CRISPR / Cas12a-based GETV detection kit and detection method

Abstract

The present invention belongs to the field of biotechnology, and specifically discloses a GETV detection kit based on CRISPR / Cas12a, including a CRISPR / Cas12a detection system suitable for GETV detection, wherein the CRISPR / Cas12a detection system includes a specific gRNA for the conserved gene of the NS2 protein of GETV, a CRISPR / Cas12a protein and an ssDNA reporter system, wherein the specific gRNA is designed and synthesized according to GETV, and the ssDNA reporter system includes an ssDNA FQ reporter for fluorescence quantitative detection and / or an ssDNA FB reporter for immunochromatographic test strip detection. The GETV detection method of the present invention has a detection limit of 2.15 copy / μL and has no cross reaction with other viruses except GETV. The present invention is the first to use CRISPR / Cas12a to detect the GETV sequence, which has the advantages of high sensitivity, strong specificity, short time consumption, and no dependence on large-scale experimental equipment. In addition, a detection method for GETV is also disclosed.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a CRISPR / Cas12a-based GETV detection kit and a detection method thereof. Background Art

[0002] Getah virus (GETV) is a member of the genus Alphavirus in the family Togaviridae. Since its initial isolation in 1955 by Scherer et al. from the mosquito Culex gelidus living in a Malaysian rubber plantation, GETV infection has been found in a variety of vertebrates, including humans, monkeys, birds, pigs, horses, and other mammals. Infection in these species is crucial for maintaining the natural transmission cycle of zoonotic diseases such as GETV. GETV was long believed to be pathogenic only in pigs and horses, causing fever, a generalized rash, and leg edema in horses, arthritis in piglets, and reproductive problems in sows. Several outbreaks of GETV infection in pigs and horses have been reported in Japan and India. While research on GETV in my country is limited, its potential for latent infection should be given high attention. An outbreak of GETV would cause severe losses to the Chinese pig industry. At present, Geta disease has been reported all over the world. It has become one of the global infectious diseases of pigs, affecting the normal production of the world's pig industry. Related research on Geta disease, such as investigation, diagnosis, prevention and vaccine development, deserves the attention of researchers.

[0003] Currently, the main methods for GETV detection include viral isolation and culture, immunological methods, and nucleic acid detection. Although known as the "gold standard," viral isolation and culture is technically complex, time-consuming, typically requiring more than a week, and relatively expensive, making it unsuitable for early diagnosis. Immunological methods are currently the most widely used, but they require a certain amount of antigen and high-quality specific antibodies. Immunological testing alone is prone to false negatives, making the results difficult to interpret. The most commonly used molecular diagnostic method for nucleic acid detection is RT-PCR, which uses primers designed to amplify the highly conserved gene sequence of GETV. However, its high cost and the requirement for specialized equipment and techniques have limited its widespread adoption.

[0004] CRISPR / Cas (Clustered regularly interspaced short palindromic repeats, CRISPRs) is an adaptive immune system in bacteria. Cas proteins target and degrade foreign nucleic acids through RNA-guided nucleases. Based on this characteristic, researchers have developed a variety of nucleic acid detection systems, including CRISPR / Cas9, CRISPR / Cas12, and CRISPR / Cas13. The CRISPR / Cas12a nucleic acid detection system extracts genomic DNA from clinical samples to be tested and performs loop-mediated isothermal nucleic acid amplification (LAMP) under isothermal conditions. The CRISPR / Cas12a-gRNA complex binds to and cleaves the target DNA, activating trans-cleavage of ssDNA. The fluorescent reporter molecule coupled to the ssDNA produces a fluorescent signal during cleavage. This new method, called DNA endonuclease-targeted CRISPR trans-reporter gene, will provide a powerful platform for the rapid and accurate detection of GETV nucleic acids.

[0005] Immunochromatographic strip testing is a highly effective technology for rapid clinical testing. Its short reaction time, long-term stability, and relatively low cost make it widely applicable for the rapid and specific detection of GETV nucleic acids. Summary of the Invention

[0006] In order to overcome the defects of the prior art, the present invention provides a CRISPR / Cas12a-based GETV detection kit and a detection method thereof.

