Primer and kit for detecting citrus broken leaf virus based on RPA-CRISPR-Cas12b and application of primer and kit

Abstract

The invention discloses a primer and a kit for detecting a citrus broken leaf virus based on RPA-CRISPR-Cas12b and application of the primer and the kit, and relates to the technical field of citrus pathogen detection. The invention develops an isothermal amplification detection method based on RPA and CRISPR for common CTLV pathogen targets in citrus, gets rid of the dependence of a traditional molecular biological method on a complex instrument and equipment platform, and greatly simplifies a detection platform; through system establishment and verification, the total detection time of the CTLV target is shortened to be within half an hour; meanwhile, a verification test result shows that the detection method developed by the invention has relatively good sensitivity and specificity, and an actual sample verification result is consistent with a result of an RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method.

Description

Technical Field

[0001] The present invention relates to the technical field of citrus pathogen detection, and particularly to primers, kits and applications for detecting Citrus tatter leaf virus based on RPA-CRISPR-Cas12b. Background Art

[0002] Citrus tatter leaf virus (CTLV) is one of the important pathogens infecting citrus. The CTLV virions are flexuous rods, about 600-700 nm × 13-15 nm in size. The thermal inactivation point is 65-70 °C (10 min), the dilution end point is 10-4-10-5, and the in vitro virus retention period is 4-8 d

[11] . It is mainly transmitted through grafting and contaminated tools, and the tatter leaf virus has a wide range of hosts in citrus. There are latent symptom phenomena in many citrus varieties, and symptoms only appear in some sensitive varieties, and there are certain differences in symptoms caused by different varieties, different inoculation conditions and different inoculation methods. After CTLV infects hybrids of trifoliate orange such as trifoliate orange, rough lemon, kumquat trifoliata, and varieties such as thick-skinned lime, it causes yellowing, graft incompatibility or abnormality, weak growth, poor fruit setting, and withering in severe cases.

[0003] One of the traditional detection methods for important graft-transmitted pathogens of citrus is the indicator plant identification method, which is quite time-consuming. At present, rapid detection methods for single pathogens have been established for the above-mentioned pathogens, including serological methods, RT-PCR / qPCR and detection methods based on loop-mediated isothermal nucleic acid amplification technology, etc. Among them, serological detection is a commonly used method for detecting common citrus viruses.

[0004] The CRISPR detection technology has high sensitivity and high specificity, and can quickly and accurately detect target nucleic acids under isothermal conditions, and is an ideal nucleic acid detection platform. Currently, CRISPR / Cas systems with trans-cleavage activity are found to include Cas13, Cas12a and Cas12b and other CRISPR / Cas systems. Compared with Cas12a, AaCas12b has a wider temperature tolerance range, and it has good detection activity in the temperature range of 37-65 °C, and is more suitable for synchronous isothermal amplification reactions such as RPA and LAMP.

[0005] Therefore, the present invention has developed an isothermal amplification detection method based on RPA and CRISPR for CTLV targets to get rid of the dependence on complex instrument equipment platforms by traditional molecular biology methods and greatly simplify the detection platform. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides primers, kits and applications for detecting Citrus tatter leaf virus based on RPA-CRISPR-Cas12b.

[0007] To achieve the above object, the technical solution of the present invention is as follows:

[0008] The present invention provides sgRNAs for detecting Citrus tristeza virus (CTLV). The sgRNAs for detecting CTLV are one of CTLV-94rev with the nucleotide sequence shown in SEQ ID NO: 1, CTLV-116forw with the nucleotide sequence shown in SEQ ID NO: 2, CTLV-39forw with the nucleotide sequence shown in SEQ ID NO: 3, CTLV-149forw with the nucleotide sequence shown in SEQ ID NO: 4, and CTLV-52rev with the nucleotide sequence shown in SEQ ID NO: 5.

[0009] The present invention also provides a reagent combination for detecting CTLV based on RPA-CRISPR-Cas12b, including RPA primer sequences and the above sgRNA sequences.

[0010] Preferably, the RPA primers include: upstream primer CTLVT6-RPA-F1 with the nucleotide sequence shown in SEQ ID NO: 6, upstream primer CTLVT6-RPA-F2 with the nucleotide sequence shown in SEQ ID NO: 7, upstream primer CTLVT6-RPA-F3 with the nucleotide sequence shown in SEQ ID NO: 8, downstream primer CTLVT6-RPA-R3 with the nucleotide sequence shown in SEQ ID NO: 9, and downstream primer CTLVT6-RPA-R21 with the nucleotide sequence shown in SEQ ID NO: 10.

