RT-RAA-CRISPR / Cas12a-based visual detection kit for passiflora edulis virus and application of RT-RAA-CRISPR / Cas12a-based visual detection kit
By using the RT-RAA-CRISPR/Cas12a system, combined with specific RAA primers and crRNA, a rapid, accurate, and convenient detection of passion fruit virus in East Asia has been achieved. This solves the detection problems in existing technologies, improves sensitivity and specificity, and is suitable for the detection of passion fruit virus in resource-scarce areas.
Patent Information
- Application Number
- CN202511001744.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-21
- Publication Date
- 2025-10-28
AI Technical Summary
Existing technologies are insufficient for quickly, accurately, and easily distinguishing and detecting East Asian passion fruit virus from other passion fruit viruses, especially in resource-scarce regions. Furthermore, existing methods are susceptible to false positives and are highly dependent on laboratory facilities.
The reverse transcription recombinase-mediated amplification (RT-RAA) technology combined with the CRISPR/Cas12a system was used. Specific RAA primer pairs and crRNA were designed, and Cas12a protein was used for detection. Visual detection was achieved by combining fluorescent reporter DNA.
It achieves rapid, accurate, simple, and highly specific detection of passion fruit virus in East Asia, can distinguish multiple passion fruit viruses, has a sensitivity 100 times higher than existing methods, requires no complex instruments, shortens the detection time, and provides reliable results.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a visualization detection kit for East Asian passion fruit virus based on RT-RAA-CRISPR / Cas12a and its application. Background Technology
[0002] Passionflower ( Passiflora edulis Passion fruit, also known as granadilla, belongs to the genus Passiflora in the family Passifloraceae. Passifora Passionflower is a perennial vine. As an important economic crop, it has edible, medicinal, and ornamental value, playing a key role in promoting rural industrial revitalization and possessing significant economic importance. It is currently widely distributed and cultivated in tropical and subtropical regions. Passionflower was introduced to my country in 1913 and is now cultivated in provinces and autonomous regions such as Guangxi, Guangdong, Guizhou, and Fujian.
[0003] East Asian Passiflora virus (EAPV) is a virus belonging to the Potato Virus Y family (Potatovirus Y). Potyviridae Potato virus Y ( ) Potyvirus EAPV (Extracorporeal Antiviral Disease) is a member of the EAPV family, first reported on passion fruit in Japan, and now distributed in Japan, South Korea, Vietnam, Malaysia, Uganda, and southern my country. In recent years, with the large-scale cultivation of passion fruit, viral diseases have become increasingly serious. Symptoms often include stunted growth of the plant as a whole, mottled, yellowing, wrinkled, and mosaic-like leaves, and shrunken, deformed, and lignified fruits. In severe cases, it can cause plant stunting or even death. EAPV poses a significant threat to passion fruit production safety, causing mosaic leaves, deformed and spotted fruits, discoloration of the peel, and lignification. It can cause passion fruit lignification disease, resulting in severe damage and almost complete loss of commercial value. Using virus-free seeds and seedlings that have undergone rigorous virus testing is the fundamental measure to break the transmission chain of passion fruit viral diseases. Therefore, developing a simple, rapid, and accurate EAPV detection method is crucial for early diagnosis of EAPV, ensuring virus-free seedlings, and effective field control.
[0004] Passion fruit has a high incidence of viruses, and co-infection is severe. The symptoms caused by various viruses (leaf mottling, curling, wrinkling, fruit malformation, lignification, etc.) are extremely similar, making it difficult to distinguish East Asian Passion Fruit Virus (EAPV) from other passion fruit viruses based on symptoms alone. Currently, the main methods used for detecting and identifying EAPV include tube capture reverse transcription-polymerase chain reaction (TC-RT-PCR), reverse transcription-polymerase chain reaction (RT-PCR), and reverse transcription loop-mediated isothermal amplification (RT-LAMP). Because PCR methods require sophisticated laboratory infrastructure and involve steps such as pre-denaturation, denaturation, annealing, and extension, they are time-consuming and limit their use in resource-scarce grassroots units. While RT-LAMP, a widely used isothermal amplification technique in recent years, does not require complex laboratory conditions, is quick, and simple to operate, it also suffers from problems such as complex primer design and the risk of false positives. Therefore, there is an urgent need to establish a simple, rapid, accurate, highly sensitive, and highly specific method for the rapid on-site detection of passion fruit virus in East Asia. Summary of the Invention
[0005] The purpose of this invention is to provide a simple, rapid, accurate, highly sensitive, and highly specific method for the rapid on-site detection of East Asian passion fruit virus. This method can not only accurately distinguish East Asian passion fruit virus from other Potato Y virus viruses in the same genus, such as telosma mosaic virus (TeMV), turnip mosaic virus (TuMV), and passion fruit severe mottle-associated virus (PFSMaV), but also accurately distinguish East Asian passion fruit virus from other viruses in the same genus that are infectious to passion fruit, such as hibiscus latent Fort Pierce virus (HLFPV), cucumber mosaic virus (CMV), and passion fruit latent virus (PLV).
[0006] To achieve the above objectives, the present invention first provides a complete set of reagents for identifying or assisting in the identification of East Asian passion fruit virus.
