A method for detecting rotavirus based on DNA-guided CRISPR / Cas12a

Abstract

The application belongs to the technical field of virus detection, and provides a method for detecting rotavirus based on DNA-guided CRISPR / Cas12a, wherein a rotavirus CRISPR / Cas12a reaction system comprises crDNA, RT-RAA primers, Cas12a protein and ssDNA-FQ reporter molecules, and the nucleotide sequence of the crDNA is shown as SEQ ID NO. 1. In the application, the DNA guide is used to replace the RNA guide, can interact with the PAM binding domain of Cas12a, activate specific recognition of the RNA target and non-specific trans-cleavage activity, so as to realize nucleic acid detection of rotavirus; and the application can realize rapid detection, simple operation, low cost and flexible result interpretation.

Description

Technical Field

[0001] This invention belongs to the field of virus detection technology, and in particular relates to a DNA-guided CRISPR / Cas12a method for detecting rotavirus. Background Technology

[0002] In the swine industry, porcine rotavirus (PoRV) is one of the leading causes of viral diarrhea in piglets. After infection, it often co-infects with other enteric pathogens such as porcine epidemic diarrhea virus (PEDV), exacerbating clinical symptoms, leading to increased piglet mortality and causing significant economic losses to the pig farming industry. Furthermore, rotavirus has the ability to recombine genes, potentially generating new genotypes and posing a risk of cross-species transmission to humans and other animals, thus also posing a public health concern. Currently, there are no specific antiviral drugs, and prevention and control mainly rely on vaccines. However, rotavirus has numerous genotypes (42 G types and multiple P types have been identified so far), and cross-immunity between different genotypes is limited. The protective effect of vaccines depends on genotype matching of circulating strains. Therefore, rapid and accurate nucleic acid detection of rotavirus is of great significance for epidemic monitoring, vaccine selection, and the formulation of prevention and control strategies.

[0003] Currently, laboratory detection methods for rotavirus mainly include virus isolation and culture, serological detection, nucleic acid amplification technology, and the emerging CRISPR / Cas molecular detection system. While virus isolation and culture are accurate and reliable, they are cumbersome, time-consuming (usually several days), and require stringent experimental conditions, making them unsuitable for rapid diagnosis. Serological methods such as ELISA and latex agglutination assays are simple to perform and low in cost, but their sensitivity is limited, they cannot detect low viral loads, cannot distinguish between different genotypes, and are susceptible to cross-reactivity. Nucleic acid amplification-based detection technologies are currently the mainstream approach. Conventional RT-PCR and real-time quantitative RT-qPCR have the advantages of high sensitivity and specificity and have been widely used for laboratory diagnosis of rotavirus. However, these methods rely on sophisticated thermal cyclers and professional operators, have long reaction times (usually 1.5-2 hours), and are not suitable for rapid on-site testing or in resource-scarce areas. Isothermal amplification techniques such as RT-RAA (reverse transcription-recombinase-mediated amplification) can rapidly amplify RNA targets under isothermal conditions (37-42℃) (20-30 minutes), have low equipment requirements, and have good potential for on-site applications. However, isothermal amplification itself tends to produce non-specific amplification, easily generating false positive signals, and is difficult to achieve accurate genotype differentiation when used alone.

[0004] In recent years, CRISPR / Cas systems have been developed for nucleic acid detection. Among them, the Cas12a-based detection platform specifically recognizes target DNA through crRNA (RNA guide), activating non-specific trans-cleavage activity to cleave fluorescent reporter molecules and generate a signal. This method has high specificity, but it has significant drawbacks: First, crRNA is an RNA molecule, which is easily degraded by nucleases and has poor stability; second, traditional Cas12a can only recognize and cleave DNA targets. For RNA viruses such as rotavirus, RNA must first be reverse transcribed into DNA, increasing the number of steps and the risk of contamination; third, Cas12a recognition of DNA targets depends on PAM sequences (such as TTTN), which limits the range of primer and target selection.

