A nucleic acid composition, kit and detection method for detecting infectious bronchitis virus based on RT-RAA and CRISPR-Cas13a

Abstract

The application provides an infectious bronchitis virus detection nucleic acid composition, kit and detection method based on RT-RAA and CRISPR-Cas13a, and belongs to the technical field of nucleic acid detection. The application provides an IBV detection nucleic acid composition, kit and detection method based on RT-RAA and CRISPR-Cas13a, a novel conservative fragment nsp6-nsp7 region is mined, and the conservative fragment is used as a detection target, specific primers are designed, and excellent amplification efficiency is obtained. The application organically combines isothermal amplification and CRISPR cutting, designs a T7 RNA polymerase promoter at the 5' end of a downstream primer, and greatly improves the sensitivity, specificity and reliability of detection. The detection method disclosed in the application utilizes the double recognition mechanism of isothermal amplification and a gene editing system, exhibits high specificity, sensitivity and anti-interference ability, and can realize on-site visual detection.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid detection technology, specifically relating to a nucleic acid composition, kit, and detection method for infectious bronchitis virus based on RT-RAA and CRISPR-Cas13a. Background Technology

[0002] Infectious bronchitis (IB) is a highly contagious, acute respiratory infectious disease of birds caused by the infectious bronchitis virus (IBV). IBV belongs to the Coronaviridae family and is a single-stranded positive-sense RNA virus. Due to the lack of proofreading function in its RNA polymerase, it is highly susceptible to homologous recombination during natural infection, leading to continuous evolution of IBV during epidemics and resulting in numerous genotypes and serotypes. The high mutation rate of IBV makes traditional nucleic acid detection methods prone to off-target effects due to variations in the target sequence of the primer or probe binding region, leading to missed detections. Therefore, identifying conserved regions from the highly variable viral genome to achieve broad-spectrum detection has always been a challenge in this field.

[0003] Currently, routine laboratory testing methods for IBV mainly include virus isolation and identification, serological testing, and reverse transcription polymerase chain reaction (RT-qPCR). Virus isolation and identification are time-consuming and cumbersome; serological methods are mostly used for retrospective epidemiological surveys and are difficult to use for rapid diagnosis of the pathogen in the early stages of infection. Although RT-qPCR has high sensitivity and specificity and is widely regarded as the "gold standard" for nucleic acid testing, this technology heavily relies on expensive variable-temperature thermal cycling equipment and a professional laboratory environment, and the testing time is long, which cannot meet the needs of point-of-care testing (POCT) in grassroots farms.

[0004] In recent years, recombinase-mediated isothermal amplification (RAA / RPA) and other nucleic acid isothermal amplification technologies have demonstrated enormous potential for field applications due to their ability to rapidly achieve exponential amplification of nucleic acids under isothermal conditions, eliminating reliance on complex instruments. Furthermore, with the rapid development of gene editing technology, clustered, regularly spaced short palindromic repeats and their associated proteins (CRISPR-Cas) systems have been successfully introduced into the field of molecular diagnostics. Among them, the Cas13a protein can specifically recognize target RNA under the precise guidance of guide RNA (crRNA) and, upon activation, releases a powerful "collateral cleavage" activity, efficiently and non-specifically cleaving the reporter probe in the system. Combining the efficient amplification of RT-RAA with the precise recognition of CRISPR-Cas13a can significantly improve detection sensitivity and signal-to-noise ratio.

[0005] However, effectively integrating RT-RAA, T7 in vitro transcription, and the Cas13a cleavage system to develop a field detection kit still faces significant technical challenges. For highly variable IBV, accurately identifying conserved regions in large-sample whole-genome sequences and designing primer-crRNA combinations with low off-target risk remains a bioinformatics bottleneck for achieving "broad-spectrum detection." Summary of the Invention

[0006] This invention provides a nucleic acid composition, kit, and detection method for infectious bronchitis virus (IBV) based on RT-RAA and CRISPR-Cas13a. It adopts a novel detection target, enabling broad-spectrum detection of various IBV genotypes. It requires no complex instruments, has a short detection time, and high specificity, providing technical support for on-site IBV screening.

