Composition and method for rapidly detecting HBV RNA based on CRISPR / Cas13a combined with double crRNA and application

Abstract

The invention relates to the technical field of hepatitis B viruses, in particular to a composition and method for rapidly detecting HBV RNA based on CRISPR / Cas13a combined with double crRNA and application of the composition, the composition comprises Cas13a, the double crRNA, a reporter group molecule, an RNase inhibitor, a Cas13a reaction buffer, T7RNA polymerase, rNTP MIX and RNase-free, the invention correspondingly discloses a detection method using the composition and application of the composition, and the method for rapidly detecting HBV RNA based on CRISPR / Cas13a combined with the double crRNA comprises the Cas13a, the double crRNA, the reporter group molecule, the RNase inhibitor, the Cas13a reaction buffer, the T7RNA polymerase, the rNTP MIX and the RNase-free. The CRISPR / Cas13a combined double crRNA and RAA amplification technology composition is matched with the application of a detection method, so that the efficient detection of HBV RNA is realized without depending on a large instrument, the detection reaction can be carried out at 37 DEG C, and the detection steps are greatly simplified; and in addition, through a fluorescence report mode and a lateral flow chromatography test strip technology, visual interpretation of a result is also realized in a low-resource region.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of hepatitis B virus, in particular to a composition, method and application for rapid detection of HBV RNA based on CRISPR / Cas13a combined with double crRNA. BACKGROUND

[0002] Hepatitis B virus (HBV) infection can cause chronic hepatitis B (CHB) and may develop into severe liver disease, including cirrhosis and hepatocellular carcinoma. At present, about 250 million people worldwide are chronically infected with HBV, which is still a serious global public health challenge. To achieve the goal of "eliminating viral hepatitis by 2030" proposed by the World Health Organization (WHO), it is necessary to improve the diagnosis rate and treatment rate of HBV. There are various serum biomarkers for HBV diagnosis, which have different clinical diagnostic significance. HBV RNA, as a new type of HBV marker, can reflect the transcriptional activity of liver covalently closed circular DNA (cccDNA) and has been proven to be of great value in evaluating the antiviral efficacy of chronic hepatitis B and predicting clinical outcomes. In some patients with chronic hepatitis B who receive nucleotide / nucleoside analog (NAs) treatment, although the viral DNA level is strongly inhibited, the covalently closed circular DNA (cccDNA) in the liver cannot be completely eliminated, and rebound is prone to occur after drug withdrawal. Therefore, most patients need long-term treatment, but the safety risks, poor patient compliance and heavy economic burden associated with long-term medication are difficult problems that cannot be ignored in clinical practice, so how to accurately predict the risk of rebound after drug withdrawal is very important for the management and treatment of patients with chronic hepatitis B. Detecting serum HBV RNA levels can comprehensively evaluate the antiviral treatment effect of patients with chronic hepatitis B and predict treatment endpoints, providing meaningful individualized guidance for clinical anti-HBV treatment, selecting appropriate drug withdrawal time and analyzing prognosis. Therefore, it is of great clinical significance to develop a rapid and sensitive detection technology for HBV RNA.

[0003] Current HBV nucleic acid technology (NAT) includes quantitative real-time polymerase chain reaction (qPCR) and droplet digital PCR (ddPCR), which have excellent sensitivity and specificity, and are therefore considered the gold standard for serum HBV RNA diagnosis. However, these methods are relatively expensive and require precise equipment and professional technicians to handle, thus limiting their large-scale application. Therefore, there is an urgent need to develop a sensitive, rapid and convenient detection kit for HBV RNA, which will help clinical HBV diagnosis and infection management in resource-poor areas.

