A Trichinella detection kit based on the CRISPR / Cas12a system and click chemistry, as well as a method of use and applications thereof

Abstract

A Trichinella detection kit based on the CRISPR / Cas12a system and click chemistry, as well as its usage method and application, belong to the technical field of immunological detection. To solve the technical problems in the prior art that the Trichinella detection methods are time-consuming and laborious, have low sensitivity, are prone to missed detection, and the signal output probes of the CRISPR / Cas12a system, the complex preparation process of artificial click enzymes, and the difficulty in storing and transporting nanomaterials, the present invention provides a Trichinella detection kit, which includes: a CRISPR / Cas12a system, a click reaction system, a 96-well reaction plate, magnetic bead probes, colloidal gold probes, washing solution, CuSO4 solution, SA solution, and standards. The Trichinella detection kit provided by the present invention has the advantages of high specificity, long-term stability, strong practicability, controllable quality, simple operation, high sensitivity, easy preservation, etc., and can be applied to Trichinella detection, with good market prospects.

Description

Technical Field

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

[0002] Trichinellosis is a foodborne parasitic disease caused by Trichinella, which exists widely worldwide 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 larvae, and pigs are the main infection sources; therefore, accurately and highly sensitively detecting Trichinella - contaminated meat is crucial for ensuring food safety.

[0003] The digestion method of pooled samples recommended by the International Commission on Trichinellosis (ICT) is the "gold standard" for detecting Trichinella. However, this method has a risk of live larva infection during sample processing, and the process is time - consuming and laborious. It requires experienced technicians to perform microscopic examination, and it is prone to missed detection; 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 cost; 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 strong specificity and high stability; however, it is difficult for ELISA to detect low - concentration pathogens, and improving its sensitivity remains a major challenge for those skilled in the art. The CRISPR / Cas system detection technology has atomic - level sensitivity, accuracy, and portability. Combined with biosensing modules such as aptamers, antigen - antibody interactions, and enzyme reactions, it can be applied to the detection of non - nucleic acid targets. Those skilled in the art organically combine ELISA with the CRISPR / Cas12a system to highly sensitively detect pathogens; however, since the CRISPR / Cas12a system mainly relies on FQ probes to output signals, the above - mentioned probes have a high usage cost and are easily affected by oxidation, thus limiting the practical application of the above - mentioned method in biochemical analysis.

[0005] Click chemistry is a highly sensitive signal output method, featuring fast reaction rates, mild conditions, and high selectivity. The copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is a revolutionary click chemical reaction. However, the preparation process of current copper-based nanocatalysts for CuAAC reactions (such as copper nanoflowers, copper nanozymes, and copper MOFs) is complex and time-consuming, and the storage and transportation conditions of nanomaterials are relatively stringent, thus affecting the practical application of click chemistry in biochemical analysis. Summary of the Invention

[0006] To solve the technical problems in the prior art that the trichinella detection method is time-consuming, laborious, low in sensitivity, prone to missed detection, and the signal output probes of the CRISPR / Cas12a system and artificial click enzymes have complex preparation processes and nanomaterials are not easy to store and transport, the present invention provides a trichinella detection kit based on the CRISPR / Cas12a system and click chemistry, as well as its usage method and application.

[0007] One object of the present invention is to provide a trichinella detection kit based on the CRISPR / Cas12a system and click chemistry, and the kit includes: a CRISPR / Cas12a system, a click reaction system, a 96-well reaction plate, magnetic bead probes, colloidal gold probes, washing solution, CuSO4 solution, SA solution, and standards.

[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 100 µM DNA strand containing 20 thymine regions; the gRNA sequence is as shown in SEQ ID NO.2.