[0007] The complete set of reagents provided by this invention for identifying or assisting in the identification of East Asian passion fruit virus includes RAA primer pairs; The RAA primer pair consists of primer-F and primer-R; The primer-F is either a1) or a2) as follows: a1) The single-stranded DNA molecule shown in sequence 1; a2) A single-stranded DNA molecule that has undergone substitution and / or deletion and / or addition of one or more nucleotides of sequence 1 and has the same function as sequence 1; The primer-R is either a3) or a4) as follows: a3) The single-stranded DNA molecule shown in sequence 2; a4) A single-stranded DNA molecule that has one or more nucleotides of sequence 2 replaced and / or deleted and / or added, and has the same function as sequence 2.
[0008] In the above-mentioned reagent kit, the molar ratio of primer-F to primer-R is 1:1.
[0009] Furthermore, the kit also includes crRNA corresponding to the RAA primer pair; The crRNA is either a5) or a6) as follows: a5) The single-stranded RNA molecule shown in sequence 3; a6) A single-stranded RNA molecule with one or more nucleotides substituted and / or deleted and / or added to sequence 3 and having the same function as sequence 3.
[0010] Furthermore, the kit also includes Cas12a protein and reporter DNA.
[0011] The Cas12a protein is selected from any of the following proteins: AsCas12a, LbCas12a, Lb4Cas12a, Lb5Cas12a, FnCas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, and BoCas12a.
[0012] In some embodiments, the Cas12a protein is the LbCas12a(Cpf1) protein.
[0013] The reporter DNA is a DNA molecule with signal reporting function. When the DNA molecule is cleaved and degraded by the Cas12a protein, it can report a positive signal and be detected.
[0014] In some implementations, the reporter DNA is a single-stranded DNA with a fluorescent group attached to one end and a quencher group attached to the other end.
[0015] The fluorescent group is selected from any one of the following groups: FAM, 6-FAM, VIC, HEX, TRT, CY3, CY5, ROX, JOE, FITC, TET, NED, TAMRA, LC RED640, LC RED705, Quasar705, Texas Red.
[0016] The quenching group is selected from any one of the following groups: TAMRA, BHQ1, BHQ2, BHQ3, MGB, Dabcy1.
[0017] In a preferred embodiment, the reporter DNA is a single-stranded DNA with a FAM fluorescent group attached to its 5' end and a BHQ1 quencher group attached to its 3' end. The nucleotide sequence of the single-stranded DNA is as follows: TTATT.
[0018] To achieve the above objectives, the present invention provides new uses for the above-mentioned reagent kit, the above-mentioned RAA primer pair, or the above-mentioned crRNA.
[0019] This invention provides the application of the above-described reagent kit, the above-described RAA primer pair, or the above-described crRNA in any one of the following b1)-b8): b1) Identification or auxiliary identification of East Asian passion fruit virus or its nucleic acid; b2) Detect or assist in the detection of whether the sample to be tested contains East Asian passion fruit virus or its nucleic acid; b3) To differentiate or assist in differentiating East Asian passion fruit virus from other viruses capable of infecting passion fruit; b4) Prevention or assisted prevention of East Asian passion fruit virus; b5) Prepare products for identification or auxiliary identification of East Asian passion fruit virus or its nucleic acid; b6) Prepare products for detecting or assisting in the detection of whether the sample to be tested contains East Asian passion fruit virus or its nucleic acid; b7) Prepare products that can differentiate or help differentiate East Asian passion fruit virus from other viruses that are capable of infecting passion fruit; b8) Prepare products for the prevention or assistance in the prevention and control of East Asian passion fruit virus.
[0020] To achieve the above objectives, the present invention also provides a kit for identifying or assisting in the identification of East Asian passion fruit virus.
[0021] The kit for identifying or assisting in the identification of East Asian passion fruit virus provided by this invention contains the above-mentioned complete set of reagents; the function of the kit is any one of c1)-c4) below: c1) Identification or auxiliary identification of East Asian passion fruit virus or its nucleic acid; c2) Detect or assist in the detection of whether the sample to be tested contains East Asian passion fruit virus or its nucleic acid; c3) To differentiate or assist in differentiating East Asian passion fruit virus from other viruses capable of infecting passion fruit; c4) Prevention or assisted prevention of East Asian passion fruit virus.
[0022] Furthermore, the kit also includes other reagents for performing RT-RAA amplification, such as buffer V and magnesium acetate I.
[0023] Furthermore, the kit also includes other reagents for CRISPR / Cas12a detection, such as 10×Cas12a Reaction Buffer.
[0024] Furthermore, the kit also includes a negative control (such as an RNA sample from the leaf or fruit epidermis of a healthy passionflower) and a positive control (such as an RNA sample from the leaf or fruit epidermis of a passionflower carrying East Asian passionflower virus).
[0025] To achieve the above objectives, the present invention also provides a method for detecting or assisting in the detection of whether a sample to be tested contains East Asian passion fruit virus.
