested and the standard respectively, incubated at 37°C for 1 h, and then magnetically separated using a magnet, and washed three times with a washing solution to obtain a mixture 1; S22: Mix the mixture 1 obtained in S11 with 5 µL of 84 nM colloidal gold probe and 95 µL of PBS, incubate the mixture at 37°C for 1 h, perform magnetic separation using a magnet, and wash the mixture three times using a washing solution to obtain a mixture 2; S33: Add 10 µL of CRISPR / Cas12a system to the mixture 2 obtained in S22, and incubate at 37°C for 30 minutes; add 5 µL of 0.5 mM CuSO4 solution and 5 µL of 5 mM SA solution, and let stand for 2 minutes; add 80 µL of click reaction system, and incubate at 37°C for 10 minutes to obtain the processed sample to be tested; S44: Detect the fluorescence intensity of the sample obtained in S33, establish a standard curve with the concentration of the standard as the horizontal axis and the fluorescence intensity F460 value with an excitation wavelength of 350 nm and an emission wavelength of 460 nm as the vertical axis, and obtain the content of the crude antigen of Trichinella spiralis in the sample to be tested according to the standard curve.

[0028] Example 3: Feasibility analysis of the Trichinella spiralis detection kit (1) Design principle of the Trichinella spiralis detection kit The design principle of the Trichinella spiralis detection kit prepared in Example 1 is as follows Figure 1 As shown, when the sample to be tested contains Trichinella spiralis, the magnetic bead probe (labeled with polyclonal antibody) recognizes and enriches the Trichinella spiralis antigen cysteine ​​protease inhibitor-like protein (CLP, an antigenic component of Trichinella spiralis and excretory secretory products).

[0029] When the sample to be tested contains Trichinella spiralis, the magnetic bead probe, CLP and colloidal gold probe will form a sandwich structure, in which the ssDNA labeled with the colloidal gold probe activates the CRISPR / Cas12a system, and the Cas12a protein cuts T20 (a DNA chain containing 20 thymine regions). When the colloidal gold probe loses the T20 template, no CuNP is generated in situ, so no CuAAC reaction occurs and there is no fluorescence signal.

[0030] When the sample to be tested does not contain Trichinella spiralis, the magnetic bead probe, CLP and colloidal gold probe will not form a sandwich structure, in which the trans-cutting activity of the CRISPR / Cas12a system is not activated. Using T20 as a template, the click enzyme CuNP is generated in situ to catalyze the CuAAC reaction and produce a strong fluorescence signal.

[0031] The fluorescence F with an excitation wavelength of 350 nm and an emission wavelength of 460 nm was read by a microplate reader. 460 The concentration of the standard is the horizontal axis, and F 460 The standard curve is established with the value as the vertical coordinate, and the content of crude Trichinella antigen in the sample to be tested is obtained according to the standard curve. In this way, the CRISPR / Cas12a system and click chemistry are combined and applied to immunological detection to achieve the purpose of high-sensitivity detection of Trichinella antigen.

[0032] (2) In order to verify whether the in situ generated click enzyme CuNP in the kit prepared in Example 1 can catalyze the copper-catalyzed click reaction (CuAAC), fluorescence experiments and transmission electron microscopy (TEM) analysis were first performed in this example. The results are as follows: Figure 2 shown.

[0033] In this example, the ssDNA with the nucleotide sequence shown in SEQ ID NO.1 was used as a template to successfully generate the click enzyme CuNP in situ. Figure 2 A mixture of T20 and CuSO4 ( Figure 2 As shown in B, curve a) and the mixture of T20 and SA ( Figure 2 As shown in B, curve b) shows no fluorescence; in contrast, the mixture of T20, CuSO4 and SA shows a fluorescence peak at 650 nm ( Figure 2 As shown in B, curve c), and the mixture solution of T20, CuSO4 and SA shows red fluorescence. TEM image is shown in Figure 2 As shown in C, uniformly dispersed spherical nanoparticles are shown with an average particle size of 5.5 nm.

