CN115595382A - Primer group for detecting porcine enterocoronavirus and application thereof - Google Patents

Primer group for detecting porcine enterocoronavirus and application thereof Download PDF

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CN115595382A
CN115595382A CN202110778864.6A CN202110778864A CN115595382A CN 115595382 A CN115595382 A CN 115595382A CN 202110778864 A CN202110778864 A CN 202110778864A CN 115595382 A CN115595382 A CN 115595382A
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primer
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牛丽莉
刘佳佳
谢胜松
陈薪全
陶大刚
徐兵荣
赵叶
沈林園
朱砺
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Sichuan Agricultural University
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Abstract

The disclosure relates to the technical field of nucleic acid detection, in particular to a primer group for detecting porcine enterocoronavirus and application thereof. The kit provided by the disclosure adopts LAMP loop-mediated isothermal amplification technology, combines CRISPR technology, and has a visual detection signal for a positive result in a CRISPR-Cas12a system, thereby facilitating the realization of field detection of viral nucleic acid. Can improve the specificity, sensitivity and accuracy of molecular diagnosis, provide more accurate data and information for the diagnosis and treatment of infectious diseases of pigs, and can simultaneously detect four porcine enterocoronaviruses. Has good application prospect, and can greatly promote the rapid development of veterinary molecular diagnosis and effectively prevent and treat porcine virology diarrhea diseases by jointly generating a new analysis method.

Description

Primer group for detecting porcine enterocoronavirus and application thereof
Technical Field
The disclosure relates to the technical field of nucleic acid detection, in particular to a primer group for detecting porcine enterocoronavirus and application thereof.
Background
The pig diarrhea is a common disease in large-scale pig farms, dehydration caused by the pig diarrhea is a main cause of death of sick pigs, and serious economic loss is caused to China and even the global pig industry. Porcine Epidemic Diarrhea Virus (PEDV) has caused more than 800 million lost pigs to the swine industry in the united states alone. Porcine enterocoronavirus is the major cause of viral diarrhea in pigs and includes transmissible gastroenteritis virus (TGEV), porcine Epidemic Diarrhea Virus (PEDV), porcine deltacoronavirus (PDCoV), and recently newly discovered porcine acute diarrhea syndrome coronavirus (SADS-CoV). The clinical presentation of PEDV is indistinguishable from the other two porcine enterocoronaviruses, i.e., TGEV, PDCoV.
At present, a disease sample of a pig is mainly sent to a specialized nucleic acid detection laboratory, total RNA in a sample to be detected is extracted, and then the total RNA is detected through reverse transcription quantitative PCR to evaluate whether the sample is positive or negative, only one virus can be detected at each time, and the experimental process is complicated. It is difficult to meet the requirements of on-site rapid nucleic acid detection.
Therefore, a primer set capable of rapidly and effectively detecting four porcine enterocoronaviruses and an application thereof are needed.
Disclosure of Invention
In view of the above, the present disclosure is directed to a primer set for detecting porcine enterocoronavirus and an application thereof.
In view of the above, the present disclosure provides a primer set for detecting porcine enterocoronavirus, comprising one or more of the following combinations:
combination one (PEDV-ORF 3):
(1) The nucleotide sequence of the upstream external primer is shown as SEQ ID NO. 1; and
(2) The nucleotide sequence of the downstream external primer is shown as SEQ ID NO. 2; and
(3) The nucleotide sequence of the upstream inner primer is shown as SEQ ID NO. 3; and
(4) The nucleotide sequence of the downstream inner primer is shown as SEQ ID NO. 4; and
(5) The nucleotide sequence of the upstream loop primer is shown as SEQ ID NO. 5; and
(6) The nucleotide sequence of the downstream loop primer is shown as SEQ ID NO. 6; and
(7) The guide nucleotide sequence is shown as SEQ ID NO. 7;
and/or
Combination two (TGEV-N):
(8) The nucleotide sequence of the upstream external primer is shown as SEQ ID NO. 8; and
(9) The nucleotide sequence of the downstream external primer is shown as SEQ ID NO. 9; and
(10) The nucleotide sequence of the upstream inner primer is shown as SEQ ID NO. 10; and
(11) The nucleotide sequence of the downstream inner primer is shown as SEQ ID NO. 11; and
(12) The nucleotide sequence of the upstream loop primer is shown as SEQ ID NO. 12; and
(13) The nucleotide sequence of the downstream loop primer is shown as SEQ ID NO. 13; and
(14) The guide nucleotide sequence is shown as SEQ ID NO. 14;
and/or
Combination three (PDCoV-N):
(15) The nucleotide sequence of the upstream external primer is shown as SEQ ID NO. 15; and
(16) The nucleotide sequence of the downstream external primer is shown as SEQ ID NO. 16; and
(17) The nucleotide sequence of the upstream inner primer is shown as SEQ ID NO. 17; and
(18) The nucleotide sequence of the downstream inner primer is shown as SEQ ID NO. 18; and
(19) The nucleotide sequence of the upstream loop primer is shown as SEQ ID NO. 19; and
(20) The nucleotide sequence of the downstream loop primer is shown as SEQ ID NO. 20; and
(21) The guide nucleotide sequence is shown as SEQ ID NO. 21;
and/or
Combination four (SADS-CoV-N):
(22) The nucleotide sequence of the upstream external primer is shown as SEQ ID NO. 22; and
(23) The nucleotide sequence of the downstream external primer is shown as SEQ ID NO. 23; and
(24) The nucleotide sequence of the upstream inner primer is shown as SEQ ID NO. 24; and
(25) The nucleotide sequence of the downstream inner primer is shown as SEQ ID NO. 25; and
(26) The nucleotide sequence of the upstream loop primer is shown as SEQ ID NO. 26; and
(27) The nucleotide sequence of the downstream loop primer is shown as SEQ ID NO. 27; and
(28) And the guide nucleotide sequence is shown as SEQ ID NO. 28.
On the basis of the research, the embodiment of the disclosure also provides application of the primer group in preparation of a kit for detecting porcine enterocoronavirus. In some embodiments, the porcine enterocoronavirus is one or more of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine delta coronavirus, or porcine acute diarrhea syndrome coronavirus.
On the basis of the research, the embodiment of the disclosure also provides a kit for detecting porcine enterocoronavirus, which comprises the primer set, a guide and a common auxiliary agent. In some embodiments, the porcine enterocoronavirus is one or more of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine delta coronavirus, or porcine acute diarrhea syndrome coronavirus.
In some embodiments, the kit further comprises a Cas12a protein and a nucleic acid probe comprising a detectable label, wherein the nucleic acid probe generates a visible detection signal when the nucleic acid probe is cleaved by the Cas12a protein shunt.
In some embodiments, the nucleic acid probe is a ssDNA-reporter molecule labeled with a fluorescent reporter at the 5 'end and a fluorescent quencher at the 3' end.
In some embodiments, the fluorescent reporter is ROX; the fluorescence quenching group is BHQ2.
In some embodiments, the visually detectable signal comprises a fluorescent signal or a solution color signal.
The kit provided by the disclosure adopts LAMP (Loop-mediated isothermal amplification) technology and combines CRISPR (clustered regularly interspaced short palindromic repeats) technology, and in a CRISPR-Cas12a system, a positive result has a visual detection signal, so that the field detection of viral nucleic acid is conveniently realized. Can improve the specificity, sensitivity and accuracy of molecular diagnosis, provide more accurate data and information for the diagnosis and treatment of infectious diseases of pigs, and can simultaneously detect four porcine enterocoronaviruses. Has good application prospect, and can greatly promote the rapid development of veterinary molecular diagnosis and effectively prevent and treat porcine virology diarrhea diseases by jointly generating a new analysis method.
Drawings
FIG. 1 is a schematic diagram of the results of screening for sgRNAs with optimal activity of the target PEDV-ORF3 gene of example 1;
FIG. 2 is a schematic diagram showing the results of determining the optimal LAMP primers and conditions for the target PEDV-ORF3 gene in example 1; among them, FIG. 2 (A, B) is an optimal LAMP primer for screening and determining a target PEDV-ORF3 gene; FIG. 2 (C, E) determines the optimal temperature (T,. Degree.C.) for LAMP amplification; FIG. 2 (D, F) determines the optimal time (t, min) for LAMP amplification;
fig. 3 is a schematic diagram of the sensitivity results for the LAMP-binding CRISPR-Cas12 a-based detection of PEDV of example 1; FIG. 3 (A) is a Cas12a visualized detection LAMP amplification product; fig. 3 (B) is an enzyme-labeled assay Cas12a visualized fluorescence value (n = 3);
FIG. 4 is a graph showing the results of screening for a highly active sgRNA of a target TGEV-N gene in example 2;
FIG. 5 is a diagram showing the results of LAMP primers for screening a target TGEV-N gene in example 2; wherein, FIG. 5 (A) is agarose gel electrophoresis detection of LAMP amplification product; fig. 5 (B) shows Cas12a visualized detection of LAMP amplification product; fig. 5 (C) is an alignment of sgrnas and primer sequences;
FIG. 6 is a schematic diagram of the sensitivity results of LAMP amplification combined with Cas12a system-based visual detection of TGEV-N gene in example 2; FIG. 6 (A) is a Cas12a visualized detection LAMP amplification product; fig. 6 (B) is an enzyme-labeled assay Cas12a visualized fluorescence value (n = 3);
FIG. 7 is a diagram showing the results of screening for a high activity sgRNA of a target PDCoV-N gene in example 3;
FIG. 8 is a diagram showing the results of screening LAMP amplification products of a target PDCoV-N gene in example 3; wherein, FIG. 8 (A) is agarose gel electrophoresis detection of LAMP amplification products; fig. 8 (B) shows Cas12a visual detection of LAMP amplification product;
FIG. 9 is a diagram showing the results of example 3 for the visual detection of sensitivity of PDCoV based on LAMP amplification product binding to Cas12a system; wherein, FIG. 9 (A) shows that Cas12a can visually detect LAMP amplification products; fig. 9 (B) is an enzyme-labeled assay Cas12a visualized fluorescence value (n = 3);
FIG. 10 is a schematic diagram of the results of screening for high activity sgRNAs of target SADS-CoV-N gene of example 4;
FIG. 11 is a diagram showing the results of screening LAMP amplification products of a target SADS-CoV-N gene of example 4; wherein, FIG. 11 (A) shows the detection of LAMP amplification products by agarose gel electrophoresis; fig. 11 (B) is an enzyme-labeled assay Cas12a visualized fluorescence value (n = 3); fig. 11 (C) is an alignment of sgRNA6 with LAMP2-BIP (outer primer) sequences;
FIG. 12 is a schematic diagram showing the results of example 4 in detecting the sensitivity of SADS-CoV-N gene based on LAMP amplification combined with Cas12a system visualization; wherein, fig. 12 (a) is Cas12a visual detection of LAMP amplification product; fig. 12 (B) is an enzyme-labeled assay Cas12a visualized fluorescence value (n = 3);
FIG. 13 is a diagram showing the result of the LAMP-CRISPR/Cas12a visualization of the specificity of detecting porcine coronavirus in example 5; wherein, FIG. 13 (A) is target LAMP primer specificity; fig. 13 (B) is the specificity of sgrnas of CRISPR/Cas12a system;
fig. 14 is a diagram showing the results of the detection based on the multiplex loop-mediated isothermal amplification combined Cas12a system in example 6.