[0005] In summary, existing detection methods still have significant shortcomings in terms of stability, ease of operation, PAM dependence, and field applicability. There is an urgent need to develop a new technology that can directly identify rotavirus RNA, eliminate the need for PAM sequences, and simplify the detection process. Summary of the Invention

[0006] The purpose of this invention is to provide a DNA-guided CRISPR / Cas12a method for detecting rotavirus, aiming to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] On one hand, the present invention provides a rotavirus CRISPR / Cas12a reaction system, comprising crDNA, RT-RAA primers, Cas12a protein, and ssDNA-FQ reporter molecule. The nucleotide sequence of the crDNA is shown in SEQ ID NO.1. The RT-RAA primers include a forward primer and a reverse primer. The nucleotide sequence of the forward primer is shown in SEQ ID NO.2. The nucleotide sequence of the reverse primer is shown in SEQ ID NO.3. The nucleotide sequence of the ssDNA-FQ reporter molecule is shown in SEQ ID NO.4.

[0009] On the other hand, the present invention also provides an application of the rotavirus CRISPR / Cas12a reaction system in the detection of rotavirus for non-disease diagnosis or treatment purposes.

[0010] On the other hand, the present invention also provides the application of the rotavirus CRISPR / Cas12a reaction system in the preparation of rotavirus kits.

[0011] On the other hand, the present invention also provides a DNA-guided CRISPR / Cas12a method for detecting rotavirus for non-disease diagnosis or treatment purposes, comprising the following steps:

[0012] The RNA of the test sample was reverse transcribed and amplified using RT-RAA primers in the rotavirus CRISPR / Cas12a reaction system. The amplified product was transcribed into RNA, which was then mixed with Cas12a protein, crDNA, and ssDNA-FQ reporter molecules and incubated to activate the trans-cleavage activity of Cas12a and generate a detection signal. When fluorescence appeared, it indicated the presence of rotavirus in the test sample.

[0013] On the other hand, the present invention also provides a rotavirus detection kit, including a rotavirus CRISPR / Cas12a reaction system.

[0014] Compared with the prior art, the specific beneficial effects of the present invention are as follows:

[0015] This invention uses a DNA guide instead of an RNA guide. DNA contains a 3' hairpin structure (mimicking the proximal double-stranded DNA of PAM) and a 5' spacer region, which can interact with the PAM binding domain of Cas12a, activating specific recognition and non-specific trans-cleavage activity of RNA targets, thereby enabling nucleic acid detection of rotavirus. The 2'-hydroxyl group of RNA ribose can efficiently hydrolyze phosphodiester bonds through intramolecular nucleophilic attack, while DNA lacks this hydroxyl group, resulting in a very low spontaneous hydrolysis rate. In addition, RNA is more sensitive to oxidation, radiation, and RNase attacks. Therefore, DNA exhibits stronger stability from both chemical kinetics and biological half-life perspectives. Thus, the DNA guide has stronger resistance to nuclease degradation than the RNA guide, and the kit prepared using it can be transported at room temperature for short periods of time.

[0016] The reaction system or kit provided by this invention enables rapid detection, is easy to operate, is low in cost, and allows for flexible interpretation of results. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the structure of crDNA provided in an embodiment of the present invention;

[0018] Figure 2 The fluorescence detection visualization results provided in the embodiments of the present invention are, from left to right, experimental group, control group 1, control group 2, and control group 3. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0020] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0021] Example 1: A rotavirus CRISPR / Cas12a reaction system, comprising the following core components:

[0022] crDNA: Designed specifically for rotavirus, such as Figure 1 As shown, it includes: a 5' spacer region (20 nt): complementary to the corresponding genotype-specific sequence; and a 3' hairpin structure (21 nt): containing a PAM-mimicking sequence (TCTT) for binding the PAM-binding domain of Cas12a. The complete nucleotide sequence is shown in SEQ ID NO.1 (synthesized by Sangon Biotech).

[0023] Cas12a protein: derived from Lachnospiraceae bacterium (LbCas12a) (Jiangsu ADS Bio);

[0024] ssDNA-FQ reporter molecule: Single-stranded DNA (ssDNA) with fluorescent groups (such as FAM) and quenching groups (such as BHQ-1) labeled at both ends, as shown in SEQ ID NO.4 (synthesized by Sangon Biotech).

[0025] RT-RAA primers, including: a forward primer with the sequence shown in SEQ ID NO.2 and a reverse primer with the sequence shown in SEQ ID NO.3 (synthesized by Sangon Biotech).

