RAA-CRISPR / Cas12a-based trichinella spiralis rapid detection system, kit and use method of kit

Abstract

The invention discloses a rapid trichina detection system based on RAA-CRISPR / Cas12a, a kit and a use method of the kit. According to the present invention, based on the RAA technology, the isothermal amplification is performed on the ribosome small subunit gene of trichina, the reaction result is visualized by using the specific and visual CRISPR / Cas12a enzyme, and the established RAA-CRISPR / Cas12a diagnostic kit can conveniently, rapidly and efficiently diagnose trichina infection. Meanwhile, the diagnosis effectiveness evaluation result of the kit in mouse muscles, blood and feces shows that the detection kit can complete rapid detection of trichina in a sample within 60 min at 37-39 DEG C, the detection sensitivity can reach 10 fg / mu L, and the kit is suitable for slaughter houses, fields and other places without special equipment. Besides, the kit can detect that except T10, T1-T12 and other trichina genotypes have no cross reaction with toxoplasma gondii and other parasites and host genes, human trichinosis can be prevented, and instant detection of related meat foods can be realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of Trichinella spiralis detection, and specifically relates to a rapid detection system for Trichinella spiralis based on RAA-CRISPR / Cas12a, a kit, and a method for using the kit. Background Art

[0002] Trichinella spiralis is a widespread zoonotic pathogen that can infect over 150 animal species, including humans. Trichinellosis, caused by infection of farmed animals, can cause significant economic losses and food safety risks. Trichinella spiralis can parasitize a wide range of hosts, affecting nearly all mammals. Transmission among animals is believed to occur through the consumption of muscle tissue containing Trichinella cysts. The primary risk of human infection comes from the consumption of meat contaminated with Trichinella. Pork is a major source of human infection, but a growing body of research suggests that wild game (such as wild dogs, horses, and bears) may become a major vector for human trichinellosis outbreaks in the future. This is not only because food processing plants and food regulatory authorities test mainstream meat products (such as pork) for Trichinellosis, but also because wild game is often hunted and consumed by individuals or small groups. This lack of testing increases the risk of Trichinellosis infection. Trichinella spiralis can survive in meat at both room and low temperatures and remains infectious and pathogenic. Even in rotting meat and preserved meat, Trichinella spiralis can remain viable and infectious for several days. Therefore, establishing a rapid, accurate, and sensitive method for detecting Trichinella spiralis would facilitate the detection of related meat products, thereby preventing the occurrence of human trichinellosis at the source and reducing the prevalence of the disease.

[0003] Currently, detection of Trichinella spiralis in muscle samples is mostly performed using compression microscopy and pooled digestion, the latter being the gold standard for diagnosis recommended by the OIE. However, these methods require high levels of tester experience and are inefficient for detecting early-stage Trichinella infections. Serological tests (such as ELISA) have improved sensitivity and mitigated this dependency on tester experience. However, limitations persist: low efficiency for early-stage infections, false-positive cross-reactions, and the risk of missed detection in low-dose infected samples. These remain significant challenges for serological testing. Even though ELISA is the sole serological epidemiological survey method recommended by the ICT, positive ELISA results still require confirmation by Western blot (Bruschi et al., 2019). Molecular testing methods can address these shortcomings, but temperature-dependent amplification methods such as PCR require specialized instrumentation. Due to these limitations, researchers primarily use POCT for laboratory research, such as genotyping of Trichinella spiralis, rather than for testing. Due to its advantages such as no need for instruments and simplicity and convenience, isothermal amplification can become a very promising on-site detection method. It can retain the sensitivity and specificity of molecular detection while also meeting most application scenarios. For example, loop-mediated isothermal amplification (LAMP) has been successfully used for the detection of Trichinella spiralis in meat, and it can complete the detection of Trichinella spiralis DNA within 60 minutes. The invention patent application with publication number CN 119530342 A discloses a Trichinella spiralis detection kit based on the CRISPR / Cas12a system and click chemistry, as well as its use method and application. It discloses the use of CRISPR / Cas12a to detect Trichinella spiralis, but it is not a test for Trichinella spiralis DNA, but a serological test for Trichinella spiralis, and the visualization of the test results cannot be achieved. Patent publication number CN 119391887 A discloses primers, a kit, and a method for seven-fold detection of Coccidia spp. based on RAA-CRISPR / Cas12a technology. Coccidia spp. are protozoa, single-celled parasites. Detection and DNA extraction from these protozoa require a large number of protozoa, and this method uses traditional kits, which are costly, complex, and time-consuming. Currently, there are no reports on using RAA-CRISPR / Cas12a technology for Trichinella spiralis DNA detection. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings of existing Trichinella spiralis detection methods and provide a rapid detection system for Trichinella spiralis based on RAA-CRISPR / Cas12a, a detection kit and a method for using the kit, so as to achieve visual and instant detection of Trichinella spiralis at a relatively low cost.

