Product for LAMP-CRISPR detection of porcine epidemic diarrhea virus

Abstract

The invention belongs to the technical field of molecular biological detection, and particularly relates to a product for LAMP-CRISPR detection of porcine epidemic diarrhea virus, which comprises an LAMP primer group designed based on highly conservative membrane protein (M) gene in porcine epidemic diarrhea virus genome and a group of sgRNA. According to the product disclosed by the invention, an LAMP amplification system and CRISPR / Cas12b detection can be fused in one-tube constant-temperature reaction, only one reaction temperature needs to be set, a cover does not need to be opened in the whole process, and the operation is simple, convenient and efficient. And the product has high sensitivity and excellent specificity, effectively eliminates false positive interference, and is more suitable for clinical rapid and accurate detection. Meanwhile, the product disclosed by the invention can also be combined with an immune colloidal gold test strip to be better applied clinically, so that the detection of the porcine epidemic diarrhea virus does not depend on large-scale experimental equipment, and the obvious advantages of high efficiency and convenience are achieved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular biological detection, and particularly relates to a product for LAMP-CRISPR detection of porcine epidemic diarrhea virus. BACKGROUND

[0002] Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric coronavirus of pigs, which mainly causes severe watery diarrhea, vomiting and dehydration in newborn piglets (especially within one week), leading to extremely high mortality and significant economic losses including mass deaths of piglets, reproductive disorders of sows, growth and fattening obstacles and dramatic increase of prevention and control costs. At present, the clinical detection of PEDV is mainly through fluorescence quantitative PCR, which is time-consuming and dependent on large instruments. Although the isothermal amplification technology LAMP has been approved for marketing, it has high sensitivity but is prone to false positives. SUMMARY

[0003] The application aims to solve the above problems in the prior art, and provides a PEDV detection scheme which is convenient and fast, does not depend on large instruments, and has sensitive and accurate detection results and is not prone to false positives. To this end, the application provides a product for LAMP-CRISPR detection of porcine epidemic diarrhea virus.

[0004] The application provides a product for LAMP-CRISPR detection of porcine epidemic diarrhea virus, comprising a LAMP primer set and sgRNA. The LAMP primer set comprises primer PEDV-M-2-F3, primer PEDV-M-2-B3, primer PEDV-M-2-LF, primer PEDV-M-2-LB, primer PEDV-M-2-FIP and primer PEDV-M-2-BIP. The nucleotide sequence of the primer PEDV-M-2-F3 is shown as SEQ ID NO. 1. The nucleotide sequence of the primer PEDV-M-2-B3 is shown as SEQ ID NO. 2. The nucleotide sequence of the primer PEDV-M-2-LF is shown as SEQ ID NO. 3. The nucleotide sequence of the primer PEDV-M-2-LB is shown as SEQ ID NO. 4. The nucleotide sequence of the primer PEDV-M-2-FIP is shown as SEQ ID NO. 5. The nucleotide sequence of the primer PEDV-M-2-BIP is shown as SEQ ID NO. 6. The nucleotide sequence of the sgRNA includes one or more of the nucleotide sequences shown in SEQ ID NO. 7~SEQ ID NO. 16.

[0005] Preferably, the product further comprises an ssDNA FAM-BHQ1 reporter probe and / or an ssDNA FAM-Bio reporter probe.

[0006] The application also provides the use of the product described in the above technical solution in the detection of porcine epidemic diarrhea virus for non-diagnostic purposes.

[0007] The application also provides a method for detecting porcine epidemic diarrhea virus for non-diagnostic purposes, comprising the following steps: using the LAMP-CRISPR product described in the above technical solution to construct a LAMP-CRISPR detection system for LAMP amplification and CRISPR-mediated specific cleavage verification in sequence.

[0008] Preferably, the method comprises the following steps: Using the LAMP primer set in the LAMP-CRISPR product described in the above technical solution as a template, LAMP amplification is performed to obtain a LAMP amplification product; The sgRNA in the LAMP-CRISPR product described in the above technical solution is used in combination with the BrCas12b protein to specifically cleave the target sequence in the LAMP amplification product, and the result is determined through signal output detection.

[0009] Preferably, the LAMP amplification and the CRISPR detection are continuously performed in the same reaction tube; the LAMP-CRISPR detection system comprises the following components in 20 µL: 2 µL of 10×Isothermal Amplification Buffer, 2 µL of dNTP Mix, 2.5 µL of LAMP primer set, 1.2 µL of MgSO4, 1 µL of DNA polymerase, 2 µL of sgRNA, 0.5 µL of BrCas12b, 1 µL of reporter probe, and 1 µL of DNA template, and ddH2O is added to 20 µL.

[0010] Preferably, the signal output detection comprises fluorescence detection or lateral flow chromatography test strip detection; When performing fluorescence detection, ssDNA FAM-BHQ1 is used as a reporter probe; The concentration of the ssDNA FAM-BHQ1 is 300~600 nM; When performing lateral flow chromatography test strip detection, ssDNA FAM-Bio is used as a reporter probe; The concentration of the ssDNA FAM-Bio is 250-500 nM.

[0011] Preferably, the reaction temperature of the LAMP amplification and CRISPR-mediated specific cleavage verification is 60-69 DEG C, respectively. The total reaction time of the LAMP amplification and CRISPR-mediated specific cleavage verification is 10-15 min.

[0012] Preferably, the ratio of the LAMP primer group (primer PEDV-M-2-F3 / primer PEDV-M-2-B3) (primer PEDV-M-2-LF / primer PEDV-M-2-LB) (primer PEDV-M-2-FIP / primer PEDV-M-2-BIP) is 1:2: (2-10).

