An era-crispr-cas12a-based porcine epidemic diarrhea virus detection method

Abstract

The present application relates to the field of animal epidemic molecular diagnosis, and particularly to a porcine epidemic diarrhea virus detection method based on ERA-CRISPR-Cas12a, which takes the conserved region of the porcine epidemic diarrhea virus PEDV N gene as a target, and performs constant temperature amplification on the ERA primer after reverse transcription of the RNA of a sample to be tested; the amplification product is mixed with LbCas12a protein, specific crRNA, single-strand DNA fluorescent reporter probe and reaction buffer, and Cas12a recognizes the PEDV target sequence through the guidance of the crRNA. When the target exists, Cas12a produces trans cleavage activity and cuts the fluorescent reporter probe, releases the fluorescent signal, and accordingly determines whether the sample contains PEDV. The method has the characteristics of high sensitivity, good specificity, short reaction time and low dependence on large instruments, and is suitable for laboratory detection and on-site rapid screening of PEDV.

Description

Technical Field

[0001] This invention relates to the field of molecular diagnostic technology for animal diseases, specifically to a method for detecting porcine epidemic diarrhea virus based on ERA-CRISPR-Cas12a. Background Technology

[0002] Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that infects pigs, belonging to the family Coronaviridae and subfamily Orthocoronaviruses. PEDV is an enveloped, single-stranded, positive-sense RNA virus with a genome approximately 28 kb in length, encoding the spike protein S, membrane protein M, envelope protein E, nucleocapsid protein N, and several non-structural proteins. PEDV infection can cause clinical symptoms in pigs such as vomiting, watery diarrhea, dehydration, and anorexia, and is particularly pathogenic to suckling piglets, often resulting in high morbidity and mortality. Significant genetic variation exists in circulating PEDV strains, with some strains evolving from genotype I to the more pathogenic genotype II, posing a continuous challenge to disease surveillance, early diagnosis, and control in pig farms.

[0003] Currently, PEDV detection commonly employs techniques such as virus isolation, antigen detection, enzyme-linked immunosorbent assay (ELISA), conventional PCR, real-time quantitative PCR, and loop-mediated isothermal amplification. Among these, real-time quantitative PCR offers high sensitivity and specificity, but its detection process relies on nucleic acid amplification instruments, quantitative PCR instruments, and corresponding specialized operating conditions, resulting in a long detection cycle and high instrument and maintenance costs. Conventional PCR products typically require agarose gel electrophoresis analysis, which involves numerous steps, is prone to amplified product contamination, and is difficult to adapt to rapid screening in aquaculture settings. Isothermal amplification technology can complete nucleic acid amplification at a constant temperature, reducing reliance on thermal cycling equipment; however, when using isothermal amplification alone for endpoint interpretation, it may still be affected by non-specific amplification or subjective result interpretation.

[0004] Therefore, existing technologies need to provide a nucleic acid detection solution suitable for on-site detection of PEDV. This solution should complete target amplification and specific identification under relatively low isothermal conditions to reduce reliance on large and precision instruments. At the same time, it should have a low detection limit and good pathogen differentiation ability, and be able to achieve objective interpretation through fluorescence signals, so as to meet the application needs of farms, grassroots animal disease detection institutions and laboratories for rapid screening of PEDV. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for detecting porcine epidemic diarrhea virus (PEDV) based on ERA-CRISPR-Cas12a. This method uses the conserved region of the PEDV N gene as the detection target, performs isothermal pre-amplification of the target sequence using ERA, and utilizes the crRNA-guided LbCas12a protein to specifically recognize the amplification product. When the target is present, LbCas12a is activated and generates trans-cleavage activity, cleaving the fluorescent reporter probe, thereby outputting a fluorescent signal that can be collected and interpreted.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for detecting porcine epidemic diarrhea virus based on ERA-CRISPR-Cas12a, comprising the following steps: (1) Extract nucleic acid from the pig sample to be tested, and reverse transcribe the extracted RNA as a template to obtain cDNA; (2) Using the conserved region of the N gene of porcine epidemic diarrhea virus (PEDV) as the target, the cDNA was enzymatically recombinated and isothermally amplified using ERA primers to obtain the ERA amplification product. (3) The ERA amplification product is mixed with LbCas12a protein, crRNA that recognizes the target sequence of PEDV-N gene, single-stranded DNA fluorescent reporter probe and reaction buffer to form a CRISPR / Cas12a detection reaction system. (4) The CRISPR / Cas12a detection reaction system is incubated under constant temperature conditions so that the LbCas12a protein recognizes the target sequence in the ERA amplification product under the guidance of crRNA and generates trans-cleavage activity to cleave the single-stranded DNA fluorescent reporter probe. (5) Collect the fluorescence signal of the detection reaction system, and use the fluorescence signal of the test sample being higher than that of the negative control group as the basis for determining the detection of PEDV.