[0005] To achieve its purpose, the present invention adopts the following technical solutions:

[0006] The present invention provides a rapid detection system for Trichinella spiralis based on RAA-CRISPR / Cas12a, which comprises a RAA isothermal amplification system and a CRISPR / Cas12a trans-cleavage detection system;

[0007] The RAA isothermal amplification system includes a RAA forward primer TSssrRAA-F1 and a reverse primer TSssrRAA-R2 designed based on Trichinella spiralis nucleic acid; the nucleotide sequence of the forward primer is shown in SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.2;

[0008] The CRISPR / Cas12a trans-cleavage detection system includes CrRNA2 designed based on Trichinella spiralis nucleic acid, and its nucleotide sequence is shown in SEQ ID NO.3.

[0009] As a further preferred embodiment of the technical solution of the present invention, the RAA isothermal amplification system contains 2 μL of a forward primer (10 μM) and 2 μL of a reverse primer (10 μM).

[0010] Furthermore, the CRISPR / Cas12a trans-cleavage detection system contains 4 μL of CrRNA2 (1 μM) and also includes:

[0011] 4 μL EnGen Lba Cas12a protein (1 μM), 5 μL ssDNA-FQ (1 μM), 3 μL DEPC water, 2 μL 10× loading buffer, 2 μL RAA amplification product.

[0012] The present invention provides a Trichinella spiralis detection kit based on RAA-CRISPR / Cas12a, which includes the above-mentioned RAA isothermal amplification system and CRISPR / Cas12a trans-cleavage detection system.

[0013] The method for using the above-mentioned RAA-CRISPR / Cas12a-based Trichinella spiralis detection kit comprises the following steps:

[0014] S1: Extract the DNA of the sample to be tested, add it to the RAA isothermal amplification system for RT-RPA isothermal amplification, and obtain the RAA amplification product;

[0015] S2: Add the RAA amplified product to the CRISPR / Cas12a detection system for reaction;

[0016] S3: Real-time detection of the fluorescence changes of the CRISPR / Cas12a detection system in S2, and plotting of a fluorescence curve; in the plotted fluorescence curve, samples with a fluorescence value higher than 2371 (au) are determined to be positive for Trichinella spiralis detection, and vice versa.

[0017] Furthermore, in step S1, the DNA extraction method for the sample to be tested is ultrasonic DNA extraction. The extraction conditions for ultrasonic DNA extraction are: ultrasonic power 200W, ultrasonication for 2 seconds, interval 2 seconds, for a total of 30 minutes; the resulting product is then added to 50 μL of DNA extraction reagent, mixed thoroughly, centrifuged at 12,000 rpm for 1 minute, and the supernatant is aspirated. The extraction is repeated three times.

[0018] Furthermore, in step S1, the RAA isothermal amplification conditions are: 39° C. water bath reaction for 25 minutes.

[0019] Furthermore, in step S2, the reaction conditions are: reaction at 37° C. for 30 min.

[0020] The one-tube method for using the RAA-CRISPR / Cas12a-based Trichinella spiralis detection kit is as follows: the RAA system and the CRISPR / Cas12a reaction system are added to the PCR reaction tube according to the dosage of the step-by-step reaction. When the test is to be performed, the DNA sample is directly added for the RAA reaction. After the reaction is completed, the PCR reaction tube is mixed to complete the CRISPR / Cas12a reaction.

[0021] By adopting the above technical solution, the beneficial effects of the present invention are:

[0022] The present invention is based on RAA technology, which performs constant-temperature amplification on the small ribosomal subunit gene of Trichinella spiralis, and then uses the specific and visual CRISPR / Cas12a enzyme to visualize the reaction results. The established RAA-CRISPR / Cas12a diagnostic kit can diagnose Trichinella spiralis infection conveniently, quickly, and efficiently. At the same time, the applicant evaluated the diagnostic effectiveness of the kit in mouse muscle, blood, and feces, aiming to apply the established RAA-CRISPR / Cas12a diagnostic kit to point-of-care testing (POCT) of Trichinella spiralis. The experimental results showed that the detection kit of the present invention can complete the reaction in one step at 37-39°C, making it suitable for places without dedicated equipment such as slaughterhouses and the wild.