Detailed Description
It is a first object of the present disclosure to provide primers in combination with guide nucleotides for the detection of four porcine enteroviruses.
The gene sequences of the 4 markers to be examined were searched and downloaded in the NCBI database (http:// www.ncbi.nlm.nih.gov. /). In the primer and probe screening aspect, 4 virus sequences are analyzed, after alignment, the sequences are compressed, and primers and sgrnas (guide RNAs) with high coverage are designed so as to adapt to the high mutation rate of RNA viruses. Through repeated primer screening and verification, 4 sets of suitable primer and guide RNA combinations are finally obtained, and are used for detecting 4 indexes as shown in Table 1.
The second purpose of the present disclosure is to provide a kit for detecting four porcine enterocoronaviruses, which comprises the primer set, the guide RNA and the auxiliary agent.
The kits described in the present disclosure comprise an amplification system and a visualization system.
In some embodiments, the AmpLification system includes 0.16U of Bst (Bacillus stearothermophilus) DNA polymerase 3.0, 10 × Isothermal AmppLication Buffer II 2.5 μ L, 8mM MgSO 4 1.4mM dNTP Mix, 2.5. Mu.L primer mixture, 1. Mu.L template and ultrapure water. Negative controls were: water is used as a template, namely, virus plasmids without porcine enterocoronavirus target fragments are used as templates.
In some embodiments, the visualization system comprises 2 μ L NEB buffer 2.1, 1000nM sgRNA,
In some embodiments, the amplification system is 25 μ L and the visualization system is 20 μ L. The amplification system was made up to 25. Mu.L with ultrapure water.
A third object of the present disclosure is to provide a method for detecting four porcine enterocoronaviruses, which uses the primer set and sgrnas described above.
In some embodiments, the detection method comprises:
(1) Extracting viral nucleic acid;
(2) Performing LAMP amplification by using corresponding primer groups at 65 ℃ for 20min to obtain amplification products;
(3) Visually detecting the amplification product obtained in the step (2) by using a Cas12a technology, wherein the reaction condition is 37 ℃ and 20min; at 98 ℃ for 2min;
(4) And (3) placing the reaction tube with the reaction solution obtained in the step (3) in a blue light or ultraviolet gel imager to observe the fluorescence intensity or the color change of the solution, distinguishing the virus types by using the corresponding sgRNAs, and indicating that a virus template exists if fluorescence exists.
In some embodiments, the LAMP amplification conditions are 57.5-67.5 ℃ and the amplification time is 15min or more.
In some embodiments, the LAMP amplification conditions are 65 ℃ and isothermal reaction for 20min.
Table 1 primers and sgrnas provided by the present disclosure
Figure BDA0003156880600000041
The method has the advantages that four main porcine enterocoronaviruses (PEDV, TGEV, PDCoV and SADS-CoV) can be rapidly and accurately identified based on LMAP isothermal amplification combined with a CRISPR-Cas12a system, and the detection sensitivity and specificity are high. The kit for detecting the porcine enterocoronavirus based on the CRISPR-Cas12a technology can be used for detecting high-sensitivity and specific pathogenic nucleic acid molecules. The detection method of the kit is more sensitive than a PCR detection method and more specific than LAMP detection, so that the detection can be more visual and convenient, and the nucleic acid pollution is reduced.
Example 1 screening and preparation of sgRNA against PEDV-ORF3 Virus
The performance of the sgRNA determines the quality of the detection effect of the kit. According to the invention, multiple groups of sgRNAs are screened aiming at a detection index PEDV-ORF3, and finally, an optimal group of sgRNAs is screened. This example is intended to illustrate the sgRNA screening process. Meanwhile, in the embodiment, a plurality of groups of LAMP amplification temperatures and times are also screened, and finally, a group of optimal temperatures and times are obtained through screening.
Designing a primer: respectively designing primer pairs for amplifying ORF3 genes aiming at the nucleic acid sequence (ID: KJ 642645.1) of the conserved gene ORF3 of the Porcine Epidemic Diarrhea Virus (PEDV); and 7 specific sgRNA sequences are designed according to the conserved region of the PCR amplified nucleic acid, PAM is TTTV, and an in vitro transcription primer pair is designed by taking an sgRNA empty vector as a template (Table 2).
TABLE 2 sgRNA sequence information for PEDV-ORF3
Figure BDA0003156880600000051
The steps for detecting nucleic acids using CRISP-Cas12 are as follows:
(1) Plasmid construction: the PEDV-ORF3 plasmid obtained by constructing the ORF3 gene fragment into the pUC57 plasmid was synthesized by Scout Biotech, and the plasmid map and the gene sequence information of the pUC 57-ligated plasmid are shown in Table 3.
(2) In vitro transcription of sgRNA: (1) and (3) PCR amplification: the template for in vitro sgRNA transfection was PCR amplified using the C12 plasmid containing T7 promoter and sgRNA scaffold as template, with T7-crRNA-F and different sgRNA-R primers (Table 3). PCR amplification was performed with T7-crRNA-F and different sgRNA-R primers. The experimental system was 20. Mu.L containing 10. Mu.L of Extaq Mix, 5ng of C12 plasmid, and 10pmoL of each of the upstream and downstream primers. The amplification procedure was: 94 ℃ for 3min; [94 ℃,30s; at 58 ℃ for 30s;72 ℃ for 12s]Circulating for 30 times; 72 ℃ for 5min; at 16 ℃ foviver. 3 mu.L of PCR amplification product was identified by electrophoresis on 2% agarose gel and the single and correct band was recovered by purification (TD 413-50, tian Mo). (2) In vitro transcription: the PCR product recovered in (2) was based on HiScribe TM The Quick T7 High Yield RNASynthesis Kit (NEB) synthesizes sgRNAs, and the reaction conditions are as follows: standing at 37 deg.C for 4-16h. (3) And (3) purifying the sgRNA, namely irradiating an operation table by ultraviolet for at least twenty minutes before purification, precooling the sgRNA to 4 ℃ by a centrifugal machine, and placing the used reagent into a refrigerator for precooling. Using phenol chloride to transcribe sgRNAPurifying by an imitation method, and storing in a refrigerator at-80 ℃ for a long time after purification.
(3) Visual detection of Cas12a system: the high-activity sgRNA is screened by using CRISPR/Cas12a technology, and in a 20 mu L reaction system, purified sgRNA (1000 nM), cas12a (500 nM), ssDNA-FQ reporter (ROX-N12-BHQ 2) (12.5 mu M) and 2 mu L NEB buffer 2.1 are added. The negative control is set as no detection target gene template, i.e. the template is water. The positive control is set to be only added with ssDNA activator, namely the corresponding sgRNA reverse primer; the experimental group is that ORF3 gene PCR amplification recovery product is about 200ng, reaction is carried out for 20min at 37 ℃, and then reaction is terminated at 98 ℃ for 2min.
The experimental results are as follows: the results of the experiment are shown in FIG. 1. As can be seen from fig. 1, among the 7 sgrnas targeting the PEDV-ORF3 gene, the color of the sgRNA5 group solution changes most significantly, and the sample containing the target gene can be clearly distinguished with naked eyes. sgRNA5 was therefore most active.
Example 2 screening and preparation of primers for PEDV-ORF3 Virus
The performance of the detection primer determines the quality of the detection effect of the kit. In this example, multiple primer combinations were screened for the detection index PEDV-ORF3, and finally an optimal primer combination was screened.
Designing a primer: 4 groups of primer pairs targeting PEDV-ORF3LAMP are designed according to the sequence of the PEDV-ORF3-sgRNA5, and the sequence of the primers is shown in Table 4.
Plasmid construction: the PEDV-ORF3 plasmid obtained by constructing the ORF3 gene fragment into the pUC57 plasmid was synthesized by Okagaku bioscience, and the plasmid map and the gene sequence information of the pUC57 universal plasmid are shown in Table 3.
TABLE 3 plasmid sequence information constructed into PUC57
Figure BDA0003156880600000061
Optimizing LAMP amplification conditions: (1) primer screening: four groups of primers (each group comprises inner primers PEDV-ORF3-FIP and PEDV-ORF3-BIP, outer primers PEDV-ORF3-F3 and PEDV-ORF3-B3, and loop primers PEDV-ORF3-LF and PEDV-ORF 3-LB) are designed aiming at the sgRNA5, and amplification is carried out by respectively taking ORF3 plasmid (10 ng) and DEPC water (1 mu L, NC) as templates. The 25. Mu.L system contained 2.5. Mu.L of amplification buffer, dNTPs (final concentration 1.4 mM), mgSO4 (final concentration 8 mM), primer Mix 2.5. Mu.L (1.6. Mu.M FIP/BIP, 0.2. Mu.M F3/B3, 0.4. Mu.M LoopF/B), 0.169 Bst3.0. Reaction at 65 deg.C for 50min, and inactivation at 85 deg.C for 5min. (2) Performing LAMP amplification temperature exploration: the LAMP primers are used for amplification, and 8 temperatures and 1 negative control are set within the range of 53-71 ℃. The specific temperature is 53, 55, 57.5, 60, 62.5, 65, 67.5, 71 (DEG C), the negative control is 65 ℃, and the reaction time is 50min; (3) the LAMP amplification time is searched: LAMP amplification is carried out by using the primers selected in the experiment and the reaction temperature, the reaction time is respectively 5, 15, 20, 25, 30, 35, 40 and 45min, and the negative control amplification time is 45min.
(3) Visual detection of Cas12a system: the 20. Mu.L CRISPR/Cas12a system contained purified sgRNA (1000 nM), cas12a (500 nM), ssDNA-FQ reporter (ROX-N12-BHQ 2) (12.5. Mu.M), 2. Mu.L NEB buffer 2.1 and 3. Mu.L of the amplification product. The reaction was stopped at 37 ℃ for 15min and then at 98 ℃ for 2min.