[0004] In recent years, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology and associated protein gene system (Cas) have been widely used in molecular diagnosis due to their unique high sensitivity and specificity. When specific CRISPR RNA (crRNA) binds to the target RNA, the CRISPR / Cas13a system exhibits strong trans-cleavage activity, which enables them to non-specifically cleave the nearby ssRNA reporter gene molecules to generate a detectable fluorescent signal. In addition, the CRISPR reporter gene can be visualized by reading the lateral flow chromatography test paper with the naked eye, thus providing a strategy for rapid diagnosis. Combined with recombinase amplification polymerase (RAA), the detection sensitivity can be significantly improved. The present method uses CRISPR / Cas13a combined with double crRNA, combined with RAA technology and lateral flow chromatography test paper, to provide an opportunity for the development of a high-sensitivity, rapid HBV RNA diagnostic kit. SUMMARY

[0005] The present application aims to provide a composition, method and application for rapid detection of HBV RNA based on CRISPR / Cas13a combined with double crRNA, to meet the needs of high stability, high sensitivity and low cost detection of HBV RNA in resource-poor areas.

[0006] To achieve the above-mentioned purpose, the present application provides a composition for rapid detection of HBV RNA based on CRISPR / Cas13a combined with double crRNA, which comprises Cas13a, double crRNA, reporter group molecules, RNase inhibitors, Cas13a reaction buffer, T7 RNA polymerase, rNTPMIX and RNase-free water.

[0007] The double crRNA in the composition of the present application can specifically bind to the cas13a fixed scaffold sequence and two RAA amplification products to form a complex, thereby activating the ability of cas13a to trans-cleave the reporter probe, and significantly enhancing the detection sensitivity of the composition for HBV RNA without the need for sophisticated instruments.

[0008] The double crRNA comprises a designed and synthesized crRNA1667 and a crRNA2304, wherein the sequence of the crRNA1667 is shown in SEQ ID NO. 1, and the sequence of the crRNA2304 is shown in SEQ ID NO. 2.

[0009] The reporter molecule is at least one of ssRNA reporter-Flu and ssRNA reporter-LFA, wherein the sequence of ssRNA reporter-Flu is shown as SEQ ID NO. 3, and the sequence of ssRNA reporter-LFA is shown as SEQ ID NO. 4.

[0010] The Cas13a is LwaCas13a.

[0011] The application also discloses a detection method for rapidly detecting HBV RNA based on a CRISPR / Cas13a combined double crRNA for non-disease diagnosis and treatment purposes, which is used in combination with the composition as described above, and the detection method comprises the following steps: S1: designing primers for a conserved segment of HBV RNA and screening a primer pair; S2: designing and synthesizing a double crRNA capable of specifically binding to the target of HBV RNA; S3: adding other components of the composition to establish a CRISPR / Cas13a-based detection system; S4: using the primer pair to perform RAA amplification on the sample to be tested, and putting the generated amplification product into the detection system for visual detection.

[0012] The primer pair has two groups, which are RT-RAA-FP1556 with the sequence shown as SEQ ID NO. 5 and RT-RAA-RP1794 with the sequence shown as SEQ ID NO. 6, and RT-RAA-FP2226 with the sequence shown as SEQ ID NO. 7 and RT-RAA-RP2394 with the sequence shown as SEQ ID NO. 8.

[0013] The 5 end of the RAA upstream primer RT-RAA-FP designed for the conserved sequence segment is designed with a T7 promoter.

[0014] The detection system is at least one of a fluorescent visual detection system and a lateral flow chromatography test strip visual detection system.

[0015] The application also discloses a kit for detecting HBV RNA, which comprises the composition as described above.

[0016] The kit also comprises a kit for RAA amplification.

[0017] This invention provides a composition, method, and application for rapid detection of HBV RNA based on CRISPR / Cas13a combined with dual crRNA. It is the first to disclose a composition utilizing CRISPR / Cas13a combined with dual crRNA and RAA amplification technology. This composition can be used in conjunction with detection methods to achieve efficient detection of HBV RNA without relying on large instruments, and can also achieve visual interpretation of HBV RNA in low-resource areas. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, 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.