[0007] The present invention provides a nucleic acid composition for detecting infectious bronchitis virus, comprising a specific primer pair for amplifying the conserved regions nsp6-nsp7 of infectious bronchitis virus and crRNA targeting the conserved region nsp7 of said infectious bronchitis virus. The downstream primer of the specific primer pair is connected to a T7 RNA polymerase promoter sequence at one end.

[0008] In one specific embodiment of the present invention, the specific primer pair includes an upstream primer with a nucleotide sequence as shown in SEQ ID No. 13 and a downstream primer with a nucleotide sequence as shown in SEQ ID No. 14 or SEQ ID No. 15.

[0009] In one specific embodiment of the present invention, the nucleotide sequence of the crRNA is shown in SEQ ID No. 11.

[0010] The present invention also provides a kit for detecting infectious bronchitis virus, comprising the above-mentioned nucleic acid composition, RT-RAA amplification reagent, T7 RNA polymerase, LwaCas13a protein, NTPs and reporter probe; The reporter probe is a single-stranded RNA consisting of 11 uracil ribonucleotides.

[0011] In one specific embodiment of the present invention, when the kit is used for fluorescence detection, a fluorescent group and a quenching group are respectively modified at both ends of the reporter probe; When the kit is used for lateral flow chromatography test strip detection, fluorescent groups and biotin are modified at both ends of the report probe, respectively.

[0012] This invention also provides a method for detecting infectious bronchitis virus for non-diagnostic and non-therapeutic purposes, comprising the following steps: (1) Using the nucleic acid of the sample to be tested as a template, RT-RAA isothermal amplification is performed using the specific primer pair in the above nucleic acid composition or the specific primer pair in the above kit to obtain the amplification product; (2) The amplification product described in step (1) is mixed with T7 RNA polymerase, LwaCas13a protein, crRNA, NTPs and reporter probe and reacted to obtain the loading solution; (3) Visually detect the sample solution to determine whether infectious bronchitis virus exists.

[0013] In one specific embodiment of the present invention, the reaction system in step (2) comprises, in 20 μL, the following components at final concentrations: LwaCas13a protein 100-400 nM, crRNA 50-200 nM, T7 RNA polymerase 1.25-7.5 U / μL, reporter probe 50 nM, NTPs 1 mM and magnesium ions 10-60 mM.

[0014] In one specific embodiment of the present invention, the reaction temperature in step (2) is 37°C and the time is 20~60 min.

[0015] The present invention also provides the use of the above-described nucleic acid composition or the above-described kit in the preparation of products for rapid diagnosis of infectious bronchitis virus.

[0016] The present invention also provides a rapid diagnostic kit for infectious bronchitis virus, comprising the above-mentioned nucleic acid composition, and further comprising RT-RAA amplification reagent, T7 RNA polymerase, LwaCas13a protein, NTPs, reporter probe and lateral flow chromatography test strip; The reporter probe is a single-stranded RNA composed of 11 uracil ribonucleotides, with one end modified with FAM or FITC and the other end modified with biotin.

[0017] Beneficial Effects: Addressing the problems of off-target false negatives, reliance on temperature-controlled instruments, difficulty in achieving both broad-spectrum coverage and point-of-care testing (POCT) in existing IBV detection technologies, and the high background noise and false negatives caused by dual-enzyme environmental conflicts in the development of single-tube isothermal CRISPR diagnostic systems, this invention provides an IBV detection nucleic acid composition, kit, and detection method based on RT-RAA and CRISPR-Cas13a. This method enables highly sensitive, highly specific, rapid, and visual broad-spectrum detection of IBV without the need for complex instruments.

[0018] This invention, through in-depth analysis of the entire IBV genome, uncovers a novel conserved fragment, the nsp6-nsp7 region. Using this conserved fragment as a detection target, specific primers are designed, exhibiting excellent amplification efficiency. In the examples, it was found that the specific primer pairs can broadly detect IBV epidemic strains with different genotypes, effectively overcoming the technical bottleneck of traditional nucleic acid detection, which is prone to missed detection due to target mutations.

