Ovine border virus RPA-Crispr cas12a detection method

Abstract

The invention discloses a sheep boundary virus RPA-Crispr cas12a detection method, which comprises the following steps: S1, obtaining and collecting related detection materials, the sheep boundary virus RPA-Crispr cas12a detection method has the beneficial effects that compared with a traditional CRISPR / Cas9 system, the sheep boundary virus RPA-Crispr cas12a detection method has the advantages that the detection efficiency is high; the Cas12a has the following technical advantages: 1, a target recognition and cutting mechanism: the Cas12a directly recognizes a target sequence (needing a T-rich PAM sequence, such as TTTN) in double-stranded DNA through a single crRNA, and does not need tracrRNA; secondly, the crRNA design process is simplified: after target combination, Cas12a activates the non-specific single-stranded DNA (ssDNA) trans-cleavage activity of Cas12a, free fluorescence-quenching labeled reporter molecules can be efficiently cleaved, and signal amplification and visual output are realized; 3, high precision and anti-interference capability: the tolerance of Cas12a to single-base mismatch is extremely low (lt); 1%), and is especially suitable for distinguishing related pathogens such as the border virus and a bovine viral diarrhea virus (BVDV) of a pestivirus genus, and the specificity gt; 99.9% of the content.

Description

Technical Field

[0001] The present invention relates to the technical field of sheep border virus detection, and specifically to a sheep border virus RPA-Crispr cas12a detection method. Background Art

[0002] Ovine border virus is a disease caused by a virus of the genus Pestovirus in the family Flaviviridae. It primarily infects sheep and occasionally goats. The virus was first discovered in the border area between Scotland and Wales, hence the name border disease.

[0003] Currently, sheep border virus (BDV) belongs to the genus Pestivirus in the family Flaviviridae. It is a single-stranded RNA virus that primarily infects pregnant ewes and fetuses, spreading vertically and horizontally. The virus induces fetal immune tolerance, leading to miscarriage, deformities, or persistent infection in lambs (such as tremors and coat abnormalities). Currently, this viral disease has a global distribution and poses a serious threat to the livestock industry. The main drawbacks of the traditional CRISPR / Cas9 system include carcinogenicity, genomic instability, and chromosome deletions. Therefore, the detection of sheep border virus is of paramount importance. Summary of the Invention

[0004] The object of the present invention is to provide a sheep border virus RPA-Crispr cas12a detection method to solve the problems raised in the above background technology.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a sheep border virus RPA-Crispr cas12a detection method, comprising the following contents:

[0006] S1: Obtain and collect relevant testing materials;

[0007] S2: Screening and designing crRNA;

[0008] S3: Results of crRNA screening;

[0009] S4: Perform RPA primer screening and design;

[0010] S5: Perform RPA reaction system work;

[0011] S6: Results of screening RPA primers;

[0012] S7: Conduct sensitivity testing;

[0013] S8: Perform specific testing work;

[0014] S9: Perform test strip detection test.

[0015] Preferably, step S1 includes the following:

[0016] S11: Obtain and collect samples of serum, whole blood, tissue homogenate or secretions;

[0017] S12: Obtain the BDV3 sequence by searching for literature related to border disease virus, perform species specificity analysis on NCBI, select specific regions as detection sites for RPA primer and crRNA design, synthesize DNA target fragments based on the designed positions of RPA primer and crRNA, and ligate them into the PUC57 vector to synthesize plasmids;

[0018] S13: Main reagents used: ① LbCas12a protease; ② Enzyme storage solution: 20mM NaAC; 500mM NaCl; 0.1mM EDTA; 0.1mM TCEP; 50% (v / v) Glycerol, pH 6.0; ③

[0019] Cleavage Buffer reaction solution: 10mM Tris-HCl, pH7.9; 50mM NaCl; 10mM MgCl; 100mL Recombinant Albumin, etc.

