CRISPR-Cas12a-based target pathogen rapid detection method

Abstract

The invention provides a CRISPR-Cas12a-based target pathogen rapid detection method, which comprises: S1, determining an LAMP primer group, and carrying out isothermal amplification on a target sequence of a target pathogen 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 to judge whether the target sequence of the target pathogen exists in the target sample or not; the LAMP primer group comprises sequences as shown in SEQ ID NO.1-SEQ ID NO.12, and the crRNA comprises sequences as shown in SEQ ID NO.13-SEQ ID NO.15. 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] This invention belongs to the technical field of biological detection, specifically relating to a rapid detection method for target pathogens based on CRISPR-Cas12a. Background Technology

[0002] Loop-mediated isothermal amplification (LAMP) involves designing four primers targeting six regions of a target gene and using strand-displacement DNA polymerase to perform nucleic acid amplification under isothermal conditions, achieving 10^10 amplifications within 15-60 minutes. 9 -10 10 The LAMP reaction generates a large amount of amplification products, namely magnesium pyrophosphate, a white precipitate. The presence of the target gene can be determined by visually observing the white precipitate. It is a "simple, rapid, accurate, and low-cost" gene amplification method. However, the rapid amplification of the LAMP reaction leads to a high concentration of the final product, which greatly increases the probability of cross-contamination. This is also a major reason for the high false positive rate of LAMP amplification.

[0003] CRISPR Cas12a (Cpf1) is a nuclease with both RNase and DNase activities. RNase activity refers to the processing of precursor crRNA into mature crRNA; DNase activity is the targeted cleavage of target DNA guided by mature crRNA. When Cas12a is activated, it can also cleave adjacent non-target DNA strands.

[0004] Leveraging these characteristics, and combining them with fluorescent reporter probes, this method can be used for pathogen detection, improving both the amplification efficiency of target genes and enhancing specific detection capabilities. A crRNA specifically targeting the gene sequence is designed to form a binary complex with Cas12a. When the reaction system contains the target gene sequence, the crRNA-Cas12a binary complex directionally cleaves the target sequence while non-specifically cleaving the fluorescent reporter probe in the system. This allows fluorescence to be observed under a specific wavelength of light, achieving simple and efficient detection of the target gene.

[0005] Porcine epidemic diarrhea virus (PEDV) is an acute intestinal infectious disease in pigs with high infectivity. Typical symptoms include watery diarrhea, vomiting, and dehydration. It can infect pigs of all ages, but newborn piglets under 7 days old are most severely affected, with a mortality rate as high as 100%. The PEDV N gene is highly conserved across different isolates, and antibody levels against the N protein are highest in the early stages of infection. This makes the N protein a key factor for rapid early diagnosis of PEDV infection; therefore, the nucleocapsid N protein gene (N) is used as the target gene for detection.

[0006] Transmissible gastroenteritis virus (TGEV) infection causes intestinal digestive system diseases, and pigs of all ages are susceptible. Affected pigs mainly exhibit clinical symptoms such as vomiting, watery diarrhea, and dehydration, with a mortality rate as high as 70%–100% in piglets under 2 weeks of age. TGEV is a member of the genus Coronavirus in the family Coronaviridae. Its viral genome is approximately 28.5 kbp, and the nucleocapsid (N) protein-coding gene is highly conserved. The encoded N protein is the only phosphorylated structural protein in TGEV and one of the most abundant viral structural proteins in infected cells. Therefore, the nucleocapsid N protein gene (N) is used as the detection target gene.

[0007] Porcine reproductive and respiratory syndrome virus (PRRSV) is an infectious disease characterized by reproductive disorders in pregnant sows and respiratory distress in pigs of all ages. Based on genomic typing, PRRSV can be divided into PRRSV-I (European type, prototype strain Lelystad virus) and PRRSV-II (North American type, prototype strain VR-2332 virus). PRRSV belongs to the Arteriviridae family and is a single-stranded positive-sense RNA virus. Its approximately 15 kbp genome is encapsulated by nucleocapsid proteins, and the virus is coated with a lipid bilayer of surface glycoproteins and membrane proteins. Glycoprotein 2 (GP2) was used as the target gene for detection through gene expression and conservation analysis.

[0008] Current pathogen detection methods mainly employ RT-qPCR, serology, and test strips. RT-qPCR requires sophisticated equipment, has long reaction times, involves multiple steps, and demands high operator skill. Serology and test strips have low sensitivity and are only suitable for individuals with obvious symptoms. However, the rapid detection system for PEDV, TGEV, and PRRSV-II based on CRISPR-Cas12a and CRISPR Cas12a described in this invention utilizes LAMP reaction to specifically and efficiently amplify the nucleic acid in the sample, increasing the amount of nucleic acid template to a detectable level for preliminary pathogen detection. In the second step, the LAMP amplification product is added to the Cas12a reaction system. Leveraging the characteristics of Cas12a, when the reaction contains a crRNA-specific target sequence, cleavage occurs, enabling secondary detection of the pathogen and improving detection accuracy. No reports have been found regarding the detection of PEDV, TGEV, and PRRSV-II using a LAMP combined with CRISPR Cas12a method.