[0007] Preferably, the ERA primer pair for the conserved region of the PEDV-N gene includes an upstream primer and a downstream primer, and the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1: 5'-ATGCGCCGTGGTGAGCGAATTGAACA-3'; The nucleotide sequence of the downstream primer is shown in SEQ ID NO.2: 5'-GACCCTGGTTATTTCCACGATTCTGT-3'.

[0008] Preferably, the crRNA includes a direct repeat sequence recognized by Cas12a and a target sequence recognition region; wherein the nucleotide sequence of the target sequence recognition region is shown in SEQ ID NO.3: 5'-CGCCTTCTTTAGCAACCCAG-3'; The complete nucleotide sequence of the crRNA is shown in SEQ ID NO.4: 5'-UAAUUUCUACUAAGUGUAGAUCGCCUUCUUUAGCAACCCAG-3'.

[0009] Preferably, in the CRISPR / Cas12a detection reaction system, the mass ratio of LbCas12a protein to crRNA is 4:1, and the initial concentrations of both LbCas12a protein and crRNA are 500 ng / μL.

[0010] Preferably, the single-stranded DNA fluorescent reporter probe is an ssDNA probe with a fluorescent group and a fluorescence quenching group, and its nucleotide sequence is: 5'-6-FAM-TTATT-BHQ1-3'. Wherein, 6-FAM is a fluorescent group located at the 5' end, and BHQ1 is a fluorescent quenching group located at the 3' end; the final concentration of the single-stranded DNA fluorescent reporter probe in the CRISPR / Cas12a detection reaction system is 300 nM.

[0011] Preferably, the reaction buffer is a 1×Reaction Buffer with a pH of 8.0 to maintain the trans-cleavage activity of the LbCas12a protein after activation of the target nucleic acid.

[0012] Preferably, the ERA amplification is performed at 40°C for 20 min; the CRISPR / Cas12a detection reaction system is incubated at 40°C for 30 min, and fluorescence signals are collected at 1 min intervals during the incubation process.

[0013] Preferably, the method further includes setting up a negative control group and a non-target crRNA control group; The negative control group was replaced with nuclease-free water instead of nucleic acid or ERA amplification product of the test sample. The non-target crRNA control group uses crRNA that is not complementary to the target sequence of the PEDV-N gene to replace the crRNA that recognizes the target sequence of the PEDV-N gene. When the test sample group produces a fluorescent signal, and the negative control group and the non-target crRNA control group do not produce a fluorescent signal equivalent to that of the test sample group, it is determined that the test sample contains PEDV nucleic acid.

[0014] This invention also provides a detection kit for a method of detecting porcine epidemic diarrhea virus, comprising: ERA primer pairs for amplifying conserved regions of the PEDV-N gene, LbCas12a protein, crRNA that recognizes the target sequence of the PEDV-N gene, single-stranded DNA fluorescent reporter probe, reaction buffer for maintaining Cas12a cleavage activity, ERA isothermal amplification reagent, reverse transcription reagent, positive control and negative control. The single-stranded DNA fluorescent reporter probe has a nucleotide sequence of 5'-6-FAM-TTATT-BHQ1-3', and the reaction buffer has a pH of 8.0.

[0015] The present invention also provides a portable detection device for detecting porcine epidemic diarrhea virus, comprising a sample processing unit, a constant temperature reaction unit, a fluorescence detection unit, a data processing unit, and a wireless communication unit; The sample processing unit is used to contain the pig samples to be tested and to provide the reaction conditions required for nucleic acid extraction, reverse transcription and ERA amplification. The isothermal reaction unit is used to maintain the ERA amplification reaction at 40°C and the CRISPR / Cas12a detection reaction at 40°C. The fluorescence detection unit includes an excitation light source and a fluorescence signal acquisition component, used to detect the fluorescence signal released after the single-stranded DNA fluorescent reporter probe is cleaved; The data processing unit is used to compare the fluorescence signal of the sample to be tested with the fluorescence signal of the negative control group and output the detection result; The wireless communication unit is used to send the detection results to the mobile terminal.