[0019] In this invention, isothermal amplification and CRISPR cleavage are organically combined during detection. In traditional isothermal amplification procedures, the amplification products are prone to carrying the viral nucleic acid template from the original sample during transfer, leading to non-specific premature activation of Cas13a. To avoid non-specific premature activation, this invention cleverly designs the T7 RNA polymerase promoter at the downstream primer end, allowing the amplified reaction product to specifically transcribe a negative-strand RNA complementary to the target. Combined with crRNA specifically complementary to this negative-strand RNA, the activation of the gene-editing enzyme in this system depends entirely on the successfully amplified nascent strand, completely shielding the system from cross-reaction to the original residual positive-strand viral template. This eliminates the risk of premature activation and false positives caused by the original nucleic acid template carried by the amplification product during the two-step method, thereby greatly improving the sensitivity, specificity, and reliability of the detection.

[0020] The detection method described in this invention utilizes a dual recognition mechanism of isothermal amplification and gene editing systems, achieving a detection limit (LOD) as low as 100 copies / reaction. Furthermore, it exhibits high specificity and resistance to interference, showing no cross-reactivity with common avian respiratory non-target pathogens such as AIV, NDV, MDV, ILTV, and IBDV. In addition, the method described in this invention not only performs both amplification and cleavage reactions under isothermal conditions, freeing it from the constraints of PCR instruments, but also allows for direct reading via lateral flow chromatography test strips. The results are intuitive and rapid, making it extremely suitable for rapid initial screening and epidemiological monitoring of diseases in frontline environments lacking standard molecular laboratories, and highly adaptable to point-of-care testing (POCT) at the grassroots level. Attached Figure Description

[0021] Figure 1 This is an agarose gel electrophoresis image of the amplification products of 14 pairs of RT-RAA primers in Example 2 of the present invention. Figure 2 This is a bar chart showing the net fluorescence growth of the amplification products of the four candidate RT-RAA primers retained in the initial screening of Example 2 of the present invention and the combined cleavage reaction with crRNA. Figure 3 This is a real-time fluorescence kinetic curve of the cleavage reaction between the amplification products of the four candidate RT-RAA primers and crRNA in Example 2 of the present invention; Figure 4This is a heatmap showing the net fluorescence growth of the system with different concentrations of Cas13a protein and crRNA in Example 3 of the present invention. Figure 5 This is a bar chart showing the net fluorescence growth of the system with different T7 RNA polymerase concentrations in Example 3 of the present invention. Figure 6 This is a bar chart showing the net fluorescence growth of the system with different magnesium ion concentrations in Example 3 of the present invention; Figure 7 This is a bar chart showing the net fluorescence growth of the detection system in Example 4 of the present invention, based on the sensitivity (LOD) test. Figure 8 This is a bar chart showing the specificity of the detection system in Example 5 of the present invention against IBV multigenotype variants and common non-target avian pathogens in clinical practice. Figure 9 This is the amplification standard curve established by the TaqMan real-time PCR gold standard system in Example 6 of the present invention; Figure 10 This invention provides a schematic diagram of the structure and detection results of an RT-RAA-Cas13a side-flow chromatography test strip for detecting IBV, where T is the detection line and C is the control line. Figure 11 This is a colorimetric result image of 38 clinical double-blind patient samples detected using a lateral flow chromatography test strip in Example 6 of the present invention. Detailed Implementation

[0022] The present invention provides a nucleic acid composition for detecting infectious bronchitis virus, comprising a specific primer pair for amplifying the conserved regions nsp6-nsp7 of infectious bronchitis virus and crRNA targeting the conserved region nsp7 of said infectious bronchitis virus. The downstream primer of the specific primer pair is connected to a T7 RNA polymerase promoter sequence at one end.

[0023] This invention uses the nsp6-nsp7 region of the IBV virus genome as the target for isothermal amplification and CRISPR targeted recognition. The isothermal amplification can be RAA or RPA. In one embodiment, RT-RAA is used as an example, but it should not be considered as the entire scope of protection of this invention.

[0024] This invention designs a specific primer pair for amplifying the conserved region nsp6-nsp7 of IBV. The specific primer pair includes an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer is shown in SEQ ID No. 13. The downstream primer has a T7 RNA polymerase promoter sequence specifically linked to its 5' end, so that the transcription product is a negative-strand RNA complementary to the target fragment. In the examples, it was found that the downstream primers shown in SEQ ID No. 14 or SEQ ID No. 15 can complete the amplification with the upstream primer and achieve good amplification results. Furthermore, it was verified that the downstream primer shown in SEQ ID No. 15 has a better amplification effect.

[0025] The present invention also designed a crRNA that targets the conserved region nsp7, the nucleotide sequence of which is shown in SEQ ID No. 11.