[0034] The CuNP click enzyme successfully generated in situ was used to catalyze the conversion of azide 1 (3-azido-7-hydroxycoumarin) and alkyne 2 (phenylacetylene). The generated triazole compound 3 showed blue fluorescence ( Figure 2 Azide 1, alkyne 2, or the mixture of azide 1 and alkyne 2 showed no fluorescence ( Figure 2 The mixture of azide 1, alkyne 2, CuSO4 and SA shows a weak fluorescence peak at 460 nm ( Figure 2 As shown in E, curve d), it shows that CuSO4 can react with SA to produce a small amount of Cu + , and then catalyzes the conversion of azide 1 and alkyne 2 into triazole compound 3, which in turn produces blue fluorescence. However, its reaction efficiency is low, so the fluorescence produced is weak. In contrast, the mixture of azide 1, alkyne 2, CuSO4, and SA+T20 shows a strong fluorescence peak at 460 nm ( Figure 2 As shown in Figure E, curve e), it shows that the in situ generated CuNPs can efficiently catalyze the conversion of azide 1 and alkyne 2 into triazole compound 3, thereby improving the efficiency of the CuAAC reaction.

[0035] It can be seen that the kit prepared in Example 1 successfully generated the CuNP click enzyme in situ, and the CuNP click enzyme has the function of efficiently catalyzing the conversion of azide 1 and alkyne 2 into triazole compound 3, thereby improving the efficiency of CuAAC reaction.

[0036] (3) This example verifies the feasibility of replacing the traditional FQ probe with the CuAAC reaction as the signal output component of the CRISPR / Cas12a system in the kit prepared in Example 1 ( Figure 3 (shown in A).

[0037] In this example, five reaction systems (reactions #1-#5) were prepared and tested, each of which contained CuSO4, SA, azide 1 or alkyne 2. Among them, reactions #1-#4 that did not contain Cas12a, gRNA, ssDNA, and T20 components, and #5 that contained all components of Cas12a, gRNA, ssDNA, and T20 ( Figure 3 (shown in B).

[0038] Reaction #5 (with all components) and reaction #4 (without T20) both produced weak fluorescence under UV light, while reactions #1-#3 all produced strong fluorescence under UV light ( Figure 3 As shown in C). It can be seen that in reactions #1, #2 and #3, which lack Cas12a, gRNA and ssDNA components respectively, Cas12a cannot exhibit trans-cleavage activity due to the lack of components generated by the CRISPR / Cas12a system, resulting in the T20 template remaining intact, while the CuNP click enzyme generated in situ by the intact T20 template normally catalyzes the production of the CuAAC reaction, thus generating strong blue fluorescence.

[0039] In reaction #4, due to the lack of T20 template, the CuNP click enzyme could not be generated in situ, and the CuAAC reaction could not be catalyzed, which reduced the reaction efficiency, so the generated blue fluorescence was weak.

[0040] In reaction #5, due to the cleavage activity of Cas12a acting on T20, the CuNP click enzyme could not be generated in situ, the occurrence of the CuAAC reaction was inhibited, and the reaction efficiency was low, so the blue fluorescence generated was weak.

[0041] It can be seen that the CuAAC reaction occurring in the kit prepared in Example 1 can be used as a signal output of the CRISPR / Cas12a system, which can sensitively reflect the presence of the target substance.

[0042] (4) In this example, in order to further study the effect of the length of the ssDNA activator on the trans-cleavage activity of the system, random sequences (such as Figure 4 Middle A and Figure 4 C, as shown in Table 1), and fluorescence detection was performed on them respectively.

[0043] The results are as follows Figure 4 Medium B and Figure 4 As shown in middle D, randomly extended M21 enhanced the activation of the CRISPR / Cas12a system, among which M21-R6 and R9-M21 extensions at the 3′ and 5′ ends showed the highest activation effect.