[0016] This invention provides a method for detecting porcine epidemic diarrhea virus based on ERA-CRISPR-Cas12a. It has the following beneficial effects: 1. This invention utilizes ERA amplification primers targeting the conserved region of the PEDV N gene and employs specific crRNA to guide LbCas12a in recognizing the ERA amplification products, forming a dual-specificity detection mechanism of ERA primer amplification recognition + Cas12a / crRNA target sequence recognition. The PEDV N gene exhibits high conservation among different prevalent PEDV strains, improving the consistency of detection across different strains. The ERA primer pairs define the target amplification region, and crRNA further recognizes the specific target sequence within the amplification product. Only when the target sequence is present and matches the crRNA recognition region is LbCas12a activated and cleaves the single-stranded DNA fluorescent reporter probe. This enhances the specificity of the detection results. Verification using nucleic acid detection of porcine pathogens such as PEDV, PRRSV, and ASFV showed that PEDV samples produced significant fluorescence signals, while the fluorescence signals of non-PEDV pathogen samples such as PRRSV and ASFV were close to those of the negative control group, indicating that the method of this invention has good pathogen differentiation capabilities.

[0017] 2. This invention combines ERA isothermal nucleic acid amplification with CRISPR-Cas12a trans-cleavage signal amplification. After ERA amplification for 20 min at 40℃, the amplified product is added to a detection system containing LbCas12a, crRNA, and a fluorescent reporter probe, and incubated at 40℃ for 30 min to complete fluorescence detection. ERA can rapidly enrich the PEDV N gene target sequence under isothermal conditions. After recognizing the target, Cas12a can continuously cleave multiple single-stranded DNA reporter probes with 6-FAM fluorescent groups and BHQ1 quenching groups, converting the target recognition event into an accumulative fluorescence output. The detection mechanism formed by amplification enrichment and trans-cleavage signal accumulation improves the detection capability of low-concentration PEDV nucleic acid; when using pClone007-PEDV-N standard positive plasmid for serial dilution detection, the detection limit of this invention reaches 2×10⁻⁶. 1 The number of copies / μL is sufficient to meet the nucleic acid screening requirements for low-load PEDV samples. Attached Figure Description

[0018] Figure 1 This is a schematic diagram illustrating the principle of PEDV detection based on RT-ERA-CRISPR-Cas12a in this invention; Figure 2 A schematic diagram of the design of ERA primers and crRNA target sites in the conserved region of the PEDV N gene; Figure 3 Agarose gel electrophoresis results of amplification of PEDV N gene fragments using different ERA primer pairs; Figure 4 Figure 1 shows the SDS-PAGE results of LbCas12a protein expression and purification. Figure 5 The figure shows the validation results of the necessity of Cas12a, crRNA, target template, and fluorescent reporter probe for the detection system. Figure 6 The fluorescence screening results of different crRNAs for detecting the PEDV N gene target are shown in the figure. Figure 7 Figure showing the results of optimizing the LbCas12a to crRNA ratio; Figure 8 Figure showing the results of optimizing the concentration of single-stranded DNA fluorescent reporter probes; Figure 9 The graph shows the fluorescence detection results under different pH reaction buffer conditions; Figure 10 Figure showing the results of ERA reaction temperature optimization; Figure 11 The graph shows the results of ERA reaction time optimization. Figure 12 The graph shows the sensitivity test results of the detection method of the present invention. Figure 13 This is a graph showing the specificity test results of the detection method of the present invention for PEDV, PRRSV, ASFV and negative control. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1: This invention provides a method for detecting porcine epidemic diarrhea virus based on ERA-CRISPR-Cas12a, as detailed below: I. Identification of PEDV N gene target region, ERA primer design and screening Whole genome sequences of PEDV strains from different regions and at different epidemic stages were downloaded from the NCBI database. The N gene sequence of each strain was extracted and multiple sequence alignment was performed. Regions with less variation among different strains and whose continuous conserved lengths met the requirements for ERA amplification and crRNA design were selected as detection target regions.