[0026] The present invention also provides a kit for detecting infectious bronchitis virus, comprising the above-mentioned nucleic acid composition, RT-RAA amplification reagent, T7 RNA polymerase, LwaCas13a protein (Cas13a for short), NTPs and reporter probe; The reporter probe is a single-stranded RNA consisting of 11 uracil ribonucleotides.

[0027] This invention targets the strong paraphyletic cleavage activity of Cas13a protein on uracil (U)-containing single-stranded RNA after activation. A single-stranded RNA probe composed of 11 uracil ribonucleotides was designed and modified with differentiated functional groups according to different application platforms. When applied to a fluorescence platform, the sequence structure of the reporter probe is: 5'-FAM-UUUUUUUUUUU-BHQ1-3'; when the probe is intact, the fluorescence of FAM is quenched by BHQ1; when the probe is cleaved by Cas13a, the FAM fluorescence signal is released. When applied to a lateral flow chromatography test strip platform, it can be used for on-site detection. The sequence structure of the reporter probe is: 5'-FAM / FITC-UUUUUUUUUUU-Biotin-3'; it is used for bidirectional interception by streptavidin on the test strip with anti-FITC antibody or antibody that recognizes FAM / FITC labeling, thereby achieving visualized on-site detection. In this embodiment of the invention, the Cas13a protein used was purchased from Beyotime Biotechnology Co., Ltd. (D0517S).

[0028] This invention also provides a method for detecting infectious bronchitis virus for non-diagnostic and non-therapeutic purposes, comprising the following steps: (1) Using the nucleic acid of the sample to be tested as a template, RT-RAA isothermal amplification is performed using the specific primer pair in the above nucleic acid composition or the specific primer pair in the above kit to obtain the amplification product; (2) The amplification product described in step (1) is mixed with T7 RNA polymerase, LwaCas13a protein, crRNA, NTPs and reporter probe and reacted to obtain the loading solution; (3) Visually detect the sample solution to determine whether infectious bronchitis virus exists.

[0029] This invention first prepares an RT-RAA isothermal amplification system, wherein the RT-RAA isothermal amplification system, in 50 μL increments, includes RT-RAA amplification reagents and specific primer pairs, and the final concentration of the specific primer pairs in the RT-RAA isothermal amplification system is 0.4 μM. The RT-RAA isothermal amplification temperature of this invention is 42℃, and the time is 30 min. After RT-RAA isothermal amplification, a DNA amplification product containing the T7 RNA polymerase promoter sequence is obtained; subsequently, under the action of T7 RNA polymerase, it is transcribed to generate target RNA complementary to the IBV positive strand fragment, which is then recognized by the crRNA-LwaCas13a complex and activated for paracleavage activity.

[0030] This invention utilizes the amplified product to reconfigure a Cas enzyme cleavage system. The cleavage system, in 20 μL units, comprises the following components at final concentrations: LwaCas13a protein 100–400 nM, crRNA 50–200 nM, T7 RNA polymerase 1.25–7.5 U / μL, reporter probe 50 nM, NTPs 1 mM, and magnesium ions 10–60 mM. In the examples, the system exhibits the highest sensitivity with the following final concentrations: LwaCas13a protein 400 nM, crRNA 200 nM, T7 RNA polymerase 3.75 U / μL, reporter probe 50 nM, NTPs 1 mM, and magnesium ions 10 mM in the reaction buffer. The reaction buffer is the T7 RNA polymerase-compatible reaction buffer, and the final magnesium ion concentration is adjusted to 10–60 mM by adding MgCl2. After configuring the cleavage system, the cleavage reaction is performed at 37°C for 20–60 min. Experimental results in the examples demonstrate that, under the parameters described in this invention, the buffering conditions of the multi-enzyme cascade reaction can be effectively coordinated, resulting in the optimal net increase in the target signal. This provides an extremely abundant cutting product when the system is transferred from the fluorescence platform to the side-flow chromatography strip, ensuring high sensitivity and clarity of visual readings.

