CRISPR-Cas12a-based target gene rapid detection method

Abstract

The invention provides a CRISPR-Cas12a-based target gene rapid detection method, which comprises: S1, determining an LAMP primer group, and carrying out isothermal amplification on a target sequence of a target gene in a to-be-detected sample through the LAMP primer group; s2, determining crRNA, a fluorescent reporter probe and Cas12a protein matched with the target sequence, and detecting the target sequence through the crRNA, the fluorescent reporter probe and the Cas12a protein so as to judge whether the target sequence of the target gene exists in the target sample or not; the LAMP primer group comprises sequences as shown in SEQ ID NO.1-SEQ ID NO.16, and the crRNA comprises sequences as shown in SEQ ID NO.17-SEQ ID NO.20. According to the invention, high-sensitivity and high-specificity detection of target nucleic acid is realized, the detection accuracy is high, the operation is simple and rapid, and the requirements on instruments and equipment are low.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological detection, and particularly relates to a target gene rapid detection method based on CRISPR-Cas12a. BACKGROUND

[0002] Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification reaction under isothermal conditions using four primers designed for six regions on the target gene and a strand displacement DNA polymerase. It can achieve 10 9 -10 10 times amplification in 15-60 minutes, and a large amount of amplification product, i.e. magnesium pyrophosphate white precipitate, can be generated. The presence or absence of the target gene can be determined by observing the white precipitate with the naked eye. LAMP is a "simple, rapid, accurate and low-cost" gene amplification method. However, the rapid amplification of LAMP results in a high concentration of end products, greatly increasing the probability of cross contamination, which is also a major reason for the high false positive rate of LAMP amplification.

[0003] CRISPR Cas12a (Cpf1) is a system with RNase activity and DNase activity. The RNase activity refers to the processing of precursor crRNA into mature crRNA. The DNase activity is the targeted cleavage of target DNA under the guidance of mature crRNA. When Cas12a is activated, it can also cut the adjacent non-target DNA strand.

[0004] Using the above characteristics, combined with a fluorescent reporter probe, it can be used for pathogen detection, which improves the amplification efficiency of the target gene and enhances the specific detection capability. The crRNA specifically targeting the detection gene sequence is designed to form a binary complex with Cas12a. When the reaction system contains the target detection gene sequence, the crRNA-Cas12a binary complex will directionally cut the target sequence and non-specifically cut the fluorescent reporter probe in the system, so that the presence of fluorescence can be observed under a specific wavelength of light, realizing simple and efficient detection of the target gene.

[0005] Shrimp hemocyte iridescent virus (SHIV) belongs to the DNA virus of the iridovirus family of Decapoda iridovirus, which is the pathogen causing "white head disease" of Macrobrachium rosenbergii. Its highest mortality rate in Macrobrachium rosenbergii can reach more than 80%, which seriously restricts the development of Macrobrachium rosenbergii aquaculture industry. The virus particle contains a single linear double-stranded DNA of 140-303 Kbp. According to the particle size, host range, DNA cross-hybridization, presence of methyltransferase and sequence characteristics of the major coat protein family, it is classified accordingly. Therefore, the major coat protein gene (MCP) is used as the detection target gene.

[0006] Macrobrachium rosenbergii nodavirus (MrNV), mainly harm Macrobrachium rosenbergii dilution fry, the disease shrimp showed white, or muscle white spots white turbidity, can die within a few days, the mortality rate of 50% ~ 70%, up to 90% above, in a very short time become an international epidemic, causing serious losses to the global Macrobrachium rosenbergii breeding industry, the international animal health group must be declared important epidemic, Macrobrachium rosenbergii white tail disease has been listed as China's aquatic animal class 2 disease, Macrobrachium rosenbergii nodavirus is a must check the pathogen, through the analysis of the virus gene expression and conservation, using the coat protein gene (CP43) as a detection target gene.