TABLE 4 LAMP primer sequence information of PEDV-ORF3
Figure BDA0003156880600000071
The experimental results are as follows: the results are shown in FIG. 2. FIG. 2 (A, B) is an optimal LAMP primer set for screening and determining the target PEDV-ORF3 gene. It can be seen that the LAMP-2 primer group has the best Cas12a visualization effect compared to the other groups. FIG. 2 (C, E) is a graph for determining the optimal temperature (T,. Degree. C.) for LAMP amplification. Wherein, 1-9 are respectively: 53. 55, 57.5, 60, 62.5, 65, 67.5, 71 and a negative control (65 ℃). It can be seen that after the PEDV-ORF3 plasmid reacts at the temperature of 53-67.5 ℃, the LAMP amplification product has a good visualization effect after being detected by a Cas12a visualization detection system (FIG. 2C), but the reaction temperature is better between 57.5-67.5 when the LAMP amplification product is combined with an agarose gel electrophoresis strip. The optimal LAMP amplification temperature was 65 deg.C (FIG. 2E).
The results of the investigation of the optimal amplification time under the conditions in which the optimal reaction temperature was determined to be 65 ℃ are shown in FIG. 2 (D, F). It can be seen that when the amplification time is greater than or equal to 15 minutes, the amplification product can achieve the effect of visual detection after the visual detection of Cas12a (FIG. 2D), but when the amplification time reaches 20 minutes, the optimal time is shown by combining with the gel electrophoresis strip (FIG. 2F).
The results show that the method for visually detecting PEDV based on the multiple loop-mediated isothermal amplification combined CRISPR-Cas12A system is basically established (FIGS. 2A and 2B).
Example 3 kit sensitivity assessment for PEDV-ORF3 Virus
To verify the sensitivity of the kit for visual detection of viral PEDV-ORF3 based on LAMP amplification in combination with CRISPR/Cas12a, the PEDV-ORF3 viral plasmid was diluted to 0.1 copies/. Mu.L (i.e., 1X 10^ (-1)) in a ten-fold gradient. And amplifying the plasmid template diluted in a gradient manner by using the LAMP primer aiming at the PEDV-ORF3 virus, and then detecting the kit.
Template dilution: the copy number of the PEDV-ORF3 plasmid was calculated using the formula (copy number (. Mu.L) = (amount of DNA (ng) × 6.022 × 1023)/(fragment size (bp) × 1 × 109 × 660) (https:// toptiptpbio.com/dnacopy-number-qpcr /), and then it was diluted to 1X 10-1 copy/. Mu.L with a ten-fold gradient.
Visual detection of LAMP combined Cas12a system: (1) LAMP amplification. The diluted plasmids were LAMP-amplified using PEDV-LAMP-2 primers, respectively, in a 25. Mu.L system containing 2.5. Mu.L of amplification buffer, dNTPs (final concentration 1.4 mM), mgSO4 (final concentration 8 mM), primer Mix 2.5. Mu.L (1.6. Mu.M FIP/BIP, 0.2. Mu.M F3/B3, 0.4. Mu.M LoopF/B), 0.169 Bst3.0. The reaction program is 65 ℃ and 20min;85 ℃ and 5min. (2) Cas12a system was visually detected. The 20. Mu.L CRISPR/Cas12a system contained purified sgRNA (1000 nM), cas12a (500 nM), ssDNA-FQ reporter (ROX-N12-BHQ 2) (12.5. Mu.M), 2. Mu.L NEB buffer 2.1 and 3. Mu.L of the amplification product. The reaction was stopped at 37 ℃ for 10min and then at 98 ℃ for 2min. And (3) directly placing the centrifuge tube containing the reaction solution in a blue light gel cutting instrument and an ultraviolet gel imaging instrument for observing fluorescence intensity or directly observing with naked eyes, simultaneously measuring a fluorescence value by using an enzyme label, uniformly mixing 20 mu L of Cas12a reaction solution and 80 mu L of DEPC water, adding the mixture into a 96-hole cell plate for enzyme label measurement (n = 3), setting fluorescence detection exciting light to be 576nm, and emitting light to be 601nm.
The experimental results are as follows: the results are shown in FIG. 3. Wherein, 1: 8X 10 4 Copy/. Mu.L; 2: 8X 10 3 Copy/. Mu.L; 3: 8X 10 2 Copy/. Mu.L; 4: 8X 10 1 Copy/. Mu.L; 5: 8X 10 0 Copy/. Mu.L; 6:1 x 10 0 Copy/. Mu.L; 7: 1X 10 -1 Copy/. Mu.L; 8: no Template Control (NTC). As can be seen, after the LAMP amplification product is visually detected by Cas12a, the detection limit can reach 1 copy/. Mu.L (FIG. 3A). Fluorescent signals are collected by a microplate reader (figure 3B), which further indicates that the visualized detection PEDV based on the combination of the loop-mediated isothermal amplification and the CRISPR-Cas12a system has high sensitivity and can meet the requirement of early detection of viruses.
Example 4 screening and preparation of sgRNA against TGEV-N Virus
The performance of the sgRNA determines the quality of the detection effect of the kit. According to the invention, multiple groups of sgRNAs are screened aiming at the detection index TGEV-N, and finally, an optimal group of sgRNAs is screened. This example is intended to illustrate the sgRNA screening process.
Designing a primer: respectively designing primer pairs for amplifying TGEV-N genes aiming at a nucleotide sequence (ID: GQ 374566.1) of a conserved gene N of the porcine transmissible gastroenteritis virus (TEGV) (Table 5); and 6 specific sgRNA sequences were designed according to the conserved region of the PCR amplified nucleic acid, PAM was TTTV, and the sgRNA empty vector was used as a template to design in vitro transcription primer pairs (Table 5).
Table 5 sgRNA sequence information of targeted TGEV N genes
Figure BDA0003156880600000081
Plasmid construction: TGEV-N plasmid obtained by constructing the N gene fragment into pUC57 plasmid is synthesized by Scout Biotech, and the plasmid map and the gene sequence information of the inserted pUC57 universal plasmid are shown in Table 6.
In vitro transcription of sgRNA: (1) and (3) PCR amplification: the template for the in vitro transcription of sgrnas was PCR amplified using T7-crRNA-F and different sgRNA-R primers (table 5) with the C12 plasmid containing T7 promoter and sgRNA scaffold as template. PCR amplification was performed with T7-crRNA-F and different sgRNA-R primers. Experimental bodyThe resulting mixture contained 10. Mu.L of Extaq Mix, 5ng of C12 plasmid, and 10pmoL of each of the upstream and downstream primers, to 20. Mu.L. The amplification procedure was: 94 ℃,3min; [94 ℃,30s; at 58 ℃ for 30s;72 ℃ for 12s]Circulating for 30 times; 72 ℃ for 5min; at 16 ℃ foviver. 3 μ L of PCR amplification product was identified by electrophoresis on 2% agarose gel to obtain a single and correct band for purification and recovery (TD 413-50, tianmo). (2) In vitro transcription: the PCR product recovered in (2) was based on HiScribe TM The Quick T7 High Yield RNASynthesis Kit (NEB) synthesizes sgRNAs, and the reaction conditions are as follows: standing at 37 deg.C for 4-16h. (3) And (3) purifying the sgRNA, namely irradiating an operation table by ultraviolet for at least twenty minutes before purification, precooling the sgRNA to 4 ℃ by a centrifugal machine, and placing the used reagent in a refrigerator for precooling. And purifying the transcribed sgRNA by a phenol chloroform method, and storing the sgRNA in a refrigerator at the temperature of-80 ℃ for a long time after purification.
Visual detection of Cas12a system: the high-activity sgRNA is screened by using CRISPR/Cas12a technology, and in a 20 mu L reaction system, purified sgRNA (1000 nM), cas12a (500 nM), ssDNA-FQ reporter (ROX-N12-BHQ 2) (12.5 mu M) and 2 mu L NEB buffer 2.1 are added. The negative control is set without adding the detection target gene template, namely the template is water. The positive control is set to be only added with ssDNA activator, namely the corresponding sgRNA reverse primer; the experimental group consisted of about 500ng of plasmid DNA, reaction at 37 ℃ for 20min, and then termination at 98 ℃ for 2min.
TABLE 6 TGEV N Gene sequences constructed into the PUC57 plasmid
Figure BDA0003156880600000091
The experimental results are as follows: the results are shown in FIG. 4. It can be seen that several sgRNA activities other than TGEV-sgRNA-2 were significantly different in single and double strand; in the naked eye condition, when a single strand is used as a template in four groups of the sgrnas 1 and 3 to 5, no difference exists between the single strand and a template-free control, while the effect of using a plasmid as the template in the sgRNA6 group is poor. Therefore, sgRNA2 activity was optimal.
Example 5 screening and preparation of primers for TGEV-N Virus
The performance of the detection primer determines the quality of the detection effect of the kit. According to the invention, multiple groups of primer combinations are screened for the detection index TGEV-N, and finally, an optimal primer combination is screened. This example is intended to illustrate the screening process of TGEV-N viral primers.
Designing a primer: 5 groups of LAMP primer pairs targeting TGEV-N genes are designed according to the screened TGEV-N-sgRNA2 sequences, and the primer sequences are shown in Table 7.
Plasmid construction: the plasmid PEDV-ORF3 obtained by constructing the N gene fragment into the pUC57 plasmid was synthesized by Scout Biotech, and the plasmid map and the gene sequence information of the pUC 57-ligated plasmid are shown in Table 6.
Visual detection of LAMP combined with Cas12a system: (1) LAMP amplification: 5 sets of primers were designed for sgRNA2 and amplified using TGEV-N plasmid (10 ng) and DEPC water (1. Mu.L, NC) as templates, respectively. The 25. Mu.L system contained 2.5. Mu.L of amplification buffer, dNTPs (final concentration 1.4 mM), mgSO4 (final concentration 8 mM), primer Mix 2.5. Mu.L (1.6. Mu.M FIP/BIP, 0.2. Mu.M F3/B3, 0.4. Mu.M LoopF/B), 0.1uu Bst3.0. Reacting at 65 deg.C for 20min, and inactivating at 85 deg.C for 5min. (2) Cas12a system visual detection reaction: the 20. Mu.L CRISPR/Cas12a system contained purified sgRNA (1000 nM), cas12a (500 nM), ssDNA-FQ reporter (ROX-N12-BHQ 2) (12.5. Mu.M), 2. Mu.L NEB buffer 2.1 and 3. Mu.L of the amplification product. The reaction was stopped at 37 ℃ for 15min and then at 98 ℃ for 2min.