[0011] Preferably, the pairing mode of the RPA primers is that upstream primer CTLVT6-RPA-F1 and downstream primer CTLVT6-RPA-R3 are used in pair, or upstream primer CTLVT6-RPA-F1 and downstream primer CTLVT6-RPA-R21 are used in pair, or upstream primer CTLVT6-RPA-F2 and downstream primer CTLVT6-RPA-R3 are used in pair, or upstream primer CTLVT6-RPA-F2 and downstream primer CTLVT6-RPA-R21 are used in pair, or upstream primer CTLVT6-RPA-F3 and downstream primer CTLVT6-RPA-R3 are used in pair, or upstream primer CTLVT6-RPA-F3 and downstream primer CTLVT6-RPA-R21 are used in pair, or upstream primers CTLVT6-RPA-F1, CTLVT6-RPA-F2, CTLVT6-RPA-F3 and downstream primers CTLVT6-RPA-R3, CTLVT6-RPA-R21 are used together.

[0012] The present invention also provides a kit for detecting Citrus tristeza virus based on RPA-CRISPR-Cas12b, including the above reagent combination.

[0013] The present invention also provides the application of the above kit in the detection of Citrus tristeza virus.

[0014] The present invention also provides a method for detecting Citrus tristeza virus based on RPA-CRISPR-Cas12b, comprising the following steps:

[0015] S1. Extract total RNA from the sample to be tested and reverse transcribe it into cDNA;

[0016] S2. Using the total cDNA in step S1 as a template, perform RPA isothermal amplification with the above RPA primers to obtain RPA amplification products;

[0017] S3. Using the RPA amplification products obtained in step S2 as a template, add them to the Cas12b detection reaction system to perform CRISPR reaction;

[0018] S4. After the CRISPR reaction is completed, read the detection signal and determine the detection result according to the detection signal.

[0019] The judgment criterion of the present invention is that using water template as a negative control, compare whether there is a significant difference between the detection data and the negative control data. If there is no significant difference, it is determined to be negative; if there is a significant difference, it is determined to be positive.

[0020] Preferably, the Cas12b detection reaction system is 2 μL of 2.5 μM SynSorAaCas12b, 1 μL of 100 ng / μL SynSor sgRNA, 5 μL of 10×AaCas12b Buffer, 0.5 μL of SynSor CRISPR ssDNA Reporter, and supplemented with water to 50 μL.

[0021] The present invention also provides the application of the above method in the detection of Citrus tristeza virus.

[0022] In summary, compared with the prior art, the solution of the present invention has the following beneficial effects:

[0023] The isothermal amplification detection method based on RPA and CRISPR provided by the present invention gets rid of the dependence on complex instrument equipment platforms of traditional molecular biology methods, greatly simplifies the detection platform; through the establishment and verification of the system, the overall detection time of the CTLV target is shortened to within half an hour; at the same time, the verification test results show that the detection method developed by the present invention has good sensitivity and specificity, and the verification results of actual samples are consistent with the results of the RT-PCR method. Description of the Drawings

[0024] Figure 1 It is the screening result diagram of CTLV target sgRNA in the present invention (NTC is the negative control, and CTLV-116forw, CTLV-39forw, CTLV-149forw, CTLV-94rev, and CTLV-52rev are all sgRNAs);

[0025] Figure 2 It is the screening result diagram of CTLV target RPA primers in the present invention (NTC is the negative control, and CTLV-RPA-F1R3, CTLV-RPA-F1R21, CTLV-RPA-F2R3, CTLV-RPA-F2R21, CTLV-RPA-F3R3, and CTLV-RPA-F3R21 are all primer combinations);

[0026] Figure 3 It is the sensitivity verification result diagram of CTLV target in the present invention (NTC is the negative control, CEVd is citrus exocortis viroid, CTV is citrus tristeza virus, and CTLV is citrus tatter leaf virus);