[0026] The method for detecting or assisting in the detection of whether a sample contains Passifloxacin virus provided by the present invention includes the following steps: extracting RNA from the sample to be tested; using the RNA of the sample to be tested as a template, performing an RT-RAA amplification reaction using the above-mentioned RAA primer pair to obtain an RT-RAA amplification product; adding the above-mentioned crRNA, the above-mentioned Cas12a protein and the above-mentioned reporter DNA to the RT-RAA amplification product to perform a CRISPR / Cas12a detection reaction; and determining whether the sample contains Passifloxacin virus based on the CRISPR / Cas12a detection reaction result.
[0027] In the above-mentioned method for detecting or assisting in the detection of whether a sample contains Passifloxacin, the method for determining whether a sample contains Passifloxacin based on the CRISPR / Cas12a detection reaction results is as follows: After the reaction is completed, observe under ultraviolet light. If the CRISPR / Cas12a detection reaction system is green, the sample contains (or carries) Passifloxacin; if the CRISPR / Cas12a detection reaction system is colorless and transparent, the sample does not contain (or does not carry) Passifloxacin.
[0028] To achieve the above objectives, the present invention finally provides a method for distinguishing or assisting in distinguishing whether the virus to be tested in a virus-infected passion fruit sample is East Asian passion fruit virus or other viruses capable of infecting passion fruit.
[0029] The method provided by this invention for distinguishing or assisting in distinguishing whether a virus to be tested in a passion fruit sample infected with a virus is East Asian passion fruit virus or other viruses capable of infecting passion fruit includes the following steps: extracting RNA of the virus to be tested from the passion fruit sample; using the RNA of the virus to be tested as a template, performing an RT-RAA amplification reaction using the above-mentioned RAA primer pair to obtain an RT-RAA amplification product; adding the above-mentioned crRNA, Cas12a protein and reporter DNA to the RT-RAA amplification product to perform a CRISPR / Cas12a detection reaction; and determining whether the virus to be tested is East Asian passion fruit virus or other viruses capable of infecting passion fruit based on the results of the CRISPR / Cas12a detection reaction.
[0030] The method described above for distinguishing or assisting in distinguishing whether the virus to be tested in a passion fruit sample infected with a virus is East Asian passion fruit virus or other viruses capable of infecting passion fruit, wherein the method for determining whether the virus to be tested is East Asian passion fruit virus or other viruses capable of infecting passion fruit based on the CRISPR / Cas12a detection reaction results is as follows: After the reaction is completed, observe under ultraviolet light. If the CRISPR / Cas12a detection reaction system is green, then the virus to be tested is East Asian passion fruit virus; if the CRISPR / Cas12a detection reaction system is colorless and transparent, then the virus to be tested is other viruses capable of infecting passion fruit.
[0031] The system for any of the above-described RT-RAA amplification reactions includes buffer V, primer-F, primer-R, and magnesium acetate I. The final concentration of primer-F and primer-R in the RT-RAA amplification reaction system is preferably 0.5 μmol·L⁻¹. -1 .
[0032] In some embodiments, the RT-RAA amplification reaction is performed as follows: buffer V 25 μL, primer-F (10 µmol·L⁻¹) -1 2.5 μL of the above primer-R (10 µmol·L⁻¹) -1 2.5 μL, magnesium acetate I 5 μL, template RNA 2 μL, nuclease-free water 13 μL.
[0033] The conditions for any of the above-mentioned RT-RAA amplification reactions are 37-45℃ for 10-40 min, preferably 43℃ for 20 min.
[0034] The system of any of the above-described CRISPR / Cas12a detection reactions includes 10×Cas12a Reaction Buffer, the above-described Cas12a protein, the above-described crRNA, and the above-described reporter DNA.
[0035] The final concentration of the crRNA in the CRISPR / Cas12a detection reaction system is preferably 120 nmol·L⁻¹. -1 .
[0036] The final concentration of the Cas12a protein in the CRISPR / Cas12a detection reaction system is preferably 100 nmol·L⁻¹. -1 .
[0037] The final concentration of the reporter DNA in the CRISPR / Cas12a detection reaction system is preferably 800 nmol·L⁻¹. -1 .
[0038] In some embodiments, the CRISPR / Cas12a detection reaction system is as follows: 2.5 μL of 10×Cas12aReaction Buffer, and the above-mentioned Cas12a protein (5 µmol·L⁻¹). -1 0.5 μL of the above crRNA (10 µmol·L⁻¹) -1 0.3 μL of the above reporter DNA (10 µmol·L⁻¹) -1 2 μL of RT-RAA amplification product and 17.7 μL of ddH2O.
[0039] The conditions for any of the above-mentioned CRISPR / Cas12a detection reactions can be 41-45℃ for 5-20 min, preferably 43℃ for 20 min.
[0040] In any of the above-described applications, kits, or methods, the test sample includes different tissue samples from passion fruit that can be infected by East Asian passion fruit virus, specifically leaf samples or fruit epidermal samples from passion fruit.
[0041] In any of the above-described applications, kits, or methods, the other viruses capable of infecting passion fruit include other viruses of the Potato Y genus that belong to the same genus as East Asian Passion Fruit Virus and other viruses of other virus genera capable of infecting passion fruit.
[0042] In some embodiments, the other Potato Virus Y genus viruses belonging to the same genus as East Asian Passion Fruit Virus include, but are not limited to, at least one of the following viruses: telosma mosaic virus (TeMV), turnip mosaic virus (TuMV), and passion fruit severe mottle-associated virus (PFSMaV).