[0009] In a preferred embodiment of the present invention, the click reaction system includes: 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 labeled with polyclonal antibodies, and the preparation method of the magnetic bead probe is:

[0011] S1: Wash 10 mg of COOH-modified magnetic beads three times with 0.05M, pH = 6 MES buffer;

[0012] S2: Add 500 µL of 4 mg / mL EDC and 5.5 mg of sulfo-NHS to the magnetic beads obtained in S1, and incubate at room temperature for 15 min for activation;

[0013] S3: Wash the activated magnetic beads in S2 three times with 500 μL of coupling solution; the pH of the coupling solution is 7.5 and it is obtained by configuring 0.1 M Na3PO4 and 0.15 M NaCl;

[0014] S4: Mix 500 μL of coupling solution and 0.5 mg of polyclonal antibody evenly, and shake the above mixture at room temperature for 4 h;

[0015] S5: Use the activated magnetic beads in S3 to magnetically separate the unbound polyclonal antibody in S4, then add 500 μL of 0.1% BSA, and shake at room temperature for 30 min to obtain magnetic bead probes;

[0016] S6: Wash the magnetic bead probes obtained in S5 three times with 10 mM PBS with pH = 7.2, resuspend in 1 mL of PBS, and store at 4°C.

[0017] In a preferred embodiment of the present invention, the colloidal gold probe is a probe labeled with ssDNA and monoclonal antibody, and the preparation method of the colloidal gold probe is as follows:

[0018] Mix 1 mL of 4.2 nM AuNP with 4 μL of 100 mM K2CO3, and shake at room temperature for 3 min; then add 10 μL of 0.1 mM labeled ssDNA and 6 μg of monoclonal antibody to obtain a probe; freeze at -20°C for 1 h, after thawing, centrifuge the above probe three times, each time centrifuge at 12800 g for 20 min, and resuspend the precipitate in 10 mM PBS with pH = 7.4.

[0019] In a preferred embodiment of the present invention, the monoclonal antibody is Ts-WN10-1H9, and the Ts-WN10-1H9 monoclonal antibody is prepared from the hybridoma cell line WN10-1H9, and the preservation number of the hybridoma cell line WN10-1H9 is CGMCC No. 18316.

[0020] In a preferred embodiment of the present invention, the sequence of the ssDNA is as shown in SEQ ID NO.1.

[0021] In a preferred embodiment of the present invention, the washing solution is PBST, the standard product uses PBS as a solvent, and contains Trichinella crude antigens with concentrations of 0, 3.125, 6.25, 12.5, 25, 50, and 100 ng / mL respectively.

[0022] The second object of the present invention is to provide a method for using the above kit for non-diagnostic purposes, and the method specifically includes the following steps:

[0023] S11: Mix 15 µL of the magnetic bead probe at 10 mg / mL with 1 mL of the sample to be tested and the standard respectively, incubate at 37 °C for 1 h, then perform magnetic separation using a magnet, and wash 3 times with the washing solution to obtain Mixture 1;

[0024] S22: Mix the Mixture 1 obtained in S11 evenly with 5 µL of the colloidal gold probe at 84 nM and 95 µL of PBS, incubate the above mixture at 37 °C for 1 h, then perform magnetic separation using a magnet, and wash 3 times with the washing solution to obtain Mixture 2;

[0025] S33: Add 10 µL of the CRISPR / Cas12a system to the Mixture 2 obtained in S22, incubate at 37 °C for 30 minutes; add 5 µL of the 0.5 mM CuSO4 solution and 5 µL of the 5 mM SA solution, let stand for 2 min; add 80 µL of the click reaction system, incubate at 37 °C for 10 min to obtain the processed sample to be tested;

[0026] S44: Detect the fluorescence intensity of the sample obtained in S33, use the standard concentration as the abscissa, and use the fluorescence intensity F460 value read at the excitation wavelength of 350 nm and the emission wavelength of 460 nm as the ordinate to establish a standard curve, and obtain the content of the Trichinella crude antigen in the sample to be tested according to the standard curve.

[0027] The third object of the present invention is to provide the application of the above kit in the detection of Trichinella.