[0023] Specifically, in order to achieve the portability of Trichinella diagnostic equipment, the present invention has developed an easy-to-operate RAA-CRISPR / Cas12a detection system. The system can complete rapid detection of the target within 60 minutes at a constant temperature of 37-39°C. Sensitivity analysis shows that the detection threshold of RAA-CRISPR / Cas12a can reach 10fg / μL, and there is no cross-reactivity with other parasites. Unlike serological tests, which can detect Trichinella infection as early as the seventeenth day (Zhao et al., 2023). RAA-CRISPR / Cas12a can detect Trichinella in the intestine as early as the first day, and can visually detect Trichinella infection in the muscle on the fourteenth day, which has advantages in the diagnosis of early Trichinella infection. The results of the detection of Trichinella in mouse muscle samples showed that while there was no cross-specificity with muscle tissue, it also had a high detection rate.

[0024] Furthermore, the RAA-CRISPR / Cas12a detection kit established by the present invention has achieved good results in detecting Trichinella pathogens. First, Trichinella is different from other parasites (such as Toxoplasma gondii, Trichomonas gallinae and Echinococcus), and the development and reproduction of both adults and muscle larvae are completed in the same host; After the larvae are released, they develop into adults in the small intestine and the newborn larvae produced by mating are distributed to the host's various tissues through the lymphatic-blood circulation, forming cysts in the striated muscle. Therefore, the applicant's detection is not only suitable for collecting diaphragmatic angles and tongue muscle tissue for detection, but also can be used to collect small intestinal tissue or feces to identify early infection (1dpi-4dpi) of Trichinella. Secondly, Trichinella mainly parasitizes the muscles and intestinal parts of animals such as pigs and dogs, especially the largest number of parasites in the diaphragm, and traditional DNA extraction methods (such as using tissue sample DNA extraction kits) are cumbersome due to the complexity of muscle tissue. In order to meet the needs of rapid detection, the applicant compared and studied two DNA extraction methods, achieving rapid processing of complex samples under the premise of high efficiency and simplicity, making each step of the detection operation faster, thereby lowering the threshold for clinical and on-site detection. At the same time, the present invention uses the small ribosomal subunit RNA gene as the target gene for molecular detection of Trichinella for the first time. Unlike the previous use of mitochondria or other coding regions, this target can cover almost all Trichinella species except T10 and T13, especially the T1 and T2 species that are prevalent globally and highly prevalent in my country; in mouse experiments, the applicant was able to detect worms in the intestine on the first day after infection, and in the tongue muscle, diaphragm, blood, myocardium and forelimb skeletal muscle on the fourth day, which is far ahead of the time window of traditional detection methods. In addition, to improve practicality and detection efficiency, the applicant established and optimized a one-tube reaction system. The entire detection process can be completed in only 55 minutes, the lowest visual detection limit can reach 100fg / μL, and the lid only needs to be opened once, which not only reduces the number of operating steps but also greatly reduces the risk of cross-contamination. Although low-concentration target gene detection currently still relies on fluorescent instruments as a key link, in the future, test strips can be used to visualize the results, thereby providing a more convenient auxiliary solution for clinical nucleic acid testing that does not require complex instruments. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The screening results of crRNA and RAA primers; A is the RAA amplification result of different primer combinations; B is the result of F1R1, F1R2 and F1R3 after RAA-crispr / Cas12a reaction; C is the fluorescence intensity of F1R1, F1R2 and F1R3 after RAA-crispr / Cas12a reaction; D is the fluorescence intensity result of crRNA1-crRNA8; E is fluorescence visualization under blue light;

[0026] Figure 2The results of the optimization of RAA-CRISPR / Cas12a detection conditions are shown in Figure 2. A represents the fluorescence intensity of the reaction between RAA and CRISPR / Cas12a after different times; B represents the fluorescence intensity of the reaction between crRNA and Cas12a at different concentrations.