[0043] In some embodiments, the other viral genera capable of infecting passion fruit are other viral genera capable of infecting passion fruit besides potato virus Y, including but not limited to at least one of the following genera: tobacco mosaic virus (Tobacco Mosaic Virus). Tobamovirus ), Cucumber mosaic virus genus ( Cucumovirus ) and Carnation Latent Virus ( Carlavirus The tobacco mosaic virus genus ( ). Tobamovirus The representative species is Hibiscus latent Fort Pierce virus (HLFPV). The genus Cucumber Mosaic Virus (HLFPV) is also mentioned. Cucumovirus The representative species is cucumber mosaic virus (CMV). The carnation latent virus genus (…) Carlavirus The representative species is Passiflora latentvirus (PLV).
[0044] This invention develops a highly efficient RT-RAA-CRISPR / Cas12a visualization detection method for EAPV based on reverse transcription recombinase-mediated amplification (RT-RAA) technology and the CRISPR / Cas12a system. Optimal primer and probe concentrations, reaction temperature, and reaction time were determined through optimization experiments. Experiments demonstrate that the EAPV visualization detection method based on RT-RAA-CRISPR / Cas12a developed in this invention exhibits strong specificity and high sensitivity. It can accurately distinguish East Asian passion fruit virus from other viruses capable of infecting passion fruit, and its sensitivity is 100 times higher than that of RT-PCR and RT-RAA methods. Furthermore, this method eliminates the need for complex operations and sophisticated instruments such as agarose gel electrophoresis. The entire detection process can be completed in just 20 minutes at 43°C, significantly shortening the detection time compared to RT-PCR. Visual detection can be achieved under ultraviolet or blue light, resulting in a significant improvement in virus detection efficiency. This invention further employs this method and RT-PCR to detect 63 passion fruit samples suspected of being infected with EAPV collected from the field. The results from both methods were completely consistent, demonstrating the accuracy and reliability of the method presented in this invention. In summary, the RT-RAA-CRISPR / Cas12a visual detection method for EAPV established in this invention is simple, rapid, accurate, highly sensitive, specific, and visual. It can not only accurately distinguish East Asian passion fruit virus from other potato virus genera (Y virus) within the same genus, but also accurately distinguish East Asian passion fruit virus from other virus genera capable of infecting passion fruit. This provides a more convenient and efficient technical means for field diagnosis and forecasting of EAPV in my country. Furthermore, to prevent the spread and diffusion of passion fruit virus, a rapid virus detection platform can be built based on this invention's detection method, combined with various control measures, such as the propagation of virus-free seedlings, the promotion and planting of virus-free seedlings, and field virus control, providing technical support and assurance for the sustainable and healthy development of my country's passion fruit industry. Attached Figure Description
[0045] Figure 1 Optimization of the RT-RAA amplification system. A: Optimization of primer concentration. B: Optimization of reaction temperature. C: Optimization of reaction time. NC: Negative control, CK: Blank control.
[0046] Figure 2 Optimization of the RT-RAA-CRISPR / Cas12a detection system. A: Optimization of reaction temperature. B: Optimization of the Cas12a / crRNA concentration ratio. C: Optimization of reporter DNA concentration. NC: Negative control.
[0047] Figure 3This study tested the specificity of the RT-RAA-CRISPR / Cas12a visualization detection method. Among the viruses identified, EAPV is East Asian Passionflower Virus, TeMV is Night-blooming Jasmine Mosaic Virus, TuMV is Turnip Mosaic Virus, HLFPV is Hibiscus Latent Piersburg Virus, PFSMaV is Passionflower Severe Mottle-Associated Virus, CMV is Cucumber Mosaic Virus, and PLV is Passionflower Latent Virus.
[0048] Figure 4 Sensitivity testing of the RT-RAA-CRISPR / Cas12a visualization detection method. A shows the sensitivity test of the RT-PCR detection method. B shows the sensitivity test of the RT-RAA detection method. C shows the sensitivity test of the RT-RAA-CRISPR / Cas12a detection method.
[0049] Figure 5 Repeatability testing of the RT-RAA-CRISPR / Cas12a visualization detection method.
[0050] Figure 6 Actual sample testing using the RT-RAA-CRISPR / Cas12a visualization detection method. A shows the results of the RT-PCR detection method. B shows the results of the RT-RAA-CRISPR / Cas12a detection method. Where 1-63: test samples; NC: negative control. Detailed Implementation
[0051] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.
[0052] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0053] The main reagents involved in the following examples and their sources or preparation methods are as follows: LbCas12a(Cpf1) is a product of Guangzhou Bolais Biotechnology Co., Ltd., catalog number: M20301-0500; 10×Cas12a Reaction Buffer is a product of Guangzhou Bolais Biotechnology Co., Ltd., catalog number: M20301-0500; Recombinase polymerase isothermal amplification (RT-RAA) nucleic acid amplification kit is a product of Qitian Gene Biotechnology (Jiangsu) Co., Ltd., catalog number: B00R00A-48; Phenol / chloroform (1:1; V:V) was prepared by the following method: 20 mL of phenol (manufacturer: Xilong Scientific Co., Ltd.; catalog number: 108-95-2) completely dissolved at 50℃ and 20 mL of chloroform (manufacturer: Sinopharm Chemical Reagent Co., Ltd.; catalog number: 67-66-3) were mixed evenly and stored at 4℃ in the dark.