The experimental results are as follows: see FIG. 5 for results. As can be seen, the LAMP1/2/3/4 group showed good visualization effect, but the no-template control in the LAMP2/3/4 group also showed positive results (FIG. 5B). The gel electrophoresis image shows that no pollution is produced in the amplification process (FIG. 5A), and after sequence alignment, the Loop primer (LB) sequence in the LAMP2/3/4 group primer can exactly have a reverse complementary relation with the TGEV-sgRNA2 sequence (containing PAM end) (FIG. 5C). Therefore, the LAMP-1 primer had the best amplification effect on TGEV N gene.
TABLE 7 targeting TGEV N Gene LAMP primer sequences
Figure BDA0003156880600000101
Example 6 kit sensitivity assessment for TGEV-N Virus
To verify the sensitivity of the kit for visual detection of viral TGEV-N based on LAMP amplification in combination with CRISPR/Cas12a, TGEV-N viral plasmid was diluted to 0.1 copies/. Mu.L (i.e., 1X 10^ (-1)) in a ten-fold gradient. And (3) amplifying the gradient diluted plasmid template by using the LAMP primer aiming at the TGEV-N virus, and then detecting the kit.
Template dilution: the method is applied with (copy number (copy/. Mu.L) = (DNA amount (ng) × 6.022 × 1) 023 ) /(fragment size (bp) × 1 × 10) 9 X 660) (https:// toptipbio.com/dna-copy-number-qpcr /) this equation calculates the copy number of the TGEV-N plasmid, which is then diluted in a tenfold gradient to 1X 10 -1 Copies/. Mu.L.
Visual detection of LAMP combined Cas12a system: (1) LAMP amplification: the diluted plasmids were subjected to LAMP amplification using TGEV-LAMP-1 primers, respectively, and the 25. Mu.L system contained 2.5. Mu.L of amplification buffer, dNTPs (final concentration 1.4 mM), mgSO4 (final concentration 8 mM), primer Mix 2.5. Mu.L (1.6. Mu.M FIP/BIP, 0.2. Mu.M F3/B3, 0.4. Mu.M LoopF/B), and 0.16U Bst3.0. The reaction program is 65 ℃ and 20min;85 ℃ for 5min. (2) Visualization detection of Cas12a system: the 20. Mu.L CRISPR/Cas12a system contained purified sgRNA (1000 nM), cas12a (500 nM), ssDNA-FQ reporter (ROX-N12-BHQ 2) (12.5. Mu.M), 2. Mu.L NEB buffer 2.1 and 3. Mu.L of the amplification product. The reaction was stopped at 37 ℃ for 10min and then at 98 ℃ for 2min. And (3) directly placing the centrifuge tube containing the reaction solution in a blue light gel cutting instrument and an ultraviolet gel imaging instrument for observing fluorescence intensity or directly observing with naked eyes, simultaneously measuring a fluorescence value by using an enzyme label, uniformly mixing 20 mu L of Cas12a reaction solution and 80 mu L of DEPC water, adding the mixture into a 96-hole cell plate for enzyme label measurement (n = 3), setting fluorescence detection exciting light to be 576nm, and emitting light to be 601nm.
The experimental results are as follows: see FIG. 6 for results. Wherein, 1: 3X 10 4 Copy/. Mu.L; 2:3 x 10 3 Copy/. Mu.L; 3:3 x 10 2 Copy/. Mu.L; 4: 3X 10 1 Copy/. Mu.L; 5: 3X 10 0 Copy/. Mu.L; 6: 1X 10 0 Copy/. Mu.L; 7: 1X 10 -1 Copy/. Mu.L; 8: no Template Control (NTC). As can be seen from fig. 6, the sensitivity of visual detection of TGEV by LAMP in combination with CRISPR-Cas12a is 1 copy/μ L (fig. 6A and 6B), and the fluorescence value is very high (fig. 6B).
Example 7 selection and preparation of sgRNA against PDCOV-N Virus
The performance of the sgRNA determines the quality of the detection effect of the kit. According to the invention, multiple groups of sgRNAs are screened aiming at a detection index PDCOV-N, and finally, an optimal group of sgRNAs is screened. This example is intended to illustrate the sgRNA screening process.
Designing a primer: respectively designing primer pairs for amplifying a porcine delta coronavirus (PDCoV) conserved gene N nucleic acid sequence (ID: KY 586149.1); and 12 specific sgRNA sequences were designed based on the conserved region of the PCR amplified nucleic acid, PAM was TTTV, and the sgRNA empty vector was used as a template to design primer pairs for in vitro transcription (table 8).
Plasmid construction: the plasmid PDCoV-N obtained by constructing the N gene fragment into the plasmid pUC57 is synthesized in Scout biosciences, and the plasmid map and the gene sequence information of the universal plasmid inoculated into the pUC57 are shown in Table 9.
In vitro transcription of sgRNA: (1) and (3) PCR amplification: the template for in vitro sgRNA transfection was PCR amplified using the C12 plasmid containing T7 promoter and sgRNA scaffold as template, with T7-crRNA-F and different sgRNA-R primers (Table 8). PCR amplification was performed with T7-crRNA-F and different sgRNA-R primers. The experimental system was 20. Mu.L containing 10. Mu.L of Extaq Mix, 5ng of C12 plasmid, and 10pmoL of each of the upstream and downstream primers. The amplification procedure was: 94 ℃ for 3min; [94 ℃,30s; at 58 ℃ for 30s;72 ℃ for 12s]Circulating for 30 times; 72 ℃ for 5min; at 16 ℃ foviver. 3 μ L of PCR amplification product was identified by electrophoresis on 2% agarose gel to obtain a single and correct band for purification and recovery (TD 413-50, tianmo). (2) In vitro transcription: the PCR product recovered in (2) was based on HiScribe TM
TABLE 9 PDCoV N Gene sequences constructed into the PUC57 plasmid
Figure BDA0003156880600000111
The Quick T7 High Yield RNASynthesis Kit (NEB) synthesizes sgRNAs, and the reaction conditions are as follows: standing at 37 deg.C for 4-16h. (3) And (3) purifying the sgRNA, namely irradiating an operation table by ultraviolet for at least twenty minutes before purification, precooling the sgRNA to 4 ℃ by a centrifugal machine, and placing the used reagent in a refrigerator for precooling. The transcribed sgRNA was purified by phenol chloroform and stored in a refrigerator at-80 ℃ for a long period after purification.
Visualization detection of Cas12a system: high-activity sgRNA was screened using CRISPR/Cas12a technique purified sgRNA (1000 nM), cas12a (500 nM), ssDNA-FQ reporter (ROX-N12-BHQ 2) (12.5. Mu.M), 2. Mu.L NEB buffer 2.1 were added to a 20. Mu.L reaction. The negative control is set without adding the detection target gene template, namely the template is water. The positive control is set to be added with ssDNA activator, namely the corresponding sgRNA reverse primer; the experimental group consisted of about 500ng of plasmid DNA, reaction at 37 ℃ for 20min, and then termination at 98 ℃ for 2min.
The experimental results are as follows: the results are shown in FIG. 7. As can be seen from fig. 7A, the sgrnas 4 and 9 have a significant naked-eye effect. Further comparison of sgRNA4 and sgRNA9 gave the results shown in fig. 7B. As can be seen, the single-stranded activity of sgRNA9 was significantly higher than that of sgRNA4. Therefore, sgRNA9 was most active on single strands.
Example 8 screening and preparation of primers for PDCOV-N Virus
The performance of the detection primer determines the quality of the detection effect of the kit. The invention screens a plurality of groups of primer combinations aiming at the detection index PDCOV-N, and finally screens a group of optimal primer combinations. This example is intended to illustrate the screening process of PDCOV-N viral primers.
Designing a primer: two groups of primer pairs targeting LAMP of PDCOV-N are designed according to the screened PDCoV-N-sgRNA9 sequences, and the primer sequences are shown in a table 10.
Table 8 construction of primer sequences targeting sgRNA of PDCoV N gene
Figure BDA0003156880600000121
Plasmid construction: the plasmid PDCoV-N obtained by constructing the N gene fragment into the plasmid pUC57 is synthesized in Scout biosciences, and the plasmid map and the gene sequence information of the universal plasmid inoculated into the pUC57 are shown in Table 9.
(2) Visual detection of LAMP combined with Cas12a system: (1) LAMP amplification: 2 sets of primers were designed for sgRNA9 and amplified using PDCoV-N plasmid (10 ng) and DEPC water (1 μ L, NC) as templates, respectively. The 25. Mu.L system contained 2.5. Mu.L of amplification buffer, dNTPs (final concentration 1.4 mM), mgSO4 (final concentration 8 mM), primer Mix 2.5. Mu.L (1.6. Mu.M FIP/BIP, 0.2. Mu.M F3/B3, 0.4. Mu.M LoopF/B), 0.1uu Bst3.0. Reacting at 65 deg.C for 20min, and inactivating at 85 deg.C for 5min. (2) Cas12a system visual detection reaction: the 20. Mu.L CRISPR/Cas12a system contained purified sgRNA (1000 nM), cas12a (500 nM), ssDNA-FQ reporter (ROX-N12-BHQ 2) (12.5. Mu.M), 2. Mu.L NEB buffer 2.1 and 3. Mu.L of the amplification product. The reaction was stopped at 37 ℃ for 15min and then at 98 ℃ for 2min.
The experimental results are as follows: please refer to fig. 8. As can be seen, from FIG. 8B, there is no significant difference in amplification efficiency between the two sets of primers, but in FIG. 8A, the effect of PDCoV-N-LAMP-1 is slightly better than that of PDCoV-N-LAMP-2. Therefore, the PDCoV-N-LAMP-1 has better effect.
TABLE 10 LAMP primer sequences of target PDCoV N genes
Figure BDA0003156880600000131
Example 9 kit sensitivity assessment for PDCOV-N Virus
In order to verify the sensitivity of the kit for visually detecting virus PDCOV-N based on LAMP amplification combined with CRISPR/Cas12a, the PDCOV-N virus plasmid is subjected to tenfold gradient dilution to 0.1 copy/mu L (namely 1 multiplied by 10^ (-1)). And (3) amplifying the gradient diluted plasmid template by using the LAMP primer aiming at the PDCOV-N virus, and then detecting the kit.
Template dilution: application of (copy number (copy/. Mu.L) = (amount of DNA (ng) × 6.022 × 10) 23 ) /(fragment size (bp) × 1 × 10) 9 X 660) (https:// toptipbio.com/dna-copy-number-qpcr /) this equation calculates the copy number of the PDCoV-N plasmid, which is then diluted ten-fold gradiently to 1 x 10 -1 Copy/. Mu.L.