[0028] The beneficial effects of the present invention: The present invention provides a kit for detecting Trichinella, and the kit is a Trichinella antigen detection platform constructed based on the CRISPR / Cas12a system and click chemistry. 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, an SA solution, and a standard product.

[0029] Magnetic bead probes (labeled with polyclonal antibodies) are used to promote antigen enrichment and reduce the reaction volume of the CRISPR / Cas12a system, thereby achieving the amplification of the first-layer signal; colloidal gold probes are labeled with ssDNA and monoclonal antibodies, and a large number of activator ssDNAs labeled on the colloidal gold are used to activate the CRISPR / Cas12a system and achieve the amplification of the second-layer signal; in order to activate more CRISPR / Cas12a systems to cleave the target T20 (a DNA strand composed of 20 consecutive thymine bases), the present invention designs an activator ssDNA with a repeat sequence (shown in SEQ ID NO.1) to achieve the amplification of the third-layer signal; CuNP generated in situ using T20 as a template serves as a click enzyme to effectively catalyze the CuAAC reaction, improve the efficiency of the CuAAC reaction and generate a fluorescence signal, thereby achieving the amplification of the fourth-layer signal.

[0030] In the present invention, a sandwich structure is formed by a magnetic bead probe labeled with a polyclonal antibody, a cysteine protease inhibitor-like protein (CLP, an antigenic component of Trichinella spiralis and its excretory-secretory products), and a colloidal gold probe (labeled with ssDNA and a monoclonal antibody); for the first time, a click enzyme-mediated CuAAC reaction generated in situ is introduced into the CRISPR / Cas12a system to achieve signal amplification and stable signal readout. Moreover, compared with the copper-based nanocatalysts used for the CuAAC reaction in the prior art, the present invention solves the problem of relatively harsh storage and transportation conditions of nanomaterials and improves the practical application of click chemistry in biochemical analysis.

[0031] The Trichinella spiralis detection kit provided by the present invention adopts a four-layer signal amplification strategy. Compared with traditional FQ probes, the click enzyme generated in situ is more cost-effective and stable, and eliminates the challenges in pre-preparation and storage; compared with traditional ELISA, the detection sensitivity is increased by three orders of magnitude.

[0032] The Trichinella spiralis detection kit provided by the present invention based on CRISPR / Cas12a-mediated click chemistry immunoassay does not require elaborate preparation of artificial click enzymes or complex covalent fixation steps, and still can achieve the purpose of ultrasensitive detection of Trichinella spiralis in meat samples. The kit has the advantages of controllable quality, simple operation, high sensitivity, etc. At the same time, it does not cross-react with other parasites during detection, and has high specificity; and it has the characteristics of long-term stability, strong practicability, easy storage, has market development value and broad promotion space, and has a good market prospect.

[0033]

Biological Deposit Information

[0034] Figure 1 It is the schematic diagram of the trichinella detection kit based on the CRISPR / Cas12a system and click chemistry in Example 3; A is the schematic diagram of probe construction; B is the schematic diagram of kit design; MNPs are magnetic bead probes, AuNP is a colloidal gold probe, Trichinella spiralis CLP Ag is the trichinella CLP antigen, CLP is the antigen component of trichinella and excretory-secretory products, CLP PcAb is a polyclonal antibody, CLP McAb is a monoclonal antibody, ssDNA is single-stranded DNA; Wavelength is the wavelength, FL Intensity is the fluorescence intensity;

[0035] Figure 2 It is the detection diagram related to the CuAAC reaction in Example 3; A is the schematic diagram of in-situ generation of CuNP using the T20 template; B is the fluorescence spectrum of in-situ formation of CuNP using T20 as the template; C is the TEM image of CuNP, with the abscissa Diameter being the diameter and the ordinate Frequency being the frequency; D is the schematic diagram of the CuAAC reaction catalyzed by CuNP; E is the fluorescence spectrum of the CuAAC reaction catalyzed by CuNP; Wavelength is the wavelength; FL Intensity is the fluorescence intensity; T20 is a DNA strand containing 20 thymine regions, CuNP is the CuNP click enzyme, Azide 1 is azide 1, and Alkyne 2 is alkyne 2;