[0027] Figure 3 The results of the efficiency comparison and detection threshold determination of different DNA extraction methods are shown; A is the visual inspection result after UV long-term light irradiation after RAA-CRISPR / Cas12a detection; B is the fluorescence value of the two methods; C is the receiver operating characteristic (ROC) curve of the RAA-CRISPR / Cas12 method for detecting Trichinella spiralis; D is the critical value of fluorescence detection using the Youden index;

[0028] Figure 4 The results show a comparison of the sensitivity and specificity of the RAA-CRISPR / Cas12a detection method for Trichinella spiralis; A is the fluorescence detection result of the RAA amplification combined with CRISPR / Cas12a detection method; B is the visual inspection result of the RAA amplification combined with CRISPR / Cas12a detection result after UV light irradiation. NC: negative control; C is the PCR result; D is the fluorescence detection result of the RAA-CRISPR / Cas12a detection method for different types of parasites; E is the visual inspection result of the RAA-CRISPR / Cas12a detection result for different types of parasites after UV light irradiation; F is the detection result of RAA-CRISPR / Cas12a for single worms in pork of different qualities; G is the visual inspection result of RAA-CRISPR / Cas12a for single worms in pork of different qualities after UV light irradiation. ***p<0.001, **p<0.01;

[0029] Figure 5 The following are the detection results of the artificially infected samples using the RAA-CRISPR / Cas12a method for Trichinella spiralis; A is the PCR detection of different tissue samples at different times, using ImageJ to scan and measure the PCR nucleic acid gel electrophoresis band intensity, and the bar graph represents the PCR nucleic acid gel electrophoresis band intensity of the corresponding tissue or time; B is the fluorescence visual inspection result of RAA-CRISPR / Cas12a for different tissue samples at different times; 1: tongue muscle; 2: forelimb; 3: heart; 4: diaphragm; 5: duodenum; 6: jejunum; 7: ileum; 8: venous blood; PC: positive control; NC: negative control; C is the fluorescence value of different samples at different times. D is the correlation analysis of the fluorescence value of the RAA-CRISPR / Cas12a method and the grayscale value of PCR; E is the Bland-Altman analysis of the microscopy method and RAA-CRISPR / Cas12a; F is the Bland-Altman analysis of PCR and RAA-CRISPR / Cas12a.

[0030] Figure 6 A RAA-CRISPR / Cas12a one-tube detection method was established; wherein, A is a RAA-CRISPR / Cas12a one-tube detection process; B is the influence of various reagents on the one-tube detection method by visual fluorescence inspection; C: the influence of various reagents on the one-tube detection method by fluorescence value; D: gray value of different RAA reaction time; E: fluorescence value of different CRISPR / Cas12a reaction time; F: visual results of one-tube RAA-CRISPR / Cas12a detection results after UV irradiation; G: fluorescence detection results of one-tube RAA-CRISPR / Cas12a detection method. NC: negative control. DETAILED DESCRIPTION

[0031] The application will be described in detail below in conjunction with the drawings.

[0032] 1. Materials and methods

[0033] 1.1 Biological materials

[0034] The application uses the Trichinella spiralis Jilin isolate preserved in the laboratory of the applicant, and the rest of the Trichinella species (T. nativa, T. britovi, T. pseudospiralis, T. murrelli, T6, T. nelsoni, T8, T9, T. papuae, T. zimbabwvensis, T. patagoniensis) are donated by Professor Liu Mingyuan of the College of Animal Medicine of Jilin University. Echinococcus granulosus, Toxoplasma gondii, Ascaris suum, Sarcocystis tenella, Echinococcus multilocularis, Fasciola hepatica, Fasciola gigantica, Taenia mulliceps, Taenia hydatigena and Hydatigen taeniaeformis are preserved in the National Key Laboratory of Animal Disease Prevention and Control.

[0035] 1.2 Reagents and instruments

[0036] The RAA nucleic acid amplification kit (basic type) used in the present invention was purchased from Hangzhou Zhongce Biotechnology Co., Ltd., the blood cell tissue genomic DNA extraction kit (DP304) was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., the pepsin powder was purchased from Sigma-Aldrich, and the DNA extraction solution (AC13309) was purchased from

[0037] Shanghai Acmec Biochemical Technology Co., Ltd., all RAA and PCR primers and ssDNA-FQ were synthesized by Sangon Biotech (Shanghai) Co., Ltd., crRNA was synthesized by Suzhou Genetron Health Co., Ltd., EnGen Lba Cas12a (cpf1) and 10× NE Buffer were purchased from New England Biolabs (NEB). The qPCR instrument (CFX96) used to test the fluorescence value of RAA-CRISPR / Cas12a in this experiment was purchased from BIO-RAD, and visualization was performed using a DUT-48 blue light gel cutter (Hangzhou Miou Instrument Co., Ltd.). The ultrasonic cell disruptor (SCIENTZ-IID) was purchased from Ningbo Xinzhi Biotechnology Co., Ltd.