Visual detection of LAMP combined with Cas12a system: (1) LAMP amplification: the diluted plasmids were LAMP amplified using PDCoV-LAMP-1 primers, respectively, and the 25. Mu.L system contained 2.5. Mu.L of amplification buffer, dNTPs (final concentration 1.4 mM), mgSO4 (final concentration 8 mM), primer Mix 2.5. Mu.L (1.6. Mu.M FIP/BIP, 0.2. Mu.M F3/B3, 0.4. Mu.M loopF/B), 0.16U Bst3.0. The reaction program is 65 ℃ and 20min;85 ℃ for 5min. (2) Visualization detection of Cas12a system: the 20. Mu.L CRISPR/Cas12a system contained purified sgRNA (1000 nM), cas12a (500 nM), ssDNA-FQ reporter (ROX-N12-BHQ 2) (12.5. Mu.M), 2. Mu.L NEB buffer 2.1 and 3. Mu.L of the amplification product. The reaction was stopped at 37 ℃ for 10min and then at 98 ℃ for 2min. And (3) directly placing the centrifuge tube containing the reaction solution in a blue light gel cutting instrument and an ultraviolet gel imaging instrument for observing fluorescence intensity or directly observing with naked eyes, simultaneously measuring the fluorescence value by using an enzyme label, uniformly mixing 20 mu LCas12a reaction solution and 80 mu L DEPC water, adding the mixture into a 96-hole cell plate for enzyme label measurement (n = 3), setting the excitation light for fluorescence detection at 576nm, and emitting light at 601nm.
The experimental results are as follows: the results are shown in FIG. 9. Wherein, 1: 4X 10 4 Copy/. Mu.L; 2: 4X 10 3 Copy/. Mu.L; 3: 4X 10 2 Copy/. Mu.L; 4: 4X 10 1 Copy/. Mu.L; 5: 4X 10 0 Copy/. Mu.L; 6: 1X 10 0 Copy/. Mu.L; 7: 1X 10 -1 Copy/. Mu.L; 8: no Template Control (NTC). As can be seen, after the LAMP amplification product is visually detected by Cas12a, the detection limit can reach 1 copy/. Mu.L (FIG. 9A). Fluorescent signals are collected by a microplate reader (figure 9B), which further indicates that the PDCoV-N kit for visually detecting the PDCoV-N based on the combination of loop-mediated isothermal amplification and CRISPR-Cas12a system has high sensitivity and can meet the requirement of early detection of viruses.
Example 10 screening and preparation of sgRNA against SADS-COV-N Virus
The performance of the sgRNA determines the quality of the detection effect of the kit. In this embodiment, multiple groups of sgrnas are screened for the detection index SADS-COV-N, and finally, an optimal group of sgrnas is screened.
Designing a primer: aiming at the conserved gene N nucleic acid sequence (ID: MG 775253.1) of the acute diarrhea syndrome coronavirus (SADS-CoV), primer pairs for amplifying the SADS-COV-N gene are respectively designed (Table 11); and according to the conserved region of the PCR amplified nucleic acid, 14 specific sgRNA sequences are designed, PAM is TTTV, and an in vitro transcription primer pair is designed by taking an sgRNA empty vector as a template (Table 11).
Plasmid construction: the SADS-CoV-N plasmid obtained by constructing the N gene fragment into the pUC57 plasmid was synthesized by Scout Biotech, and the plasmid map and the gene sequence information of the pUC57 universal plasmid are shown in Table 12.
TABLE 11 construction of sgRNA primer sequences targeting the SADS-CoV N Gene
Figure BDA0003156880600000141
In vitro transcription of sgRNA: (1) and (3) PCR amplification: the template for in vitro sgRNA transfection was PCR amplified using the C12 plasmid containing T7 promoter and sgRNA scaffold as template, with T7-crRNA-F and different sgRNA-R primers (Table 10). PCR amplification was performed with T7-crRNA-F and different sgRNA-R primers. The experimental system was 20. Mu.L containing 10. Mu.L of Extaq Mix, 5ng of C12 plasmid, and 10pmoL of each of the upstream and downstream primers. The amplification procedure was: 94 ℃ for 3min; [94 ℃,30s; at 58 ℃ for 30s;72 ℃ for 12s]Circulating for 30 times; 72 ℃ for 5min; at 16 ℃ foviver. 3 μ L of PCR amplification product was identified by electrophoresis on 2% agarose gel to obtain a single and correct band for purification and recovery (TD 413-50, tianmo). (2) In vitro transcription: the PCR product recovered in (2) was based on HiScribe TM The Quick T7 High Yield RNASynthesis Kit (NEB) synthesizes sgRNAs, and the reaction conditions are as follows: standing at 37 deg.C for 4-16h. (3) And (3) purifying the sgRNA, namely irradiating an operation table by ultraviolet for at least twenty minutes before purification, precooling the sgRNA to 4 ℃ by a centrifugal machine, and placing the used reagent in a refrigerator for precooling. The transcribed sgRNA was purified by phenol chloroform and stored in a refrigerator at-80 ℃ for a long period after purification.
Visualization detection of Cas12a system: the high-activity sgRNA is screened by using CRISPR/Cas12a technology, and in a 20 mu L reaction system, purified sgRNA (1000 nM), cas12a (500 nM), ssDNA-FQ reporter (ROX-N12-BHQ 2) (12.5 mu M) and 2 mu L NEB buffer 2.1 are added. The negative control is set as no detection target gene template, i.e. the template is water. The positive control is set to be only added with ssDNA activator, namely the corresponding sgRNA reverse primer; the experimental group consisted of about 500ng of plasmid DNA, reaction at 37 ℃ for 20min, and then termination at 98 ℃ for 2min.
TABLE 12 SADS-CoV N Gene sequences constructed into PUC57 plasmid
Figure BDA0003156880600000151
The experimental results are as follows: please refer to fig. 10. Wherein, 1 to 15 are sgRNA1 to sgRNA15 respectively. It can be seen that sgRNA3 and sgRNA15 are completely inactive, whereas sgRNA1, sgRNA8, sgRNA12 and sgRNA14 are significantly less active than the other sgrnas (fig. 10A). Further screening of sgRNA2, sgRNA6, sgRNA10, sgRNA11, and sgRNA13 revealed that the activities of sgRNA2 and sgRNA10 were significantly lower when targeting single strand than other sgrnas (fig. 10B). When a double-stranded template is targeted (i.e., a plasmid), there is no significant difference between sgRNA6, sgRNA11, and sgRNA13, but the naked eye effect of sgRNA6 is the best when the single-stranded template is targeted (fig. 10B), and thus the effect of SADS-CoV-sgRNA6 is the best.
Example 11 screening and preparation of primers for SADS-COV-N Virus
The performance of the detection primer determines the quality of the detection effect of the kit. In this example, multiple primer combinations were screened for the detection index SADS-COV-N, and finally an optimal primer combination was screened.
Designing a primer: 3 sets of primer pairs targeting SADS-COV-N were designed according to the selected SADS-CoV-sgRNA6 sequence, and the primer sequences are shown in Table 13.
Plasmid construction: SADS-CoV-N plasmid obtained by constructing N gene fragment into pUC57 plasmid was synthesized by Oncorks Biotech, inc., which
TABLE 13 LAMP primers targeting SADS-CoV-N Gene
Figure BDA0003156880600000161
The plasmid map and the information on the gene sequence of the pUC 57-ligated universal plasmid are shown in Table 12.
Visual detection of LAMP combined with Cas12a system: (1) LAMP amplification: 3 sets of primers were designed for sgRNA6 and amplified using PDCoV-N plasmid (10 ng) and DEPC water (1. Mu.L, NC) as templates, respectively. The 25. Mu.L system contained 2.5. Mu.L of amplification buffer, dNTPs (final concentration 1.4 mM), mgSO4 (final concentration 8 mM), primer Mix (1.6. Mu.M FIP/BIP, 0.2. Mu.M F3/B3, 0.4. Mu.M oopF/B), 0.1697U Bst3.0. Reacting at 65 deg.C for 20min, and inactivating at 85 deg.C for 5min. (2) Cas12a system visual detection reaction: the 20 μ LCRISPR/Cas12a system contained purified sgRNA (1000 nM), cas12a (500 nM), ssDNA-FQ reporter (ROX-N12-BHQ 2) (12.5 μ M), 2 μ L NEB buffer 2.1 and 3 μ L of amplification product. The reaction was stopped at 37 ℃ for 15min and then at 98 ℃ for 2min.
The experimental results are as follows: the amplification products were detected, and the results are shown in FIG. 11. As can be seen, the LAMP-2 primer set is suspected to be contaminated (FIG. 11B), but the result of agarose gel electrophoresis combined with the LAMP-2 primer set suggests that the amplification process is free from contamination (FIG. 11A). After aligning the sequences (fig. 11C), the LAMP2 primer sequences were found to have no overlap with the PAM-terminal sequence of the SADS-CoV-sgRNA6, but another PAM sequence (TTTG) was included in the SADS-CoV-sgRNA6 sequence and to have an inverse complementary relationship with B1C in the SAD-N-LAMP2-BIP sequence. Compared with the LAMP-3 primer, the agarose gel electrophoresis effect of the LAMP-1 primer is slightly better than that of the LAMP-3 primer. Therefore, LAMP-1 is the best primer for SADS-CoV.
Example 12 kit sensitivity assessment for SADS-COV-N Virus
To verify the sensitivity of the kit for visual detection of the virus SADS-COV-N based on LAMP amplification combined with CRISPR/Cas12a, the SADS-COV-N virus plasmid was subjected to tenfold gradient dilution to 0.1 copy/. Mu.L (i.e., 1 × 10^ (-1)). And amplifying the gradient diluted plasmid template by using the LAMP primer aiming at the SADS-COV-N virus, and detecting the kit.
Template dilution: application of (copy number (copy/. Mu.L) = (amount of DNA (ng) × 6.022 × 10) 23 ) /(fragment size (bp) × 1 × 10) 9 X 660) calculating the copy number of the SADS-CoV-N plasmid, and then performing ten-fold gradient dilution to 1X 10 -1 Copies/. Mu.L.