[0019] Figure 1 This is a schematic diagram of the detection principle of a fluorescence and lateral flow chromatography test strip visualization method for detecting HBV RNA based on CRISPR / Cas13a combined with dual crRNA.

[0020] Figure 2 The results are electrophoresis results used to screen and validate the optimal primers for different target regions of RAA. (A) RT-RAA primer screening results for the 1500-1800 segment; (B) RT-RAA primer screening results for the 2000-2400 segment.

[0021] Figure 3 This is the result of the optimal crRNA screening for different regions; (A) Fluorescence dynamics curve of the optimal crRNA screening in the 1500-1800 range; (B) Comparison of fluorescence endpoint results of the optimal crRNA screening in the 1500-1800 range at 60 min; (C) Fluorescence dynamics curve of the optimal crRNA screening in the 2000-2400 range; (D) Comparison of fluorescence endpoint results of the optimal crRNA screening in the 2000-2400 range at 60 min.

[0022] Figure 4 This is a graph showing the sensitivity results of CRISPR / Cas13a combined with dual crRNA detection of HBV RNA; (A) Comparison of 60-minute endpoint fluorescence values ​​of HBV plasmids with different concentrations detected by dual detection of crRNA1667+crRNA2304; (B) Specificity evaluation of the RT-RAA-CRISPR / Cas13a combined dual crRNA detection method.

[0023] Figure 5is the visual result of the RT-RAA-CRISPR / Cas13a combined double crRNA fluorescent detection method.

[0024] Figure 6 is the visual result of the RT-RAA-CRISPR / Cas13a combined double crRNA test strip detection method.

[0025] Figure 7 is the schematic diagram of the RT-RAA-CRISPR / Cas13a combined double crRNA test strip detection method. DETAILED DESCRIPTION

[0026] The following examples facilitate a better understanding of the present application, but do not limit the present application. In the following examples, the experimental methods are conventional methods, unless otherwise specified. In the following examples, the test materials used are commercially available from conventional biochemical reagent stores, unless otherwise specified.

[0027] Example 1: Construction of HBV standard plasmid HBV genotype C (AF533983.1) with the sequence shown in SEQ ID NO 9 was cloned into a puc57 vector to construct an HBV standard plasmid as a standard product, as a plasmid standard product, which was synthesized by Shanghai Shengong Biotechnology Co., Ltd.

[0028] Example 2: Establishment of a fluorescent visualization system for detecting HBV RNA based on CRISPR / Cas13a combined double crRNA; More than 10,000 sequences containing HBV 8 genotypes were downloaded from the HBVdatabase database (Note that the HBVdatabase database link is https: / / hbvdb.lyon.inserm.fr / HBVdb / HBVdbIndex, and this time the version of the database is 62.0, and the update time is August 3, 2025), and Jalview software was used for conservation analysis, and the highly conserved 1556-1794 segment and 2226-2359 segment (conservation rate higher than 95%) were selected as target amplification segments, while avoiding the main splice body SP1 region (2447-489) of HBV RNA. Based on the database for the target segment, specific RAA primers FP and RP were designed, wherein a T7 promoter sequence (5'-TAATACGACTCACTATAGGG-3') was added to the 5' end of RT-RAA-FP, the primer sequences are shown in Table 1, and a DNA constant temperature rapid amplification reagent kit (basic type, Amply Future Co., Ltd.) was used for RAA amplification, and the HBV genotype C standard plasmid was amplified by RAA to obtain the target fragment.

[0029] Table 1 The RAA amplification reaction system includes the following components: 29.4 μL Abuffer dissolved resuspended RT-RAA freeze-dried particles, 2 μl RT-RAA-FP1556 (10 μM), 2 μl RT-RAA-RP1794 (10 μM), 2 μl RT-RAA-FP2226 (10 μM), 2 μl RT-RAA-RP2394 (10 μM) added to the resuspended RAA mixture for double amplification, the primer-reverse transcription isothermal amplification enzyme mixture (18.5 μl) is divided into two 1.5 ml EP tubes, 5 μL of RNA template is added. Finally, 1.5 μL of Bbuffer is added to start the amplification reaction, mix well, and react at 42°C for 30 min.