[0036] Figure 3 It is the detection diagram related to the CRISPR / Cas12a system in Example 3; A is the schematic diagram of the principle of action of the CRISPR / Cas12a system; B is the detection diagram related to the combination of the CRISPR / Cas12a system and the CuAAC reaction; Under LED blue light is the detection under LED blue light; C is the fluorescence spectrum of the combination of the CRISPR / Cas12a system and the CuAAC reaction; Contain all components means containing all components;

[0037] Figure 4Comparison diagram of the effects of different single-stranded DNA activators on the CRISPR / Cas12a system in Example 3: A is the structural schematic diagram of the M21-Ry activator; B is the action diagram of the M21-Ry activator; C is the structural schematic diagram of the Ry-M21 activator; D is the action diagram of the Ry-M21 activator; E is the structural schematic diagram of the repeat sequence activator; F is the action diagram of the repeat sequence activator;

[0038] Figure 5 Performance evaluation diagram in Example 4; A is the linear relationship diagram between fluorescence intensity and concentration of Trichinella spiralis crude protein; B is the specificity detection diagram, Trichinella spiralis is Trichinella spiralis, Clonorchis sinensis is Clonorchis sinensis, Cryptosporidiumn Tyzzer is Cryptosporidium, 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, Rabbit is rabbit. Detailed implementation manners

[0039] Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate modifications and combinations to the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technical solution of the present invention.

[0040] In order to make the purpose, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with specific implementation manners and the accompanying drawings of the specification. The test methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art unless otherwise specified, and those skilled in the art can obtain them through commercial channels.

[0041] Example 1: A Trichinella detection kit based on the CRISPR / Cas12a system and click chemistry biosensor

[0042] The Trichinella detection kit described in this example includes: a CRISPR / Cas12a system, a click reaction system, a 96-well reaction plate, magnetic bead probes, colloidal gold probes, washing solution, CuSO4 solution, SA solution and standards;

[0043] 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 strand (T20) containing 20 thymine regions; the sequence of the gRNA is shown as SEQ ID NO.2;

[0044] The click reaction system includes: 0.25 mM of 3-azido-7-hydroxycoumarin and 2000-fold diluted phenylethylene;

[0045] The magnetic bead probe is a probe labeled with polyclonal antibody, and the preparation method of the magnetic bead probe is as follows:

[0046] S1: Wash 10 mg of COOH-modified magnetic beads three times with 0.05 M MES buffer at pH = 6;

[0047] S2: Add 500 µL of 4 mg / mL EDC and 5.5 mg of sulfo-NHS to the magnetic beads obtained in S1, and incubate at room temperature for 15 min for activation;

[0048] S3: Wash the activated magnetic beads in S2 three times with 500 µL of coupling solution (pH = 7.5, 0.1 M Na3PO4, 0.15 M NaC);

[0049] S4: Mix 500 µL of coupling solution and 0.5 mg of polyclonal antibody evenly, and shake the above mixture at room temperature for 4 h;

[0050] S5: Use the activated magnetic beads in S3 to magnetically separate the unbound polyclonal antibody in S4, then add 500 µL of 0.1% BSA, and shake at room temperature for 30 min to obtain the magnetic bead probe;

[0051] S6: Wash the magnetic bead probe obtained in S5 three times with 10 mM PBS at pH = 7.2, resuspend in 1 mL of PBS, and store at 4°C;

[0052] The colloidal gold probe is a probe labeled with ssDNA and monoclonal antibody, and the preparation method of the colloidal gold probe is as follows:

[0053] Mix 1 mL of 4.2 nM AuNP with 4 μL of 100 mM K2CO3 and oscillate at room temperature for 3 min; then add 10 μL of 0.1 mM ssDNA and 6 μg of monoclonal antibody to obtain a probe, and freeze it at -20 °C for 1 h; after thawing, centrifuge the above probe three times, each time at 12,800 g for 20 min, and resuspend the precipitate in 10 mM PBS with pH = 7.4;

[0054] The monoclonal antibody is Ts-WN10-1H9, and the Ts-WN10-1H9 monoclonal antibody is prepared from the hybridoma cell line WN10-1H9, and the preservation number of the hybridoma cell line WN10-1H9 is CGMCC No. 18316;

[0055] The sequence of the ssDNA is shown as SEQ ID NO.1;

[0056] The washing solution is PBST, and the standard product uses PBS as a solvent and includes Trichinella crude antigens with concentrations of 0, 3.125, 6.25, 12.5, 25, 50, and 100 ng / mL respectively.

[0057] Example 2: Usage method of the Trichinella detection kit

[0058] S11: Mix 15 μL of 10 mg / mL magnetic bead probe with 1 mL of the sample to be tested and the standard product respectively, incubate at 37 °C for 1 h, then perform magnetic separation using a magnet, and wash 3 times with the washing solution to obtain mixture 1;

[0059] S22: Mix the mixture 1 obtained in S11 evenly with 5 μL of 84 nM colloidal gold probe and 95 μL of PBS, incubate the above mixture at 37 °C for 1 h, then perform magnetic separation using a magnet, and wash 3 times with the washing solution to obtain mixture 2;

[0060] S33: Add 10 μL of CRISPR / Cas12a system to the mixture 2 obtained in S22, incubate at 37 °C for 30 minutes; add 5 μL of 0.5 mM CuSO4 solution and 5 μL of 5 mM SA solution, let it stand for 2 min; add 80 μL of click reaction system, incubate at 37 °C for 10 min to obtain the processed sample to be tested;

[0061] S44: Detect the fluorescence intensity of the sample obtained in S33. Using the standard product concentration as the abscissa and the fluorescence intensity F460 value read at the excitation wavelength of 350 nm and the emission wavelength of 460 nm as the ordinate, establish a standard curve, and obtain the content of Trichinella crude antigen in the sample to be tested according to the standard curve.

[0062] Example 3: Feasibility analysis of the Trichinella detection kit

[0063] (1) Design principle of the Trichinella detection kit

[0064] The design principle of the Trichinella detection kit prepared in Example 1 is as Figure 1 shown. When the sample to be tested contains Trichinella, the magnetic bead probe (labeled with polyclonal antibody) recognizes and enriches the Trichinella antigen cystatin-like protein (CLP, an antigen component of Trichinella and its excretory-secretory products).

[0065] When the sample to be tested contains Trichinella, the magnetic bead probe, CLP, and colloidal gold probe will form a sandwich structure. Among them, the ssDNA labeled by the colloidal gold probe activates the CRISPR / Cas12a system, and the Cas12a protein cleaves T20 (a DNA strand containing 20 thymine regions). When the colloidal gold probe loses the T20 template, no in-situ generation of CuNP occurs. Therefore, the CuAAC reaction does not occur and no fluorescence signal is generated.

[0066] When the sample to be tested does not contain Trichinella, the magnetic bead probe, CLP, and colloidal gold probe will not form a sandwich structure. Among them, the trans-cleavage activity of the CRISPR / Cas12a system is not activated. Using T20 as the template, click enzyme CuNP is generated in-situ, catalyzing the occurrence of the CuAAC reaction and generating a strong fluorescence signal.

[0067] Read the fluorescence F 460 value at the excitation wavelength of 350 nm and the emission wavelength of 460 nm through an enzyme-labeled instrument. Using the standard product concentration as the abscissa and the F 460 value as the ordinate, establish a standard curve, and obtain the content of Trichinella crude antigen in the sample to be tested 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 highly sensitive detection of Trichinella antigen.