[0038] 1.3 Extraction of Trichinella spiralis DNA

[0039] Using the Jilin strain of Trichinella spiralis preserved in the applicant's laboratory, six-week-old SPF female Kunming mice were experimentally infected. The mice were then skinned and placed in a 1L pepsin solution (containing 10ml of concentrated hydrochloric acid and 10g of pepsin powder). The mice were stirred at 42°C for 2 hours, then allowed to settle for 2 hours to collect the parasites. The parasites were counted and DNA was extracted. The present invention employs two methods (a kit method and ultrasonic fragmentation) to extract Trichinella spiralis DNA. The kit method uses the Tiangen Blood Cell Tissue Genomic DNA Extraction Kit (DP304) according to the manufacturer's instructions for DNA extraction, and DNA is eluted in 50μL of elution buffer. The ultrasonic fragmentation method utilizes an ultrasonic cell disruptor for 30 minutes at 200W power, followed by three extractions with DNA extraction solution.

[0040] 1.4 Selection of crRNA and Preparation of RAA Primers

[0041] The nucleotide sequence of the small subunit ribosoma RNA gene of Trichinella spiralis was downloaded from NCBI GenBank (https: / / www.ncbi.nlm.nih.gov / genbank / ) (GenBank: JQ823166.1) and named TSssr. Using this gene as a template, eight crRNAs were synthesized based on the complementary pairing characteristics of crRNA and target sequence to screen the best crRNA. They were named crRNA1-crRNA8. After screening the best crRNA, three pairs of upper and reverse primers were designed with crRNA as the center, named F1, F2, F3 and R1, R2, R3, respectively. They were combined in pairs to further screen the best primer pairing. The crRNA sequence, RAA primer sequence and length, as well as the primer sequences of the tag ssDNA-FQ non-specifically cut by Cas12a and the traditional PCR method are shown in Table 1.

[0042] Table 1 CrRNA and RAA primer sequences

[0043]

[0044]

[0045] 1.5 Preliminary establishment of the RAA-CRISPR / Cas12a system

[0046] The RAA reaction was performed according to the instructions of the RAA Nucleic Acid Amplification Kit (Hangzhou Zhongce Biotechnology Co., Ltd.). The system consisted of 25 μL ABuffer, 13.5 μL nuclease-free water, 2.5 μL B Buffer, 2 μL forward primer (10 μM), 2 μL reverse primer (10 μM), and 5 μL DNA sample. The reaction was carried out in a 39°C water bath for 30 minutes. 50 μL DNA extract was added to the obtained amplified product. After centrifugation at 12000 rpm for 5 minutes, the supernatant was collected. The obtained supernatant was the amplified product. The RAA amplified product was added to the CRISPR / Cas12a reaction system. The CRISPR / Cas12a reaction system was 4 μL EnGen Lba Cas12a protein (1 μM), 4 μL CcrRNA (1 μM), 5 μL ssDNA-FQ (1 μM), 3 μL DEPC water, 2 μL 10× loading buffer, and 2 μL RAA amplified product. Then, place it in a qPCR instrument at 37°C for 30 minutes, determine the results in the FAM channel, collect the fluorescence signal every 5 seconds, and repeat 3 times. Dilute the required reagents to the corresponding concentration with DEPC water.

[0047] 1.6 Sensitivity and specificity analysis of RAA-CRISPR / Cas12a detection system

[0048] First, DNA was extracted from a piece of Trichinella spiralis using a blood cell tissue genomic DNA extraction kit. The obtained DNA was diluted to different concentrations, and RAA-CRISPR / Cas12a reaction was performed, and the fluorescence value was recorded. In addition, 1 g of Trichinella spiralis was added to different weights of pork, and the results were observed and the fluorescence value was recorded.

[0049] The DNA of Echinococcus granulosus, Toxoplasma gondii, Ascaris suum, Sarcocystis hominis, Echinococcus multilocularis, Fasciola hepatica, Fasciola gigantica, Taenia multiceps, Cysticocercus pisiformis and Taenia taeniaeformis was extracted using a blood cell tissue genomic DNA extraction kit, and the DNA was used as a template for RAA-CRISPR / Cas12a reaction, and the results were observed.