[0027] Figure 4 It is the specificity verification result diagram of CTLV target in the present invention (NTC is the negative control, 5.62E+00 represents 5.62×10 0 、5.62E-01 represents 5.62×10 -1 、5.62E-02 represents 5.62×10 -2 、5.62E-03 represents 5.62×10 -3 、5.62E-04 represents 5.62×10 -4 、5.62E-05 represents 5.62×10 -5 、5.62E-06 represents 5.62×10 -6 、5.62E-07 represents 5.62×10 -7 、5.62E-08 represents 5.62×10 -8 and 5.62E-09 represents 5.62×10 -9 ; * indicates p<0.05, and ns indicates no statistical significance);

[0028] Figure 5 It is the detection result diagram of CTLV target citrus leaf samples in the present invention (NTC is the negative control, CEVd is citrus exocortis viroid, CTV is citrus tristeza virus, and CTLV is citrus tatter leaf virus). Detailed implementation manners

[0029] To enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments and drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative work shall fall within the scope of protection of the present invention.

[0030] Example 1 Design of sgRNA

[0031] According to the commonly used target regions of citrus tristeza virus and combined with the host background genome to be avoided, bioinformatics algorithms are used to score the design of sgRNA from the perspectives of PAM position, GC%, internal dimer structure, fragment structure openness, base position preference, specificity, etc. The scoring is understood in terms of the success probability. Three candidate fragments with scores above 40 are retained. If the scores are generally lower than 40, the number of candidate fragments needs to be increased to improve the success probability. If the sequence in the spacer has a risk of disrupting the secondary structure of the backbone, it will be directly excluded from the candidate list to avoid risks of specificity and sensitivity.

[0032] Based on the above design principles, sgRNA is designed for each selected region. The spacer sequences of the designed sgRNA are analyzed for coverage and specificity using NCBI primer - BLAST. The alignment results show that the spacer sequences of the designed sgRNA have a 100% coverage within the detected species and good inclusiveness; the number of bases matching across species is <15 nt, indicating good specificity. In this example, the sgRNA in Table 1 is selected for synthesis.

[0033] Table 1 Information of sgRNA used

[0034]

[0035]

[0036] Note: The underlined part is the spacer region of sgRNA.

[0037] Example 2 Verification of sgRNA Performance

[0038] In this example, the fluorescence intensity of the CRISPR reaction is measured for the amplification products of each sgRNA and the corresponding templates designed in Example 1.

[0039] 1. Design of PCR primers

[0040] According to the designed position of the sgRNA, regions within 100 bp upstream and downstream of the sgRNA are selected to design PCR primers (see Table 2). Commonly used software for designing PCR primers is utilized for primer design, and the primer length is controlled within 20 - 25 bp. If there are multiple sgRNA design sites within the same target fragment, the PCR amplification product (with the fragment length controlled within 500 bp) will contain all the sgRNA binding sites, enabling performance verification of all sgRNAs under the same template.

[0041] Table 2 Specific Primers for Template Amplification

[0042]

[0043] 2. Template Amplification Verification

[0044] Use the template DNA and the corresponding primers for PCR amplification to obtain a high - concentration PCR product.

[0045] PCR reaction system: 44 μL of 1.1× GoldMix (Green), 2 μL each of the upstream primer / downstream primer (10 μM), 2 μL of plasmid template or genomic / cDNA template, and water is added to make up to 50 μL.

[0046] PCR reaction program: Pre - denaturation at 98°C for 2 min, denaturation at 98°C for 10 sec, annealing at 50°C for 15 sec, extension at 72°C for 10 sec, 30 cycles, final extension at 72°C for 5 min, and incubation at 10°C for storage.

[0047] 3. sgRNA Verification

[0048] Configure the CRISPR system: 2 μL of SynSor AaCas12b (2.5 μM), 1 μL of SynSor sgRNA (100 ng / μL), 5 μL of 10× AaCas12b Buffer, 0.5 μL of SynSor CRISPR ssDNA Reporter (12b - FAM), and water is added to make up to 50 μL.

[0049] Take 1 μL of the PCR amplification product and mix it with the CRISPR system. Set the reaction at 43°C for 15 min in a qPCR instrument, and collect FAM fluorescence signals every minute. According to the curve changes of the fluorescence signals, conduct a preliminary verification of the sgRNA performance or specificity. There should be a significant difference in the end points where the fluorescence value curves of the target amplification product and the negative control stop rising.

[0050] The calculation formula for the fluorescence intensity growth rate is as follows:

[0051]

[0052] Among them, Fluorescence Slope represents the fluorescence growth rate in the present invention, Rn represents the fluorescence signal at the nth minute, R1 represents the fluorescence signal at the first minute, and n represents the reaction minute number.