[0030] After the reaction is completed, an equal volume of DNA extraction solution (Tris-saturated phenol / chloroform / isoamyl alcohol=25:24:1) is added and mixed, centrifuged at 12000g at room temperature for 5 min, and the supernatant is carefully aspirated to obtain the amplification product with excess protein removed. Prepare a 2% agarose gel: weigh 1 g of agarose and dissolve in 50 ml of 1×TAE solution, heat in a microwave oven until the agarose is completely dissolved, then add goldview nucleic acid dye, shake well, pour the prepared solution into a mold, cool to form a 2% agarose gel, and place it in an electrophoresis tank. Mix the purified different segment primer pair amplification products with LoadingBuffer and add them to the sample well of the agarose gel, U=120V, electrophoresis for 30 min; place the gel in a gel imager for imaging. See Figure 2 Under ultraviolet irradiation, observe the brightness and position of the target band in the agarose gel, and select the primer pair with the best amplification efficiency.

[0031] A plurality of crRNAs are designed and synthesized for RAA amplification products 1 and 2 of different high-conserved segments, see Figure 4 to screen out the best crRNA1667 and crRNA2304 that can specifically bind to the target and highly activate Cas13a, wherein the sequence of RAA amplification product 1 is shown in SEQ ID NO. 10, and the sequence of RAA amplification product 2 is shown in SEQ ID NO. 11. RAA amplification products 1 and 2 are added to the CRISPR / Cas13a detection system at the same time, under the action of T7 RNA polymerase, target ssRNA is transcribed, different segment target ssRNA is base-paired with the corresponding crRNA, and then combined with Cas13a to form a ternary complex, which activates the enzyme activity of Cas13a, and trans-cleaves the fluorescence reporter group molecule (ssRNA reporter-Flu) in the system.

[0032] The components of the CRISPR / Cas13a combined double crRNA fluorescence detection system are as follows: LwaCas13a (100 nM) 2.4 μL, crRNA1667 (100 nM) 1.2 μL, crRNA2304 (100 nM) 1.2 μL, ssRNA reporter-Flu (10 μM) 0.8 μL, RNase inhibitor 0.5 μL, 10x Cas13a reaction buffer 2 μL, T7 RNA polymerase 0.5 μL, rNTP MIX (25 mM) 0.8 μL, RNase-free water to 20 μL, RT-RAA amplification product 12 μL, RT-RAA amplification product 22 μL.

[0033] LwaCas13a, 10x Cas13a reaction buffer were purchased from Guangzhou Meigao Biotechnology; T7 RNA polymerase was purchased from NEB (catalog number: M0251S), rNTP MIX was purchased from NEB (catalog number: N0466S), RNase inhibitor was purchased from Nanjing Novozyme Biotech Co., Ltd. (catalog number: R301-02), crRNA1667, crRNA2304, ssRNA reporter molecule were synthesized by Shanghai Sangon Biological Engineering Co., Ltd., and the specific sequences are shown in Table 2.

[0034] Table 2 The sensitivity and specificity of the established CRISPR / Cas13a were verified. For sensitivity verification, the RNA transcribed from the HBV standard plasmid by T7 RNA polymerase was diluted by 10 times successively, ranging from 6.57x (10 4 -10 -1 ) copies / μL, and RAA amplification was followed by CRISPR / Cas13a reaction. Referring to Figure 4 , the fluorescence detection results showed that the double RT-RAA-CRISPR detection system could detect 6.57x10 0 copies / μL of plasmid amplification product, with a fluorescence signal intensity of 6751±1493 a.u., which had a statistical difference compared with the negative control. For specificity verification of the method, serum samples of patients infected with hepatitis B virus and other hepatitis viruses, including hepatitis A virus (HAE), hepatitis C virus (HCV) and hepatitis E virus (HEV), were collected, and HBV RNA in the serum samples was extracted, and CRISPR / Cas13a detection was performed after RT-RAA amplification. The specificity verification results showed that the established CRISPR / Cas13a method had high specificity for hepatitis B virus, and there was no cross reaction with other hepatitis viruses.