[0007] The technical solution adopted by the present invention to solve its technical problem is: a CRISPR / Cas12a-based GETV detection kit, including a CRISPR / Cas12a detection system suitable for porcine GETV detection.

[0008] The CRISPR / Cas12a detection system includes a specific gRNA for the NS2 conserved gene of porcine GETV, a CRISPR / Cas12a protein, and an ssDNA reporter system.

[0009] Furthermore, the specific gRNA is targeted at porcine GETV, and its sequence is shown in SEQ ID NO. 1.

[0010] Furthermore, the ssDNA reporter system includes an ssDNA FQ reporter for fluorescence quantitative detection and / or an ssDNA FB reporter for immunochromatographic test strip detection.

[0011] Furthermore, the ssDNA FQ reporter is ssDNA labeled with 6-carboxyfluorescein and a fluorescence quencher, and the labeled product is as follows: 5'-FAM-TTATTATT-BHQ-3', named ssDNA FQ reporter / 5'-FAM-TTATTATT-BHQ-3'; the sequence of ssDNA in the ssDNA FQ reporter is SEQ ID NO. 2;

[0012] The ssDNA FB reporter is ssDNA labeled with fluorescein and biotin. The labeled product is as follows: 5'-FAM-TTATTATT-Biotin-3', named ssDNA FB reporter / 5'-FAM-TTATTATT-Biotin-3'; the sequence of ssDNA in the ssDNA FB reporter is SEQ ID NO. 3.

[0013] Furthermore, the kit also includes an immunochromatographic test strip, which detects that the 5' end and 3' end of ssDNA are modified with a FAM group and a biotin group, respectively, and the immunochromatographic test strip contains an anti-fluorescein antibody labeled with gold nanoparticles, and the anti-fluorescein antibody can bind to the fluorescein-modified end of the ssDNA FB reporter. The quality control line (C line) of the immunochromatographic test strip contains streptavidin, and the streptavidin can bind to the biotin group. The detection line (T line) of the immunochromatographic test strip contains an anti-fluorescein antibody.

[0014] Furthermore, the method for preparing the specific gRNA includes: searching for a targeting sequence containing the CRISPR / Cas12a recognition sequence (PAM) TTTN for the conserved GETV NS2 gene, designing a 48nt gRNA, and after the design is completed, the biological company synthesizes the target gRNA.

[0015] Furthermore, the CRISPR / Cas12a protein is produced by NEB (New England Biotechnology (Beijing) Co., Ltd.).

[0016] That is, the CRISPR / Cas12a-based GETV detection kit provided by the present invention can be used for both fluorescence quantitative detection and immunochromatographic test strips. When using fluorescence quantitative detection, the DNA (ssDNA) reporter system in the CRISPR / Cas12a detection system is an ssDNA FQ reporter; when using immunochromatographic test strips, the DNA (ssDNA) reporter system is an ssDNA FB reporter.

[0017] First, when using fluorescence quantitative detection, when the GETV gene is present in the CRISPR / Cas12a detection system, the nuclease activity of the CRISPR / Cas12a protein is specifically activated under the guidance of GETV-specific gRNA. The activated CRISPR / Cas12a protein cleaves the ssDNA FQ reporter labeled with a fluorescent group and a quencher, releasing the activated fluorescent group, which can be detected using fluorescence quantitative detection. Conversely, when the GETV gene sequence is absent in the sample being tested, the fluorescence reading is displayed as a baseline value.

[0018] Secondly, when using the immunochromatographic test strip for detection, after the sample to be tested after CRISPR / Cas12a cutting is added to the immunochromatographic test strip, the anti-fluorescein antibody labeled with colloidal gold combines with the fluorescein-labeled ssDNA reporter system, and the complex moves from the quality control line to the detection line along the liquid flow direction; the streptavidin on the quality control line saturates the capture of the ssDNA reporter system labeled with biotin, thereby displaying the band; when CRISPR / Cas12a detects the GETV gene, it will cut off the ssDNA reporter system labeled with fluorescein and biotin, so that the fluorescein-labeled ssDNA fragments will be captured and colored by the fluorescein antibody on the detection line; when CRISPR / Cas12a cannot detect the gene sequence of GETV, the ssDNA reporter system labeled with fluorescein and biotin cannot be cut off, so that the fluorescein-labeled ssDNA fragments will not be captured by the detection line and colored.