[0020] Preferably, step S2 includes the following:

[0021] S21: Screen crRNA components and design reaction steps;

[0022] S22: The reaction procedure was 40°C, and fluorescence was collected every 1 min for a total of 30 min.

[0023] Preferably, step S3 includes the following:

[0024] S31: Through screening, it was found that crRNA3 has the fastest fluorescence accumulation rate, that is, the highest activity, followed by crRNA2 and crRNA4, and crRNA1 also has high fluorescence.

[0025] Preferably, step S4 includes the following:

[0026] S41: Perform RPA amplification primer combinations and obtain final information based on different combinations.

[0027] Preferably, step S5 includes the following contents:

[0028] S51: extract 10 μL of the reaction product for electrophoresis verification;

[0029] S52: Set the temperature to 40°C and the reaction time to 30 minutes.

[0030] Preferably, step S6 includes the following contents:

[0031] S61: Based on the crRNA screening results, RPA primers are designed for the most efficient crRNA3 and the more efficient crRNA2 and crRNA4, and the RPA primers are screened after permutation and combination;

[0032] S62: By Figure 7 The gel electrophoresis results show that the bands amplified by RPA primers No. 5 and 6 are in the correct position and have good amplification effects. The corresponding crRNAs are cr2 and cr4. Among them, the RPA primer pair No. 6 has a better amplification effect and the fluorescence value of cr2 is higher. That is, the RPA primer pair F3R4 and crRNA2 are selected for the next experiment.

[0033] Preferably, step S7 includes the following contents:

[0034] S71: Dilute the target from its original concentration to 1000 copies / μL and 100 copies / μL to verify the sensitivity;

[0035] S72: The reaction procedure is: temperature 40°C, time 30 min;

[0036] S73: After the reaction is completed, 5 μL of RPA product is pipetted into the cleavage system;

[0037] S74: Results by Figure 8 and Figure 9 As shown, the target concentration is 1000 copies / μL, and samples with 100 copies / μL can be detected.

[0038] Preferably, step S8 includes the following:

[0039] S81: Seven sequences of bovine viral diarrhea virus (BVDV), Japanese encephalitis (JEV), goat pox virus (GPTV), goat enterovirus (EVG), sheep parainfluenza (CPV), peste des petits ruminants (PPV), and Brucella (BC) with clinical symptoms similar to BDV were selected for specificity testing.

[0040] Preferably, step S9 includes the following contents:

[0041] S91: The optimal RPA primer pair (F3R4) and optimal crRNA (crRNA2) screened by the test strip test were used for test strip test, as well as 10,000, 1,000, and 100 copies / μL targets;

[0042] S92: Reaction procedure: temperature 40°C, time 30 min;

[0043] S93: After the reaction is completed, add 20 μL of DEPC H2O to the tube to a total volume of 50 μL, mix well by pipetting, insert the test strip conjugate pad into the tube, wait 1 to 2 minutes until the quality control line (C line) develops color, remove the test strip and observe the results within 10 minutes.

[0044] Compared with the prior art, the present invention has the following advantages over the traditional CRISPR / Cas9 system:

[0045] First: Target recognition and cleavage mechanism: Cas12a directly recognizes the target sequence in double-stranded DNA through a single crRNA (requires a T-rich PAM sequence, such as TTTN), without the need for tracrRNA;

[0046] Second: It simplifies the crRNA design process: after target binding, Cas12a activates its non-specific single-stranded DNA (ssDNA) trans-cleavage activity, which can efficiently cut free fluorescent-quenched labeled reporter molecules to achieve signal amplification and visual output;

[0047] Third: High precision and anti-interference ability: Cas12a has an extremely low tolerance for single-base mismatches (<1%), making it particularly suitable for distinguishing closely related pathogens such as ovine border virus and bovine viral diarrhea virus (BVDV) of the same pestivirus genus. It has a specificity of >99.9%, and its cleavage activity is not interfered with by common inhibitors in samples (such as heme and polysaccharides), allowing direct detection of unpurified clinical samples.