[0044] like Figure 4 As shown in E (as shown in Table 1), the matching sequence was repeated in this example, and the effect of the random sequence length of the matching repeat sequence on the trans-cleavage activity of the system was explored; the results are shown in Figure 4 As shown in F, the matching repeat sequence provided in this example significantly enhanced the activation effect of the CRISPR / Cas12a system, among which the R9-M21-R27-M21-R6 matching repeat sequence had the most significant activation effect on CRISPR / Cas12a. Therefore, the R9-M21-R27-M21-R6 matching repeat sequence was selected as the ssDNA activator, and the nucleotide sequence of the R9-M21-R27-M21-R6 matching repeat sequence was shown in SEQ ID NO.1.

[0045]

[0046] Example 4: Performance evaluation of the Trichinella spiralis detection kit (1) The test sample was tested using the kit prepared in Example 1, and a standard curve of fluorescence intensity and Trichinella crude protein concentration was constructed (eg Figure 5 As shown in A, when the crude protein concentration of Trichinella spiralis increased from 3.125 ng / mL to 100 ng / mL, the fluorescence intensity increased significantly. The fluorescence intensity (Y) was linearly related to the crude protein content of Trichinella spiralis (X), and the equation was Y=21.45X+3381.46 (R²=0.9747), with a detection limit of 0.35 ng / mL.

[0047] (2) In this example, the specificity of the kit prepared in Example 1 was verified. The kit prepared in Example 1 was used to specifically detect Trichinella spiralis, Clonorchis sinensis, Cryptosporidium and Leishmania parasites (the above-mentioned Trichinella spiralis and Leishmania were extracted from seed mice in this laboratory, Clonorchis sinensis was extracted from fish in this laboratory, and Cryptosporidium was donated by other laboratories). The results are shown in FIG. Figure 5 As shown in B, the fluorescence values ​​of the parasite samples of Trichodina sinensis, Clonorchis sinensis, Cryptosporidium and Leishmania were highly close to those of the blank control group, and were significantly higher than the low fluorescence value of the Trichinella spiralis sample, indicating that the kit provided by the present invention has excellent specificity.

[0048] (3) To further verify the detection capability of the kit prepared in Example 1 for a single Trichinella spiralis, this example tested 100 g of pork samples containing a known number of Trichinella spiralis (each sample contained 0, 1 or 5 Trichinella spiralis, n=3). The results are as follows: Figure 5 As shown in C, compared with the sample group containing 0 Trichinella, the fluorescence intensity of the sample containing 1 Trichinella is significantly reduced, proving that the Trichinella detection kit provided by the present invention can achieve detection at the single microorganism level.

[0049] (4) This example further evaluated the detection effect of the Trichinella spiralis detection kit based on the CRISPR / Cas12a system and click chemistry prepared in Example 1 in detecting Trichinella spiralis in different mammalian samples to verify its practicality. In this example, five Trichinella spiralis were added to pig, dog, rat and rabbit samples (100 g each, n=3), and the kit prepared in Example 1 was used for detection. The results are shown in Figure 1. Figure 5 As shown in D, the fluorescence intensity of the samples containing Trichinella spiralis from pigs, dogs, rats and rabbits was significantly lower than that of the blank control without Trichinella spiralis, indicating that the Trichinella spiralis detection kit provided by the present invention can be used to detect Trichinella spiralis in different mammalian species and has good practicality.

[0050] The contents not described in detail in the specification of the present invention are well-known technologies for those skilled in the art. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be based on the definition of the claims.

Claims

1. A Trichinella detection kit based on CRISPR / Cas12a system and click chemistry, characterized in that: The kit includes: a CRISPR / Cas12a system, a click reaction system, a 96-well reaction plate, a magnetic bead probe, a colloidal gold probe, a washing solution, a CuSO4 solution, a SA solution and a standard.

2. The kit according to claim 1, characterized in that The CRISPR / Cas12a system includes: 3.6 mL of 1X NEB2.1 buffer, 5 μL of 20 μM gRNA, 10 μL of 10 μM Cas12a, and 36 μL of a 100 μM DNA chain containing 20 thymine regions; the sequence of the gRNA is shown in SEQ ID NO.

2.