Visual detection of LAMP combined with Cas12a system: (1) LAMP amplification: the diluted plasmids were LAMP amplified using SADS-CoV-LAMP-1 primers, respectively, in a 25. Mu.L system containing 2.5. Mu.L of amplification buffer, dNTPs (final concentration 1.4 mM), mgSO4 (final concentration 8 mM), primer Mix 2.5. Mu.L (1.6. Mu.M FIP/BIP, 0.2. Mu.M F3/B3, 0.4. Mu.M loopF/B), 0.169 Bst3.0. The reaction program is 65 ℃ and 20min;85 ℃ for 5min. (2) Visualization detection of Cas12a system: the 20. Mu.L CRISPR/Cas12a system contained purified sgRNA (1000 nM), cas12a (500 nM), ssDNA-FQ reporter (ROX-N12-BHQ 2) (12.5. Mu.M), 2. Mu.L NEB buffer 2.1 and 3. Mu.L of the amplification product. The reaction was stopped at 37 ℃ for 10min and then at 98 ℃ for 2min. Directly placing a centrifuge tube containing reaction liquid in a blue light gel cutting instrument and an ultraviolet gel imager to observe fluorescence intensity or directly observing with naked eyes, simultaneously utilizing enzyme labeling to measure fluorescence value, uniformly mixing 20 mu L of Cas12a reaction liquid and 80 mu L of DEPC water, adding a 96-hole cell plate to carry out enzyme labeling measurement (n = 3), setting fluorescence detection exciting light to be 576nm, and emitting light to be 601nm.
The experimental results are as follows:
the results of the experiment are shown in FIG. 12. Wherein, 1: 9X 10 4 Copy/. Mu.L; 2: 9X 10 3 Copy/. Mu.L; 3: 9X 10 2 Copy/. Mu.L; 4: 9X 10 1 Copy/. Mu.L; 5: 9X 10 0 Copy/. Mu.L; 6:1 x 10 0 Copy/. Mu.L; 7:1 x 10 -1 Copy/. Mu.L; 8: no Template Control (NTC). After the LAMP amplification product is visually detected by Cas12A, the detection limit can reach 1 copy/mu L (FIG. 12A), and the result of fluorescence signal collection by a microplate reader also shows that the detection limit reaches 1 copy/mu L (FIG. 12B).
Example 13 evaluation of specificity of kits for four viruses PEDV-ORF3, TGEV-N, PDCoV-N and SADS-CoV-N
The specificity of the LAMP primer and the specificity of the sgRNA directly determine the detection accuracy of the kit. In addition to the convenience and sensitivity of the detection, the most important criteria for the evaluation of the kit is the accuracy of the detection, which is determined by the specificity. This example is directed to the detection of kits.
(1) Primer specificity: about 10ng of PEDV-ORF3 plasmid, TGEV-N plasmid, PDCoV-N plasmid, SADS-CoV plasmid and negative control using water as a template were amplified using LAMP primers corresponding to PEDV, TGEV, PDCoV and SADS-CoV, respectively. The reaction conditions are 65 ℃ and 20min;85 ℃ and 5min. The amplification products were detected by electrophoresis on a 1.5% agarose gel.
The experimental results are as follows: FIG. 13 shows the specific amplification results shown in FIG. 13A. Wherein, 1: the PEDV-ORF3 plasmid; 2: TGEV-N plasmid; 3: a PDCoV-N plasmid; 4: SADS-CoV-N plasmid; 5: no template control. PEDV-ORF3-LAMP2 primers are used for respectively amplifying PEDV-ORF3 plasmids, TGEV-N plasmids, PDCoV-N plasmids, SADS-CoV plasmids and water, and amplification products only contain PEDV-ORF3 plasmid bands through detection, so that the PEDV-LAMP2 primers have good specificity. The TGEV-N-LAMP1 primer is used for amplifying PEDV-ORF3 plasmid, TGEV-N plasmid, PDCoV-N plasmid, SADS-CoV plasmid and water respectively, and an amplification product only contains a TGEV-N plasmid strip through detection, so that the TGEV-N-LAMP1 primer has good specificity. The PDCoV-N-LAMP1 primer is used for respectively amplifying PEDV-ORF3 plasmid, TGEV-N plasmid, PDCoV-N plasmid, SADS-CoV plasmid and water, and the amplification product only contains PDCoV-N plasmid strip through detection, which indicates that the PDCoV-N-LAMP1 primer has good specificity. The SADS-CoV-N-LAMP3 primer is used for respectively amplifying PEDV-ORF3 plasmid, TGEV-N plasmid, PDCoV-N plasmid, SADS-CoV plasmid and water, and the amplification product only contains SADS-CoV plasmid band by detection, which shows that the SADS-CoV-N-LAMP3 primer has good specificity.
(2) specificity of sgRNA: PEDV-sgRNA5, TGEV-sgRNA2, PDCoV-sgRNA9 and SADS-CoV-sgRNA6 are respectively applied to a Cas12a system to detect the amplified product of the amplified band in the step (1) and a corresponding template-free control, and the reaction conditions are (37 ℃ and 10 min). 1,5 for the PEDV group in fig. 13A; 2,5 of TGEV group; eight samples, namely 3,5 of the PDCoV group and 4,5 of the SADS-CoV group, are selected, and then the SgRNA of the four viruses is used for visually identifying Cas12 a.
(3) As a result: the results are shown in FIG. 13B. Wherein, 1: PEDV LAMP amplification product; 2: TGEV LAMP amplification product; 3: PDCoV LAMP amplification products; 4: SADS-CoV LAMP amplification products; 5: template-free LAMP products of sgRNA corresponding viruses; it can be seen that only the amplification products corresponding to sgrnas gave a positive result. Thus, all sgrnas in the kit are specific. The virus detection method established in the experiment has the advantages of dual specificity of LAMP primers and sgRNA specificity, and has no cross reactivity, so that the method has the characteristic of high accuracy in virus detection.
Example 14 method for visual detection of four porcine coronaviruses based on multiple LAMP combined CRISPR-Cas12a
A plurality of viruses are amplified simultaneously, and different viruses are distinguished by the advantage that sgRNA in a CRISPR/Cas12a system can specifically recognize target genes. The specific embodiment is as follows:
(1) Template dilution: PEDV-ORF3 plasmid, TGEV-N plasmid, PDCoV-N plasmid and SADS-CoV-N plasmid were each diluted to 10 ng/. Mu.L with DEPC water.
(2) Visualization detection of the multiplex loop-mediated isothermal amplification combined with CRISPR-Cas12a system: (1) multiplex LAMP amplification: quadruple amplification is realized by adjusting the addition amount of primers of four viruses in a reaction system; the additive amount of Primer MIX is 1 μ L (PEDV), 0.6 μ L (TGEV) and 1.5 μ L (PDCoV and SADS-CoV) times respectively, each viral plasmid is 10ng, and the amplification condition is 65 ℃ and 20min;85 ℃ for 5min. (2) Visualization detection of Cas12a system: the 20. Mu.L CRISPR/Cas12a system contained purified sgRNA (1000 nM), cas12a (500 nM), ssDNA-FQ reporter (ROX-N12-BHQ 2) (12.5. Mu.M), 2. Mu.L NEB buffer 2.1 and 3. Mu.L of the amplification product. The reaction condition is 37 ℃ and 20min; at 98 deg.C for 2min.
(3) As a result: the results are shown in FIG. 14. Of these, 2 is a negative control. Therefore, the visualized detection method of the multiple loop-mediated isothermal amplification combined CRISPR-Cas12a system disclosed by the embodiment of the disclosure has better accuracy of the detection result after the addition amount of each virus primer is adjusted.
Therefore, the visualized detection method combining the multiple loop-mediated isothermal amplification with the CRISPR-Cas12a system is established, the method firstly amplifies nucleic acid by using LAMP technology, and then visually detects LAMP amplification products by using the Cas12a system, and the method has good accuracy and specificity.
The disclosed embodiments are intended to embrace all such alternatives, modifications and variances which fall within the broad scope of the appended claims.