[0019] The present invention also provides a method for detecting GETV, using the above-mentioned CRISPR / Cas12a-based GETV detection kit.

[0020] Preferably, the GETV detection method comprises the following steps:

[0021] Step a: using a nucleic acid rapid release reagent to release nucleic acid in the sample to be tested;

[0022] Step b: Amplify the nucleic acid in the sample using isothermal amplification primers: Add the product obtained in step a to the LAMP isothermal amplification system using GETV specific primers (SEQ ID NO. 4-7) and react at 64°C for 30 minutes to amplify the specific product;

[0023] Step c: Detection of GETV using the CRISPR / Cas12a detection system: The product from step b was added to the CRISPR / Cas12a detection system and reacted at 37°C for 30 minutes;

[0024] Step d: Detect GETV in the sample using a fluorescent quantitative detection method or an immunochromatographic test strip.

[0025] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0026] 1. The present invention utilizes CRISPR / Cas12a to specifically recognize nucleic acids and combines it with immunochromatography technology to achieve high sensitivity, high specificity, and rapid visual detection of GETV. Research has shown that the NS2 gene of GETV is highly conserved, and the NS2 protein gene was selected as the target sequence for detection. Based on the characteristics of CRISPR / Cas12a recognizing specific PAM sequences, a specific gRNA was designed for the NS2 protein gene target sequence. Testing revealed that the gRNA had a high sensitivity for detecting GETV, and a CRISPR / Cas12a rapid GETV detection system was further established.

[0027] 2. The present invention is a GETV detection tool based on CRISPR / Cas12a, which includes an immunochromatographic test strip test, which can achieve convenient and fast result interpretation.

[0028] 3. This invention utilizes CRISPR / Cas12a for specific sequence cleavage and immunochromatography to achieve rapid, highly specific, highly sensitive, and visual detection of GETV. GETV RNA can be detected at 2.15 copies / μL. The GETV detection method established in this invention provides an accurate, rapid, and simple method for clinical diagnosis and laboratory research.

[0029] 4. The present invention discloses a series of CRISPR / Cas12a reaction systems, gRNA and LAMP amplification primers for GETV detection, and their sequences are shown in SEQ ID NO. 1 to SEQ ID NO. 7. The combination of CRISPR / Cas12a, gRNA and LAMP amplification primers can be used for GETV detection. The present invention is the first to use CRISPR / Cas12a to detect GETV, which has the advantages of high sensitivity, strong specificity, short time consumption, high throughput, and no dependence on large-scale experimental equipment. These advantages make the CRISPR / Cas12a-based immunochromatographic test strip detection method developed by the present invention convenient for rapid detection and diagnosis of GETV in laboratories and clinical medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Graph showing the fluorescence detection results (50 min) of GETV detection by CRISPR / Cas12a-based gRNA in an embodiment of the present invention;

[0031] Figure 2In the embodiment of the present invention, when GETV is detected by fluorescence quantitative method based on CRISPR / Cas12a, the GETV RNA concentration is 2.15×10 4 , 2.15×10 3 , 2.15×10 2 , 2.15×10 1 , 2.15×10 0 and sensitivity test results of negative controls;

[0032] Figure 3 In the embodiment of the present invention, when GETV is detected by fluorescence quantitative method based on CRISPR / Cas12a, the GETV RNA concentration is 2.15×10 4 , 2.15×10 3 , 2.15×10 2 , 2.15×10 1 , 2.15×10 0 and a bar graph of the sensitivity test results of negative controls;

[0033] Figure 4 In the embodiment of the present invention, when GETV is detected by immunochromatographic test strips based on CRISPR / Cas12a, the GETV RNA concentration is 2.15×10 4 , 2.15×10 3 , 2.15×10 2 , 2.15×10 1 , 2.15×10 0 and negative control detection diagram;