[0048] Fourth, it is applicable to multiple scenarios: the constant temperature reaction conditions (37–42°C) are seamlessly compatible with the RPA amplification system, eliminating the need for a thermal cycler and making it suitable for deployment in multiple scenarios, including fields, laboratories, and mobile testing vehicles.

[0049] Fifth: Supports dual-mode output (fluorescence meter quantitative + test strip qualitative), meeting differentiated needs from scientific research-level precise quantification to grassroots rapid screening;

[0050] Sixth: Technical Scalability: By designing multi-target crRNAs or combining them with microfluidic chips, it is possible to simultaneously detect co-infecting pathogens such as sheep border virus (BDV) and foot-and-mouth disease virus (FMDV) and peste des petits ruminants (PPR) virus, achieving "one-tube, multiple-test" capabilities. Previously, based on the synergistic effect of recombinase polymerase amplification (RPA) and the CRISPR / Cas12a system, an RPA-CRISPR / Cas12a colloidal gold test strip for sheep border virus type 3 was constructed. This method has a sensitivity of 100 copies / reaction, a detection time of less than 1 hour, and good specificity. It showed no cross-reactivity with seven other pathogens with clinically similar symptoms to BDV: bovine viral diarrhea virus, Japanese encephalitis, sheep pox, sheep enterovirus, sheep parainfluenza, peste des petits ruminants (PPR), and Brucella. Further testing of clinical samples demonstrated a 100% positive coincidence rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Schematic diagram of the process of the present invention;

[0052] Figure 2 Schematic diagram of the crRNA screening reaction system of the present invention;

[0053] Figure 3 This is a schematic diagram of the BDV crRNA screening trend of the present invention;

[0054] Figure 4 This is a schematic diagram of crRNA screening of the present invention;

[0055] Figure 5 Schematic diagram of the combination of RPA amplification primers of the present invention;

[0056] Figure 6 Schematic diagram of the RPA reaction procedure of the present invention;

[0057] Figure 7 This is a schematic diagram of gel electrophoresis of the RPA amplification product of the present invention;

[0058] Figure 8 This is a schematic diagram of the BDV sensitivity detection trend of the present invention;

[0059] Figure 9 This is a schematic diagram of BDV sensitivity detection of the present invention;

[0060] Figure 10 This is a schematic diagram of the specific detection trend of the present invention;

[0061] Figure 11 Schematic diagram of specific detection of the present invention;

[0062] Figure 12 Schematic diagram of the test results of the test strip of the present invention. DETAILED DESCRIPTION

[0063] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0064] See also Figures 1 to 12 The present invention provides a technical solution: a sheep border virus RPA-Crispr cas12a detection method, comprising the following contents:

[0065] S1: Obtain and collect relevant testing materials;

[0066] S2: Screening and designing crRNA;

[0067] S3: Results of crRNA screening;

[0068] S4: Perform RPA primer screening and design;

[0069] S5: Perform RPA reaction system work;

[0070] S6: Results of screening RPA primers;

[0071] S7: Conduct sensitivity testing;

[0072] S8: Perform specific testing work;

[0073] S9: Perform test strip detection test.

[0074] Wherein, the step S1 includes the following contents:

[0075] S11: Obtain and collect samples of serum, whole blood, tissue homogenate or secretions;

[0076] S12: Obtain the BDV3 sequence by searching for literature related to border disease virus, perform species specificity analysis on NCBI, select specific regions as detection sites for RPA primer and crRNA design, synthesize DNA target fragments based on the designed positions of RPA primer and crRNA, and ligate them into the PUC57 vector to synthesize plasmids;

[0077] S13: Main reagents used: ① LbCas12a protease; ② Enzyme storage solution: 20mM NaAC; 500mM NaCl; 0.1mM EDTA; 0.1mM TCEP; 50% (v / v) Glycerol, pH 6.0; ③

[0078] Cleavage Buffer reaction solution: 10mM Tris-HCl, pH7.9; 50mM NaCl; 10mM MgCl; 100mL Recombinant Albumin, etc.