[0050] 1.7 Clinical application of Trichinella spiralis RAA-CRISPR / Cas12a detection method

[0051] Applicants first detected 7 parts (tongue, forelimb, heart, diaphragm, duodenum, jejunum and ileum) and blood in artificially infected mice of different days using RAA-CRISPR / Cas12a. 500 Trichinella spiralis ML were orally infected with 6-week-old Kunming mice, and at 1 dpi, 4 dpi, 7 dpi, 14 dpi, 21 dpi, and 35 dpi, the corresponding tissues were taken, and the tissue DNA was extracted using a blood cell tissue genomic DNA extraction kit. After 5 μL was taken for RAA-CRISPR / Cas12a reaction. In addition, T. spiralis, T. nativa, T. britovi, T. pseudospiralis, T. murrelli, T6, T. nelsoni, T8, T9, T. papuae, T. zimbabwvensis and T. patagoniensis were detected, and the DNA of different species of Trichinella was extracted for RAA-CRISPR / Cas12a reaction. At the end of the experiment, artificial digestion method was used to confirm that all mice were infected with Trichinella ML.

[0052] Applicants collected 124 dog tongue and dog foreleg samples from the market and dog farms, stored them in PBS, and extracted the tissue DNA using a blood cell tissue genomic DNA extraction kit. Routine PCR and RAA-CRISPR / Cas12a were used for detection. After RAA-CRISPR / Cas12a results were analyzed by qPCR instrument, standard artificial digestion method and PCR were compared and analyzed by SPSS 27.0.

[0053] 2 Results

[0054] 2.1 Screening of CrRNA and RAA primer

[0055] The applicants first screened 8 designed CrRNAs. The results showed that the fluorescence intensity of CrRNA2, CrRNA5, CrRNA6, CrRNA7 and CrRNA8 was higher, and the fluorescence could be detected by naked eye. However Figure 1

[0056] The fluorescence intensity of CrRNA2 was the highest, so the applicants selected CrRNA2 as the best CrRNA design in subsequent experiments. The RAA primer targeting the conserved region of Trichinella spiralis mitochondrial sequence was selected to amplify the screened CrRNA. The designed upper reverse primer was combined for amplification, and 3 pairs of primers (F1R1, F1R2, F1R3) were preliminarily screened for subsequent use. Through RAA-CRISPR / Cas12a experiment, F1R2 was selected for subsequent experiment.

[0057] 2.2 Optimization of RAA and CRISPR / Cas12a detection method

[0058] In order to improve the detection efficiency of CRISPR / Cas12a, the applicants set 5 groups of Cas12a protein concentration (100 nM, 250 nM, 500 nM, 750 nM, 1000 nM) and 5 groups of crRNA concentration (100 nM, 250 nM, 500 nM, 750 nM, 1000 nM) for orthogonal experiment. According to the results shown in Figure 2 , the applicants selected the best concentration of Cas12a protein and crRNA as 1000 nM. With 10 ng / μL Trichinella spiralis DNA as template for RAA amplification, RAA reaction time (5 min, 10 min, 15 min, 20 min, 25 min, 30 min) and CRISPR / Cas12a reaction time (5 min, 10 min, 15 min, 20 min, 25 min, 30 min) were set for orthogonal experiment. The change of fluorescence value was monitored to determine the appropriate reaction duration. As Figure 2 shown in the results, the RAA amplification time of 25 minutes and the CRISPR / Cas12a reaction time of 30 minutes were finally selected as the best reaction time of CRISPR / Cas12a system.

[0059] 2.3 Comparison of DNA extraction methods and establishment of detection threshold

[0060] In order to conveniently obtain nucleic acid for detection, the applicants compared the traditional kit DNA extraction method (Tiangen tissue DNA extraction kit) with the ultrasonic fragmentation DNA extraction method Figure 3 , as Figure 3 ​As shown in A, DNA extracted using two methods was subjected to RAA-CRISPR / cas12a reaction respectively, and the results showed no significant difference. Figure 3 The fluorescence value results of B also illustrate this point. It is worth noting that since the DNA extraction using the tissue DNA extraction kit takes a long time (more than 3 days), and the DNA extraction using the ultrasonic fragmentation method takes a shorter time (1h-4h), the applicant subsequently used the ultrasonic fragmentation method for DNA extraction from tissue samples. In addition, in order to find the detection threshold of the established RAA-CRISPR / Cas12a method, the applicant tested 52 known positive samples and 62 known negative samples. Figure 3 As shown, the Area under the curve (AUC value) is 0.9171, and the Youden index reaches a maximum value of 0.7119 when the fluorescence value is 2371 (au). Based on this, the applicant determines that the samples with a fluorescence value higher than 2371 (au) are positive for the Trichinella spiralis test.