[0068] (2) In this example, in order to verify whether the click enzyme CuNP generated in-situ in the kit prepared in Example 1 can catalyze the copper-catalyzed click reaction (CuAAC), this example first carried out fluorescence experiments and transmission electron microscopy (TEM) analysis, and the results are as Figure 2 shown.

[0069] In this example, ssDNA with the nucleotide sequence shown in SEQ ID NO.1 was used as a template, and click enzyme CuNP was successfully generated in situ ( Figure 2 as shown in A). The 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) did not show fluorescence; in contrast, the mixture of T20, CuSO4 and SA showed a fluorescence peak at 650 nm ( Figure 2 as shown in B, curve c), and the solution of the mixture of T20, CuSO4 and SA showed red fluorescence. The TEM image is as shown in Figure 2 C, showing uniformly dispersed spherical nanoparticles with an average particle size of 5.5 nm.

[0070] The above-mentioned successfully generated CuNP click enzyme in situ was used to catalyze the conversion reaction of azide 1 (3-azido-7-hydroxycoumarin) and alkyne 2 (phenylacetylene), and the generated triazole compound 3 showed blue fluorescence ( Figure 2 as shown in D). Neither azide 1, alkyne 2, nor the mixture of azide 1 and alkyne 2 showed fluorescence ( Figure 2 as shown in E, curves a–c); the mixture of azide 1, alkyne 2, CuSO4 and SA showed a weak fluorescence peak at 460 nm ( Figure 2 as shown in E, curve d), indicating that CuSO4 can react with SA to produce a small amount of Cu + , which then catalyzes the conversion of azide 1 and alkyne 2 into triazole compound 3, thereby producing blue fluorescence, but its reaction efficiency is low, so the fluorescence generated is weak; in contrast, the mixture of azide 1, alkyne 2, CuSO4, SA+T20 showed a strong fluorescence peak at 460 nm ( Figure 2 as shown in E, curve e), indicating that the in-situ generated CuNP can efficiently catalyze the conversion of azide 1 and alkyne 2 into triazole compound 3, improving the efficiency of the CuAAC reaction.

[0071] It can be seen that the kit prepared in Example 1 successfully generated 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 and improving the efficiency of the CuAAC reaction.

[0072] (3) In this example, in order to verify 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 as shown in A).

[0073] In this example, five reaction systems (Reaction #1 - #5) were prepared and tested. Each system contained CuSO4, SA, azide 1 or alkyne 2. Among them, Reaction #1 - #4 separately did not contain Cas12a, gRNA, ssDNA, and T20 components individually, and #5 contained all components of Cas12a, gRNA, ssDNA, and T20 ( Figure 3 as shown in B).

[0074] Both Reaction #5 (containing all components) and Reaction #4 (without T20) produced weak fluorescence under ultraviolet light conditions, while Reactions #1 - #3 all produced strong fluorescence under ultraviolet light conditions ( Figure 3 as shown in C). It can be seen that in Reactions #1, #2, and #3 that separately lacked Cas12a, gRNA, and ssDNA components individually, due to the lack of components for the CRISPR / Cas12a system to occur, Cas12a could not exhibit trans - cleavage activity, resulting in the integrity of the T20 template. And the CuNP click enzyme generated in situ from the intact T20 template normally catalyzed the occurrence of the CuAAC reaction, so strong blue fluorescence was produced.

[0075] In Reaction #4, due to the lack of the T20 template, the CuNP click enzyme could not be generated in situ, the CuAAC reaction could not be catalyzed to occur, the reaction efficiency was reduced, so the blue fluorescence produced was weak.

[0076] 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 produced was weak.

[0077] Thus, it can be seen that the CuAAC reaction occurring in the kit prepared in Example 1 can be used as the signal output of the CRISPR / Cas12a system and can sensitively reflect the presence of the target substance.