[0079] Wherein, the step S2 includes the following contents:

[0080] S21: Screen crRNA components and design reaction steps;

[0081] S22: The reaction procedure was 40°C, and fluorescence was collected every 1 min for a total of 30 min.

[0082] Wherein, the step S3 includes the following contents:

[0083] S31: Through screening, it was found that crRNA3 has the fastest fluorescence accumulation rate, that is, the highest activity, followed by crRNA2 and crRNA4, and crRNA1 also has high fluorescence.

[0084] Wherein, the step S4 includes the following contents:

[0085] S41: Perform RPA amplification primer combinations and obtain final information based on different combinations.

[0086] Wherein, the step S5 includes the following contents:

[0087] S51: extract 10 μL of the reaction product for electrophoresis verification;

[0088] S52: Set the temperature to 40°C and the reaction time to 30 minutes.

[0089] Wherein, the step S6 includes the following contents:

[0090] S61: Based on the crRNA screening results, RPA primers are designed for the most efficient crRNA3 and the more efficient crRNA2 and crRNA4, and the RPA primers are screened after permutation and combination;

[0091] S62: By Figure 7 The gel electrophoresis results show that the bands amplified by RPA primers No. 5 and 6 are in the correct position and have good amplification effects. The corresponding crRNAs are cr2 and cr4. Among them, the RPA primer pair No. 6 has a better amplification effect and the fluorescence value of cr2 is higher. That is, the RPA primer pair F3R4 and crRNA2 are selected for the next experiment.

[0092] Wherein, the step S7 includes the following contents:

[0093] S71: Dilute the target from its original concentration to 1000 copies / μL and 100 copies / μL to verify the sensitivity;

[0094] S72: The reaction procedure is: temperature 40°C, time 30 min;

[0095] S73: After the reaction is completed, 5 μL of RPA product is pipetted into the cleavage system;

[0096] S74: Results by Figure 8 and Figure 9 As shown, the target concentration is 1000 copies / μL, and samples with 100 copies / μL can be detected.

[0097] Wherein, the step S8 includes the following contents:

[0098] S81: Seven sequences with clinical symptoms similar to BDV (BDV plasmid sequence as shown in SEQ ID NO.1) including bovine viral diarrhea virus (BVDV, as shown in SEQ ID NO.3), Japanese encephalitis (JEV, as shown in SEQ ID NO.8), sheep pox (GPTV, as shown in SEQ ID NO.6), sheep enterovirus (EVG, as shown in SEQ ID NO.5), sheep parainfluenza (CPV, as shown in SEQ ID NO.7), peste des petits ruminants (PPV, as shown in SEQ ID NO.4), and Brucella (BC, as shown in SEQ ID NO.2) were selected for specificity testing.

[0099] Wherein, the step S9 includes the following contents:

[0100] S91: The optimal RPA primer pair (F3R4) and optimal crRNA (crRNA2) screened by the test strip test were used for test strip test, as well as 10,000, 1,000, and 100 copies / μL targets;

[0101] S92: Reaction procedure: temperature 40°C, time 30 min;

[0102] S93: After the reaction is completed, add 20 μL of DEPC H2O to the tube to a total volume of 50 μL, mix well by pipetting, insert the test strip conjugate pad into the tube, wait 1 to 2 minutes until the quality control line (C line) develops color, remove the test strip and observe the results within 10 minutes.