[0007] Macrobrachium rosenbergii extra small virus (XSV) can cause Macrobrachium rosenbergii white tail disease, often found in the same time as Macrobrachium rosenbergii nodavirus in the body of the disease shrimp, the virus particles only 15 nm in diameter, the genome may include 0.9 kbp and 0.85 kbp two single-stranded RNA, suspected to be a new satellite virus. Through the analysis of the virus gene expression and conservation, using the coat protein gene (CP17) as a detection target gene.

[0008] Enterocytozoon hepatopenaei (EHP) is a ubiquitous obligate intracellular parasite, which can cause hepatopancreas microsporidiosis in shrimp, directly interfere with the growth and development of shrimp, eventually lead to the growth and development of shrimp slow, and easy to merge infection with other pathogens caused by white feces or cause shrimp death. Through the analysis of the pathogen gene expression and conservation, using the PTP2 gene as a detection target gene.

[0009] The existing pathogen detection methods mainly adopt RT-qPCR, serology, test strip and the like. The RT-qPCR has high requirements on instruments and equipment, long reaction time, multiple detection steps and high requirements on personnel operation; the serology and test strip have low detection sensitivity and are only suitable for personnel with obvious symptoms. However, the CRISPR-Cas12a and CRISPR Cas12a-based SHIV, MrNV, XSV and EHP rapid detection system in the application specifically and efficiently amplifies the nucleic acid of the sample to be detected through the LAMP reaction, amplifies the nucleic acid template with a small content to a detectable level, and completes the preliminary detection of the pathogen. In the second step, the LAMP amplification product is added to the Cas12a reaction system, and the Cas12a has the characteristics that when the reaction contains the crRNA specific target sequence, the shearing action is started, and the secondary detection of the pathogen is realized, thereby improving the accuracy of the pathogen detection. There is no report on the detection of SHIV, MrNV, XSV and EHP based on the LAMP combined with the CRISPR Cas12a method.

[0010] Therefore, it is necessary to provide a rapid detection method based on corresponding LAMP primers and crRNA and for SHIV, MrNV, XSV and EHP. SUMMARY

[0011] In order to solve the above technical problems, the application provides a CRISPR-Cas12a-based target gene rapid detection method to solve the problems in the background art.

[0012] In one aspect, the application provides the following technical scheme, a CRISPR-Cas12a-based target gene rapid detection method, the target gene includes SHIV, MrNV, XSV and EHP, comprising: S1, determining a LAMP primer set, and isothermal amplifying a target sequence of the target gene in a sample to be detected through the LAMP primer set; S2, determining a crRNA, a fluorescent reporter probe and a Cas12a protein matched with the target sequence, and detecting the target sequence through the crRNA, the fluorescent reporter probe and the Cas12a protein to determine whether the target sequence of the target gene exists in the target sample; The LAMP primer set includes the sequences shown in SEQ ID NO. 1 to SEQ ID NO. 16, and the crRNA includes the sequences shown in SEQ ID NO. 17 to SEQ ID NO. 20.

[0013] The CRISPR-Cas12a-based target gene rapid detection method provided by the application has the following beneficial effects: The present application relies on two parts: (1) using LAMP isothermal amplification method to specifically amplify the nucleic acid of the sample to be tested to complete the primary detection; (2) using CRISPR Cas12a protein to detect whether the target sequence exists in the amplification product of the nucleic acid of the sample to be tested. LAMP is a reaction that can be carried out at a constant temperature, and has high amplification efficiency, simple operation, rapidness, low requirement for instrument equipment, and high specificity. Whether the target sequence exists can be preliminarily judged according to whether there is an amplification product. Then, a fluorescent reporter probe is added in the CRISPR Cas12a system, the enzyme activity is activated by specific targeting of the gene sequence by crRNA, and the pathogen can be visually detected by using specific light.

[0014] Currently, pathogen detection mainly uses RT-qPCR, serology, test strip and the like. RT-qPCR has high requirement for instrument equipment, long reaction time, multiple detection steps, high requirement for personnel operation, low detection sensitivity of serology and test strip. In recent years, LAMP method has also been used to complete the detection of pathogens, but only one detection exists, and there is an accuracy problem. In the present application, LAMP isothermal amplification technology and Cas12a protein are combined to realize high-sensitivity and high-specificity detection of the target nucleic acid, improve the accuracy of SHIV, MrNV, XSV and EHP detection, and the operation is simple, rapid and has low requirement for instrument equipment. The present application is also helpful for developing in vitro detection methods for other fish and shrimp pathogens, and can be applied to primary breeding institutions without experimental conditions and trained personnel.