[0021] ERA primers were designed within the conserved region of the PEDV N gene using Primer Premier 5 software. To screen primer combinations suitable for ERA amplification, four pairs of primers were designed around each of the two candidate crRNA target regions, resulting in a total of eight candidate ERA primer pairs, each with a length of 26–28 nt. Each primer pair was evaluated for dimerization, hairpin structure, and non-specific binding before synthesis.

[0022] Using the constructed pClone007-PEDV-N standard positive plasmid as a template, the amplification performance of candidate primer pairs was evaluated. Each ERA amplification reaction tube contained 2.5 μL of 10 μM upstream primer, 2.5 μL of 10 μM downstream primer, 2 μL of positive standard plasmid, 2 μL of activator, and ERA lyophilized reaction microspheres. Nuclease-free water was then added to the volume specified in the kit. After briefly centrifuging to mix, the reaction tubes were incubated at 40°C for 25 min.

[0023] After amplification, 10 μL of the amplification product was subjected to 2.5% agarose gel electrophoresis, with the reaction system without template serving as a negative control. Electrophoresis results showed that multiple primer sets designed for the crRNA1 target region all produced the target band. The primer set consisting of SEQ ID NO.1 and SEQ ID NO.2 showed a clear and bright band, while the negative control showed no target band. Therefore, this primer set was identified as the primer pair used for PEDV N gene ERA amplification in subsequent experiments.

[0024] II. Design, preparation and screening of PEDV target crRNA like Figure 2 As shown, based on the Cas12a target sequence recognition requirements, target sites matching the conserved sequence of the PEDV N gene were selected within the ERA amplification region, and two candidate target crRNAs were designed, denoted as crRNA1 and crRNA2, respectively; another non-target crRNA with no complementary relationship to the PEDV N gene was designed, denoted as no-target crRNA.

[0025] The DNA template sense strand sequence of crRNA1 is: 5'-GAAATTAATACGACTCACTATAGGTAATTTCTACTAAGTGTAGATCGCCTTCTTTAGCAACCCAG-3'; Its complementary strand sequence is: 5'-CTGGGTTGCTAAAGAAGGCGATCTACACTTAGTAGAAATTACCTATAGTGAGTCGTATTAATTTC-3'.

[0026] The DNA template sense strand sequence of crRNA2 is: 5'-GAAATTAATACGACTCACTATAGGTAATTTCTACTAAGTGTAGATGAGATGCGGAATTTGTCGAA-3'; Its complementary strand sequence is: 5'-TTCGACAAATTCCGCATCTCATCTACACTTAGTAGAAATTACCTATAGTGAGTCGTATTAATTTC-3'.

[0027] Each pair of complementary DNA oligonucleotides was dissolved separately in annealing buffer to ensure a uniform final concentration. After heating at 95°C for 5 min, the mixture was slowly cooled to room temperature to form a double-stranded DNA template. T7 in vitro transcription was performed using the annealed product as a template to obtain crude crRNA. After transcription, DNase I was added, and the mixture was digested at 37°C for 10 min to remove the DNA template. The RNA product was then purified using an RNA purification and recovery kit to obtain crRNA for Cas12a detection.

[0028] Using the PEDV-N standard positive plasmid as a template, crRNA1, crRNA2, and no-target crRNA were added after ERA amplification for Cas12a fluorescence detection. The results showed that the real-time fluorescence intensity and endpoint fluorescence value of the crRNA1 group were higher than those of the crRNA2 group, while the no-target crRNA group did not produce a fluorescence signal comparable to that of the target crRNA group. Therefore, crRNA1 was selected as the specific recognition RNA in the PEDV detection system.

[0029] III. Construction of PEDV N gene standard positive plasmid The target fragment of the PEDV N gene was amplified by PCR. After PCR amplification, the amplified product was confirmed by agarose gel electrophoresis, and the target band was excised and purified by gel extraction. The purified PEDV N gene fragment was ligated with the pClone007 vector at 25℃ for 20 min to obtain the recombinant ligation product.

[0030] The recombinant ligation product was transformed into competent *E. coli* cells, plated on LB agar plates containing the appropriate antibiotics, and incubated overnight at 37°C. Single colonies were picked for colony PCR identification using universal primers M13F and M13R. PCR-positive colonies were sequenced for confirmation, and the recombinant plasmid pClone007-PEDV-N with the correct insert sequence was obtained through screening.