[0078] (4) In this example, to further study the effect of the length of the ssDNA activator on the trans - cleavage activity of the system on the colloidal gold probe, random sequences were added to the 3′ and 5′ ends of M21 based on the RESET effect (random extension sequence enhances trans - cleavage activity) (as shown in A and Figure 4 C, as shown in Table 1), and fluorescence detection was carried out on them respectively. Figure 4 The results are as shown in B and

[0079] D. The randomly extended M21 enhanced the activation of the CRISPR / Cas12a system. Among them, the extensions at the 3′ and 5′ ends of M21 - R6 and R9 - M21 showed the highest activation effect. Figure 4 as shown in B and Figure 4 D.

[0080] As Figure 4 shown in E (as shown in Table 1), in this embodiment, the matching sequence was repeated, and the influence of the random sequence length of the matching repeated sequence on the trans-cleavage activity of the system was explored; the results are as Figure 4 shown in F. The matching repeated sequence provided in this example significantly enhanced the activation effect of the CRISPR / Cas12a system. Among them, the R9-M21-R27-M21-R6 matching repeated sequence had the most significant activation effect on CRISPR / Cas12a. Therefore, the R9-M21-R27-M21-R6 matching repeated sequence was selected as the ssDNA activator, and the nucleotide sequence of the R9-M21-R27-M21-R6 matching repeated sequence is shown in SEQ ID NO.1.

[0081]

[0082] Example 4: Performance Evaluation of Trichinella Detection Kit

[0083] (1) The kit prepared in Example 1 was used to detect the test sample, and a standard curve of fluorescence intensity vs. Trichinella crude protein concentration was constructed (as Figure 5 shown in A). When the Trichinella crude protein concentration increased from 3.125 ng / mL to 100 ng / mL, the fluorescence intensity increased significantly. The fluorescence intensity (Y) and the Trichinella crude protein content (X) showed a linear relationship, and the equation was Y = 21.45X + 3381.46 (R² = 0.9747), and the detection limit was 0.35 ng / mL.

[0084] (2) To verify the specificity of the kit prepared in Example 1 in this embodiment, the kit prepared in Example 1 was used to specifically detect Trichinella, Clonorchis sinensis, Cryptosporidium, and Leishmania parasites (the above-mentioned Trichinella and Leishmania were obtained by extracting from the preserved mice in this laboratory, Clonorchis sinensis was extracted from fish in this laboratory, and Cryptosporidium was obtained as a gift from other laboratories), and the results are as Figure 5 shown in B. The detection fluorescence values of the samples of Clonorchis sinensis, Clonorchis sinensis, Cryptosporidium, and Leishmania parasites were highly close to those of the blank control group, and were significantly higher than the low fluorescence values of the Trichinella samples, indicating that the kit provided by the present invention has excellent specificity.

[0085] (3) To further verify the detection ability of the kit prepared in Example 1 for individual Trichinella, in this embodiment, 100-gram pork samples containing a known number of Trichinella (each sample contained 0, 1, or 5 Trichinella, n = 3) were tested, and the results are as Figure 5As shown in C, compared with the group of samples containing 0 Trichinella spiralis, the fluorescence intensity of the samples containing 1 Trichinella spiralis was significantly reduced, proving that the Trichinella spiralis detection kit provided by the present invention can achieve detection at the single microorganism level.

[0086] (4) In this example, the detection effect of the Trichinella spiralis detection kit based on the CRISPR / Cas12a system and click chemistry prepared in Example 1 was further evaluated in detecting Trichinella spiralis in different mammalian samples to verify its practicability. In this example, five Trichinella spiralis were added to pig, dog, rat, and rabbit samples (100 grams each, n = 3), and the kit prepared in Example 1 was used for detection. The results are as Figure 5 shown in D. The fluorescence intensities of the pig, dog, rat, and rabbit samples containing Trichinella spiralis were all significantly lower than those of the blank control without Trichinella spiralis, indicating that the Trichinella spiralis detection kit provided by the present invention can be applied to detect Trichinella spiralis in different mammalian species and has good practicability.