[0061] 2.4 Sensitivity and specificity of the RAA-CRISPR / Cas12a detection method

[0062] Based on the RAA-CRISPR / Cas12a system initially established in 1.5, the applicant further evaluated the detection sensitivity by performing gradient dilution of the Trichinella spiralis muscle larvae genome ( Figure 4 ). Figure 4 The results showed that the RAA-CRISPR / Cas12a detection system could achieve a minimum detection concentration of 10 fg / μL when diluting Trichinella spiralis DNA at different concentrations, but the lowest detection threshold observed by the naked eye was 1 pg / μL. Furthermore, to further investigate the impact of porcine genomic background DNA on RAA-CRISPR / Cas12a fluorescence analysis, the applicants added single Trichinella spiralis to porcine diaphragm samples of varying mass, extracted tissue DNA, and then performed RAA-CRISPR / Cas12a assays. The results showed that background DNA did not interfere with the assay.

[0063] In order to evaluate the specificity of the RAA-CRISPR / Cas12a method, DNA from nine parasites, including Trichinella spiralis and Ascaris suum, was used as templates for RAA amplification. The fluorescence value of the amplified products was detected using the CRISPR / Cas12a system. Figure 4 B shows that only Trichinella spiralis is a positive result, and the fluorescence values ​​of other parasites are similar to those of the negative control. Under blue light irradiation, fluorescence can only be observed in the tube where the template is Trichinella spiralis, indicating that this detection method has good detection specificity.

[0064] 2.5RAA-CRISPR / Cas12a mouse infection model test results

[0065] Furthermore, in order to expand the application scope of the Cas12a system, the applicant applied the established Trichinella diagnostic system to clinical sample detection. The applicant first evaluated whether RAA-CRISPR / Cas12a can detect all Trichinella genotypes. After extracting the genes of T.spiralis, T.nativa, T.britovi, Tpseudospiralis, T.murrelli, T6, T.nelsoni, T8, T9, T.papuae, T.zimbabwvensis, and T.patagoniensis, the genomes with a concentration of 10ng / μL were used as templates, and the RAA-CRISPR / Cas12a system was used for visual detection and traditional PCR detection. Figure 5 In the study, it was found that the detection efficiency of RAA-CRISPR / Cas12a for different species of Trichinella was higher than that of nucleic acid gel electrophoresis. At the same time, the present invention applied the RAA-CRISPR / Cas12a diagnostic method for Trichinella and traditional PCR to detect different tissue samples of mice infected with Trichinella at different stages, and evaluated the sensitivity of this method to different tissue samples of mice infected with different doses of Trichinella. It can be found that the RAA-CRISPR / Cas12a diagnostic method and the traditional PCR diagnostic method are consistent with each other, and the kappa value of RAA-CRISPR / Cas12a and conventional PCR is equal to 0.831. Starting from the 7th day, the fluorescence phenomenon can be observed with the naked eye in mice infected with 500 Trichinella muscle larvae. At the same time, the fluorescence value of Trichinella in the diaphragm is higher. As in previous studies, the number of Trichinella parasites in the diaphragm is relatively large.

[0066] 2.6 Clinical Sample Testing

[0067] The applicants tested 124 samples of dog meat infected with Trichinella spiralis using the RPA-CRISPR / Cas12a fluorescence detection method they developed, comparing the results with those from compression microscopy and polymerase chain reaction (PCR) testing for Trichinella spiralis. The results showed that RPA-CRISPR / Cas12a detected 4 positive samples and 120 negative samples among the 124 clinical samples, consistent with the results from compression microscopy and PCR. The sensitivity, specificity, and consistency of this research platform were 100.0%.

[0068] Table 2 Test results of dog meat samples from different regions

[0069]

[0070] 2.7 Preliminary Study on One-Tube Detection of RAA-CRISPR / Cas12a

[0071] One-tube testing process Figure 6 ( Figure 6 A), respectively, add the RAA system and CRISPR / Cas12a reaction system to the PCR reaction tube according to the dosage of the step-by-step reaction. When the test is ready, directly add the DNA sample to perform the RAA reaction. After the test is completed, mix the PCR reaction tube.