[0013] Preferably, the concentration of MgSO4 in the LAMP-CRISPR detection system is 6-12 mM; the concentration of dNTP Mix in the LAMP-CRISPR detection system is 0.4-1.6 mM; the concentration of BrCas12b in the LAMP-CRISPR detection system is 250-500 nM; and the concentration of sgRNA in the LAMP-CRISPR detection system is 250-1000 nM.

[0014] Beneficial effects: The application provides a product for LAMP-CRISPR detection of porcine epidemic diarrhea virus, which comprises a LAMP primer group and sgRNA, wherein the LAMP primer group comprises primer PEDV-M-2-F3, primer PEDV-M-2-B3, primer PEDV-M-2-LF, primer PEDV-M-2-LB, primer PEDV-M-2-FIP and primer PEDV-M-2-BIP, and the nucleotide sequences are shown in SEQ ID NO. 1-SEQ ID NO. 6; and the nucleotide sequence of the sgRNA comprises one or more of the nucleotide sequences shown in SEQ ID NO. 7-SEQ ID NO. 16. The LAMP-CRISPR product can fuse the LAMP amplification system and the CRISPR / Cas12b detection in one constant-temperature reaction, only one reaction temperature needs to be set, and the whole process does not need to be opened, so that the operation is simple and efficient.

[0015] Meanwhile, the application also designs a LAMP primer set by targeting the highly conserved membrane protein (M) gene in the genome of porcine epidemic diarrhea virus, which significantly improves the broad-spectrum and specificity of the detection system. Combined with the target cleavage activity of CRISPR / Cas12b, the overall LAMP-CRISPR product of the application has high sensitivity (can detect as low as 1 copy / μL of DNA template) and excellent specificity (no cross reaction with nucleic acids of common pig disease viruses such as PDCoV, PRV, PRRSV and SADS-CoV), and effectively eliminates false positive interference, making it more suitable for clinical rapid and accurate detection.

[0016] Further, the LAMP-CRISPR product of the application can also be combined with an immunocolloidal gold test strip to be compatible with the high sensitivity of the LAMP-CRISPR detection system and the visualization of the reagent strip, significantly improving the detection limit of the existing immunocolloidal gold test strip, and better applying to the clinic, so that the detection of porcine epidemic diarrhea virus is less dependent on large experimental equipment, and has the advantages of high efficiency and convenience. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced below.

[0018] Figure 1 LAMP amplification nucleic acid electrophoresis result map for M gene targeting primer; Figure 2 LAMP nucleic acid electrophoresis result map at different temperatures; Figure 3 LAMP nucleic acid electrophoresis result map of different dNTP Mix concentrations; Figure 4 LAMP nucleic acid electrophoresis result map of different primer concentration ratios; Figure 5 LAMP nucleic acid electrophoresis result map of 10 8 ~10 0 copies of DNA; Figure 6 M gene sgRNA target cleavage nucleic acid electrophoresis map; Figure 7 M gene different sgRNA with cleavage activity fluorescence map; Figure 8 Different sgRNA fluorescence detection result map; Figure 9 Different Mg 2+ concentration fluorescence detection result map; Figure 10 Different sgRNA and BrCas12b concentration ratio fluorescence detection result map; Figure 11 Fig. 3 is a fluorescence detection result diagram for different ssDNA FAM-BHQ1 reporter concentrations; Figure 12 Fig. 4 is a fluorescence detection result diagram for different copy number DNA templates; Figure 13 Fig. 5 is a LAMP detection result diagram for different copy number DNA templates; Figure 14 Fig. 6 is a specificity fluorescence detection result diagram; Figure 15 Fig. 7 is a test strip visual detection effect diagram. DETAILED DESCRIPTION

[0019] The application provides a product for detecting porcine epidemic diarrhea virus by LAMP-CRISPR, which comprises a LAMP primer group and sgRNA. The LAMP primer group comprises primer PEDV-M-2-F3, primer PEDV-M-2-B3, primer PEDV-M-2-LF, primer PEDV-M-2-LB, primer PEDV-M-2-FIP and primer PEDV-M-2-BIP. The nucleotide sequence of the primer PEDV-M-2-F3 is shown as SEQ ID NO. 1. The nucleotide sequence of the primer PEDV-M-2-B3 is shown as SEQ ID NO. 2. The nucleotide sequence of the primer PEDV-M-2-LF is shown as SEQ ID NO. 3. The nucleotide sequence of the primer PEDV-M-2-LB is shown as SEQ ID NO. 4. The nucleotide sequence of the primer PEDV-M-2-FIP is shown as SEQ ID NO. 5. The nucleotide sequence of the primer PEDV-M-2-BIP is shown as SEQ ID NO. 6.

[0020] As an embodiment, the product comprises a reagent or a kit. As an embodiment, the SEQ ID NO. 1 of the present application is specifically CTGCATTCCAGTGCTTGG. As an embodiment, the SEQ ID NO. 2 of the present application is specifically CCAACCAGTGCCAGATGA. As an embodiment, the SEQ ID NO. 3 of the present application is specifically CAAGCAATGTACCACTAAGGAGTGT. As an embodiment, the SEQ ID NO. 4 of the present application is specifically AAGGCCACTACAACAATTGTCTACG. As an embodiment, the SEQ ID NO. 5 of the present application is specifically GCCAGTAGCAACCTTATAGCCCGCACCAACTGGTGTAACG. As an embodiment, the SEQ ID NO. 6 of the present application is specifically ACCTAATTTCGTCACAGTCGCCACTGAACGACCAACACGT. As an embodiment, the LAMP primer set is designed by targeting the highly conserved membrane protein (M) gene in the genome of porcine epidemic diarrhea virus, which significantly improves the broad-spectrum and specificity of the detection system. As an embodiment, the amplification reaction temperature of the LAMP primer set of the present application is 60℃-69℃. As an embodiment, the corresponding LAMP reaction in the range of 60℃-69℃ of the LAMP primer set of the present application is enhanced with the increase of temperature, and the band of the amplification product is most obvious when the electrophoresis detection is carried out at 65℃, and the band starts to weaken at 69℃. As an embodiment, the ratio of (primer PEDV-M-2-F3 / primer PEDV-M-2-B3):(primer PEDV-M-2-LF / primer PEDV-M-2-LB):(primer PEDV-M-2-FIP / primer PEDV-M-2-BIP) in the LAMP primer set of the present application can be 1:2:(2-10). As an embodiment, when the M gene of porcine epidemic diarrhea virus is amplified by using the LAMP primer set of the present application, the ratio of (primer PEDV-M-2-F3 / primer PEDV-M-2-B3):(primer PEDV-M-2-LF / primer PEDV-M-2-LB):(primer PEDV-M-2-FIP / primer PEDV-M-2-BIP) can be 1:2:6.