[0009] Therefore, there is a need for a rapid detection method for PEDV, TGEV, and PRRSV-II based on corresponding LAMP primers and crRNA. Summary of the Invention

[0010] To address the aforementioned technical problems, this invention provides a rapid detection method for target pathogens based on CRISPR-Cas12a, thereby resolving the issues described in the background section.

[0011] On the one hand, this invention provides the following technical solution: a rapid detection method for target pathogens based on CRISPR-Cas12a, wherein the target pathogens include PEDV, TGEV, and PRRSV-II, comprising: S1. Determine the LAMP primer set, and isothermally amplify the target sequence of the target pathogen 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 pathogen's target sequence exists in the target sample; The LAMP primer set includes sequences as shown in SEQ ID NO.1 to SEQ ID NO.12, and the crRNA includes sequences as shown in SEQ ID NO.13 to SEQ ID NO.15.

[0012] The beneficial effects of the rapid detection method for target pathogens based on CRISPR-Cas12a proposed in this invention are as follows: This invention relies on two parts: (1) using the LAMP isothermal amplification method to specifically amplify the nucleic acid of the sample to be tested for the first detection; (2) using CRISPR Cas12a protein to perform a second detection on 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. It has high amplification efficiency, is simple and fast to operate, has low requirements for instruments and equipment, and has high specificity. The presence or absence of amplification products can be used to make a preliminary judgment on the presence or absence of target sequences. Next, a fluorescent reporter probe is added to the CRISPR Cas12a system. The enzyme activity is activated by specifically targeting the gene sequence through crRNA. The pathogen can be detected by visual light using specific light. Current pathogen detection methods mainly employ RT-qPCR, serology, and test strips. RT-qPCR requires sophisticated equipment, has long reaction times, involves multiple detection steps, and demands high operator skill. Serology and test strips have low sensitivity. While LAMP has been used for pathogen detection in recent years, it only provides single-stage detection and suffers from accuracy issues. This invention combines LAMP isothermal amplification technology with Cas12a protein to achieve highly sensitive and specific detection of target nucleic acids, improving the accuracy of PEDV, TGEV, and PRRSV-II detection. Furthermore, it is simple, rapid, and requires minimal equipment. This invention also contributes to the development of in vitro detection methods for other swine pathogens, applicable to grassroots farms lacking experimental facilities and trained personnel.

[0013] In addition, the rapid detection method for target pathogens based on CRISPR-Cas12a provided by the present invention may also have the following additional technical features: Preferably, 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 by the fluorescent reporter probe and releases fluorescence for detection.

[0014] Preferably, the sequence of the fluorescent reporter probe is 5'-FAM-TTTTTT-BHQ1-3'.

[0015] Preferably, the Cas12a protein is specifically the AsCas12a protein.

[0016] Preferably, the target gene for PEDV detection is the N gene, the target gene for TGEV detection is the N gene, and the target gene for PRRSV-II detection is the GP2 gene. Attached Figure Description

[0017] 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.

[0018] Figure 1 A detection system diagram of a rapid detection method for target pathogens based on CRISPR-Cas12a provided in an embodiment of the present invention; Figure 2 The PEDV-N sensitivity evaluation diagram provided in the embodiments of the present invention; Figure 3 The TGEV-N sensitivity evaluation diagram provided in the embodiments of the present invention; Figure 4 This is a sensitivity evaluation chart of PRRSV-Ⅱ-GP2 provided in an embodiment of the present invention; Figure 5 This is a PRRSV-Ⅱ-crRNA specificity evaluation diagram provided in an embodiment of the present invention.

[0019] The present invention will be further described below with reference to the accompanying drawings and description. Detailed Implementation

[0020] 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.

[0021] This invention provides a rapid detection method for target pathogens 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.

[0022] like Figure 1 As shown, for the rapid detection method for target pathogens based on CRISPR-Cas12a of the present invention, the target pathogens include PEDV, TGEV, and PRRSV-II, including: S1. Determine the LAMP primer set, and isothermally amplify the target sequence of the target pathogen using the LAMP primer set; Specifically, in step S1, the nucleic acid of the sample to be tested is specifically amplified using the LAMP isothermal amplification method to complete the initial detection. Therefore, step S1 is the initial detection process, and a preliminary judgment can be made on the presence of the target sequence based on whether there are amplification products.