[0031] The positive bacterial culture with the correct sequence was mixed with an equal volume of 50% glycerol solution and stored at -80°C. The recombinant plasmid was extracted and its concentration was determined. The copy number was calculated based on the plasmid molecular weight for subsequent sensitivity evaluation, primer screening, and system optimization.

[0032] IV. Expression and purification of recombinant LbCas12a protein Using the 6His-MBP-TEV-huLbCpf1 plasmid as a template, the LbCpf1 gene fragment was amplified using primers containing BamHI and SalI restriction sites. The amplification primers were: pET-28a-LbCpf1-F. 5'-CGCGGATCCATGAGCAAGCTGGAGAAGTTTACAAACTGC-3'; pET-28a-LbCpf1-R: 5'-GCGTCGACGTGCTTCACGCTGGTCTGGGCGTACTC-3'.

[0033] The LbCpf1 gene fragment obtained by PCR amplification and the pET-28a vector were double-digested with BamHI and SalI, respectively. The target fragment was recovered and ligated to construct the recombinant expression plasmid pET-28a-His-huLbCpf1. The ligation product was transformed into DH5α competent cells, and positive clones were screened and verified by colony PCR and sequencing.

[0034] The correctly sequenced recombinant plasmid was transformed into BL21 competent cells. Single colonies were picked and inoculated into 4 mL of LB broth and cultured at 37°C and 220 rpm for 12–16 h. The seed culture was then transferred to 400 mL of LB broth and cultured at 37°C and 220 rpm until the OD600 reached 0.6–0.8. IPTG was added to a final concentration of 1 mM, and expression was induced for 16 h at 16°C and 220 rpm.

[0035] After induction, the bacterial cell pellet was collected by centrifugation, and the bacterial cell resuspended in lysis buffer. The pellet was then sonicated to release soluble protein. The lysis buffer was centrifuged, and the supernatant was collected. His-tagged protein was purified using a Ni-NTA affinity chromatography column. The purified protein was further dialyzed to replace the soluble protein with a suitable storage buffer for Cas12a detection. SDS-PAGE analysis of the purified product confirmed the acquisition of LbCas12a protein with the expected molecular weight. The results are as follows: Figure 4 As shown.

[0036] The purified LbCas12a protein and the commercially available LbCas12a protein were used separately in the PEDV-N target detection reaction, and the results are as follows: Figure 6 As shown, both can produce obvious fluorescence signals in the presence of the target and specific crRNA, indicating that the prepared protein has Cas12a trans-cleavage activity.

[0037] V. Establishment of CRISPR-Cas12a detection system and verification of necessary components The CRISPR / Cas12a reaction system includes 2 μL of 500 ng / μL Cas12a, 2 μL of 500 ng / μL crRNA, 2 μL of 10 μMssDNA probe, and 9 μL of 1*Reaction Buffer. This mixture is added to 50 μL of ERA amplification product, thoroughly mixed, and incubated at 40°C for 30 min. Fluorescence is recorded every 1 min using the thermostat, and the real-time fluorescence intensity is recorded using 30 recordings. Finally, the final fluorescence intensity is observed visually or photographed using a gel imaging system. The results are as follows: Figure 5As shown. Using standard positive plasmids as templates, target crRNAs were screened, and a negative control group was set up. The optimal crRNA was selected based on the endpoint fluorescence intensity for the experiment, and each experiment was repeated three times.

[0038] VI. Optimization of the ERA-CRISPR / Cas12a detection method Optimization of the Cas12a / crRNA concentration ratio: Cas12a and crRNA were diluted to a uniform concentration, and different volumes were set up, requiring a total of 5 sets of experiments. The effect of different Cas12a and crRNA concentration ratios on fluorescence intensity was observed to determine the optimal ratio.

[0039] The initial concentrations of LbCas12a protein and crRNA were both prepared at 500 ng / μL, with LbCas12a / crRNA mass ratios set at 4:1, 2:1, 1:1, 1:2, and 1:4. Equal volumes of PEDV N gene ERA amplification product were added to each group, and the reporter probe, reaction buffer, and incubation conditions were kept consistent. Fluorescence signals were collected every 1 minute using a thermostatic fluorescence detector for a total of 30 minutes. Results are as follows: Figure 7 As shown, the fluorescence intensity was highest when the mass ratio was 4:1, and this ratio was selected as the Cas12a and crRNA ratio for the subsequent detection system.