[0026] The specific reaction system is as follows:

[0027] The RT-RAA mixture (RT-RAA kit purchased from TwistAmp®) is shown in Table 1:

[0028] Table 1

[0029] RT-RAA forward primer (10 μM) 2.4 μL RT-RAA reverse primer (10 μM) 2.4 μL Test sample 1 μL water 12.2 μL MgOAc (Magnesium Acetate) (280mM) 2.5 μL

[0030] The in vitro transcription reaction system is shown in Table 2:

[0031] Table 2

[0032] The Cas12a detection mixture system is shown in Table 3:

[0033] Table 3

[0034] crDNA 200 nM ssDNA-FQ reporter molecule (FQ probe) 500 nM Enzyme digestion buffer (purchased from New England Biolabs) 5μL

[0035] Example 2: A method for detecting rotavirus based on DNA-guided CRISPR / Cas12a, comprising the following steps:

[0036] (1) RT-RAA amplification: Take the RNA sample to be tested and mix it with the RT-RAA mixture (Table 1) except for MgOAc at the bottom of the reaction tube. Add the lyophilized enzyme ball (included in the RT-RAA kit, containing reverse transcriptase, recombinase, single-strand binding protein, and DNA polymerase) and mix. Then add MgOAc, tighten the tube cap, and incubate at 37°C for 20 minutes to complete the reverse transcription and amplification reaction and obtain the amplification product (DNA). Then transcribe it into RNA. The specific operation is as follows: add the amplification product (or after purification) to the in vitro transcription reaction system (Table 2) and incubate at 37°C for an appropriate time to transcribe the DNA into RNA (RNA target).

[0037] (5) Cas12a detection: The transcribed RNA was mixed with the Cas12a detection mixture (Table 3) and incubated at 37°C for 30 minutes to activate the trans-cleavage activity of Cas12a, generating a detection signal. Fluorescence detection was performed, and green fluorescence was visually observed under a blue light transilluminator (470 nm). Figure 2 As shown, this indicates that rotavirus was detected;

[0038] Using the crDNA+FQ probe+Cas12a protein+RNA target from Example 2 as the experimental group, and simultaneously setting up FQ probe+Cas12a+RNA target, crDNA+FQ probe+RNA target, and crDNA+FQ probe+Cas12a as control groups 1-3, the detection process was carried out, and the results are as follows. Figure 2 As shown, this indicates that without the necessary components, rotavirus RNA cannot be cleaved, making accurate detection impossible.

[0039] The detection process in this embodiment of the invention starts from obtaining the rotavirus sample, firstly performing RT-RAA amplification, which takes 10 minutes, then transcribing it into RNA, and finally detecting it using the CRISPR-Cas12a system, which takes 15 minutes. The process is short and can achieve rapid detection.

[0040] The above description is only 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 should be included within the protection scope of the present invention.

Claims

1. A rotavirus CRISPR / Cas12a reaction system, characterized in that, The invention includes crDNA, RT-RAA primers, Cas12a protein, and ssDNA-FQ reporter molecule. The nucleotide sequence of the crDNA is shown in SEQ ID NO.

1. The RT-RAA primers include a forward primer and a reverse primer. The nucleotide sequence of the forward primer is shown in SEQ ID NO.

2. The nucleotide sequence of the reverse primer is shown in SEQ ID NO.

3. The nucleotide sequence of the ssDNA-FQ reporter molecule is shown in SEQ ID NO.

4.

2. The application of the rotavirus CRISPR / Cas12a reaction system as described in claim 1 in the detection of rotavirus for purposes other than disease diagnosis or treatment.

3. The application of the rotavirus CRISPR / Cas12a reaction system as described in claim 1 in the preparation of a rotavirus detection kit.

4. A method for detecting rotavirus based on DNA-guided CRISPR / Cas12a for non-disease diagnosis or treatment purposes, characterized in that, Includes the following steps: The RNA of the test sample is reverse transcribed and amplified using the RT-RAA primers in the rotavirus CRISPR / Cas12a reaction system as described in claim 1. The amplified product is transcribed into RNA, and then the RNA is mixed with Cas12a protein, crDNA, and ssDNA-FQ reporter molecule and incubated to activate the trans-cleavage activity of Cas12a and generate a detection signal. When fluorescence appears, it indicates that rotavirus is present in the test sample.

5. A rotavirus detection kit, characterized in that, Includes the rotavirus CRISPR / Cas12a reaction system as described in claim 1.

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