[0054] The negative controls (NC) in the following examples are all total RNA extracted from the epidermis of healthy passionflower leaves or fruits.
[0055] The blank control (CK) in the following examples is ddH2O.
[0056] The total RNA extraction method for the samples in the following examples is as follows: Take 100 mg of positive samples carrying EAPV (passion fruit leaves or fruit peel), extract total RNA using a high-purity RNA extraction kit (manufacturer: Beijing TransGen Biotech Co., Ltd.), and store the total RNA at -80℃ for later use.
[0057] The positive samples carrying EAPV in the following examples are passion fruit leaves or fruit peels carrying EAPV.
[0058] Example 1: RT-RAA primer pairs, crRNA sequences, and kits for detecting EAPV based on RT-RAA-CRISPR / Cas12a. I. Design of RT-RAA primer pairs and crRNA sequences This invention uses the NCBI website's Basic Local Alignment Search Tool (BLAST) to perform extensive sequence analysis, thereby obtaining the target region of EAPV. RT-RAA primers and crRNA are then designed for this target region. The specific primer sequences are shown in Table 1.
[0059] After repeated screening and optimization, the RT-RAA primer pair for detecting EAPV based on RT-RAA-CRISPR / Cas12a was finally determined to consist of RT-RAA-EAPV-F and RT-RAA-EAPV-R, with the crRNA being EAPV-crRNA.
[0060] Table 1
[0061] II. EAPV Visualization Detection Kit Based on RT-RAA-CRISPR / Cas12a The EAPV visualization detection kit based on RT-RAA-CRISPR / Cas12a of this invention includes primers RT-RAA-EAPV-F, RT-RAA-EAPV-R, EAPV-crRNA, reporter DNA, and Cas12a protein (such as LbCas12a(Cpf1)) in step one.
[0062] Example 2: Establishment and optimization of an EAPV visualization detection method based on RT-RAA-CRISPR / Cas12a This invention provides a visualization detection method for EAPV based on RT-RAA-CRISPR / Cas12a, comprising an RT-RAA amplification reaction and a CRISPR / Cas12a detection reaction. The reaction systems and conditions for both the RT-RAA amplification reaction and the CRISPR / Cas12a detection reaction were optimized. The specific steps are as follows: I. Optimization of RT-RAA Amplification Reactions 1. RT-RAA amplification reaction system and conditions Using total RNA from positive samples carrying EAPV as a template, the RT-RAA amplification reaction system was prepared using an RT-RAA nucleic acid amplification kit.
[0063] The RT-RAA amplification reaction system is as follows: Buffer V 25 μL, RT-RAA-EAPV-F (10 µmol·L⁻¹) -1 ) 2μL, RT-RAA-EAPV-R (10μmol·L -1 2 μL of nuclease-free water, 14 μL of magnesium acetate I, and 2 μL of RNA template were added. After the RT-RAA amplification reaction system was prepared, it was gently mixed and briefly centrifuged. Then, it was reacted at 37 °C for 40 min to obtain the RT-RAA amplification product.
[0064] 2. Optimization of RT-RAA amplification reaction system and conditions The RT-RAA amplification reaction system and conditions were optimized using a single-variable method. Gradient settings were applied to primer concentration, reaction temperature, and reaction time to analyze their effects on RT-RAA amplification efficiency. Specifically, the final primer concentration was set to 0.1 µmol·L⁻¹. -1 0.2 µmol·L -1 0.3 µmol·L -1 0.4 µmol·L -1 0.5 µmol·L -1 0.6 µmol·L -1 The reaction temperatures were set sequentially to 35℃, 37℃, 39℃, 41℃, 43℃, and 45℃; the reaction times were set sequentially to 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, and 40 min. After the RT-RAA amplification reaction was completed, the RT-RAA amplification products were purified using an equal volume of Tris-saturated phenol / chloroform (1:1; V:V), and the amplification results were observed and analyzed by 2% agarose gel electrophoresis. Based on the clarity and specificity of the electrophoretic bands, the optimal RT-RAA amplification conditions were determined through comparative analysis.
[0065] The results showed that the RT-RAA primer concentration was 0.5 µmol·L⁻¹. -1 The amplification efficiency is highest at 37℃; the expected band size can be amplified within the reaction temperature range of 37~45℃, with the highest amplification efficiency at 37℃, 39℃, or 43℃; the expected band size can be amplified within 10~40 min, with the optimal reaction time being 20 min, 25 min, and 30 min. Figure 1 AC). Considering cost, amplification efficiency, and the LbCas12a(Cpf1) reaction temperature (45℃), the optimal RT-RAA amplification system was finally determined as follows: buffer V 25μL, primer RT-RAA-EAPV-F (10µmol·L⁻¹). -1 ) 2.5μL, RT-RAA-EAPV-R (10μmol·L -1 2.5 μL of magnesium acetate I, 5 μL of template RNA, 2 μL of nuclease-free water, and 13 μL of water were mixed thoroughly and reacted at a constant temperature of 43℃ for 20 min.