[0035] Example 3: CRISPR / Cas13a combined with double crRNA to detect HBV RNA lateral flow chromatographic test strip visualization system.

[0036] The combination of lateral flow chromatographic test strip technology visualizes the detection results, making the method applicable to on-site detection and diagnosis in resource-poor areas. Based on the fluorescence visualization method, other system conditions remain unchanged, the ssRNA test strip reporter molecule (ssRNA reporter-LFA) is designed, and the 6-carboxyfluorescein (6-FAM) and biotin-labeled nucleic acid test strip of Wobio Biotech Co., Ltd. is used for visualization detection. The principle is as follows: the lateral chromatographic test strip is a sample pad, a conjugate pad, a quality control line (C), a detection line (T), and an absorbent pad in turn along the flow direction. The colloidal gold-labeled anti-FAM monoclonal antibody is used, the streptavidin (SA) is coated on the C line, and the goat anti-mouse secondary antibody is coated on the T line. When there is a large amount of HBV RNA target in the reaction system, the Cas13a cleavage activity is activated, the ssRNA test strip reporter molecule is completely cut, the colloidal gold-labeled anti-FAM monoclonal antibody binds to the FAM group and flows to the T line, which is captured by the secondary antibody, and the test strip shows a single T line, which is positive. When there is a small amount of HBV RNA target in the reaction system, Cas13a cannot completely cut the ssRNA test strip reporter molecule, and there is a part of the intact ssRNA test strip reporter molecule in the system. This part of the intact reporter molecule can be captured by streptavidin on the C line to develop color, and the cut reporter FAM binds to the anti-FAM monoclonal antibody on the T line to develop color. At this time, the test strip develops color on the C line and the T line, and the result is positive. When there is no HBV RNA target in the reaction system, the intact reporter molecule is completely captured on the C line, and the C line develops color to be negative. When neither the C line nor the T line develops color, it indicates that the operation is wrong or the test strip is invalid. The reaction is carried out at room temperature, and the result can be judged in 5-10 min. The principle diagram of the test strip can be seen in Figure 7 .

[0037] The RAA amplification product is added to the CRISPR / Cas13a test strip detection system, the T7 RNA polymerase transcribes the amplification product to form the target ssRNA, and the target ssRNA of different segments is base-paired with the corresponding crRNA. Then, combined with Cas13a, a ternary complex is formed, which activates the Cas13a enzyme activity and trans-cleaves the test strip reporter molecule in the system.

[0038] The sequence of the test strip reporter molecule (ssRNA reporter-LFA) is 6-FAM-TrUrUrUrUrUrC-Biotin.

[0039] The components of the CRISPR / Cas13a combined double crRNA test strip detection system are as follows: LwaCas13a (100 nM) 2.4 μL, crRNA1667 (100 nM) 1.2 μL, crRNA2304 (100 nM) 1.2 μL, ssRNA reporter-LFA (10 μM) 0.8 μL, RNase inhibitor 0.5 μL, 10x Cas13a reaction buffer 2 μL, T7 RNA polymerase 0.5 μL, rNTP MIX (25 mM) 0.8 μL, RNase-free water to 20 μL, RT-RAA amplification product 12 μL, RT-RAA amplification product 22 μL.