[0021] The nucleotide sequence of the sgRNA comprises one or more of the nucleotide sequences shown in SEQ ID NO. 7~SEQ ID NO. 16. As an embodiment, the sgRNA of the present application comprises M-sgRNA-1 with a PAM site of TTA, and the nucleotide sequence is shown in SEQ ID NO. 7, specifically: TAGCCCTCTACAAGCAATGT. As an embodiment, the sgRNA of the present application comprises M-sgRNA-2 with a PAM site of TTA, and the nucleotide sequence is shown in SEQ ID NO. 8, specifically: CCTGTACGCCAGTAGCAACC. As an embodiment, the sgRNA of the present application comprises M-sgRNA-3 with a PAM site of TTG, and the nucleotide sequence is shown in SEQ ID NO. 9, specifically: ACTTACCTGTACGCCAGTAG. As an embodiment, the sgRNA of the present application comprises M-sgRNA-4 with a PAM site of TTA, and the nucleotide sequence is shown in SEQ ID NO. 10, specifically: CCTAATTTCGTCACAGTCGC. As an embodiment, the sgRNA of the present application comprises M-sgRNA-5 with a PAM site of TTG, and the nucleotide sequence is shown in SEQ ID NO. 11, specifically: CGTCACAGTCGCCAAGGCCA. As an embodiment, the sgRNA of the present application comprises M-NT-sgRNA-1 with a PAM site of ATG, and the nucleotide sequence is shown in SEQ ID NO. 12, specifically: TACCACTAAGGAGTGTTAGC. As an embodiment, the sgRNA of the present application comprises M-NT-sgRNA-2 with a PAM site of ATA, and the nucleotide sequence is shown in SEQ ID NO. 13, specifically: GCCCTCTACAAGCAATGTAC. As an embodiment, the sgRNA of the present application comprises M-NT-sgRNA-3 with a PAM site of ATT, and the nucleotide sequence is shown in SEQ ID NO. 14, specifically: GACTTACCTGTACGCCAGTA. As an embodiment, the sgRNA of the present application comprises M-NT-sgRNA-4 with a PAM site of ATT, and the nucleotide sequence is shown in SEQ ID NO. 15, specifically: TCGTCACAGTCGCCAAGGCC. As an embodiment, the sgRNA of the present application comprises M-NT-sgRNA-5 with a PAM site of ATT, and the nucleotide sequence is shown in SEQ ID NO. 16, specifically: GACTGAACGACCAACACGTC. As an embodiment, the PAM site of the sgRNA of the present application comprises a classic PAM site (TTN) and a non-classic PAM site (ATN).As an embodiment, the classical sgRNAs described in the application can all target and cut within 10 min, and the substrate is cut to form product bands of different sizes, wherein the classical sgRNAs represented by M-sgRNA-1 and M-sgRNA-3 show strong cutting activity. As an embodiment, the M-NT-sgRNA-1 and M-NT-sgRNA-3 among the non-classical sgRNAs described in the application have weak cutting ability of the substrate within 10 min, and only a small amount of product bands are present. The weak cutting ability of sgRNA helps to reduce the competition of target DNA when LAMP is fused, improve the content of amplification product of target DNA, improve the detection ability of low-abundance virus, and also reduce the detection time. As an embodiment, the M-NT-sgRNA-1 described in the application has weak target cutting ability, and is accompanied by strong cutting ability, which can better play the amplification role when fused with LAMP, amplify the fluorescence signal synchronously, and can efficiently detect low-abundance virus templates to diagnose virus infection in a shorter time.

[0022] As an embodiment, the product described in the application further comprises an ssDNA FAM-BHQ1 reporter probe and / or an ssDNA FAM-Bio reporter probe.

[0023] The application also provides the use of the product described in the above technical solution for the detection of porcine epidemic diarrhea virus for non-diagnostic purposes. As an embodiment, the LAMP-CRISPR product described in the application can fuse the LAMP amplification system and the CRISPR / Cas12b detection in one tube constant temperature reaction, only one reaction temperature needs to be set, and the whole process does not need to be opened, which is simple and efficient. As an embodiment, the LAMP-CRISPR product described in the application designs LAMP primer sets by targeting the highly conserved membrane protein (M) gene in the porcine epidemic diarrhea virus genome, which significantly improves the broad-spectrum and specificity of the detection system, and combines the target cutting activity of CRISPR / Cas12b, so that the whole LAMP-CRISPR product of the application has high sensitivity and excellent specificity, and effectively eliminates false positive interference, making it more suitable for clinical rapid and accurate detection. As an embodiment, the LAMP-CRISPR product described in the application can be combined with an immunocolloidal gold test strip to be compatible with the high sensitivity of the LAMP-CRISPR detection system and the visualization of the reagent strip, significantly improving the detection limit of the existing immunocolloidal gold test strip, and better applying to the clinic, so that the detection of porcine epidemic diarrhea virus is less dependent on large experimental equipment, and has significant advantages of high efficiency and convenience.