[0040] Based on the above proportions, the final concentrations of the reporting probe were set to 200 nM, 300 nM, 400 nM, and 500 nM, respectively. The results are as follows... Figure 8 As shown, the fluorescence signal of the system is enhanced with the increase of the reporter probe concentration; considering the fluorescence intensity, reaction stability and reagent cost, 300 nM was selected as the optimal final concentration of the reporter probe.

[0041] The pH of the 1×Reaction Buffer was adjusted to 7.0, 7.5, 8.0, and 8.5, respectively, and the results were measured under the same other reaction conditions. The results are as follows: Figure 9 As shown, the highest endpoint fluorescence value and a more obvious real-time fluorescence growth curve were obtained at pH 8.0. Therefore, pH 8.0 was determined as the buffer condition for the Cas12a detection system of this invention.

[0042] The ERA reaction temperatures were set at 37℃, 39℃, 40℃, 42℃, and 44℃, and ERA amplification was performed using pClone007-PEDV-N plasmid with the same copy number as a template. After amplification, an equal amount of amplification product was added to the optimized CRISPR-Cas12a detection system, incubated at 40℃ for 30 min, and fluorescence signals were collected.

[0043] Experimental results are as follows Figure 10As shown, the ERA amplification products at 40℃ produced a strong fluorescence signal in subsequent Cas12a detection, indicating that the ERA amplification efficiency was good at this temperature. Therefore, 40℃ was selected as the ERA reaction temperature.

[0044] At 40℃, ERA amplification times were set to 10 min, 15 min, 20 min, 25 min, and 30 min, respectively. The results are as follows: Figure 11 As shown, the fluorescence signal gradually increases with the extension of amplification time; a stable and obvious positive fluorescence signal was obtained at 20 min, and further extending the reaction time had limited effect on improving the detection results. Therefore, this invention determines 20 min as the preferred ERA amplification time.

[0045] Example 2: This invention illustrates the detection principle of Cas12a and the detection method of PEDV using the ERA-CRISPR / Cas12a detection system: Firstly, CRISPR nucleic acid detection technology combines high sensitivity, simplicity, and low cost, making it an ideal choice for disease diagnosis. For example... Figure 1 As shown, Cas12a, guided by crRNA, can precisely recognize and cleave target nucleic acids. Cas12a forms a protein-nucleic acid complex (RNP) with crRNA. The crRNA recognizes the target and guides Cas12a to perform cis- and trans-cleavage. The presence of the target can be confirmed by detecting whether the probe has been cleaved. When Cas12a is activated, it begins to cleave the probe extensively, and the cleaved probe emits a fluorescent signal. Cas12a can only be activated and cleave the probe in the presence of the target.

[0046] By integrating isothermal amplification with the CRISPR / Cas system, the number of target nucleic acids is rapidly increased through isothermal amplification, and then the CRISPR / Cas system is used for specific recognition and signal amplification, thereby achieving highly sensitive and highly specific detection.

[0047] Based on the conserved sequence of PEDV-N, eight pairs of primers and two crRNAs were designed, with their positions as follows: Figure 2 As shown. To determine the optimal primers for ERA amplification, the target gene amplification effect of eight pairs of RPA primers was analyzed by 2.5% agarose gel electrophoresis, as shown. Figure 3 As shown.

[0048] To analyze the necessity of the reaction components and verify the trans-cleavage activity of Cas12, all components including Cas12a, crRNA, template, and reporter were individually omitted and replaced with an equal volume of enzyme-free water. Fluorescence signals and endpoint fluorescence data were collected and analyzed. The results showed that Cas12a, crRNA, template, and reporter are crucial for the ERA / Cas12a reaction. The absence of these components did not result in significant differences compared to the control group. In conclusion, the presence of Cas12a, crRNA, RPA mixture, and reporter is essential for the ERA / Cas12a method.

[0049] The results showed that primer pairs targeting crRNA1 produced bands of similar brightness. The control group showed no band, therefore F1 / R1 was selected as the optimal primer pair. The F5 / R5 primer pair targeting crRNA2 produced the brightest band, while the control group showed no band. Subsequently, crRNA1, crRNA2, and non-target crRNAs were screened, and fluorescence signals and endpoint fluorescence data were recorded after ERA-CRISPR / Cas12a assays. Compared to the negative control, crRNA1 showed the highest fluorescence value, and the no-target-crRNA group did not produce visible fluorescence. Based on these results, the F1 / R1 primer pair and crRNA1 were selected for further detection.