[0031] This invention employs a unique reverse transcription and cross-recognition design to eliminate template-borne noise. In the isothermal amplification-CRISPR cleavage two-step detection system, the amplification product is highly susceptible to carrying the viral nucleic acid template from the original sample during transfer, thus triggering non-specific premature activation of Cas13a. This invention cleverly designs the T7 RNA polymerase promoter at the downstream primer end, allowing the DNA product after the amplification reaction to specifically transcribe a negative-strand RNA complementary to the target. Combined with crRNA specifically complementary to this negative-strand RNA, the activation of the gene-editing enzyme in the cleavage system depends entirely on the successfully amplified nascent strand. This completely shields the system from cross-reaction to the original residual positive-strand viral template, eliminating the risk of premature activation and false positives caused by the original nucleic acid template carried by the amplification product during the two-step system, thereby greatly improving the sensitivity, specificity, and reliability of the detection.

[0032] This invention provides a visual detection method for the products after the cleavage reaction. This visual detection includes fluorescence detection or test strip detection. When the reporter probe is modified with a fluorescent group and a quencher group at both ends, fluorescence detection can be used. For example, when the fluorescent group is FAM and the quencher group is BHQ1, the detection wavelength is: excitation wavelength 485~495 nm, emission wavelength 515~525 nm; preferably, the excitation wavelength is 495 nm and the emission wavelength is 520 nm. A positive result is a significant increase in the fluorescence signal of the FAM channel with reaction time, or a significantly higher fluorescence intensity / net fluorescence increase at the reaction endpoint than the negative control, manifested as a significant enhancement of the green fluorescence signal. A negative result is that LwaCas13a is not activated by the target, the reporter probe is not cleaved, the FAM fluorescence is still quenched by BHQ1, the FAM channel fluorescence signal does not increase significantly, or the fluorescence intensity is basically the same as the negative control. When the two ends of the reporter probe are modified with FAM / FITC and biotin, respectively, a test strip can be used for detection. In this embodiment, the lateral flow chromatography test strip used is a disposable CRISPR nucleic acid detection test strip (single target), product code R104ZC, purchased from Hangzhou Zhongce Biotechnology Co., Ltd. The test strip has a lateral flow chromatography structure; the C-line is coated with streptavidin, the T-line is coated with goat anti-mouse secondary antibody, and the conjugate pad contains latex microsphere-labeled anti-FITC antibody. It is suitable for detecting CRISPR reporter probes with one end labeled with biotin and the other end labeled with FAM or FITC. In this invention, an equivalent lateral flow chromatography test strip with the same detection principle and the same line position interpretation method as the above test strip can also be used. During lateral flow chromatography detection, the product after the cleavage reaction is diluted with enzyme-free water and added dropwise to the sample well of the test strip. The colorimetric reaction time is 5-10 minutes, and the result is read within 10 minutes. In one embodiment of the present invention, the two ends of the reporter probe are modified with a FAM fluorescent group and biotin, respectively. After the colorimetric reaction, if the control area C line is colored and a clearly visible red band appears on the detection area T line, or only the detection area T line shows a clearly visible red band, the result is considered positive, indicating that the Cas enzyme is activated, the nucleic acid probe is cleaved, and the IBV target nucleic acid is present in the sample. If the IBV target nucleic acid is not present in the sample, LwaCas13a is not activated, the reporter probe is not cleaved, and the intact FAM-Biotin reporter probe is captured by the C line. Therefore, after the colorimetric reaction, if the control area C line is colored but the detection area T line is not colored, the result is considered negative, indicating that the Cas enzyme is not activated, the nucleic acid probe is not cleaved, and the IBV target nucleic acid is not detected in the sample. If no band appears on either the control area C line or the detection area T line, the result is considered invalid, indicating that the test strip is invalid, damp, damaged, or the operation is incorrect, and the sample should be resampled or re-amplified before testing.

[0033] The present invention also provides the use of the above-described nucleic acid composition or the above-described kit in the preparation of products for rapid diagnosis of infectious bronchitis virus.

[0034] The present invention utilizes the above-mentioned nucleic acid composition, kit and detection method to perform on-site visual detection of IBV with high specificity and high sensitivity. Experiments in the examples have shown that the limit of detection (LOD) is as low as 100 copies / reaction, and there is no cross-reaction with common non-target avian respiratory pathogens such as AIV, NDV, MDV, ILTV and IBDV, demonstrating high specificity and anti-interference ability.