[0034] Figure 5 This is a specific detection diagram corresponding to different viruses detected by fluorescence quantitative method based on CRISPR / Cas12a in an embodiment of the present invention;

[0035] Figure 6 This is a bar graph showing the results of detecting different viruses using fluorescence quantitative method based on CRISPR / Cas12a in this embodiment;

[0036] Figure 7 This is a graph showing the results of detecting different viruses using immunochromatographic test strips based on CRISPR / Cas12a in an embodiment of the present invention. DETAILED DESCRIPTION

[0037] The following is a further description of specific embodiments of the present invention in conjunction with the accompanying drawings. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0038] In this invention, the loop-mediated isothermal nucleic acid amplification kit (LAMP) and CRISPR / Cas12a protein were purchased from New England Biolabs; gRNA, loop-mediated amplification primer nucleic acid, and ssDNA probe were synthesized by Sangon Biotechnology Co., Ltd.; and pre-treated nucleic acid was obtained using a rapid nucleic acid release agent purchased from Novozymes. GETV was isolated, identified, and stored in the laboratory.

[0039] Rapid and sensitive detection of GETV fragments

[0040] 1.1 Nucleic acid preparation

[0041] The GETV gene fragment in this case is a strain isolated and identified by the laboratory (laboratory self-preservation).

[0042] The present invention is used to centrifuge the supernatant of virus cell culture to remove the 10 7 Cell debris from 0.5 mL of culture supernatant containing 100 plaque-forming units (pFU) was removed, and viral RNA was extracted using a Norvegian virus extraction kit according to the manufacturer's protocol. To assess the limit of detection, the amount of GETV RNA was determined spectrophotometrically and converted to molecular copies using a published formula. Reverse transcription experiments were performed using the HiScript II 1st Strand cDNA Synthesis Kit (Norvegian) using 10-fold diluted GETV RNA as a template. The reverse transcription reaction system and reaction process are shown in Table 1:

[0043] Table 1 Reverse transcription system

[0044]

[0045] 1.0 μL of reverse-transcribed GETV RNA sample was subjected to LAMP amplification reactions: the LAMP reaction mixture contained 1.0 μL each of 5 mM F3 and B3 primers, 1.0 μL each of 40 mM FIP and BIP primers, 2.5 μL of 10× reaction buffer, 1.0 μL of 8 U / μL Bst DNA polymerase (New England Biolabs), 3.5 μL of 10 mM dNTP mix (Takara), 5.0 μL of 30 mM MgSO₄ (Sigma), 1 μL of reverse-transcribed GETV RNA, and 3.0 μL of nuclease-free ultrapure water. The reaction was incubated at 64°C for 30 min, and samples were obtained for subsequent nucleic acid detection. The sequences of the 5 mM F3 and B3 primers and the 40 mM FIP and BIP primers (SEQ ID NOs. 4-7) are shown in Table 2.

[0046] Table 2. GETV LAMP primer design

[0047]

[0048] 1.2 Design and preparation of GETV-specific gRNA

[0049] gRNA preparation was performed according to the following protocol. A targeting sequence containing the CRISPR / Cas12a recognition sequence (PAM) TTTN was identified within the conserved region of the porcine GETV NS2 gene, and a 48-nt gRNA (SEQ ID NO. 1) was designed. After design, the gRNA was directly synthesized at Sangon Biotech.

[0050] The GETV gRNA provided by the present invention is SEQ ID NO. 1, and the specific target recognition region information is shown in Table 3.

[0051] Table 3. GETV-specific gRNA

[0052]

[0053] This test uses a 20 μL system as shown in Table 4, but is not limited thereto, including adjustments to the proportions of the corresponding components:

[0054] Table 4. GETV CRISPR / Cas12a detection system

[0055]

[0056] The ssDNA reporter system is ssDNA FQ reporter and / or ssDNAFB reporter.