Sequence listing
<110> Sichuan university of agriculture
<120> primer set for detecting porcine enterocoronavirus and application thereof
<130> FI210586
<160> 136
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gcactgttta aagcgtctt 19
<210> 2
<211> 21
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gaggaaagaa agtgtcgtag t 21
<210> 3
<211> 46
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gcaccacaat aatataaaag tgggcggcgc aattatatta tgttgg 46
<210> 4
<211> 41
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ttgttgcaca cttattggca gaagagcgca tttttatagc g 41
<210> 5
<211> 22
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
aagaacaatg acagcaaaac gc 22
<210> 6
<211> 24
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tgtttagtct gcttttactc ctgg 24
<210> 7
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atagcgccag gagtaaaagc agacatctac aacagtagaa at 42
<210> 8
<211> 22
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ggcaacaatc caataacaag aa 22
<210> 9
<211> 19
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
tgaactgctt ctagctcca 19
<210> 10
<211> 43
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gttgctgttg tttttctgtg tcagatgaca gtgtagaaca agc 43
<210> 11
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
actctaaaac aagagatact acgcccacaa cctttacctg ca 42
<210> 12
<211> 24
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
acctaacttt ttaagtgcgg caag 24
<210> 13
<211> 25
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gaatgaaaac aaacacacct ggaag 25
<210> 14
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
agaacaagct gttcttgccg cactatctac aacagtagaa at 42
<210> 15
<211> 19
<212>DNA/RNA
<213> Artificial Sequence-unrelated dsRNA (Artificial Sequence)
<400> 15
gcaatcagcc caggaaacg 19
<210> 16
<211> 18
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
aacaccaggc acatgtcc 18
<210> 17
<211> 39
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
tcacccttgg gtaaagtccg ccagctgcgg tacgtcgta 39
<210> 18
<211> 40
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
atgtcggctc tgcagacact ggctagagcc atgatgcgag 40
<210> 19
<211> 17
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
tgggagcttg atgctgg 17
<210> 20
<211> 18
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
agacgggtat ggctgatc 18
<210> 21
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
tggcaccagt acgagaccgg ttgcatctac aacagtagaa at 42
<210> 22
<211> 20
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
acaagctttg gcagacttgg 20
<210> 23
<211> 20
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
agaaactgag cgaggaccaa 20
<210> 24
<211> 41
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
tgcaggtgag acagctctgc ttagcttcta gccagtccag g 41
<210> 25
<211> 39
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
tgcccctaaa ccggctcgta acgcacgtcc tcctcagaa 39
<210> 26
<211> 17
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
tcttggtttg ggtgtat 17
<210> 27
<211> 20
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
caaacctgaa tggaagcgtg 20
<210> 28
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
tagccagtcc aggcctcaaa gtggatctac aacagtagaa at 42
<210> 29
<211> 20
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
gaggaaagaa agtgtcgtag 20
<210> 30
<211> 18
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
gacgggtttt cttttcac 18
<210> 31
<211> 20
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
tcgctattac gccagctggc 20
<210> 32
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
agtctgcttt tactcctggc gctaatctac aacagtagaa at 42
<210> 33
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
tctgctttta ctcctggcgc tataatctac aacagtagaa at 42
<210> 34
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
gcgcattttt atagcgccag gagtatctac aacagtagaa at 42
<210> 35
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
agcgcatttt tatagcgcca ggagatctac aacagtagaa at 42
<210> 36
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
atagcgccag gagtaaaagc agacatctac aacagtagaa at 42
<210> 37
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
caaagcctgc caataagtgt gcaaatctac aacagtagaa at 42
<210> 38
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
cgccaggagt aaaagcagac taaaatctac aacagtagaa at 42
<210> 39
<211> 12
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
gtatccagtg cg 12
<210> 40
<211> 320
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence) -PEDV-ORF 3 Sequence constructed into pUC57 plasmid
<400> 40
agacaagctt caaatgtgac gggttttctt ttcaccagtg tttttatcta cttctttgca 60
ctgtttaaag cgtcttcttt gaggcgcaat tatattatgt tggcagcgcg ttttgctgtc 120
attgttcttt attgcccact tttatattat tgtggtgcat ttttagatgc aactattatt 180
tgttgcacac ttattggcag gctttgttta gtctgctttt actcctggcg ctataaaaat 240
gcgctcttta ttatctttaa tactacgaca ctttctttcc tcaatggtaa agcagcttat 300
tatgacggca aatccattgt 320
<210> 41
<211> 111
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence) -C12
<400> 41
ggatccctaa tacgactcac tatagggaat ttctactgtt gtagattgag accgagagag 60
ggtctcattt tttaaagggc ccgtcgactg cagaggcctg catgcaagct t 111
<210> 42
<211> 19
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
gcactgttta aagcgtctt 19
<210> 43
<211> 20
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
aggaaagaaa gtgtcgtagt 20
<210> 44
<211> 46
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
gcaccacaat aatataaaag tgggcggcgc aattatatta tgttgg 46
<210> 45
<211> 41
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
ttgttgcaca cttattggca gaagagcgca tttttatagc g 41
<210> 46
<211> 22
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
aagaacaatg acagcaaaac gc 22
<210> 47
<211> 24
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
tgtttagtct gcttttactc ctgg 24
<210> 48
<211> 21
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
gtttaaagcg tcttctttga g 21
<210> 49
<211> 20
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
aggaaagaaa gtgtcgtagt 20
<210> 50
<211> 46
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
gcaccacaat aatataaaag tgggcgcgca attatattat gttggc 46
<210> 51
<211> 40
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
ttgttgcaca cttattggca gagagcgcat ttttatagcg 40
<210> 52
<211> 22
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
aagaacaatg acagcaaaac gc 22
<210> 53
<211> 24
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
tgtttagtct gcttttactc ctgg 24
<210> 54
<211> 21
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
gtttaaagcg tcttctttga g 21
<210> 55
<211> 21
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
gaggaaagaa agtgtcgtag t 21
<210> 56
<211> 46
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
gcaccacaat aatataaaag tgggcgcgca attatattat gttggc 46
<210> 57
<211> 41
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
ttgttgcaca cttattggca gaagagcgca tttttatagc g 41
<210> 58
<211> 22
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
aagaacaatg acagcaaaac gc 22
<210> 59
<211> 24
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
tgtttagtct gcttttactc ctgg 24
<210> 60
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
gctctcaatc tagatctcgg tctaatctac aacagtagaa at 42
<210> 61
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
agaacaagct gttcttgccg cactatctac aacagtagaa at 42
<210> 62
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
aaaacaacag caacgttctc gttcatctac aacagtagaa at 42
<210> 63
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
taaatctaaa gaacgtagta actcatctac aacagtagaa at 42
<210> 64
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
cttaaaaagt taggtgttga cacaatctac aacagtagaa at 42
<210> 65
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
cccttgactg gttaacttca agttatctac aacagtagaa at 42
<210> 66
<211> 750
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence) -TGEV N Gene Sequence constructed into PUC57 plasmid
<400> 66
aacttatgtc cgagagactt tgtacccaaa ggaataggta acagggatca gcagattggt 60
tattggaata gacaaactcg ctatcgcatg gtgaagggcc aacgtaaaga gcttcctgaa 120
aggtggttct tctactactt aggtactgga cctcatgcag atgccaaatt taaagataaa 180
ttagatggag ttgtctgggt tgccaaggat ggtgccatga acaaaccaac cacgcttggt 240
agtcgtggtg ctaataatga atccaaagct ttgaaattcg atggtaaagt gccaggcgaa 300
tttcaacttg aagttaacca gtcaagggac aattcaaggt cacgctctca atctagatct 360
cggtctagaa acagatctca atctagaggc aggcaacaat ccaataacaa gaaggatgac 420
agtgtagaac aagctgttct tgccgcactt aaaaagttag gtgttgacac agaaaaacaa 480
cagcaacgtt ctcgttctaa atctaaagaa cgtagtaact ctaaaacaag agatactacg 540
cctaagaatg aaaacaaaca cacctggaag agaactgcag gtaaaggtga tgtgacaaga 600
ttttatggag ctagaagcag ttcagccaat tttggtgaca gtgacctcgt tgccaatggg 660
agcagtgcca agcattaccc acaattggct gaatgtgttc catctgtgtc tagcattttg 720
tttggaagct attggacttc aaaggaagat 750
<210> 67
<211> 23
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
aatttcaact tgaagttaac cag 23
<210> 68
<211> 18
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
aacgagaacg ttgctgtt 18
<210> 69
<211> 44
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
tgcctctaga ttgagatctg tttcttcaag ggacaattca aggt 44
<210> 70
<211> 46
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
agaaggatga cagtgtagaa caagcgtttt tctgtgtcaa caccta 46
<210> 71
<211> 22
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
cgagatctag attgagagcg tg 22
<210> 72
<211> 17
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
gttcttgccg cacttaa 17
<210> 73
<211> 19
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
ccaggcgaat ttcaacttg 19
<210> 74
<211> 18
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
gaacgagaac gttgctgt 18
<210> 75
<211> 45
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
tgcctctaga ttgagatctg tttcttcaag ggacaattca aggtc 45
<210> 76
<211> 45
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
agaaggatga cagtgtagaa caagctgttt ttctgtgtca acacc 45
<210> 77
<211> 23
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
gaccgagatc tagattgaga gcg 23
<210> 78
<211> 24
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
tgttcttgcc gcacttaaaa agtt 24
<210> 79
<211> 20
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
ggcgaatttc aacttgaagt 20
<210> 80
<211> 18
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
gaacgagaac gttgctgt 18
<210> 81
<211> 44
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
gcctctagat tgagatctgt ttctgtcaag ggacaattca aggt 44
<210> 82
<211> 44
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
gaaggatgac agtgtagaac aagctgtttt tctgtgtcaa cacc 44
<210> 83
<211> 23
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
ccgagatcta gattgagagc gtg 23
<210> 84
<211> 23
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
tgttcttgcc gcacttaaaa agt 23
<210> 85
<211> 18
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
tcaaggtcac gctctcaa 18
<210> 86
<211> 21
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
gtttgttttc attcttaggc g 21
<210> 87
<211> 46
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
gcttgttcta cactgtcatc cttctatctc ggtctagaaa cagatc 46
<210> 88
<211> 50
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
gttaggtgtt gacacagaaa aacaaatctc ttgttttaga gttactacgt 50
<210> 89
<211> 21
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
tgttgcctgc ctctagattg a 21
<210> 90
<211> 24
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
cagcaacgtt ctcgttctaa atct 24
<210> 91
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
tctagcgttg aaggggtcaa ctctatctac aacagtagaa at 42
<210> 92
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
gccggacatg tgcctggtgt tcagatctac aacagtagaa at 42
<210> 93
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
ctgagaaatg gtttcaccct tgggatctac aacagtagaa at 42
<210> 94
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
cagaggcaca ggcaatcagc ccagatctac aacagtagaa at 42
<210> 95
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
cacttctatt aaacctcatg ttgcatctac aacagtagaa at 42
<210> 96
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
atccttaagt ctcccatagt caggatctac aacagtagaa at 42
<210> 97
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 97
catccttaag tctcccatag tcagatctac aacagtagaa at 42
<210> 98
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 98
gggttcggga gctgacactt ctatatctac aacagtagaa at 42
<210> 99