3. The kit according to claim 1, characterized in that The click reaction system includes: 0.25 mM 3-azido-7-hydroxycoumarin and 2000-fold diluted phenylacetylene.

4. The kit according to claim 1, characterized in that The magnetic bead probe is a probe for labeling polyclonal antibodies, and the preparation method of the magnetic bead probe is as follows: S1: Wash 10 mg of COOH-modified magnetic beads three times with 0.05 M, pH 6 MES buffer; S2: 500 μL of 4 mg / mL EDC and 5.5 mg of sulfo-NHS were added to the magnetic beads obtained in S1 and incubated at room temperature for 15 min for activation; S3: Wash the activated magnetic beads in S2 three times with 500 μL coupling solution; the coupling solution has a pH of 7.5 and is prepared from 0.1 M Na3PO4 and 0.15 M NaCl; S4: Mix 500 μL of coupling solution and 0.5 mg of polyclonal antibody, and shake the mixture at room temperature for 4 h; S5: Use the magnetic beads activated in S3 to magnetically separate the unbound polyclonal antibodies in S4, then add 500 μL of 0.1% BSA, shake at room temperature for 30 min, and obtain the magnetic bead probe; S6: Wash the magnetic bead probe obtained in S5 three times with 10 mM PBS, pH = 7.2, resuspend in 1 mL PBS, and store at 4°C.

5. The kit according to claim 1, characterized in that The colloidal gold probe is a probe for labeling ssDNA and monoclonal antibodies. The preparation method of the colloidal gold probe is as follows: Mix 1mL 4.2nM AuNP with 4μL 100mM K2CO3 and oscillate at room temperature for 3min; then add 10μL 0.1mM ssDNA and 6μg monoclonal antibody to obtain the probe; freeze it at -20℃ for 1h, and after thawing, perform three rounds of centrifugation on the probe, each round of centrifugation at 12800g for 20min, and resuspend the precipitate in 10mM PBS, pH=7.

4.

6. The kit according to claim 5, characterized in that The monoclonal antibody is Ts-WN10-1H9, which is prepared by hybridoma cell line WN10-1H9. The deposit number of the hybridoma cell line WN10-1H9 is CGMCC No.18316.

7. The kit according to claim 5, characterized in that The sequence of the ssDNA is shown in SEQ ID NO.

1.

8. The kit according to claim 1, characterized in that The washing solution is PBST, the standard uses PBS as a solvent, and contains crude Trichinella spiralis antigens with concentrations of 0, 3.125, 6.25, 12.5, 25, 50 and 100 ng / mL, respectively.

9. A method for using the kit according to any one of claims 1 to 8 for non-diagnostic purposes, characterized in that: The method of use specifically comprises the following steps: S11: 15 μL of 10 mg / mL magnetic bead probe was mixed with 1 mL of the sample to be tested and the standard respectively, incubated at 37°C for 1 h, and then magnetically separated using a magnet, and washed three times with a washing solution to obtain a mixture 1; S22: Mix the mixture 1 obtained in S11, 5 μL of 84 nM colloidal gold probe and 95 μL of PBS, incubate the mixture at 37° C. for 1 h, perform magnetic separation using a magnet, and wash the mixture three times using a washing solution to obtain a mixture 2; S33: Add 10 μL of CRISPR / Cas12a system to the mixture 2 obtained in S22, and incubate at 37°C for 30 minutes; add 5 μL of 0.5 mM CuSO4 solution and 5 μL of 5 mM SA solution, and let stand for 2 minutes; add 80 μL of click reaction system, and incubate at 37°C for 10 minutes to obtain the treated sample to be tested; S44: Detect the fluorescence intensity of the sample obtained in S33, establish a standard curve with the concentration of the standard as the horizontal axis and the fluorescence intensity F460 value with an excitation wavelength of 350nm and an emission wavelength of 460nm as the vertical axis, and obtain the content of the crude antigen of Trichinella spiralis in the sample to be tested according to the standard curve.

10. Use of the kit according to any one of claims 1 to 8 in the detection of Trichinella spiralis.

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