[0057] ssDNA FQ reporter is ssDNA labeled with 6-carboxyfluorescein and a fluorescence quencher. The labeled product is as follows: 5'-FAM-TTATTATT-BHQ-3', named ssDNA FQ reporter / 5'-FAM-TTATTATT-BHQ-3'; the sequence of ssDNA in ssDNA FQ reporter is SEQ ID NO. 2;

[0058] ssDNA FB reporter is ssDNA labeled with fluorescein and biotin. The labeled product is as follows: 5'-FAM-TTATTATT-Biotin-3', named ssDNA FB reporter / 5'-FAM-TTATTATT-Biotin-3'; the sequence of ssDNA in ssDNAFBreporter is SEQ ID NO. 3.

[0059] The ssDNA in ssDNA FQ reporter and ssDNA FB reporter are shown in Table 5:

[0060] Table 5.

[0061]

[0062] In addition, the kit also includes an immunochromatographic test strip. The immunochromatographic test strip detects ssDNA with the 5' and 3' ends modified with FAM and biotin groups, respectively. The immunochromatographic test strip contains anti-fluorescein antibodies labeled with gold nanoparticles. The anti-fluorescein antibodies can bind to the fluorescein-modified end of the ssDNA FB reporter. The quality control line of the immunochromatographic test strip contains streptavidin, which can bind to the biotin group.

[0063] 1.3 Fluorescence quantitative detection

[0064] In the fluorescence quantitative assay, CRISPR / Cas12a sequentially adds various components to the target gene detection system. After mixing, the components are incubated at 37°C for 30 seconds per cycle, and the fluorescence signal is detected for 100 cycles.

[0065] Fluorescence detection was used to determine the activity of the CRISPR / Cas12a detection system. Fluorescence quantification was used to measure the fluorescence of the detection reaction. The kinetics of gRNA cleavage detection of GETV were monitored (100 cycles). During GETV detection, the gRNA cleavage detection kinetics of GETV were monitored. This invention uses a fluorescence method to determine the results, which can achieve GETV detection.

[0066] 1.4 Immunochromatographic test strips

[0067] In the immunochromatographic strip test, the CRISPR / Cas12a target gene detection system is sequentially added to the various components. After mixing thoroughly, the components are incubated at 37°C for 30 minutes.

[0068] The immunochromatographic test strip assay procedure is as follows: 20 μL of CRISPR / Cas12a cleavage product is mixed with 30 μL of ultrapure water. The test strip is immersed in the mixture and allowed to react for 5 minutes. The result can be visually determined.

[0069] Example 1

[0070] Fluorescence quantification is used to measure and detect CRISPR / Cas12a reaction kinetics monitoring, as shown in the attached Figure 1 As shown in Figure 2, kinetic monitoring of gRNA cleavage detection for GETV (100 cycles); as shown in Figure 2 Figure 1As shown, in the GETV detection, it was found that gRNA had a higher fluorescence value for the detection of GETV.

[0071] According to the results obtained in Example 1, the gRNA has a high fluorescence value for the detected GETV gene, so this specific gRNA is used for subsequent detection. This detection uses a 20 μL system as shown in Table 4, but is not limited thereto.

[0072] Example 2

[0073] CRISPR / Cas12a detects GETV sensitivity

[0074] In the sensitivity test case, the GETV cultured in the cell was lysed and extracted RNA was converted to copy number according to the molecular weight, and a 10-fold serial dilution was performed to obtain RNA containing 2.15×10 4 , 2.15×10 3 , 2.15×10 2 , 2.15×10 1 , 2.15×10 0 Copy number (copy / μL), negative control, and reverse transcription were performed separately. 1 μL of serially diluted sample was subjected to LAMP amplification reaction: the LAMP reaction mixture contained 1.0 μL each of 5 mM F3 and B3 primers, 1.0 μL each of 40 mM BIP and FIP primers, 2.5 μL 10× reaction buffer, 1.0 μL 8 U / μL Bst DNA polymerase (New England Biolabs), 3.5 μL 10 mM dNTP mix (Takara), 5.0 μL 30 mM MgSO4 (Sigma), 1 μL reverse-transcribed GETV RNA, and 3.0 μL nuclease-free ultrapure water. Mix well, react at 64°C for 30 minutes, and obtain samples for nucleic acid detection.