[0087] The content not described in detail in the specification of the present invention is well-known technology to those skilled in the art. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A Trichinella detection kit based on the CRISPR / Cas12a system and click chemistry, characterized in that, The kit 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 product; The magnetic bead probe is a probe labeled with 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 MES buffer solution with pH = 6; S2: Add 500 μL of 4 mg / mL EDC and 5.5 mg of sulfo-NHS to the magnetic beads obtained in S1, and incubate 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 evenly, and shake the above mixture at room temperature for 4 h; S5: Use the activated magnetic beads in S3 to magnetically separate the unbound polyclonal antibody in S4, then add 500 μL of 0.1% BSA, and shake at room temperature for 30 min to obtain magnetic bead probe; S6: Wash the magnetic bead probe obtained in S5 three times with 10 mM PBS with pH = 7.2, resuspend in 1 mL of PBS, and store at 4°C; The colloidal gold probe is a probe labeled with ssDNA and monoclonal antibody, and the preparation method of the colloidal gold probe is: Mix 1 mL of 4.2 nM AuNP with 4 μL of 100 mM K2CO3, and shake at room temperature for 3 min; then add 10 μL of 0.1 mM ssDNA and 6 μg of monoclonal antibody to obtain the probe; freeze at -20°C for 1 h, after thawing, centrifuge the above probe three times, each time centrifuge at 12800 g for 20 min, and resuspend the precipitate in 10 mM PBS with pH = 7.4; The monoclonal antibody is Ts-WN10-1H9, and the Ts-WN10-1H9 monoclonal antibody is prepared from the hybridoma cell line WN10-1H9, and the preservation number of the hybridoma cell line WN10-1H9 is CGMCC No. 18316; The sequence of the ssDNA is shown as SEQ ID NO.1; The CRISPR / Cas12a system includes: 3.6 mL of 1X NEB 2.1 buffer solution, 5 μL of 20 μM gRNA, 10 μL of 10 μM Cas12a and 36 μL of 100 μM DNA strand containing 20 thymine regions; the sequence of the gRNA is shown as SEQ ID NO.2; 2. The kit according to claim 1, wherein The click reaction system includes: 0.25 mM 3-azido-7-hydroxycoumarin and 2000-fold diluted phenylacetylene.

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

4. A method of using the kit according to any one of claims 1 to 3 for non-diagnostic purposes, characterized in that, The specific usage method includes the following steps: S11: Mix 15 μL of the magnetic bead probe at 10 mg / mL with 1 mL of the sample to be tested and the standard products respectively, incubate at 37°C for 1 h, then perform magnetic separation using a magnet, and wash 3 times with the washing solution to obtain Mixture 1; S22: Mix the Mixture 1 obtained in S11 evenly with 5 μL of the colloidal gold probe at 84 nM and 95 μL of PBS, incubate the above mixture at 37°C for 1 h, then perform magnetic separation using a magnet, and wash 3 times with the washing solution to obtain Mixture 2; S33: Add 10 μL of the CRISPR / Cas12a system to the Mixture 2 obtained in S22, incubate at 37°C for 30 minutes; add 5 μL of the CuSO4 solution at 0.5 mM and 5 μL of the SA solution at 5 mM, let it stand for 2 min; add 80 μL of the click reaction system, incubate at 37°C for 10 min to obtain the processed sample to be tested; S44: Detect the fluorescence intensity of the sample obtained in S33. Use the standard product concentration as the abscissa and the fluorescence intensity F460 value read at the excitation wavelength of 350 nm and the emission wavelength of 460 nm as the ordinate to establish a standard curve, and obtain the content of the crude Trichinella antigen in the sample to be tested according to the standard curve.

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