[0024] The application further provides a method for detecting porcine epidemic diarrhea virus for non-diagnostic purposes, comprising the following steps: using the LAMP-CRISPR product described in the above technical solution to construct a LAMP-CRISPR detection system to sequentially perform LAMP amplification and CRISPR-mediated specific cleavage verification.

[0025] As an embodiment, the method of the application comprises the following steps: Using the LAMP primer set in the LAMP-CRISPR product described in the above technical solution as a template, LAMP amplification is performed to obtain a LAMP amplification product; The sgRNA in the LAMP-CRISPR product described in the above technical solution is used in combination with the BrCas12b protein to specifically cleave the target sequence in the LAMP amplification product, and the result is determined through signal output detection.

[0026] As an embodiment, the LAMP amplification and CRISPR detection of the application are continuously performed in the same reaction tube; the LAMP-CRISPR detection system comprises the following components in 20 μL: 2 μL of 10x Isothermal Amplification Buffer, 2 μL of dNTP Mix, 2.5 μL of LAMP primer set, 1.2 μL of MgSO4, 1 μL of DNA polymerase, 2 μL of sgRNA, 0.5 μL of BrCas12b, 1 μL of reporter probe and 1 μL of DNA template, and is supplemented with ddH2O to 20 μL.

[0027] As an embodiment, the reaction temperature of the LAMP amplification and CRISPR-mediated specific cleavage verification of the application is 60℃-69℃; the total reaction time of the LAMP amplification and CRISPR-mediated specific cleavage verification is 10-15 min. As an embodiment, the reaction temperature of the LAMP amplification and CRISPR-mediated specific cleavage verification of the application is 65℃.

[0028] As an implementation form, the LAMP primer set in the application is (primer PEDV-M-2-F3 / primer PEDV-M-2-B3): (primer PEDV-M-2-LF / primer PEDV-M-2-LB): (primer PEDV-M-2-FIP / primer PEDV-M-2-BIP) is 1:2: (2-10). As an implementation form, the LAMP primer set in the application is (primer PEDV-M-2-F3 / primer PEDV-M-2-B3): (primer PEDV-M-2-LF / primer PEDV-M-2-LB): (primer PEDV-M-2-FIP / primer PEDV-M-2-BIP) is 1:2:6.

[0029] As an implementation form, the concentration of MgSO4 in the LAMP-CRISPR detection system of the application is 6-12 mM; the concentration of dNTP Mix in the LAMP-CRISPR detection system is 0.4-1.6 mM; the concentration of BrCas12b in the LAMP-CRISPR detection system is 250-500 nM; the concentration of sgRNA in the LAMP-CRISPR detection system is 250-1000 nM. As an implementation form, the concentration of MgSO4 in the LAMP-CRISPR detection system of the application is 8 mM; the concentration of dNTP Mix in the LAMP-CRISPR detection system is 1 mM; the concentration of BrCas12b in the LAMP-CRISPR detection system is 250 nM; the concentration of sgRNA in the LAMP-CRISPR detection system is 500 nM.

[0030] As an implementation form, the signal output detection of the application comprises fluorescence detection or lateral flow chromatographic test strip detection; when the fluorescence detection is performed, ssDNA FAM-BHQ1 is used as a reporter probe; the concentration of the ssDNA FAM-BHQ1 is 300-600 nM; when the lateral flow chromatographic test strip detection is performed, ssDNA FAM-Bio is used as a reporter probe; the concentration of the ssDNA FAM-Bio is 250-500 nM. As another implementation form, when the fluorescence detection is performed, ssDNA FAM-BHQ1 is used as a reporter probe; the concentration of the ssDNA FAM-BHQ1 is 500 nM; the concentration of the ssDNA FAM-Bio of the application is 250 nM. As an implementation form, the application uses the lateral flow chromatographic test strip detection to perform signal output detection, which is compatible with the high sensitivity of the LAMP-CRISPR detection system and the visualization of the reagent strip, significantly improves the detection limit of the existing immunocolloidal gold test strip, and is better applied to the clinic, so that the detection of the porcine epidemic diarrhea virus is less dependent on large experimental equipment, and has the advantages of high efficiency and convenience.

[0031] In order to further illustrate the application, a product for detecting the porcine epidemic diarrhea virus by using the LAMP-CRISPR detection system is described in detail below in combination with the accompanying drawings and examples, but they should not be understood as limiting the protection scope of the application.

[0032] Example 1 LAMP primer design and verification of amplification effect The completely conserved sequence in the M gene of PEDV is selected, the LAMP primer is designed by using the online website PrimerExplorer V5, is synthesized by Shanghai Shengong Bioengineering Co., Ltd., and the M gene is amplified by using the synthesized LAMP primer, the amplification kit is purchased from Beijing NEB Company, the DNA template of the M gene is diluted to 10 0 ~10 5 copies / μL, 1 μL is taken as a template, the reaction system is shown in Table 1 under the condition of 65℃, the reaction is performed for 40 min, wherein the primer Mix (LAMP primer premix) is 100 μL, which comprises: 16 μM FIP, 16 μM BIP, 2 μM F3, 2 μM B3, 4 μM LF and 4 μM LB.