[0066] II. Optimization of CRISPR / Cas12a detection reaction 1. Establishment of the RT-RAA-CRISPR / Cas12a detection reaction system After the RT-RAA amplification reaction is completed, the RT-RAA amplification product does not need to be purified and can be directly used to prepare the CRISPR / Cas12a detection reaction system.
[0067] The CRISPR / Cas12a detection reaction system (25 μL) is as follows: 10×Cas12a Reaction Buffer 2.5 μL, LbCas12a(Cpf1) (5 μmol·L⁻¹) -1 )0.5μL, EAPV-crRNA (10μmol·L -1 0.3 μL, reporter DNA (10 μmol·L⁻¹) -1 1 μL, 2 μL of unpurified RT-RAA amplification product, and 18.7 μL of nuclease-free water.
[0068] The CRISPR / Cas12a detection reaction system was prepared on ice, mixed thoroughly, and then placed in an LC96 real-time PCR instrument, set to detect FAM fluorescence signals. The CRISPR / Cas12a detection reaction conditions were as follows: reaction at 45℃ for 20 min, with fluorescence data acquired every minute.
[0069] 2. Optimization of reaction temperature for RT-RAA-CRISPR / Cas12a detection The RT-RAA-CRISPR / Cas12a detection reaction system was subjected to reactions at five different temperatures (41℃, 42℃, 43℃, 44℃, and 45℃).
[0070] The results showed that the RT-RAA-CRISPR / Cas12a detection system could achieve good green fluorescence intensity in the range of 41–45 °C. Figure 2 (A) This is consistent with the reported results. To achieve one-tube operation of the detection system, 43℃ was selected as the optimal reaction temperature for the detection system.
[0071] 3. Optimization of Cas12a / crRNA concentration ratio To optimize the concentration ratio of Cas12a and crRNA, a two-factor orthogonal experimental design was used to design the concentration gradient of Cas12a and crRNA. A total of 16 experiments were required, as shown in Table 2. The effects of different concentration ratios of Cas12a and crRNA on the fluorescence signal intensity were observed, thereby determining the optimal concentration ratio scheme.
[0072] Table 2
[0073] The results showed that the green fluorescence intensity of the RT-RAA-CRISPR / Cas12a detection system reached its maximum when the concentration ratio of Cas12a / crRNA was 100:120. Figure 2 (B), therefore, 100 nmol·L⁻¹ is chosen. -1and 120 nmol·L -1 The optimal final concentrations for Cas12a and crRNA were determined, respectively.
[0074] 4. Optimization of reported DNA concentration After determining the optimal concentrations of Cas12a and crRNA, the concentration of reporter DNA was further optimized, with a setting of 0 nmol·L⁻¹. -1 200 nmol·L -1 400 nmol·L -1 800 nmol·L -1 1200 nmol·L -1 1600 nmol·L -1 2000 nmol·L -1 Assuming the final concentration, and keeping other conditions constant, we observed the changes in fluorescence signal intensity at different reporter DNA concentrations to determine the optimal reporter DNA concentration.
[0075] The results showed that when the final concentration of the reporter DNA was 800 nmol·L⁻¹ - At ¹, the fluorescence intensity of the detection reaction system reached its peak. Further increasing the reporter DNA concentration did not significantly increase the fluorescence intensity. Figure 2 C). Therefore, 800 nmol·L - ¹ represents the optimal final concentration of the report DNA.
[0076] Based on the above optimization experimental results, the specific components of the optimal RT-RAA-CRISPR / Cas12a detection reaction system are as follows: 10×Cas12a Reaction Buffer 2.5 μL, LbCas12a(Cpf1) (5 µmol·L⁻¹) - ¹) 0.5μL, EAPV-crRNA (10μmol·L - ¹) 0.3 μL, reporter DNA (10 µmol·L⁻¹) - ¹) 2 μL of RT-RAA amplification product and 17.7 μL of ddH₂O. The optimal reaction conditions are 43℃ for 20 min.
[0077] III. Visual Detection Method for EAPV Based on RT-RAA-CRISPR / Cas12a After optimization in steps one and two, the EAPV visualization detection method based on RT-RAA-CRISPR / Cas12a of this invention includes the following steps: 1. Using the RNA of the sample to be tested as a template, RT-RAA amplification reaction was carried out using primers RT-RAA-EAPV-F and RT-RAA-EAPV-R to obtain RT-RAA amplification products.
[0078] The RT-RAA amplification reaction system is as follows: Buffer V 25 μL, primer RT-RAA-EAPV-F (10 µmol·L⁻¹) -1 ) 2.5μL, RT-RAA-EAPV-R (10μmol·L -1 2.5 μL of magnesium acetate I, 5 μL of template RNA, and 13 μL of nuclease-free water were added. The final concentrations of primers RT-RAA-EAPV-F and RT-RAA-EAPV-R in the RT-RAA amplification reaction system were both 0.5 μmol·L⁻¹. -1 .
[0079] The RT-RAA amplification reaction conditions are as follows: reaction at a constant temperature of 43℃ for 20 min.