[0015] In addition, the target gene rapid detection method based on CRISPR-Cas12a provided by the present application can also have the following additional technical features: Preferably, one end of the fluorescent reporter probe is labeled with a quencher gene, and the other end of the fluorescent reporter probe is labeled with a fluorescent gene. The fluorescent reporter probe is cleaved and releases fluorescence for detection by the activated Cas12a protein.

[0016] Preferably, the sequence of the fluorescent reporter probe is 5'-FAM-TTTTTT-BHQ1-3'.

[0017] Preferably, the Cas12a protein is specifically LbCas12a protein.

[0018] Preferably, the target gene for SHIV detection is MCP gene, the target gene for MrNV detection is CP43 gene, the target gene for XSV detection is GP17 gene, and the target gene for EHP detection is PTP2 gene. BRIEF DESCRIPTION OF DRAWINGS

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 A detection system diagram of the rapid target gene detection method based on CRISPR-Cas12a provided in the embodiments of the present invention; Figure 2 This is a SHIV-MCP sensitivity evaluation chart provided in an embodiment of the present invention; Figure 3 The MrNV-CP43 sensitivity evaluation chart provided in this embodiment of the invention; Figure 4 The XSV-CP17 sensitivity evaluation chart provided in this embodiment of the invention; Figure 5 The sensitivity evaluation chart of EHP-PTP2 provided in the embodiments of the present invention.

[0021] The present invention will be further described below with reference to the accompanying drawings and description. Detailed Implementation

[0022] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain embodiments of the present invention, and should not be construed as limiting the present invention.

[0023] This invention provides a rapid detection method for target genes based on CRISPR-Cas12a. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The method and application of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the method and application described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0024] like Figure 1 As shown, for the rapid detection method of target genes based on CRISPR-Cas12a of the present invention, the target genes include SHIV, MrNV, XSV, and EHP, including: S1. Determine the LAMP primer set, and isothermally amplify the target sequence of the target gene using the LAMP primer set; Specifically, in step S1, the initial detection is completed by specifically amplifying the nucleic acid of the sample to be detected using the LAMP isothermal amplification method, and therefore step S1 is the initial detection process, and a preliminary judgment can be made on whether the target sequence exists according to whether there is an amplification product.

[0025] S2, determining a crRNA, a fluorescent reporter probe, and a Cas12a protein matched with the target sequence, and detecting the target sequence by the crRNA, the fluorescent reporter probe, and the Cas12a protein to determine whether the target sequence of the target gene exists in the target sample; Specifically, in step S2, the secondary detection is performed on whether the target sequence exists in the amplification product of the nucleic acid of the sample to be detected by using the CRISPR Cas12a protein.

[0026] Therefore, it can be known that the actual detection step in the application is: first, obtaining a sample to be detected, and then detecting the target sequence of the target gene in the sample to be detected by using the LAMP primer group isothermal amplification, if there is a corresponding amplification product, it indicates that there is a corresponding pathogen gene, and then the target sequence is detected by using the crRNA, the fluorescent reporter probe, and the Cas12a protein, and the secondary detection is performed by judging whether the target sequence exists, and the rapid and accurate detection of the target gene is realized by two detections.

[0027] The LAMP primer group includes sequences as shown in SEQ ID NO. 1-SEQ ID NO. 16, and the crRNA includes sequences as shown in SEQ ID NO. 17-SEQ ID NO. 20.

[0028] One end of the fluorescent reporter probe is labeled with a quencher gene, the other end of the fluorescent reporter probe is labeled with a fluorescent gene, and the fluorescent reporter probe is cleaved and releases fluorescence for detection by the activated Cas12a protein.

[0029] The sequence of the fluorescent reporter probe is 5'-FAM-TTTTTT-BHQ1-3'.