[0033] After the LAMP amplification product of the primer is obtained, the amplification product is subjected to nucleic acid electrophoresis detection, the result is shown in Figure 1 , wherein Figure 1 M in the above table is DL2,000 DNA Marker, Figure 1 1 in the above table is a negative control (ddH2O is a template),Figure 1 2 is 10 0 electrophoretic bands under the condition of 10 Figure 1 3 is 10 1 electrophoretic bands under the condition of 10 Figure 1 4 is 10 2 electrophoretic bands under the condition of 10 Figure 1 5 is 10 3 electrophoretic bands under the condition of 10 Figure 1 6 is 10 4 electrophoretic bands under the condition of 10 Figure 1 7 is 10 5 electrophoretic bands under the condition of 10 copies / μL, and the LAMP primers obtained in the application are good for M gene primers under the condition of 10 0 ~10 5 copies / μL, and can be used for subsequent LAMP amplification reaction condition optimization.

[0034] Table 1 LAMP amplification reaction system

[0035] Example 2 LAMP amplification reaction condition optimization 1. LAMP amplification reaction temperature optimization DNA template is diluted to 10 4 copies / μL as the template, and the reaction system is configured according to Table 1 in the embodiment 1, the reaction temperature gradient is 60℃, 61℃, 62℃, 63℃, 64℃, 65℃, 69℃, and the LAMP nucleic acid electrophoretic results under different temperatures are shown in Figure 2 , wherein Figure 2 M in the table is DL2,000 DNA Marker, Figure 2 1 is a negative control (ddH2O is the template), Figure 2 2 Figure 2 8 in the table are electrophoretic bands under the conditions of 60℃, 61℃, 62℃, 63℃, 64℃, 65℃ and 69℃ respectively.

[0036] According to the results shown in Figure 2 , it can be known that the LAMP reaction is enhanced with the increase of temperature, and the band is most obvious at 65℃, and the band is weakened at 69℃, therefore, the optimal reaction temperature of the LAMP amplification primer group in the application is set to 65℃.

[0037] 2. dNTP Mix concentration optimization The same 10 4DNA template at a concentration of copies / μL was used, and the reaction system was prepared according to Table 1 in Example 1. dNTP Mix concentrations were optimized using gradients of 0.4 mM, 0.6 mM, 0.8 mM, 1 mM, 1.2 mM, 1.4 mM, and 1.6 mM. The reaction was carried out at 65°C for 40 min. LAMP nucleic acid electrophoresis results for different dNTP Mix concentrations are shown below. Figure 3 As shown, where Figure 3 M in the text stands for DL2,000 DNA Marker. Figure 3 1 in the table represents the control (ddH2O is the template, and the concentration of dNTP Mix is ​​0.8 mM). Figure 3 2 in the table is the control (with template, without dNTP Mix). Figure 3 3~ Figure 3 9 in the image represents electrophoretic bands under conditions of 0.4 mM, 0.6 mM, 0.8 mM, 1 mM, 1.2 mM, 1.4 mM, and 1.6 mM.

[0038] according to Figure 3 The results show that the LAMP reaction is affected by the concentration of dNTP Mix. When the dNTP concentration is low, the reaction does not amplify. As the concentration increases, the amplification reaction is enhanced. After reaching 1 mM, the amplification reaction does not show a significant enhancement trend. Therefore, the final dNTP Mix concentration of 1 mM was chosen as the subsequent condition for the LAMP reaction.

[0039] 3. Optimization of LAMP amplification primer concentrations Using the same 10 4 DNA template at a concentration of copies / μL was used. The reaction system was prepared according to Table 1 in Example 1. LAMP amplification primer ratios F3 / B3:LF / LB:FIP / BIP were used in gradients of 1:2:2, 1:2:4, 1:2:6, 1:2:8, and 1:2:10. The reaction was carried out at 65℃ for 40 min. The LAMP nucleic acid electrophoresis results for different primer ratios are shown in Figure 4. Figure 4 M in the text refers to DL2,000 DNA Marker. Figure 4 1 in Figure 4 3 in Figure 4 5 in Figure 4 7 and Figure 4 9 in the sample is a negative control (ddH2O is the template). Figure 4 1 and Figure 4 The number 2 in the equation represents the electrophoretic band under the condition of F3 / B3:LF / LB:FIP / BIP=1:2:2. Figure 4 3 and Figure 4The band number 4 represents the electrophoretic band under the condition of F3 / B3:LF / LB:FIP / BIP = 1:2:4. Figure 4 5 and Figure 4 The band number 6 represents the electrophoretic band under the condition of F3 / B3:LF / LB:FIP / BIP = 1:2:6. Figure 4 7 and Figure 4 The band number 8 represents the electrophoretic band under the condition of F3 / B3:LF / LB:FIP / BIP = 1:2:8. Figure 4 9 and Figure 4 The 10 in the figure represents the electrophoretic bands under the condition of F3 / B3:LF / LB:FIP / BIP=1:2:10.

[0040] according to Figure 5 The results show that the LAMP reaction is affected by the primer concentration ratio. The M gene exhibits the best amplification effect at the ratio of F3 / B3:LF / LB:FIP / BIP = 1:2:6. However, the ratio of F3 / B3:LF / LB:FIP / BIP = 1:2:8 does not show a significant increasing trend; instead, it weakens at F3 / B3:LF / LB:FIP / BIP = 1:2:10. Therefore, the optimal primer ratio for LAMP amplification is determined to be F3 / B3:LF / LB:FIP / BIP = 1:2:6.