[0080] 2. Add the following reagents to 2 μL of RT-RAA amplification product: 2.5 μL of 10×Cas12a Reaction Buffer, and LbCas12a(Cpf1) (5 µmol·L⁻¹). -1 )0.5μL, EAPV-crRNA (10μmol·L -1 0.3 μL, reporter DNA (10 µmol·L⁻¹) -1 2 μL of EAPV-crRNA and 17.7 μL of ddH2O were added to obtain the CRISPR / Cas12a detection reaction system. The final concentration of EAPV-crRNA in the CRISPR / Cas12a detection reaction system was 120 nmol·L⁻¹. -1 The final concentration of Cas12a protein in the CRISPR / Cas12a detection reaction system was 100 nmol·L⁻¹. -1 The final concentration of DNA in the CRISPR / Cas12a detection reaction system was reported to be 800 nmol·L⁻¹. -1 .
[0081] The CRISPR / Cas12a detection reaction system was placed in an LC96 real-time PCR instrument and set to detect FAM fluorescence signals. Fluorescence data were acquired every minute. The CRISPR / Cas12a detection reaction conditions were as follows: reaction at 43℃ for 20 min. After the reaction, the product was transferred to a gel imaging system, and the results were observed under UV light.
[0082] 3. Result Determination If the CRISPR / Cas12a detection reaction system is green, the sample contains East Asian passion fruit virus; if the CRISPR / Cas12a detection reaction system is colorless and transparent, the sample does not contain East Asian passion fruit virus.
[0083] Example 3, Specificity Test Total RNA from positive samples carrying EAPV-related viruses (TeMV, TuMV, PFSMaV, hibiscus latent Fort Pierce virus, CMV, and PLV) was used as test samples. The EAPV visualization detection method based on RT-RAA-CRISPR / Cas12a established in this invention was used for detection.
[0084] The results showed that among all the tested samples, only the positive samples carrying EAPV produced a strong fluorescent signal, while samples carrying other viruses did not show a fluorescent signal. Figure 3 This demonstrates that the detection method established in this invention has strong specificity.
[0085] Example 4: Sensitivity Test Total RNA was extracted from positive samples carrying EAPV and serially diluted 10-fold to 4.26 ng / L. -1 4.26×10 - 1 ng·L -1 4.26×10 -2 ng·L -1 4.26×10 -3 ng·L -1 4.26×10 -4 ng·L -1 4.26×10 -5 ng·L -1 4.26×10 -6 ng·L -1RNA dilutions of different concentrations were obtained. Using the original solution and RNA dilutions of different concentrations as templates, the EAPV visualization detection method based on RT-RAA-CRISPR / Cas12a established in this invention was used for detection. RT-PCR and RT-RAA detection methods were used as controls. The limits of detection of the three detection methods (RT-PCR, RT-RAA, and RT-RAA-CRISPR / Cas12a) were compared to evaluate the differences in sensitivity.
[0086] The RT-PCR detection method includes the following steps: the extracted total EAPV RNA is reverse transcribed to obtain cDNA, the cDNA is serially diluted according to the same method, and RT-PCR detection is performed on the cDNA dilutions of different concentrations (the primer sequences of RT-PCR-EAPV-F / RT-PCR-EAPV-R are shown in Table 1).
[0087] The results showed that RT-PCR and RT-RAA methods had similar sensitivity, with a detection limit of 4.62 × 10⁻⁶. -3 ng·μL - ¹( Figure 4 (AB). When RT-RAA technology is innovatively combined with the CRISPR / Cas12a system, the detection sensitivity is significantly enhanced, with a detection limit of 4.62 × 10⁻⁶. -5 ng·μL - ¹, is 100 times faster than RT-PCR and RT-RAA methods ( Figure 4 C).
[0088] Example 5: Repeatability Test The EAPV visualization detection method based on RT-RAA-CRISPR / Cas12a established in this invention was used to detect the concentration of 4.26 × 10⁻⁶ in Example 4. -1 ng·L -1 4.26×10 -3 ng·L -1 4.26×10 -5 ng·L -1 RNA dilutions were used for testing, with each concentration tested in three independent replicates.
[0089] The results showed that the coefficient of variation of the EAPV visualization detection method based on RT-RAA-CRISPR / Cas12a established in this invention was less than 5%, and no fluorescence signal was detected in the negative control. Figure 5 This indicates that the detection method established in this invention has good repeatability.
[0090] Example 6: Actual Sample Testing RNA extracted from 63 passion fruit samples suspected of EAPV infection collected from the field was used as test samples. The EAPV visualization detection method based on RT-RAA-CRISPR / Cas12a established in this invention was used for detection. At the same time, RT-PCR was used as a verification method to explore the applicability and accuracy of the EAPV visualization detection method established in this invention in actual field detection.
[0091] The results showed that 21 positive samples were detected by the RT-PCR method, and the EAPV visualization detection method based on RT-RAA-CRISPR / Cas12a established in this invention also detected 21 positive samples (Table 3). The consistency rate between the two methods was 100%. Therefore, the EAPV visualization detection method based on RT-RAA-CRISPR / Cas12a established in this invention is accurate and reliable, and suitable for actual field detection of EAPV.