[0053] The results showed that the sgRNA effects of CTLV targets ranked from high to low as CTLV-116forw, CTLV-39forw, CTLV-149forw, CTLV-94rev, CTLV-52rev (see Figure 1 ).

[0054] Example 3 Design and Verification of PRA Primers

[0055] 1. RPA Primer Design

[0056] Within the detection target range, fragments with a length of 30 - 35 bases were selected as RPA primer candidates; to ensure stability and specificity, the GC content of RPA primers should be between 40% - 60%, and the RPA amplification fragment is usually 100 - 200 bp. Based on this primer design principle, 3 upstream primers and 3 downstream primers were designed for each template (see Table 3), and NCBI primer - BLAST was used for amplification coverage and specificity verification.

[0057] Table 3 RPA Primer Table

[0058]

[0059] 2. RPA Primer Screening

[0060] CTLV-116forw with better verification performance in Example 2 was used for RPA primer screening.

[0061] Cross - primer performance verification was carried out on the synthesized upstream primers CTLV - RPA - F1, F2, F3 and downstream primers CTLV - RPA - R3, R21 in the present invention. The results showed that the better primer screening effects for CTLV targets were: CTLV - RPA - F3 / CTLV - RPA - R21, followed by CTLV - RPA - F3 / CTLV - RPA - R3, CTLV - RPA - F1 / CTLV - RPA - R21 (see Figure 2 ).

[0062] Example 4 Sensitivity Verification of Citrus Tristeza Virus Detection System

[0063] The PAR primer CTLV - RPA - F3 / CTLV - RPA - R21 with better screening and verification in Example 3 was used for sensitivity verification with gradient - diluted plasmids. The detection limits of each primer and the corresponding target were judged according to the significant analysis of the sensitivity verification experiment results.

[0064] Plasmid samples with a concentration gradient of 5.62E-09 to 5.62E+01 ng were used to verify the sensitivity of Citrus tristeza virus. According to the sensitivity verification results (see Figure 3 ), and combined with the analysis of significant differences, the lowest detectable sample of the CTLV target per reaction was 5.62 fg.

[0065] Example 5 Specificity verification of the Citrus tristeza virus detection system

[0066] The PAR primers CTLV-RPA-F3 / CTLV-RPA-R21 with better screening and verification in Example 3 were used to verify the target specificity of three pathogens, CTLV, CTLV and CTV, respectively.

[0067] The CTLV target system only produced positive results in the CTLV target detection. The detection of CTV and CTLV plasmid DNA showed negative results, indicating that the target had good specificity (see Figure 4 ).

[0068] Example 6 Verification of samples

[0069] The RPA primers determined by screening in Example 3 for the detection of Citrus split leaf virus, the detection system established based on the above primers, and the existing RT-PCR method were used to detect and verify 22 target nucleic acid samples. The target plasmid sample was used as a positive control, and ddH2O was used as a negative control. Each sample was subjected to a repeated verification experiment twice.

[0070] The RT-PCR method is specifically as follows:

[0071] Using the extracted total RNA of the sample as a template, cDNA was reverse transcribed. The system was: dNTP Mix (10 mM) 1 μL, 5×M-MLV buffer 4 μL, M-MLV reverse transcriptase (200 U·μl -1 , PROMEGA) 0.5 μL, RNaseinhibitor (40 U·μl -1 , PROMEGA) 0.5 μL, random hexamer primer (10 μM) 0.5 μL, Oligo(dT)18 primer (10 μM) 0.5 μL, and made up to 20 μL with sterilized ddH2O. After mixing, it was incubated at 37°C for 1 h to synthesize cDNA.

[0072] PCR reaction system: 1 μL of cDNA, 7.5 μL of 2× Taq mix (A005-100, Kangrun Biotech), 0.6 μL of CTLV-F (5'-CCCTCTCAGCTAGAATTGAA-3', concentration 10 μM), 0.6 μL of CTLV-R (5'-AGAGTGGACAAACTCTAGAC-3', concentration 10 μM), made up to 15 μL with sterilized ddH2O, expected fragment size 889 bp; reaction conditions: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 sec, annealing at 54°C for 30 sec, extension at 72°C for 50 sec, 35 cycles; further extension at 72°C for 10 min, analyzed by 1% agarose gel electrophoresis.