[0072] In the CRISPR / Cas12a reaction, the components of the RAA system are introduced, so it is necessary to re-explore the optimal reaction time of RAA and CRISPR / Cas12a in a one-tube test. Figure 6 B and 6C, the applicant found that the optimal RAA and CRISPR reaction time is consistent with the conclusion of 2.3.5 (RAA reaction 25min, CRISPR reaction 30min). As mentioned above, the applicant obtained that the minimum sensitivity of the RAA-CRISPR / Cas12a two-step detection was 10fg. In the preliminary exploration of one-tube detection, the applicant selected 1pg / μL, 100fg / μL, 10fg / μL and 5fg / μL four concentrations to evaluate the sensitivity of the one-tube detection. The experimental results showed that at 100fg / μL, the fluorescence difference of visual detection was the largest. Therefore, the applicant used 100fg / μL as

[0073] Minimum sensitivity of the RAA-CRISPR / Cas12a one-tube visual assay.

Claims

1. A rapid detection system for Trichinella spiralis based on RAA-CRISPR / Cas12a, characterized in that: The detection system includes an RAA isothermal amplification system and a CRISPR / Cas12a trans-cleavage detection system; The RAA isothermal amplification system includes a RAA forward primer TSssrRAA-F1 and a reverse primer TSssrRAA-R2 designed based on Trichinella spiralis nucleic acid; the nucleotide sequence of the forward primer is shown in SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.2; The CRISPR / Cas12a trans-cleavage detection system includes CrRNA2 designed based on Trichinella spiralis nucleic acid, and its nucleotide sequence is shown in SEQ ID NO.

3.

2. A rapid detection system for Trichinella spiralis based on RAA-CRISPR / Cas12a according to claim 1, characterized in that The RAA isothermal amplification system contains 2 μL of a forward primer (10 μM) and 2 μL of a reverse primer (10 μM).

3. A rapid detection system for Trichinella spiralis based on RAA-CRISPR / Cas12a according to claim 2, characterized in that The CRISPR / Cas12a trans-cleavage detection system contains 4 μL of CrRNA2 (1 μM) and also includes: 4 μL EnGen Lba Cas12a protein (1 μM), 5 μL ssDNA-FQ (1 μM), 3 μL DEPC water, 2 μL 10× loading buffer, 2 μL RAA amplification product.

4. A Trichinella spiralis detection kit based on RAA-CRISPR / Cas12a, characterized in that The kit comprises the RAA isothermal amplification system and the CRISPR / Cas12a trans-cleavage detection system according to any one of claims 1 to 3.

5. The method for using the RAA-CRISPR / Cas12a-based Trichinella spiralis detection kit according to claim 4, wherein: The following steps are involved: S1: Extract the DNA of the sample to be tested, add it to the RAA isothermal amplification system for RT-RPA isothermal amplification, and obtain the RAA amplification product; S2: Add the RAA amplified product to the CRISPR / Cas12a detection system for reaction; S3: Real-time detection of fluorescence changes of the CRISPR / Cas12a detection system in S2 and drawing of fluorescence curves; In the fluorescence curve drawn, samples with fluorescence values ​​higher than 2371 (au) were determined to be positive for Trichinella spiralis, and vice versa.

6. The method for using the RAA-CRISPR / Cas12a-based Trichinella spiralis detection kit according to claim 5, wherein: In step S1, the DNA extraction method of the sample to be tested is ultrasonic fragmentation DNA extraction method.

7. The method for using the RAA-CRISPR / Cas12a-based Trichinella spiralis detection kit according to claim 6, wherein: The extraction conditions of the ultrasonic DNA extraction method are as follows: ultrasonic power 200W, ultrasonication 2s, interval 2s, a total of 30min; then the obtained product is added to 50μL of DNA extraction reagent, mixed evenly, centrifuged at 12,000rpm for 1min, and the supernatant is aspirated, and the extraction is repeated 3 times.

8. The method for using the RAA-CRISPR / Cas12a-based Trichinella spiralis detection kit according to claim 7, wherein: In step S1, the RAA isothermal amplification conditions are: 39° C. water bath reaction for 25 minutes.

9. The method for using the RAA-CRISPR / Cas12a-based Trichinella spiralis detection kit according to claim 8, wherein in step S2, the reaction conditions are: 37°C for 30 minutes.

10. The one-tube method for using the RAA-CRISPR / Cas12a-based Trichinella spiralis detection kit according to claim 4, wherein: Add the RAA system and CRISPR / Cas12a reaction system to the PCR reaction tube according to the dosage of the step-by-step reaction. When testing, directly add the DNA sample to perform the RAA reaction. After the reaction is completed, mix the PCR reaction tube to complete the CRISPR / Cas12a reaction.

Images