[0041] 4. LAMP reaction with plasmid templates of different copy numbers Using the optimized reaction system described above, and referring to Table 1 in Example 1, the DNA template copy number was calculated, and a 10-fold serial dilution was performed to obtain a final product of 1.0 × 10⁻⁶. 8 1.0×10 7 1.0×10 6 1.0×10 5 1.0×10 4 1.0×10 3 1.0×10 2 1.0×10 1 1.0×10 0 Using 1 μL of plasmids with different copy numbers as templates, LAMP reactions were performed sequentially at 65 °C for 40 min to obtain 10 8 ~10 0 LAMP electrophoresis results for copy number DNA are as follows: Figure 5 As shown, where Figure 5 M in the text stands for DL2,000 DNA Marker. Figure 5 1 in the diagram is the negative control (ddH2O is the template). Figure 5 Bands 2-10 in the image are electrophoretic bands representing the M gene amplification products. Figure 5 The 2 in the middle is 10 8electrophoretic bands under 1 copy condition, Figure 5 3 is 10 in 7 electrophoretic bands under 1 copy condition, Figure 5 4 is 10 in 6 electrophoretic bands under 1 copy condition, Figure 5 5 is 10 in 5 electrophoretic bands under 1 copy condition, Figure 5 6 is 10 in 4 electrophoretic bands under 1 copy condition, Figure 5 7 is 10 in 3 electrophoretic bands under 1 copy condition, Figure 5 8 is 10 in 2 electrophoretic bands under 1 copy condition, Figure 5 9 is 10 in 1 electrophoretic bands under 1 copy condition, Figure 5 10 is 10 in 0 electrophoretic bands under 1 copy condition.

[0042] According to the results shown in Figure 6 It can be seen from the results that the M gene LAMP reaction can detect as low as 1 copy / μL of plasmid, and no band appears in the negative control.

[0043] Example 3 Verification system optimization of CRISPR-mediated specific cleavage 1. sgRNA targeting cleavage activity verification Based on the PAM site of the CRISPR / Cas12b system, the sgRNA sequence was designed by using the online website (https: / / www.benchling.com / crispr) for the amplified fragment of the M gene of PEDV. Two types of PAM sites were designed: classic PAM site (TTN) and non-classic PAM site (ATN), each with 5, a total of 10 sgRNAs, as shown in Table 2.

[0044] Table 2 sgRNAs of two types of PAM sites and their sequences

[0045] Synthesized by Shanghai Shengong Bioengineering Co., Ltd. The reaction system was configured as shown in Table 3, and the reaction was carried out at 65℃ for 10 min to verify the cleavage efficiency of the target fragment.

[0046] Table 3 BrCas12b target cleavage verification reaction system

[0047] Targeted cleavage validation was performed on all sgRNAs. Using M gene DNA as a template, the mixture was reacted in a 20 μL system containing 250 nMBrCas12b and 8 mM MgSO4 at 65°C for 10 min. The results were verified by nucleic acid electrophoresis, yielding the electrophoresis results of targeted cleavage of M gene sgRNAs. Figure 6 As shown, where Figure 6 M in the text stands for DL2,000 DNA Marker. Figure 6 1 in the table represents the control group (without BrCas12b protein and sgRNA). Figure 6 2 in the table represents the control group (without sgRNA). Figure 6 3 in the table is the control (without BrCas12b protein). Figure 6 4~ Figure 6 The 13 in the image represent the electrophoretic bands of the M-sgRNA-1 targeted cleavage product, the M-sgRNA-2 targeted cleavage product, the M-sgRNA-3 targeted cleavage product, the M-sgRNA-4 targeted cleavage product, the M-sgRNA-5 targeted cleavage product, the M-NT-sgRNA-1 targeted cleavage product, the M-NT-sgRNA-2 targeted cleavage product, the M-NT-sgRNA-3 targeted cleavage product, the M-NT-sgRNA-4 targeted cleavage product, and the M-NT-sgRNA-5 targeted cleavage product, respectively.

[0048] according to Figure 7 The results show that all classic sgRNAs can target and cleave the substrate within 10 minutes, forming product bands of different sizes. Among them, classic sgRNAs M-sgRNA-1 and M-sgRNA-3, representing classic sgRNAs, exhibited strong cleavage activity. In contrast, non-classical sgRNAs M-NT-sgRNA-1 and M-NT-sgRNA-3 showed weaker substrate cleavage ability, producing only a small number of product bands. The weaker targeting ability of sgRNAs helps reduce competition for target DNA during LAMP fusion, increasing the amplification product content of target DNA, improving the detection capability of low-abundance viruses, and also reducing detection time.

[0049] 2. Verification of sgRNA cleavage activity Using M gene DNA as a template, 400 nM ssDNA FAM-BHQ1 reporter was added to the targeted cleavage reaction system shown in Table 3. The reaction was carried out at 65℃ in a real-time PCR instrument for 40 min, and fluorescence was detected. Fluorescence was collected every 1 min to evaluate the cleavage efficiency of different sgRNAs. The fluorescence detection results of the cleavage activity of different sgRNAs of the M gene are shown below.Figure 7 Results are shown.

[0050] According to Figure 8 The results show that different sgRNAs have different collateral cleavage activities. The sgRNAs of the M gene (M-sgRNA-1, M-sgRNA-2 and M-NT-sgRNA-1) all have high cleavage activities, and the non-canonical M-NT-sgRNA-1 has the strongest collateral cleavage activity. Selecting DNA with strong collateral cleavage can maximize the amplification of the fluorescent signal, which helps to shorten the detection time and improve the detection efficiency. Considering the above factors, M-NT-sgRNA-1 has weak targeted cleavage ability and strong collateral cleavage ability, and can be better combined with LAMP to amplify and activate the fluorescent signal simultaneously, which can efficiently detect low-abundance viral templates and diagnose viral infection in a shorter time.

[0051] 3. Screening of sgRNA in LAMP-CRISPR / Cas12b system 10 4 copies / μL M gene DNA as a template, and the reaction system was configured according to Table 4. The fluorescence cleavage reaction was further detected by selecting M-sgRNA-1, M-sgRNA-2 and M-NT-sgRNA-1 according to the LAMP and CRISPR one-pot method. The fluorescence was collected every 1 min for 40 min at 65°C in the fluorescence quantitative qPCR. The fluorescence detection results of different sgRNAs are shown in Figure 8 According to the results shown in Figure 9 The results show that the cleavage efficiency of M-NT-sgRNA-1 is the strongest, so M-NT-sgRNA-1 is selected for subsequent reaction system optimization and establishment of the CRISPR / Cas12b detection method.