[0103] Specifically, when using the present invention, first obtain and collect sample serum, whole blood, tissue homogenate or secretion, obtain the BDV3 sequence by searching the border disease virus-related literature, perform interspecies specificity analysis on NCBI, and select the specific region as the detection site for RPA primer and crRNA design. According to the RPA primer and crRNA design position, the DNA target fragment is synthesized and connected to the PUC57 vector to synthesize the plasmid. The main reagents used are ①LbCas12a protease; ②Enzyme storage solution: 20mM NaAC; 500mM NaCl; 0.1mM EDTA; 0.1mM TCEP; 50% (v / v) Glycerol, pH 6.0; ③Cleavage Buffer reaction solution: 10mM Tris-HCl, pH 7.9; 50mM NaCl; 10mM MgCl; 100mLRecombinant Albumin, etc., according to test needs, a commercial nucleic acid extraction kit (such as a magnetic bead method or column method) can be used to extract viral RNA / DNA. If it is an RNA virus, reverse transcription (RT) must be performed first to generate cDNA, which can be used as an auxiliary work, and then the crRNA components are screened and the reaction steps are designed. The reaction program is 40°C, and fluorescence is collected every 1 minute for a total of 30 minutes (such as Figure 2 As shown in Figure 2), the screening work showed that crRNA3 had the fastest fluorescence accumulation rate, that is, the highest activity, followed by crRNA2 and crRNA4, and crRNA1 also had high fluorescence (as shown in Figure 2). Figure 3 and Figure 4 As shown), the RPA amplification primers are matched and combined, and the final information is obtained according to different matching combinations (as shown Figure 5 ), extract 10 μL of the reaction product for electrophoresis verification, set the temperature to 40°C, and the reaction time to 30 min (as shown Figure 6 As shown), according to the crRNA screening results, RPA primers were designed for the most efficient crRNA3 and the more efficient crRNA2 and crRNA4, and the RPA primers were screened after permutation and combination. Figure 7 The gel electrophoresis results show that the bands amplified by RPA primers No. 5 and 6 are in the correct position and have good amplification effects. The corresponding crRNAs are cr2 and cr4. Among them, the RPA primer pair No. 6 has a better amplification effect and the fluorescence value of cr2 is higher. That is, the RPA primer pair F3R4 and crRNA2 are selected for the next experiment. The target is diluted from the original concentration to 1000 copies / μL and 100 copies / μL to verify the sensitivity. The reaction procedure is: temperature 40°C, time 30min, and after the reaction, 5μL of RPA product is drawn and added to the cutting system. The results are shown in FIG. Figure 8 and Figure 9As shown, the target concentration was 1000 copies / μL, and samples with 100 copies / μL could be detected. Seven sequences of bovine viral diarrhea virus (BVDV), Japanese encephalitis (JEV), goat pox (GPTV), goat enterovirus (EVG), sheep parainfluenza (CPV), peste des petits ruminants (PPV), and Brucella (BC) with similar clinical symptoms to BDV were selected for specificity testing (e.g. Figure 10 and Figure 11 As shown), the optimal RPA primer pair (F3R4) and optimal crRNA (crRNA2) screened out by the test strip test, as well as 10000, 1000 and 100 copies / μL targets were tested for test strip detection: Reaction procedure: temperature 40°C, time 30 min, after the reaction, add 20 μL DEPC H2O to the tube to a total volume of 50 μL, pipette to mix, insert the test strip conjugate pad into the tube, wait 1 to 2 minutes until the quality control line (C line) develops color, remove the test strip and observe the results within 10 minutes (as shown Figure 12 shown).

[0104] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A sheep border virus RPA-Crispr cas12a detection method, characterized in that Includes the following: S1: Obtain and collect relevant testing materials; S2: Screening and designing crRNA; S3: Results of crRNA screening; S4: Perform RPA primer screening and design; S5: Perform RPA reaction system work; S6: Results of screening RPA primers; S7: Conduct sensitivity testing; S8: Perform specific testing work; S9: Perform test strip detection test.