[0040] After mixing the above reaction system, incubate at 37°C for 30 min, then add 30 μL of RNase-free water and mix, insert the CRISPR / Cas13a detection test strip into the reaction solution, and observe and record the detection results after 5-10 min. The CRISPR / Cas13a combined test strip detection results show that when the target RNA is detected at > 6.57x10 3 copies / μL, the test strip has a single band on the T line, indicating that at this concentration, after the RT-RAA-CRISPR reaction, there is a large amount of target RNA in the system to activate the Cas13a protein, resulting in all ssRNA test strip reporter molecules being cut, which is a strong positive; when 6.57x10 2 copies / μL target RNA is detected, the test strip has T and C lines, indicating that at this concentration, the Cas13a protein activity is partially activated, and only part of the reporter molecules can be cut, which is a weak positive; when ≤ 6.57x10 1 copies / μL target RNA is detected, the test strip only has C line color development, indicating that HBV RNA cannot be detected in the sample, and the result is negative. Figure 6 Therefore, the detection sensitivity of the RT-RAA-CRISPR test strip is 6.57x10 2 copies / μL.

[0041] Therefore, the present application successfully establishes a new technology for detecting HBV RNA based on CRISPR / Cas13a combined double crRNA detection fluorescence and test strip visualization, and the kit can be applied to the detection of HBV RNA infection in low-resource areas.

[0042] The above disclosed is only a preferred embodiment of the present application, of course, cannot be limited by this to limit the scope of the present application, the person skilled in the art can understand that the implementation of all or part of the above-mentioned processes, and according to the equivalent changes of the claims of the present application, still belong to the scope covered by the present application.

Claims

1. A composition for rapid detection of HBV RNA based on CRISPR / Cas13a combined with double crRNA, characterized in that, the composition comprises Cas13a, double crRNA, reporter molecule, RNase inhibitor, Cas13a reaction buffer, T7 RNA polymerase, rNTP MIX and RNase-free water. 2.The composition of claim 1, characterized in that, the double crRNA comprises designed and synthesized crRNA1667 and crRNA2304, wherein the sequence of crRNA1667 is shown in SEQ ID NO.1 and the sequence of crRNA2304 is shown in SEQ ID NO.

2. 3.The composition of claim 1 or 2, characterized in that, the reporter molecule is at least one of ssRNA reporter-Flu and ssRNA reporter-LFA, wherein the sequence of ssRNA reporter-Flu is shown in SEQ ID NO.3 and the sequence of ssRNA reporter-LFA is shown in SEQ ID NO.

4. 4.The composition of claim 1 or 2, characterized in that, the Cas13a is LwaCas13a. 5.A detection method for rapid detection of HBV RNA based on CRISPR / Cas13a combined with double crRNA for non-disease diagnosis and treatment purposes, the detection method comprising the following steps: S1: designing primers for the conserved segment of HBV RNA and screening a primer pair; S2: designing and synthesizing double crRNA that can specifically bind to the target of HBV RNA; S3: adding Cas13a, reporter molecule, RNase inhibitor, Cas13a reaction buffer, T7 RNA polymerase, rNTP MIX and RNase-free water to the double crRNA to establish a detection system based on CRISPR / Cas13a; S4: using the primer pair to perform RAA amplification on the sample to be tested, and putting the generated amplification product into the detection system for visual detection. 6.The detection method of claim 5, characterized in that, the primer pair has two groups, which are RT-RAA-FP1556 with the sequence shown in SEQ ID NO.5 and RT-RAA-RP1794 with the sequence shown in SEQ ID NO.6, and RT-RAA-FP2226 with the sequence shown in SEQ ID NO.7 and RT-RAA-RP2394 with the sequence shown in SEQ ID NO.

8. 7.The detection method of claim 5, characterized in that, the detection system is at least one of a fluorescent visual detection system and a lateral flow chromatography test strip visual detection system. 8.A kit for detecting HBV RNA, carrying the composition of any one of claims 1-4.

9. The kit of claim 8, wherein Also included are kits for RAA amplification.

10. Use of a composition according to any one of claims 1 to 4 for non-diagnostic and therapeutic purposes in the detection of HBV RNA.

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