[0023] 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 pathogen's target sequence exists in the target sample; Specifically, in step S2, the CRISPR Cas12a protein is used to perform a secondary detection on whether the target sequence exists in the amplification product of the nucleic acid of the test sample.

[0024] Therefore, the actual detection steps in this application are as follows: First, obtain the sample to be tested, and isothermally amplify the target sequence of the target pathogen in the sample to be tested using the LAMP primer set. If the corresponding amplification product is present, it indicates the presence of the corresponding pathogen. Then, the target sequence is detected using the crRNA, the fluorescent reporter probe, and the Cas12a protein. A second detection is performed by determining whether the target sequence exists. The rapid and accurate detection of the target pathogen is achieved through two detections.

[0025] The LAMP primer set includes sequences as shown in SEQ ID NO.1 to SEQ ID NO.12, and the crRNA includes sequences as shown in SEQ ID NO.13 to SEQ ID NO.15.

[0026] The fluorescent reporter probe is labeled with a quenching gene at one end and a fluorescent gene at the other end. The activated Cas12a protein is cleaved by the fluorescent reporter probe and releases fluorescence for detection.

[0027] The sequence of the fluorescent reporter probe is 5'-FAM-TTTTTT-BHQ1-3'.

[0028] Specifically, the Cas12a protein is the AsCas12a protein.

[0029] The target gene for PEDV detection is the N gene, the target gene for TGEV detection is the N gene, and the target gene for PRRSV-II detection is the GP2 gene.

[0030] The reagents and consumables used in this invention are all commercially available products. The invention is further illustrated below with reference to the embodiments: The main reagents used in this invention are as follows: DNA annealing buffer (5X) (D2810) was purchased from Solarbio; DEPC water (R0022) was purchased from Beyotime Biotech; T7 High Yield RNA Transcription kit (TR101-01) was purchased from Vazyme; NucleoSpin® RNA Clean-up (740609.5) was purchased from MAHEREY-NAGEL; Bst 4.0 DNA / RNA polymerase (A3805) was purchased from XinHai Gene; 10mM dNTPs (TKR-4019) were purchased from Takara. The Bio-lifesci CRISPR-Cas12a (AsCpf1 gene editing protein (M20303-0500) and fluorescent probe (5'-FAM-TTTTTT-BHQ1-3') were purchased from Guangzhou Bolais Biotechnology Co., Ltd.; the plasmid containing PEDV-N, TGEV-N, and PRRSV-Ⅱ-GP2 genes was synthesized by Qingke.

[0031] Example 1: LAMP primer and crRNA design and preparation 1. LAMP primer design: Conservation analysis was performed on the PEDV, TGEV, and PRRSV-II sequences. LAMP primer target regions were selected in the relatively conserved regions of PEDV, TGEV, and PRRSV-II, respectively. Then, based on the analysis results and design requirements, LAMP primers targeting the PEDV, TGEV, and PRRSV-II target genes were designed. The specific sequences are shown in Table 1. The primers were synthesized by Genewiz.

[0032] 2. crRNA design and preparation After identifying the target regions of the PEDV, TGEV, and PRRSV-II genes using the LAMP primer set, corresponding crRNAs were designed within these regions. The specific sequences are shown in Table 1.

[0033] The preparation of crRNA includes DNA synthesis, Oligo annealing, in vitro transcription, and RNA purification steps, finally yielding the corresponding crRNA. The DNA primers were synthesized by Genewiz and consist of an upstream T7 sequence (SEQ ID NO.16) and a downstream T7 sequence, structural sequence, and corresponding target sequence (SEQ ID NO.17~SEQ ID NO.19), as shown in Table 1.

[0034] The primers were diluted to 100 μM with 1X annealing buffer and annealed with primer Oligo. The annealing reaction system is shown in Table 2, and the reaction conditions are shown in Table 3. The annealed products were transcribed in vitro according to the instructions of the Vazyme in vitro transcription kit. The reaction system is shown in Table 4, and the reaction conditions are shown in Table 5. After agarose gel electrophoresis, the in vitro transcribed products were purified according to the instructions of the RNA purification kit. After elution with DEPC water, the concentration of the purified crRNA was determined and aliquoted at -80℃ for use.