[0035] The present invention also provides a rapid diagnostic kit for infectious bronchitis virus, comprising the above-mentioned nucleic acid composition, and further comprising RT-RAA amplification reagent, T7 RNA polymerase, LwaCas13a protein, NTPs, reporter probe and lateral flow chromatography test strip; The reporter probe is a single-stranded RNA composed of 11 uracil ribonucleotides, with one end modified with FAM or FITC and the other end modified with biotin.

[0036] The kit described in this invention is a kit for on-site visual detection of IBV, and the two ends of the reporter probe are modified with a fluorescent group and biotin, respectively. The sequence structure of the reporter probe is: 5'-FAM / FITC-UUUUUUUUUU-Biotin-3'.

[0037] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a nucleic acid composition, kit, and detection method for detecting infectious bronchitis virus based on RT-RAA and CRISPR-Cas13a provided by the present invention, shall not be construed as limiting the scope of protection of the present invention.

[0038] Example 1: IBV cleavage target establishment and primer sequence design 1.1 Large-scale full-length genome sequence screening and target region identification for IBV We obtained 805 publicly available full-length IBV genome sequences from public databases, wrote scripts to compare and analyze the results, and searched for highly consistent sequences between different genotypes. Finally, we successfully identified regions within the IBV genome that theoretically meet the conditions for cleavage reaction and used them as ideal targets for subsequent isothermal amplification and CRISPR targeting.

[0039] 1.2 Cross-matrix design of candidate crRNA and RT-RAA amplification primers Based on the identified conserved fragments, and strictly adhering to the design principles of isothermal amplification primers and the targeting recognition principles of CRISPR-Cas13a, a systematic candidate sequence design strategy was formulated: Three potentially highly active candidate guide RNAs were designed for the core sequence within the conserved target region, named cr1, cr2, and cr3, respectively. Simultaneously, to address the amplification region limitations of isothermal amplification technology, multiple isothermal amplification forward primers (F) and reverse primers (R) were designed around the recognition sites of the three crRNAs. Specifically, two forward primers (F1, F2) and two reverse primers (R1, R2) were designed for each crRNA target region. The primers were cross-combined to obtain 12 candidate RT-RAA amplification primer pairs (the specific combination matrix is: cr1-F1R1, cr1-F1R2, cr1-F2R1, cr1-F2R2; cr2-F1R1, cr2-F1R2, cr2-F2R1, cr2-F2R2; cr3-F1R1, cr3-F1R2, cr3-F2R1, cr3-F2R2). Detailed information on all candidate guide RNAs and amplification primers is shown in Table 1.

[0040] Table 1. Detailed information on candidate guide RNAs and amplification primers

[0041] 1.3 Design and Synthesis of Reporting Probes To capitalize on the strong paraphyletic cleavage activity of Cas13a protein on uracil (U)-containing single-stranded RNA after activation, a single-stranded RNA probe consisting of 11 uracil ribonucleotides was designed, and differentiated group modifications were performed according to different application platforms (all synthesized by Shanghai Bioscient Biotechnology Co., Ltd.): The fluorescent platform reporter probe (for laboratory validation) has the following sequence structure: 5'-FAM-UUUUUUUUUU-BHQ1-3'.

[0042] The side-flow chromatography test strip platform report probe (for on-site testing) has the following sequence structure: 5'-FAM / FITC-UUUUUUUUUU-Biotin-3'.

[0043] Example 2: Stepwise screening and verification of the optimal RT-RAA primer and crRNA combination To screen for core sequences with extremely high amplification efficiency, no primer dimers, and the strongest targeted cleavage activity from the candidate sequence library designed in Example 1, a two-stage verification strategy of "initial screening based on isothermal amplification performance - secondary screening based on CRISPR targeted cleavage" was adopted to rigorously screen candidate sequences. The coronavirus ORF1ab gene encodes various non-structural proteins, among which nsp3 and nsp12 are widely considered to be highly conserved detection targets in existing technologies. Isothermal primers were designed directly using sequences from existing technologies that target the conserved regions of nsp3 and classic nsp12, and used as controls in the screening verification experiment.

[0044] 2.1 First Stage: Initial Screening of RT-RAA Amplification Performance of Candidate Primers IBV-positive nucleic acid samples identified by the laboratory were extracted as templates, and isothermal amplification experiments were performed using 12 pairs of candidate RT-RAA primers designed in Example 1 and primers targeting the conserved regions of nsp3 and classical nsp12 in the prior art.