[0050] Seven validation reaction systems were designed. The first three systems contained complete reaction systems with purified Cas12a, crRNA, reporter, and target (PEDV-N), with the first two containing specific crRNA and the third containing non-target crRNA. The remaining four systems were reaction systems lacking purified Cas12a, crRNA, target, and reporter, respectively. Each reaction component was incubated at 37°C for 30 min, and fluorescence was observed and photographed using a gel imaging system.

[0051] The ERA-CRISPR / Cas12a method was used, with 70 ng / µL of PEDV-N gene as a template, followed by amplification at 40℃ for 30 min and incubation. Fluorescence signals were collected using a thermostat.

[0052] Example 3: This invention provides a method for detecting porcine epidemic diarrhea virus (PEDV) detection performance. Sensitivity evaluation: The pClone007-PEDV-N standard positive plasmid was serially diluted 10-fold to prepare templates of different copy concentrations. Using each gradient plasmid as a template, ERA amplification and CRISPR-Cas12a fluorescence detection were performed according to the conditions described in Example 2 above.

[0053] The results are as follows Figure 12As shown, in 2×10 1 At a template concentration of 2 copies / μL, the detection system still produced a significantly higher fluorescence signal than the negative control group; when the template concentration was further reduced, the difference in fluorescence signal between the two control groups decreased. Therefore, the detection limit of the method of this invention for the PEDV N gene standard plasmid is 2×10⁻⁶. 1 copies / μL.

[0054] Specificity evaluation: Nucleic acid was extracted from samples of PEDV, porcine reproductive and respiratory syndrome virus (PRRSV), African swine fever virus (ASFV), and other porcine pathogens to be evaluated. For RNA virus samples, RNA was extracted and then reverse transcribed; for DNA virus samples, DNA was extracted directly and evaluated under the same ERA-CRISPR-Cas12a detection conditions.

[0055] Detection was performed using PEDV N gene-specific ERA primers and crRNA. Results are as follows: Figure 13 As shown, the PEDV sample group produced obvious real-time fluorescence accumulation and endpoint fluorescence signals; the fluorescence signals of the PRRSV, ASFV, and other non-PEDV pathogen sample groups were similar to those of the negative control group, and no obvious visible fluorescence was observed under UV or blue light excitation conditions. These results indicate that the method of this invention has good detection specificity for PEDV and did not show cross-reactivity with other porcine pathogen nucleic acids tested.

[0056] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for detecting porcine epidemic diarrhea virus based on ERA-CRISPR-Cas12a, characterized in that, Includes the following steps: (1) Extract nucleic acid from the pig sample to be tested, and reverse transcribe the extracted RNA as a template to obtain cDNA; (2) Using the conserved region of the N gene of porcine epidemic diarrhea virus (PEDV) as the target, the cDNA was enzymatically recombinated and isothermally amplified using ERA primers to obtain the ERA amplification product. (3) The ERA amplification product is mixed with LbCas12a protein, crRNA that recognizes the target sequence of PEDV-N gene, single-stranded DNA fluorescent reporter probe and reaction buffer to form a CRISPR / Cas12a detection reaction system. (4) The CRISPR / Cas12a detection reaction system is incubated under constant temperature conditions so that the LbCas12a protein recognizes the target sequence in the ERA amplification product under the guidance of crRNA and generates trans-cleavage activity to cleave the single-stranded DNA fluorescent reporter probe. (5) Collect the fluorescence signal of the detection reaction system, and use the fluorescence signal of the test sample being higher than that of the negative control group as the basis for determining the detection of PEDV.

2. The method for detecting porcine epidemic diarrhea virus based on ERA-CRISPR-Cas12a according to claim 1, characterized in that, The ERA primer pair for the conserved region of the PEDV-N gene includes an upstream primer and a downstream primer, and the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1: 5'-ATGCGCCGTGGTGAGCGAATTGAACA-3'; The nucleotide sequence of the downstream primer is shown in SEQ ID NO.2: 5'-GACCCTGGTTATTTCCACGATTCTGT-3'.