[0073] The results showed that positive results could be detected in the CTLV target system in all samples except D5-4 ( Figure 5 ), and were consistent with the results of the RT-PCR method.

[0074] In summary, in the present invention, the CTLV target sgRNA uses CTLV-116forw, combined with the primers CTLV-RPA-F3 / CTLV-RPA-R21, the lowest detection limit is 5.62 pg, and it is specific for the pathogens CTLV and CTV; the results in the actual sample detection are consistent with the results of the RT-PCR method.

[0075] The above-described embodiments only represent the preferred embodiments of the present invention, and their descriptions are relatively specific and detailed, but should not be construed as limiting the scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims

1. An sgRNA for detecting citrus leaf crushing virus, characterized in that: The sgRNA for detecting CTLV is one of CTLV-94rev as shown in SEQ ID NO: 1, CTLV-116forw as shown in SEQ ID NO: 2, CTLV-39forw as shown in SEQ ID NO: 3, CTLV-149forw as shown in SEQ ID NO: 4, and CTLV-52rev as shown in SEQ ID NO:

5.

2. A reagent combination for detecting citrus leaf crushing virus based on RPA-CRISPR-Cas12b, characterized in that: Comprising an RPA primer sequence and the sgRNA sequence described in claim 1.

3. The reagent combination according to claim 2, characterized in that: The RPA primers include: an upstream primer CTLVT6-RPA-F1 whose nucleotide sequence is shown in SEQ ID NO:6, an upstream primer CTLVT6-RPA-F2 whose nucleotide sequence is shown in SEQ ID NO:7, an upstream primer CTLVT6-RPA-F3 whose nucleotide sequence is shown in SEQ ID NO:8, a downstream primer CTLVT6-RPA-R3 whose nucleotide sequence is shown in SEQ ID NO:9, and a downstream primer CTLVT6-RPA-R21 whose nucleotide sequence is shown in SEQ ID NO:

10.

4. The reagent combination according to claim 2, characterized in that: The pairing of the RPA primers is as follows: the upstream primer CTLVT6-RPA-F1 is paired with the downstream primer CTLVT6-RPA-R3, or the upstream primer CTLVT6-RPA-F1 is paired with the downstream primer CTLVT6-RPA-R21, or the upstream primer CTLVT6-RPA-F2 is paired with the downstream primer CTLVT6-RPA-R3, or the upstream primer CTLVT6-RPA-F2 is paired with the downstream primer CTLVT6-RPA-R2 1, or the upstream primer CTLVT6-RPA-F3 and the downstream primer CTLVT6-RPA-R3 are used in pair, or the upstream primer CTLVT6-RPA-F3 and the downstream primer CTLVT6-RPA-R21 are used in pair, or the upstream primers CTLVT6-RPA-F1, CTLVT6-RPA-F2, CTLVT6-RPA-F3 and the downstream primers CTLVT6-RPA-R3, CTLVT6-RPA-R21 are used together.

5. A kit for detecting citrus leaf crushing virus based on RPA-CRISPR-Cas12b, characterized in that: The invention comprises the reagent combination described in any one of claims 2 to 3.

6. Use of the kit for detecting citrus leaf crushing virus based on RPA-CRISPR-Cas12b as claimed in claim 5 in the detection of citrus leaf crushing virus.

7. A method for detecting citrus leaf crushing virus based on RPA-CRISPR-Cas12b, characterized in that: The steps include: S1, extract total RNA from the sample to be tested and reverse transcribe it into cDNA; S2, using the total cDNA in step S1 as a template, performing RPA isothermal amplification using the RPA primers described in claim 3 or 4 to obtain an RPA amplification product; S3, using the RPA amplification product obtained in step S2 as a template, adding the Cas12b detection reaction system to perform CRISPR reaction; S4. After the CRISPR reaction is completed, the detection signal is read and the detection result is determined based on the detection signal.

8. The method according to claim 7, characterized in that The Cas12b detection reaction system is 2 μL of 2.5 μM SynSorAaCas12b, 1 μL of 100 ng / μL SynSor sgRNA, 5 μL of 10×AaCas12b Buffer, 0.5 μL of SynSor CRISPRssDNA Reporter and water to 50 μL.

9. Use of the method according to claim 7 or 8 in the detection of citrus leaf crushing virus.

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