2. The sheep border virus RPA-Crispr cas12a detection method according to claim 1, characterized in that: The step S1 includes the following contents: S11: Obtain and collect samples of serum, whole blood, tissue homogenate or secretions; S12: Obtain the BDV3 sequence by searching for literature related to border disease virus, perform species specificity analysis on NCBI, select specific regions as detection sites for RPA primer and crRNA design, synthesize DNA target fragments based on the designed positions of RPA primer and crRNA, and ligate them into the PUC57 vector to synthesize plasmids; S13: Main reagents used: ① LbCas12a protease; ② Enzyme storage solution: 20mM NaAC; 500mM NaCl; 0.1mM EDTA; 0.1mM TCEP; 50% (v / v) Glycerol, pH 6.0; ③ Cleavage Buffer reaction solution: 10mM Tris-HCl, pH7.9; 50mM NaCl; 10mM MgCl; 100mL Recombinant Albumin, etc.

3. The sheep border virus RPA-Crispr cas12a detection method according to claim 1, characterized in that: The step S2 includes the following contents: S21: Screen crRNA components and design reaction steps; S22: The reaction procedure was 40°C, and fluorescence was collected every 1 min for a total of 30 min.

4. The sheep border virus RPA-Crispr cas12a detection method according to claim 1, characterized in that: The step S3 includes the following contents: S31: Through screening, it was found that crRNA3 has the fastest fluorescence accumulation rate, that is, the highest activity, followed by crRNA2 and crRNA4, and crRNA1 also has high fluorescence.

5. The sheep border virus RPA-Crispr cas12a detection method according to claim 1, characterized in that: The step S4 includes the following contents: S41: Perform RPA amplification primer combinations and obtain final information based on different combinations.

6. The sheep border virus RPA-Crispr cas12a detection method according to claim 1, characterized in that: The step S5 includes the following contents: S51: extract 10 μL of the reaction product for electrophoresis verification; S52: Set the temperature to 40°C and the reaction time to 30 minutes.

7. The sheep border virus RPA-Crispr cas12a detection method according to claim 1, characterized in that: The step S6 includes the following contents: S61: Based on the crRNA screening results, RPA primers are designed for the most efficient crRNA3 and the more efficient crRNA2 and crRNA4, and the RPA primers are screened after permutation and combination; S62: From the gel electrophoresis results shown in Figure 7, it can be seen that the bands amplified by RPA primers No. 5 and 6 are in the correct position and have good amplification effects. The corresponding crRNAs are cr2 and cr4. Among them, the RPA primer pair No. 6 has a better amplification effect and the fluorescence value of cr2 is higher. That is, the RPA primer pair F3R4 and crRNA2 are selected for the next experiment.

8. The sheep border virus RPA-Crispr cas12a detection method according to claim 1, characterized in that: The step S7 includes the following contents: S71: Dilute the target from its original concentration to 1000 copies / μL and 100 copies / μL to verify the sensitivity; S72: The reaction procedure is: temperature 40°C, time 30 min; S73: After the reaction is completed, 5 μL of RPA product is pipetted into the cleavage system; S74: The results are shown in Figures 8 and 9. The target concentration is 1000 copies / μL, and samples with 100 copies / μL can be detected.

9. The sheep border virus RPA-Crispr cas12a detection method according to claim 1, characterized in that: The step S8 includes the following contents: S81: Seven sequences of bovine viral diarrhea virus (BVDV), epidemic encephalitis B, sheep pox, sheep enterovirus, sheep parainfluenza, peste des petits ruminants, and Brucella with clinical symptoms similar to BDV were selected for specificity testing.

10. The sheep border virus RPA-Crispr cas12a detection method according to claim 1, characterized in that: The step S9 includes the following contents: S91: The optimal RPA primers and optimal crRNA screened by the test strip test were used for test strip test, as well as 10,000, 1,000, and 100 copies / μL targets; S92: Reaction procedure: temperature 40°C, time 30 min; S93: After the reaction is completed, add 20 μL of DEPC H2O to the tube to a total volume of 50 μL, mix well by pipetting, insert the test strip conjugate pad into the tube, wait 1 to 2 minutes until the quality control line shows color, remove the test strip and observe the results within 10 minutes.

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