[0075] The results obtained in this Example 2 are shown in the attached Figures 2-4 As shown in the results of GETV sensitivity detection, in this embodiment, the CRISPR / Cas12a fluorescence method and the test strip method were used to detect GETV, which can achieve a sensitivity of 2.15×10 0 High-sensitivity detection of copies.

[0076] Example 3

[0077] Specificity for GETV Detection specificity was used to analyze the specificity of the established CRISPR / Cas12a detection method: PRRSV, PRV, PCV, ASFV, CSFV, JEV, PEDV and GETV.

[0078] The results obtained in Example 3 are as follows: the reaction results of the fluorescence method and the test strip method using cDNA / DNA or tissue sample grinding solution containing PRRSV, PRV, PCV, ASFV, CSFV, JEV, PEDV and GETV as templates Figures 5-7 As shown, CRISPR / Cas12a detection is highly specific.

[0079] This example performs rapid nucleic acid detection on clinical samples. All samples and operations are completed in the laboratory. In this example, the sample is a viral nucleic acid isolated and identified in the laboratory, and the nucleic acid obtained by extraction is subjected to CRISPR / Cas12a detection. This example uses a viral DNA / RNA extraction kit (column method) purchased from Novozymes to obtain pretreated nucleic acid and perform the next step of detection.

[0080] In the examples, fluorescence detection was used to determine the activity of the CRISPR / Cas12a detection system. In the CRISPR / Cas12a detection system, each component was added sequentially and mixed evenly, and then fluorescence quantification was used to measure the fluorescence of the detection reaction. The fluorescence kinetics were monitored, and the detection was performed every 30 seconds for 100 cycles. The positive judgment results were consistent with the clinical sample test results. The results showed that the nucleic acid detection technology based on CRISPR / Cas12a can achieve sensitive, rapid, and accurate detection of GETV nucleic acid.

[0081] The embodiments of the present invention are described in detail above with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. It is apparent to those skilled in the art that various changes, modifications, substitutions, and variations to these embodiments may be made without departing from the principles and spirit of the present invention, and these changes and modifications still fall within the scope of protection of the present invention.

Claims

1. A CRISPR / Cas12a-based GETV detection kit, characterized by: Includes CRISPR / Cas12a detection system suitable for GETV detection; The CRISPR / Cas12a detection system includes a specific gRNA for the NS2 conserved gene of GETV, a CRISPR / Cas12a protein, and an ssDNA reporter system; The specific gRNA is directed against porcine GETV, and its sequence is SEQ ID NO.1; The ssDNA reporter system includes an ssDNA FQ reporter for fluorescence quantitative detection or an ssDNA FB reporter for immunochromatographic test strip detection; The ssDNA FQ reporter is ssDNA labeled with 6-carboxyfluorescein and a fluorescence quencher. The labeled product is as follows: 5'-FAM-TTATTATT-BHQ-3', named ssDNA FQ reporter; the sequence of ssDNA in the ssDNA FQ reporter is: 5'-TTATTATT-3'; The ssDNA FB reporter is ssDNA labeled with fluorescein and biotin. The labeled product is as follows: 5'-FAM-TTATTATT-Biotin-3', named ssDNA FB reporter; the sequence of ssDNA in the ssDNA FB reporter is: 5'-TTATTATT-3'.

2. The CRISPR / Cas12a-based GETV detection kit according to claim 1, wherein: The kit also includes an immunochromatographic test strip, which detects that the 5' and 3' ends of ssDNA are modified with FAM and biotin groups, respectively. The immunochromatographic test strip contains an anti-fluorescein antibody labeled with gold nanoparticles, which can bind to the fluorescein-modified end of the ssDNA FB reporter. The quality control line of the immunochromatographic test strip contains streptavidin, which can bind to the biotin group.

3. The CRISPR / Cas12a-based GETV detection kit according to claim 1, wherein: The method for preparing the specific gRNA includes: searching for a targeting sequence containing the CRISPR / Cas12a recognition sequence TTTN for the conserved GETV NS2 gene, designing the gRNA, and after the design is completed, synthesizing the target gRNA by a biological company.

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