[0092] Table 3
[0093] Note: + indicates a positive sample.
[0094] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.
Claims
1. A set of reagents for identifying or assisting in the identification of East Asian passion fruit virus, said set of reagents including RAA primer pairs; The RAA primer pair consists of primer-F and primer-R; The primer-F is either a1) or a2) as follows: a1) The single-stranded DNA molecule shown in sequence 1; a2) A single-stranded DNA molecule that has undergone substitution and / or deletion and / or addition of one or more nucleotides of sequence 1 and has the same function as sequence 1; The primer-R is either a3) or a4) as follows: a3) The single-stranded DNA molecule shown in sequence 2; a4) A single-stranded DNA molecule that has one or more nucleotides of sequence 2 replaced and / or deleted and / or added, and has the same function as sequence 2.
2. The complete set of reagents according to claim 1, characterized in that: The molar ratio of primer-F to primer-R is 1:
1.
3. The complete set of reagents according to claim 1 or 2, characterized in that: The complete reagent kit also includes the crRNA corresponding to the RAA primer pair; The crRNA is either a5) or a6) as follows: a5) The single-stranded RNA molecule shown in sequence 3; a6) A single-stranded RNA molecule with one or more nucleotides substituted and / or deleted and / or added to sequence 3 and having the same function as sequence 3.
4. The complete set of reagents according to any one of claims 1-3, characterized in that: The kit also includes Cas12a protein and reporter DNA.
5. The use of the kit of any of claims 1-4, the RAA primer pair of any of claims 1-4, or the crRNA of any of claims 1-4 in any of the following b1)-b8): b1) Identification or auxiliary identification of East Asian passion fruit virus or its nucleic acid; b2) Detect or assist in the detection of whether the sample to be tested contains East Asian passion fruit virus or its nucleic acid; b3) To differentiate or assist in differentiating East Asian passion fruit virus from other viruses capable of infecting passion fruit; b4) Prevention and control or auxiliary prevention and control of East Asian passion fruit virus; b5) Prepare products for identification or auxiliary identification of East Asian passion fruit virus or its nucleic acid; b6) Prepare products for detecting or assisting in the detection of whether the sample to be tested contains East Asian passion fruit virus or its nucleic acid; b7) Prepare products that can differentiate or help differentiate East Asian passion fruit virus from other viruses that are capable of infecting passion fruit; b8) Prepare products for the prevention or assistance in the prevention and control of East Asian passion fruit virus.
6. A kit for identifying or assisting in the identification of East Asian passion fruit virus, comprising the complete set of reagents as described in any one of claims 1-4; the kit functions as any one of c1)-c4): c1) Identification or auxiliary identification of East Asian passion fruit virus or its nucleic acid; c2) Detect or assist in the detection of whether the sample to be tested contains East Asian passion fruit virus or its nucleic acid; c3) To differentiate or assist in differentiating East Asian passion fruit virus from other viruses capable of infecting passion fruit; c4) Prevention or assisted prevention of East Asian passion fruit virus.
7. A method for detecting or assisting in the detection of whether a test sample contains Passifloxacin virus, the method comprising the following steps: extracting RNA from the test sample; using the RNA of the test sample as a template, performing an RT-RAA amplification reaction using any of the RAA primer pairs described in claims 1-4 to obtain an RT-RAA amplification product; adding crRNA, Cas12a protein and reporter DNA described in any of claims 1-4 to the RT-RAA amplification product to perform a CRISPR / Cas12a detection reaction; and determining whether the test sample contains Passifloxacin virus based on the CRISPR / Cas12a detection reaction result.
8. A method for distinguishing or assisting in distinguishing whether a virus to be tested in a passion fruit sample infected with a virus is East Asian passion fruit virus or other viruses capable of infecting passion fruit, the method comprising the following steps: extracting RNA of the virus to be tested from the passion fruit sample; using the RNA of the virus to be tested as a template, performing an RT-RAA amplification reaction using any of the RAA primer pairs described in claims 1-4 to obtain an RT-RAA amplification product; adding crRNA, Cas12a protein and reporter DNA described in any of claims 1-4 to the RT-RAA amplification product to perform a CRISPR / Cas12a detection reaction; and determining whether the virus to be tested is East Asian passion fruit virus or other viruses capable of infecting passion fruit based on the CRISPR / Cas12a detection reaction results.
9. The method according to claim 7 or 8, characterized in that: The final concentrations of primer-F and primer-R in the RT-RAA amplification reaction system were both 0.5 μmol·L⁻¹. -1 ; Alternatively, the final concentration of the crRNA in the CRISPR / Cas12a detection reaction system is 120 nmol·L⁻¹. -1 ; Alternatively, the final concentration of the Cas12a protein in the CRISPR / Cas12a detection reaction system is 100 nmol·L⁻¹. -1 ; Alternatively, the final concentration of the reporter DNA in the CRISPR / Cas12a detection reaction system is 800 nmol·L⁻¹. -1 .
10. The method according to any one of claims 7-9, characterized in that: The conditions for the RT-RAA amplification reaction were 43℃ for 20 min; Alternatively, the CRISPR / Cas12a detection reaction is performed at 43°C for 20 minutes.