[0035] Table 1 LAMP primers and crRNA sequences

[0036] Table 2 Annealing reaction system

[0037] Table 3 Annealing reaction conditions

[0038] Table 4 In vitro transcription reaction system

[0039] Table 5 In vitro transcription reaction conditions

[0040] Example 2: Sensitivity evaluation of the present invention for detecting PEDV-N, TGEV-N, and PRRSV-II-GP2 genes. The synthesized PEDV-N, TGEV-N, and PRRSV-Ⅱ-GP2 gene plasmids were serially diluted, with plasmid concentrations of 10-1. 0 Up to 10 6 The copy / μL was increased 10-fold and used as a template for the LAMP reaction. The LAMP reaction was performed in a 10 μL system, as shown in Table 6, and the reaction conditions are shown in Table 7. After the LAMP reaction was completed, the LAMP amplification product was added to the CRISPR Cas12a-crRNA-fluorescent reporter probe reaction system, as shown in Table 8, and the reaction conditions are shown in Table 9. Table 6 LAMP reaction system

[0041] Table 7 LAMP Reaction Conditions

[0042] Table 8 CRISPR Cas12a-crRNA-fluorescent reporter probe reaction system

[0043] Table 9. CRISPR Cas12a-crRNA-fluorescent reporter probe reaction conditions

[0044] The results are as follows Figure 2 As shown, LAMP primers and crRNA designed for the PEDV-N gene were used. After the LAMP product was reacted with the CRISPRCas12a-crRNA-fluorescent reporter probe system, the template concentration was observed to be 10 under blue light and ultraviolet light. 2 Amplification products were observed at a concentration of 10 copies / μL, indicating that the lowest detectable template concentration for visual detection of this LAMP-CRISPR Cas12a is 10. 2 Copy / μL.

[0045] The results are as follows Figure 3 As shown, LAMP primers and crRNA designed for the TGEV-N gene were used. After the LAMP product was reacted with the CRISPRCas12a-crRNA-fluorescent reporter probe system, the template concentration was observed to be 10 under blue light and ultraviolet light. 3 Amplification products were observed at a concentration of 10 copies / μL, indicating that the lowest detectable template concentration for visual detection of this LAMP-CRISPR Cas12a is 10. 3 Copy / μL.

[0046] The results are as follows Figure 4 As shown, LAMP primers and crRNA designed for the PRRSV-II-GP2 gene were used. After the LAMP product was reacted with the CRISPR Cas12a-crRNA-fluorescent reporter probe system, a template concentration of 10 could be observed under blue light and ultraviolet light. 2 Amplification products were observed at a concentration of 10 copies / μL, indicating that the lowest detectable template concentration for visual detection of this LAMP-CRISPR Cas12a is 10. 2 Copy / μL.

[0047] Example 3 Visual detection of PRRSV-II crRNA specificity Using synthesized PEDV-N, TGEV-N, and PRRSV-Ⅱ-GP2 gene plasmids as templates, respectively, the plasmid concentration was 10. 6 Copy / μL. The LAMP reaction was performed in a 10 μL system, as shown in Table 6, and the reaction conditions are shown in Table 7. After the LAMP reaction was completed, the LAMP amplification products were added to the CRISPR Cas12a-crRNA (PRRSV-Ⅱ-crRNA)-fluorescent reporter probe reaction system, as shown in Table 8, and the reaction conditions are shown in Table 9.

[0048] The results are as follows Figure 5As shown, when LAMP reaction products using PEDV-N, TGEV-N, and PRRSV-Ⅱ-GP2 plasmids as templates were added to the CRISPR Cas12a-crRNA (PRRSV-Ⅱ-crRNA)-fluorescent reporter probe reaction system, only the LAMP product corresponding to PRRSV-Ⅱ-GP2 could activate the CRISPR Cas12a-crRNA (PRRSV-Ⅱ-crRNA)-fluorescent reporter probe reaction system. After cleaving the fluorescent reporter probe, fluorescence was observed under blue light and ultraviolet light, indicating that PRRSV-Ⅱ-crRNA has the specificity to recognize the PRRSV-Ⅱ-GP2 LAMP product.

[0049] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention are equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. A rapid detection method for target pathogens based on CRISPR-Cas12a, wherein the target pathogens include PEDV, TGEV, and PRRSV-II, characterized in that, include: S1. Determine the LAMP primer set, and isothermally amplify the target sequence of the target pathogen 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 pathogen's target sequence exists in the target sample; The LAMP primer set includes sequences as shown in SEQ ID NO.1 to SEQ ID NO.12, and the crRNA includes sequences as shown in SEQ ID NO.13 to SEQ ID NO.

15.

2. The rapid detection method for target pathogens 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 pathogens 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 pathogens based on CRISPR-Cas12a according to claim 1, characterized in that, The Cas12a protein is specifically the AsCas12a protein.

5. The rapid detection method for target pathogens based on CRISPR-Cas12a according to claim 1, characterized in that, The target gene for PEDV detection is the N gene, the target gene for TGEV detection is the N gene, and the target gene for PRRSV-II detection is the GP2 gene.

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