[0052] Table 4. LAMP and CRISPR / Cas12b one-pot detection system

[0053] 4. Optimization of MgSO4 concentration Based on the selected optimal sgRNA (M-NT-sgRNA-1), 10 4 copies / μL M gene DNA as a template, and the reaction system was configured according to Table 3. The MgSO4 concentration was further optimized in a gradient manner. The MgSO4 was used in a gradient manner of 4 mM, 6 mM, 8 mM, 10 mM and 12 mM. The fluorescence was collected every 1 min for 40 min at 65°C in the fluorescence quantitative qPCR. The fluorescence detection results of different Mg 2+ concentrations are shown in Figure 9 According to the results shown in Figure 10The results show that under different concentrations of MgSO4, the sgRNA cleavage activity is different, and no cleavage reaction occurs when the concentration of MgSO4 is 4 mM. After increasing the concentration of MgSO4, the sgRNA collateral cleavage activity is enhanced. The strongest cleavage activity is shown at 8 mM. And with the increase of the concentration to 12 mM, the fluorescence value of the cleavage does not improve. Therefore, 8 mM is the best MgSO4 concentration for the reaction.

[0054] 5. Optimization of BrCas12b and sgRNA concentration ratio To determine the optimal protein to sgRNA ratio, under the condition of fixed MgSO4 concentration (8 mM), 10 4 copies / μL M gene DNA as template, according to Table 4, the reaction system was configured, and the BrCas12b and sgRNA concentration ratio was set to 1:1, 1:2, 2:1, 1:4, 4:1, etc. Under the condition of 65℃, it was placed in the fluorescence quantitative qPCR for 40 min, and the fluorescence was collected every 1 min. The fluorescence detection results of different sgRNA and BrCas12b concentration ratios are shown in Figure 10 According to the results shown in Figure 11 It can be seen from the results shown in

[0055] 6. Optimization of ssDNA FAM-BHQ1 reporter concentration Under the above optimal conditions, 10 4 copies / μL M gene DNA as template, according to Table 4, the reaction system was configured, and the BrCas12b and sgRNA concentration ratio was set to 1:1, 1:2, 2:1, 1:4, 4:1, etc. Under the condition of 65℃, it was placed in the fluorescence quantitative qPCR for 40 min, and the fluorescence was collected every 1 min. The fluorescence detection results of different sgRNA and BrCas12b concentration ratios are shown in Figure 11 According to the results shown in Figure 12 It can be seen from the results shown in

[0056] Example 4 Sensitivity and specificity of the LAMP-CRISPR / Cas12b system 1. Sensitivity detection Referring to Table 4, the optimized LAMP and CRISPR / Cas12b nucleic acid detection reaction systems of Examples 2 and 3 were configured, using M gene DNA as a template, with the template serially diluted to 10-1. -1 ~10 6 The DNA templates were analyzed using M-NT-sgRNA-1 at copies / μL. The reaction was carried out at 65℃ in a quantitative qPCR incubator for 10 min, with fluorescence collected every 1 min. The fluorescence detection results for different copy numbers of DNA templates are shown below. Figure 13 As shown in the figure, the detection limit of this method is as low as 1 copy / µL DNA.

[0057] Using the conventional LAMP method, after configuring the optimized reaction system according to Table 1 in Example 1, M gene DNA was used as the template, and the template was serially diluted to 10⁻⁶. -1 ~10 6 The reaction was carried out at copies / μL for 10 min, and the fluorescence detection results of DNA templates with different copy numbers were obtained as follows: Figure 13 As shown, where Figure 13 M in the text stands for DL2,000 DNA Marker. Figure 13 1 in the diagram is the negative control (ddH2O is the template). Figure 13 2~ Figure 13 9 in the image represents the fluorescence detection results of M gene amplification. Specifically... Figure 13 The 2 in the middle is 10 -1 Electrophoretic bands under copy conditions Figure 13 The 3 in the middle is 10 0 Electrophoretic bands under copy conditions Figure 13 The 4 in the middle is 10 1 Electrophoretic bands under copy conditions Figure 13 The 5 in the middle is 10 2 Electrophoretic bands under copy conditions Figure 13 The 6 in the middle is 10 3 Electrophoretic bands under copy conditions Figure 13 The 7 in the middle is 10 4 Electrophoretic bands under copy conditions Figure 13 The 8 in the middle is 10 5 Electrophoretic bands under copy conditions Figure 14 The 9 in the middle is 10 6 Electrophoretic bands under copy conditions. It can be seen that the detection limit of the conventional LAMP method is 10-1. 2copies / µL DNA, which is 100 times lower than the CRISPR / Cas12b nucleic acid detection reaction.

[0058] 2. Specific detection The LAMP and CRISPR / Cas12b nucleic acid detection reaction system optimized in Example 2 and Example 3 was configured according to Table 4, and the nucleic acids of common pig viruses such as PDCoV, PRV, PRRSV, SADS-CoV, etc. were used as templates, PEDV-M-NT-sgRNA-1 was selected, and a 20 μL reaction system was configured. Fluorescence detection was performed at 65°C, and fluorescence was collected every 1 min for 40 min in the fluorescence quantitative qPCR reaction. The specific fluorescence detection results are shown in Figure 15 It can be seen that PDCoV, PRV, PRRSV, SADS-CoV, SPV, PCV and the negative control with ddH2O as the template have consistent results and do not produce fluorescence signal amplification. Only the PEDV positive group can excite a large amount of fluorescence value, indicating that the PEDV nucleic acid detection based on CRISPR / Cas12b has good specificity.