[0030] The Cas12a protein is specifically an LbCas12a protein.

[0031] The target gene for SHIV detection is the MCP gene, the target gene for MrNV detection is the CP43 gene, the target gene for XSV detection is the GP17 gene, and the target gene for EHP detection is the PTP2 gene.

[0032] The reagents and consumables used in the application are ordinary commercially available goods, and the application will be further described in conjunction with the following examples: The main reagent information used in the present application is as follows: DNA annealing buffer (5X) (D2810) is purchased from Solarbio Company; DEPC water (R0022) is purchased from Biyun Tian Company; T7 High Yield RNA Transcription kit (TR101-01) is purchased from Vazyme Company; NucleoSpin RNA Clean-up (740609.5) is purchased from MACHEREY-NAGEL Company; Bst 4.0 DNA / RNA polymerase (A3805) is purchased from Xin Hai Gene Company; 10 mM dNTP (TKR-4019) is purchased from Takara Company; Bio-lifesci CRISPR-Cas12a (LbCpf1 gene editing protein (M20301-0500), fluorescent probe (5'-FAM-TTTTTT-BHQ1-3')) are both purchased from Guangzhou Bio-lifesci Biotechnology Co., Ltd.; Plasmids containing SHIV-MCP, MrNV-CP43, XSV-CP17, and EHP-PTP2 genes are synthesized by GenScript.

[0033] Example 1 LAMP primer and crRNA design and preparation 1. Design of LAMP primers The SHIV, MrNV, XSV, and EHP sequences were analyzed for conservation, and the target regions of the LAMP primers were selected in the relatively conserved regions of SHIV, MrNV, XSV, and EHP, respectively. Then, according to the analysis results and design requirements, LAMP primers targeting the target genes of SHIV, MrNV, XSV, and EHP were designed, and the specific sequences are shown in Table 1, which were synthesized by Goldengene.

[0034] 2. Design and preparation of crRNA After confirming the target regions of SHIV, MrNV, XSV, and EHP genes by LAMP amplification, corresponding crRNAs were designed in the regions, and the specific sequences are shown in Table 1.

[0035] The preparation of crRNA includes the steps of DNA synthesis, Oligo annealing, in vitro transcription and RNA purification, and finally the corresponding crRNA is obtained. The DNA primer is synthesized by Genewiz, including T7 sequence as the upstream (SEQ ID NO. 21), and the downstream composed of T7 sequence, structure sequence and corresponding target sequence (SEQ ID NO. 22-SEQ ID NO. 25). The specific sequence is shown in Table 1. The above-mentioned primer is diluted to 100 μM with 1X annealing buffer, and the primer Oligo annealing is carried out. The annealing reaction system is shown in Table 2, and the reaction conditions are shown in Table 3. The annealing product is subjected to in vitro transcription according to the Vazyme in vitro transcription kit instruction, the reaction system is shown in Table 4, and the reaction conditions are shown in Table 5. After the in vitro transcription product is detected by agarose gel electrophoresis, it is purified according to the RNA purification kit instruction, eluted with DEPC water, and the concentration of the purified crRNA is determined. It is ready for use after being divided and stored at -80℃.

[0036] Table 1 LAMP primer and crRNA sequence

[0037] Table 2 Annealing reaction system

[0038] Table 3 Annealing reaction conditions

[0039] Table 4 In vitro transcription reaction system

[0040] Table 5 In vitro transcription reaction conditions

[0041] Example 2 Sensitivity evaluation of the application for detecting SHIV-MCP, MrNV-CP43, XSV-CP17 and EHP-PTP2 genes The synthesized SHIV-MCP, MrNV-CP43, XSV-CP17 and EHP-PTP2 gene plasmids are diluted step by step, and the plasmid concentration is 10 0 to 10 10 copies / μL in 10-fold increments, which is used as the template for LAMP reaction. The LAMP reaction adopts a 10 μL system, and the reaction system is shown in Table 6, and the reaction conditions are shown in Table 7. After the LAMP reaction, the LAMP amplification product is added to the CRISPR Cas12a-crRNA-fluorescent reporter probe reaction system, the reaction system is shown in Table 8, and the reaction conditions are shown in Table 9. Table 6 LAMP reaction system