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 99
tggcaccagt acgagaccgg ttgcatctac aacagtagaa at 42
<210> 100
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 100
cgagaccggt tgccaaatac ctgaatctac aacagtagaa at 42
<210> 101
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 101
gttgctctc aaggtggcca gcgaatctac aacagtagaa at 42
<210> 102
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 102
aagttgctct caaggtggcc agcgatctac aacagtagaa at 42
<210> 103
<211> 900
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence) -PDCoV N Gene Sequence constructed into PUC57 plasmid
<400> 103
gaaaaccatg gctactggct gcgttacacc agacaaaagc caggtggtac tccgattcct 60
ccatcctttg ccttttatta tactggcaca ggtcccagag gaaatcttaa gtatggtgaa 120
ctccctccta atgatacccc agcaaccact cgtgttactt gggttaaggg ttcgggagct 180
gacacttcta ttaaacctca tgttgccaaa cgcaacccca acaatcctaa acatcagctg 240
ctacctctcc gattcccaac cggagatggc ccagctcaag gtttcagagt tgaccccttc 300
aacgctagag gaagacctca ggagcgtgga agtggcccaa gatctcaatc tgttaactcc 360
agaggcacag gcaatcagcc caggaaacgc gaccaatctg caccagctgc ggtacgtcgt 420
aagacccagc atcaagctcc caagcggact ttacccaagg gtgaaaccat ttctcaggta 480
tttggcaacc ggtctcgtac tggtgccaat gtcggctctg cagacactga gaagacgggt 540
atggctgatc ctcgcatcat ggctctagcc ggacatgtgc ctggtgttca ggaaatgttt 600
ttcgctggcc accttgagag caactttcag gcgggggcaa ttacccttac cttctcctac 660
tcaatcacag tcaaggaggg ttttcctgac tatgggagac ttaaggatgc gctcaatacg 720
gtcgttaacc agacctatga gccacctacc aaaccaacta aggacaagaa gcctgacaaa 780
caagtccagt ctgctaaacc caaacagcag aagaaaccta aaaaggtaac tctgccagca 840
gacaaacagg attgggagtg ggatgatgct tttgagataa agcaggaatc agcagcgtag 900
<210> 104
<211> 19
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 104
gcaatcagcc caggaaacg 19
<210> 105
<211> 19
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 105
tcctgaacac caggcacat 19
<210> 106
<211> 39
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 106
tcacccttgg gtaaagtccg ccagctgcgg tacgtcgta 39
<210> 107
<211> 39
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 107
atgtcggctc tgcagacact ggtccggcta gagccatga 39
<210> 108
<211> 17
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 108
tgggagcttg atgctgg 17
<210> 109
<211> 18
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 109
ggtatggctg atcctcgc 18
<210> 110
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 110
gtctgttgac attgttgctg cagtatctac aacagtagaa at 42
<210> 111
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 111
agctgtctca cctgcacctg ccccatctac aacagtagaa at 42
<210> 112
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 112
cgtgctgaac gaggtcactg tcacatctac aacagtagaa at 42
<210> 113
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 113
tcctaattct gaggaggacg tgcgatctac aacagtagaa at 42
<210> 114
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 114
gctagaagct atgcccaagt ctgcatctac aacagtagaa at 42
<210> 115
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 115
tagccagtcc aggcctcaaa gtggatctac aacagtagaa at 42
<210> 116
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 116
atagcttcta gccagtccag gcctatctac aacagtagaa at 42
<210> 117
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 117
gctttggtcc tcgctcagtt tctaatctac aacagtagaa at 42
<210> 118
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 118
aatttctaga aactgagcga ggacatctac aacagtagaa at 42
<210> 119
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 119
taaaccggct cgtaagcaga tggaatctac aacagtagaa at 42
<210> 120
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 120
tcaggtctgg tggtaattga aaccatctac aacagtagaa at 42
<210> 121
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 121
caacagtcag gtctggtggt aattatctac aacagtagaa at 42
<210> 122
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 122
cgttcacagt ctcgttctcg caatatctac aacagtagaa at 42
<210> 123
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 123
cagtctcgtt ctcgcaatca aagtatctac aacagtagaa at 42
<210> 124
<211> 750
<212> DNA
<213> Sequence of SADS-CoV N Gene of Artificial Sequence (Artificial Sequence) constructed into PUC57 plasmid
<400> 124
ccaacgtaaa gatcagcctt ctaactggca cttttattac cttggtactg gtcctcacgc 60
agatgctcct ttcaggaaac ggattcaggg tgtgcattgg gtcgctgttg acggtgctaa 120
aactagcccc acaggtcttg gtgttcgcaa tcgtaacaaa gaacctgcta cacctcagtt 180
tgggtttcaa ttaccaccag acctgactgt tgttgaggtt acttctagaa gtgcttcacg 240
ttcacagtct cgttctcgca atcaaagtca aagccgcagt ggtgctcaga cacctcgtgc 300
tcaacagccg tcacagtctg ttgacattgt tgctgcagtt aaacaagctt tggcagactt 360
gggcatagct tctagccagt ccaggcctca aagtggtaaa aatacaccca aaccaagaag 420
cagagctgtc tcacctgcac ctgcccctaa accggctcgt aagcagatgg acaaacctga 480
atggaagcgt gttcctaatt ctgaggagga cgtgcgtaaa tgctttggtc ctcgctcagt 540
ttctagaaat tttggtgaca gtgacctcgt tcagcacggt gttgaagcta agcactttcc 600
aacaattgct gagttgcttc cgacacaagc tgcactagcc tttggtagtg aaatcacaac 660
caaagagtct ggtgaatttg tagaagtcac ctatcactat gtaatgaagg tccccaagac 720
tgataaaaat ctacccagat ttcttgagca 750
<210> 125
<211> 20
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 125
acaagctttg gcagacttgg 20
<210> 126
<211> 19
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 126
gagcgaggac caaagcatt 19
<210> 127
<211> 39
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 127
tgcaggtgag acagctctgc agcttctagc cagtccagg 39
<210> 128
<211> 39
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 128
gcccctaaac cggctcgtaa gcgcacgtcc tcctcagaa 39
<210> 129
<211> 19
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 129
cttcttggtt tgggtgtat 19
<210> 130
<211> 22
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 130
acaaacctga atggaagcgt gt 22
<210> 131
<211> 18
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 131
gctcagacac ctcgtgct 18
<210> 132
<211> 18
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 132
ccatctgctt acgagccg 18
<210> 133
<211> 42
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 133
agctatgccc aagtctgcca aaccgtcaca gtctgttgac at 42
<210> 134
<211> 40
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 134
tctagccagt ccaggcctca aaggtgcagg tgagacagct 40
<210> 135
<211> 21
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 135
gcttgtttaa ctgcagcaac a 21
<210> 136
<211> 22
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 136
aaatacaccc aaaccaagaa gc 22

Claims (9)

1. The primer group for detecting the porcine enterocoronavirus is characterized by comprising one or more than two of the following combinations:
the combination is as follows:
(1) The nucleotide sequence of the upstream external primer is shown as SEQ ID NO. 1; and
(2) The nucleotide sequence of the downstream external primer is shown as SEQ ID NO. 2; and
(3) The nucleotide sequence of the upstream inner primer is shown as SEQ ID NO. 3; and
(4) The nucleotide sequence of the downstream inner primer is shown as SEQ ID NO. 4; and
(5) The nucleotide sequence of the upstream loop primer is shown as SEQ ID NO. 5; and
(6) The nucleotide sequence of the downstream loop primer is shown as SEQ ID NO. 6; and
(7) The guide nucleotide sequence is shown as SEQ ID NO. 7;
and/or
Combining two:
(8) The nucleotide sequence of the upstream external primer is shown as SEQ ID NO. 8; and
(9) The nucleotide sequence of the downstream external primer is shown as SEQ ID NO. 9; and
(10) The nucleotide sequence of the upstream inner primer is shown as SEQ ID NO. 10; and
(11) The nucleotide sequence of the downstream inner primer is shown as SEQ ID NO. 11; and
(12) The nucleotide sequence of the upstream loop primer is shown as SEQ ID NO. 12; and
(13) The nucleotide sequence of the downstream loop primer is shown as SEQ ID NO. 13; and
(14) The guide nucleotide sequence is shown as SEQ ID NO. 14;
and/or
Combining three:
(15) The nucleotide sequence of the upstream external primer is shown as SEQ ID NO. 15; and
(16) The nucleotide sequence of the downstream external primer is shown as SEQ ID NO. 16; and
(17) The nucleotide sequence of the upstream inner primer is shown as SEQ ID NO. 17; and
(18) The nucleotide sequence of the downstream inner primer is shown as SEQ ID NO. 18; and
(19) The nucleotide sequence of the upstream loop primer is shown as SEQ ID NO. 19; and
(20) The nucleotide sequence of the downstream loop primer is shown as SEQ ID NO. 20; and
(21) The guide nucleotide sequence is shown as SEQ ID NO. 21;
and/or
And (4) combining:
(22) The nucleotide sequence of the upstream external primer is shown as SEQ ID NO. 22; and
(23) The nucleotide sequence of the downstream external primer is shown as SEQ ID NO. 23; and
(24) The nucleotide sequence of the upstream inner primer is shown as SEQ ID NO. 24; and
(25) The nucleotide sequence of the downstream inner primer is shown as SEQ ID NO. 25; and
(26) The nucleotide sequence of the upstream loop primer is shown as SEQ ID NO. 26; and
(27) The nucleotide sequence of the downstream loop primer is shown as SEQ ID NO. 27; and
(28) And the guide nucleotide sequence is shown as SEQ ID NO. 28.
2. The use of the primer set of claim 1 in the preparation of a kit for detecting porcine enteric coronavirus.
3. The use of claim 2, wherein the porcine enterocoronavirus is one or more of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine delta coronavirus, or porcine acute diarrhea syndrome coronavirus.
4. The kit for detecting porcine enterocoronavirus, which is characterized by comprising the primer set, the guide RNA and the auxiliary agent according to claim 1.
5. The kit of claim 4, wherein the porcine enteric coronavirus is one or more of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine delta coronavirus, or porcine acute diarrhea syndrome coronavirus.
6. The kit of claim 4, further comprising a Cas12a protein and a nucleic acid probe comprising a detectable label, wherein the nucleic acid probe generates a visible detection signal when it is cleaved by the Cas12a protein shunt.
7. The kit of claim 6, wherein the nucleic acid probe is ssDNA-reporter, and the 5 'end is labeled with a fluorescent reporter group and the 3' end is labeled with a fluorescent quencher group.
8. The kit of claim 7, wherein the fluorescent reporter group is ROX; the fluorescence quenching group is BHQ2.
9. The kit of claim 6, wherein the visually detectable signal comprises a fluorescent signal or a solution color signal.
CN202110778864.6A 2021-07-09 2021-07-09 Primer group for detecting porcine enterocoronavirus and application thereof Pending CN115595382A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106676197A (en) * 2016-12-27 2017-05-17 河南农业大学 Dual fluorescence RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection method for PDCoV (porcine Delta coronavirus) and PEDV (porcine epidemic diarrhea virus) and application thereof
CN108411040A (en) * 2018-05-21 2018-08-17 浙江大学 Pig acute diarrhea syndrome coronavirus Primer composition and its kit and method
CN111235232A (en) * 2020-01-19 2020-06-05 华中农业大学 Visual rapid nucleic acid detection method based on CRISPR-Cas12a system and application
CN112522444A (en) * 2020-12-22 2021-03-19 中国农业科学院兰州兽医研究所 Composition for African swine fever virus LAMP-CRISPR detection, detection kit and detection method

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106676197A (en) * 2016-12-27 2017-05-17 河南农业大学 Dual fluorescence RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection method for PDCoV (porcine Delta coronavirus) and PEDV (porcine epidemic diarrhea virus) and application thereof
CN108411040A (en) * 2018-05-21 2018-08-17 浙江大学 Pig acute diarrhea syndrome coronavirus Primer composition and its kit and method
CN111235232A (en) * 2020-01-19 2020-06-05 华中农业大学 Visual rapid nucleic acid detection method based on CRISPR-Cas12a system and application
CN112522444A (en) * 2020-12-22 2021-03-19 中国农业科学院兰州兽医研究所 Composition for African swine fever virus LAMP-CRISPR detection, detection kit and detection method

Non-Patent Citations (2)

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Title
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王恩惠: "基于RAA--CRISPR/Cas13a的猪伪狂犬病毒检测方法的建立及初步应用", 万方中国学位论文全文数据库, 27 September 2023 (2023-09-27), pages 1 - 72 *

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