3. The method for detecting porcine epidemic diarrhea virus based on ERA-CRISPR-Cas12a according to claim 1, characterized in that, The crRNA includes a direct repeat sequence recognized by Cas12a and a target sequence recognition region; wherein the nucleotide sequence of the target sequence recognition region is shown in SEQ ID NO.3: 5'-CGCCTTCTTTAGCAACCCAG-3'; The complete nucleotide sequence of the crRNA is shown in SEQ ID NO.4: 5'-UAAUUUCUACUAAGUGUAGAUCGCCUUCUUUAGCAACCCAG-3'.

4. The method for detecting porcine epidemic diarrhea virus based on ERA-CRISPR-Cas12a according to claim 1, characterized in that, In the CRISPR / Cas12a detection reaction system, the mass ratio of LbCas12a protein to crRNA is 4:1, and the initial concentrations of both LbCas12a protein and crRNA are 500 ng / μL.

5. The method for detecting porcine epidemic diarrhea virus based on ERA-CRISPR-Cas12a according to claim 1, characterized in that, The single-stranded DNA fluorescent reporter probe is an ssDNA probe with a fluorescent group and a fluorescence quenching group, and its nucleotide sequence is: 5'-6-FAM-TTATT-BHQ1-3'. Wherein, 6-FAM is a fluorescent group located at the 5' end, and BHQ1 is a fluorescent quenching group located at the 3' end; the final concentration of the single-stranded DNA fluorescent reporter probe in the CRISPR / Cas12a detection reaction system is 300 nM.

6. The method for detecting porcine epidemic diarrhea virus based on ERA-CRISPR-Cas12a according to claim 1, characterized in that, The reaction buffer is a 1×Reaction Buffer with a pH of 8.0 to maintain the trans-cleavage activity of the LbCas12a protein after activation of the target nucleic acid.

7. The method for detecting porcine epidemic diarrhea virus based on ERA-CRISPR-Cas12a according to claim 1, characterized in that, The ERA amplification was performed at 40°C for 20 min; the CRISPR / Cas12a detection reaction system was incubated at 40°C for 30 min, and fluorescence signals were collected at 1 min intervals during the incubation process.

8. The method for detecting porcine epidemic diarrhea virus based on ERA-CRISPR-Cas12a according to claim 1, characterized in that, The method also includes setting up a negative control group and a non-target crRNA control group; The negative control group was replaced with nuclease-free water instead of nucleic acid or ERA amplification product of the test sample. The non-target crRNA control group uses crRNA that is not complementary to the target sequence of the PEDV-N gene to replace the crRNA that recognizes the target sequence of the PEDV-N gene. When the test sample group produces a fluorescent signal, and the negative control group and the non-target crRNA control group do not produce a fluorescent signal equivalent to that of the test sample group, it is determined that the test sample contains PEDV nucleic acid.

9. A detection kit for implementing the porcine epidemic diarrhea virus detection method according to any one of claims 1 to 8, characterized in that, include: ERA primer pairs for amplifying conserved regions of the PEDV-N gene, LbCas12a protein, crRNA that recognizes the target sequence of the PEDV-N gene, single-stranded DNA fluorescent reporter probe, reaction buffer for maintaining Cas12a cleavage activity, ERA isothermal amplification reagent, reverse transcription reagent, positive control and negative control. The single-stranded DNA fluorescent reporter probe has a nucleotide sequence of 5'-6-FAM-TTATT-BHQ1-3', and the reaction buffer has a pH of 8.

0.

10. A portable detection device for implementing the porcine epidemic diarrhea virus detection method according to any one of claims 1 to 8, characterized in that, It includes a sample processing unit, an isothermal reaction unit, a fluorescence detection unit, a data processing unit, and a wireless communication unit; The sample processing unit is used to contain the pig samples to be tested and to provide the reaction conditions required for nucleic acid extraction, reverse transcription and ERA amplification. The isothermal reaction unit is used to maintain the ERA amplification reaction at 40°C and the CRISPR / Cas12a detection reaction at 40°C. The fluorescence detection unit includes an excitation light source and a fluorescence signal acquisition component, used to detect the fluorescence signal released after the single-stranded DNA fluorescent reporter probe is cleaved; The data processing unit is used to compare the fluorescence signal of the sample to be tested with the fluorescence signal of the negative control group and output the detection result; The wireless communication unit is used to send the detection results to the mobile terminal.

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