[0042] Table 7 LAMP reaction conditions

[0043] Table 8 CRISPR Cas12a-crRNA-fluorescent reporter probe reaction system

[0044] Table 9 CRISPR Cas12a-crRNA-fluorescent reporter probe reaction conditions

[0045] Results as shown in Figure 2 , the LAMP primers and crRNA designed for SHIV-MCP gene, the LAMP product after CRISPR Cas12a-crRNA-fluorescent reporter probe system reaction, the template concentration of 10 3 copies / μL can be observed under blue light and ultraviolet lamp, that is, the minimum detectable template concentration of LAMP-CRISPR Cas12a visual detection is 10 3 copies / μL.

[0046] Results as shown in Figure 3 , the LAMP primers and crRNA designed for MrNV-CP43 gene, the crRNA designed for MrNV-CP43 gene, the LAMP product after CRISPR Cas12a-crRNA-fluorescent reporter probe system reaction, the template concentration of 10 3 copies / μL can be observed under blue light and ultraviolet lamp, that is, the minimum detectable template concentration of LAMP-CRISPR Cas12a visual detection is 10 3 copies / μL.

[0047] Results as shown in Figure 4 , the LAMP primers and crRNA designed for XSV-CP17 gene, the crRNA designed for XSV-CP17 gene, the LAMP product after CRISPR Cas12a-crRNA-fluorescent reporter probe system reaction, the template concentration of 10 2 copies / μL can be observed under blue light and ultraviolet lamp, that is, the minimum detectable template concentration of LAMP-CRISPR Cas12a visual detection is 10 2 copies / μL.

[0048] Results as shown in Figure 5As shown, the LAMP primers and crRNA designed for the EHP-PTP2 gene, the crRNA designed for the EHP-PTP2 gene, and the LAMP product after the CRISPR Cas12a-crRNA-fluorescent reporter probe system reaction can be observed under blue light and ultraviolet light when the template concentration is 10 2 copies / μL, there is an amplification product, that is, the minimum detectable template concentration of the LAMP-CRISPR Cas12a visual detection is 10 2 copies / μL.

[0049] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, and improvement within the spirit and principle of the present application, such as equivalent replacement, are included in the protection scope of the present application.

Claims

1. A rapid detection method for target genes based on CRISPR-Cas12a, wherein the target genes include SHIV, MrNV, XSV, and EHP, characterized in that, include: S1. Determine the LAMP primer set, and isothermally amplify the target sequence of the target gene in the sample to be tested using the LAMP primer set; S2. Identify the crRNA, fluorescent reporter probe, and Cas12a protein that match the target sequence, and use the crRNA, fluorescent reporter probe, and Cas12a protein to detect the target sequence to determine whether the target gene target sequence exists in the target sample; The LAMP primer set includes sequences as shown in SEQ ID NO.1 to SEQ ID NO.16, and the crRNA includes sequences as shown in SEQ ID NO.17 to SEQ ID NO.

20.

2. The rapid detection method for target genes based on CRISPR-Cas12a according to claim 1, characterized in that, One end of the fluorescent reporter probe is labeled with a quenching gene, and the other end of the fluorescent reporter probe is labeled with a fluorescent gene. The activated Cas12a protein is cleaved and releases fluorescence for detection.

3. The rapid detection method for target genes based on CRISPR-Cas12a according to claim 1, characterized in that, The sequence of the fluorescent reporter probe is 5'-FAM-TTTTTT-BHQ1-3'.

4. The rapid detection method for target genes based on CRISPR-Cas12a according to claim 1, characterized in that, The Cas12a protein is specifically the LbCas12a protein.

5. The rapid detection method for target genes based on CRISPR-Cas12a according to claim 1, characterized in that, The target gene for SHIV detection is the MCP gene, the target gene for MrNV detection is the CP43 gene, the target gene for XSV detection is the GP17 gene, and the target gene for EHP detection is the PTP2 gene.

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