[0059] 3. Combined with test strip detection The LAMP and CRISPR / Cas12b nucleic acid detection reaction system optimized in Example 2 and Example 3 was configured according to Table 4, and 10 0 copies / µL M gene DNA was used as a template, ssDNA FAM-Bio reporter (250 nM) was used to replace ssDNA FAM-BHQ1 reporter, 1 μL of the reaction solution was diluted with Hybridetect Assay buffer at a ratio of 1:4 after being placed in a 65°C water bath for 15 min, and then dropped into the sample pad of the HybriDetect test strip. After 2-3 min, the strip of the test strip was observed, and the results are shown in Figure 15 Figure 15 The left reagent strip in the middle is the LAMP and CRISPR / Cas12b nucleic acid detection reaction result; Figure 15 The middle reagent strip shown in the middle is the non-amplification CRISPR / Cas12b nucleic acid detection reaction result; ​ The right reagent strip is the reaction result of the negative control (ddH2O as the template). It can be seen that the LAMP and CRISPR / Cas12b nucleic acid detection reaction established in the present application can well detect positive samples, while the conventional CRISPR / Cas12b nucleic acid detection reaction cannot be detected, indicating that the combined detection method can improve the low-abundance detection ability and realize visual detection, and the result can be determined without relying on a fluorescence detection instrument.

[0060] ​Although the above embodiments have been described in detail, it should be understood that these are only some embodiments of the present application, but not all embodiments. Other embodiments can be obtained on the basis of the above embodiments without creativity, and these embodiments all belong to the protection scope of the present application.

Claims

1. A product for LAMP-CRISPR detection of porcine epidemic diarrhea virus, characterized in that, The LAMP primer set and the sgRNA; The LAMP primer set comprises primer PEDV-M-2-F3, primer PEDV-M-2-B3, primer PEDV-M-2-LF, primer PEDV-M-2-LB, primer PEDV-M-2-FIP and primer PEDV-M-2-BIP; The nucleotide sequence of the primer PEDV-M-2-F3 is shown as SEQ ID NO. 1; The nucleotide sequence of the primer PEDV-M-2-B3 is shown as SEQ ID NO. 2; The nucleotide sequence of the primer PEDV-M-2-LF is shown as SEQ ID NO. 3; The nucleotide sequence of the primer PEDV-M-2-LB is shown as SEQ ID NO. 4; The nucleotide sequence of the primer PEDV-M-2-FIP is shown as SEQ ID NO. 5; The nucleotide sequence of the primer PEDV-M-2-BIP is shown as SEQ ID NO. 6; The nucleotide sequence of the sgRNA comprises one or more of the nucleotide sequences shown as SEQ ID NO. 7 to SEQ ID NO.

16.

2. The product of claim 1, wherein, The product further comprises an ssDNA FAM-BHQ1 reporter probe and / or an ssDNA FAM-Bio reporter probe.

3. Use of the product of claim 1 or 2 in the detection of porcine epidemic diarrhea virus for non-diagnostic purposes.

4. A method for detecting porcine epidemic diarrhea virus for non-diagnostic purposes, characterized in that, The method comprises the following steps: The LAMP-CRISPR detection system is constructed using the LAMP-CRISPR product of claim 1 or 2 to sequentially perform LAMP amplification and CRISPR-mediated specific cleavage verification.

5. The method of claim 4, wherein, The method comprises the following steps: Using the DNA of the sample to be detected as a template, the LAMP primer set in the LAMP-CRISPR product of claim 1 is used to perform LAMP amplification to obtain a LAMP amplification product; The sgRNA in the LAMP-CRISPR product of claim 1 is used in combination with the BrCas12b protein to specifically cleave the target sequence in the LAMP amplification product, and the result is determined through signal output detection.

6. The method of claim 5, wherein, The LAMP amplification and CRISPR detection are sequentially performed in the same reaction tube; the LAMP-CRISPR detection system comprises the following components in a total volume of 20 µL: 2 µL of 10× Isothermal Amplification Buffer, 2 µL of dNTP Mix, 2.5 µL of LAMP primer set, 1.2 µL of MgSO4, 1 µL of DNA polymerase, 2 µL of sgRNA, 0.5 µL of BrCas12b, 1 µL of reporter probe and 1 µL of DNA template, and ddH2O is added to make up to 20 µL.

7. The method of claim 5, wherein, The signal output detection comprises fluorescence detection or lateral flow chromatography test strip detection; When performing fluorescence detection, the ssDNA FAM-BHQ1 is used as a reporter probe; The concentration of the ssDNA FAM-BHQ1 is 300-600 nM; When the lateral flow chromatographic test strip detection is carried out, ssDNA FAM-Bio is used as a report probe; The concentration of the ssDNA FAM-Bio is 250-500 nM.

8. The method of claim 4, wherein, The reaction temperature of the LAMP amplification and CRISPR-mediated specific cleavage verification is 60-69 DEG C respectively. The total reaction time of the LAMP amplification and CRISPR-mediated specific cleavage verification is 10-15 min.

9. The method of claim 6, wherein, The LAMP primer group (primer PEDV-M-2-F3 / primer PEDV-M-2-B3): (primer PEDV-M-2-LF / primer PEDV-M-2-LB): (primer PEDV-M-2-FIP / primer PEDV-M-2-BIP) is 1:2: (2-10).

10. The method of claim 5, wherein, The concentration of MgSO4 in the LAMP-CRISPR detection system is 6-12 mM; the concentration of dNTP Mix in the LAMP-CRISPR detection system is 0.4-1.6 mM; the concentration of BrCas12b in the LAMP-CRISPR detection system is 250-500 nM; and the concentration of sgRNA in the LAMP-CRISPR detection system is 250-1000 nM.

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