[0045] The basic type of RT-RAA nucleic acid amplification reagent (purchased from Hangzhou Zhongce Biotechnology Co., Ltd., catalog number: S003ZC) was used for the reaction. A 50 μL standard reaction system was prepared as follows: Take 25 μL of Buffer A, add 2.0 μL each of 10 μM candidate upstream and downstream primers, 13.5 μL of nuclease-free water, and 5 μL of extracted RNA sample; add the above mixture to a detection unit tube containing the reaction powder, and finally add 2.5 μL of Buffer B to the tube cap, tighten the cap, invert to mix, and briefly centrifuge. Incubate the reaction tube at 42℃ for 30 min.

[0046] After the reaction, to remove protein components that might affect electrophoresis, 50 μL of phenol:chloroform:isoamyl alcohol (25:24:1) extraction buffer was added to the reaction tube. After thorough mixing and centrifugation, the supernatant was collected for 2% agarose gel electrophoresis analysis. Electrophoresis results. Figure 1As shown, 10 out of 14 primer pairs exhibited severe primer dimer dispersion or no amplified bands in their respective lanes and were therefore discarded. This demonstrates that the conserved target of coronavirus in conventional PCR cannot be directly translocated to an isothermal amplification system. In the isothermal amplification (RT-RAA) system, the binding of primers to the template has far more stringent requirements on the GC continuity, secondary structure, and matching degree of the sequence than in PCR. Only the amplification products of four primer pairs—cr2-F1R1, cr2-F1R2, cr3-F2R1, and cr3-F2R2—exhibited bright, single bands with molecular weights perfectly matching the expected target gel bands. To further confirm the accuracy of the amplified sequences, these four amplification products were subsequently purified by gel extraction and sent for Sanger sequencing. Sequence alignment results confirmed that the actual amplified sequences perfectly matched the expected conserved IBV fragment (100% homology), providing an absolutely precise substrate template for subsequent Cas13a targeted cleavage.

[0047] 2.2 Second Stage: CRISPR-Cas13a Targeted Cutting Kinetics Rescreening The four high-quality primer pairs retained from the initial screening correspond to the target regions of the two candidate crRNAs designed in Example 1. To evaluate the true detection efficacy of the cross-combination of these four primer pairs and the two crRNAs, a fluorescence system was introduced for verification in this example.

[0048] Add LwaCas13a protein, the corresponding candidate crRNA, T7 RNA polymerase, NTPs, reaction buffer, RNase inhibitor, fluorescent reporter probe (5'-FAM-UUUUUUUUUU-BHQ1-3'), and the corresponding amplification product obtained in the first stage to the reaction system, and add nuclease-free water to a final volume of 20 μL. Place the reaction tube in a real-time PCR instrument, set the reaction temperature to 37℃, and collect the FAM channel fluorescence signal every 1 min for 60 min.

[0049] The cutting characteristics of each combination were analyzed by real-time fluorescence kinetic curve analysis, and the results are as follows: Figure 2 and Figure 3 As shown, different primer combinations with crRNA exhibited significant differences in Cas13a cleavage activation, with some combinations showing extremely low fluorescence values.

[0050] 2.3 Determination of the optimal nucleic acid testing combination After fluorescence kinetic curve ( Figure 3 ) and fluorescence growth value ( Figure 2A comprehensive comparison revealed that the cr3-F2R2 combination exhibited significant advantages: its fluorescence signal peaked fastest, reaching an extremely high fluorescence peak within 30 minutes, while the negative control group (NTC) maintained a stable baseline for 60 minutes with no nonspecific cleavage noise. This invention ultimately determined the optimal nucleic acid combination targeting the conserved nsp6-nsp7 region of IBV (i.e., the primer pairs shown in SEQ ID No. 13 and SEQ ID No. 15, and the crRNA shown in SEQ ID No. 11). This combination achieved optimal synergy between high isothermal amplification efficiency and sensitive Cas13a targeting recognition, and was directly used for subsequent single-tube conjugation system optimization and test strip performance evaluation.

[0051] Example 3: Microenvironment optimization of CRISPR-Cas13a single-tube coupled detection system In a single-tube reaction system, the transcription of T7 RNA polymerase and the cleavage of Cas13a protein have different requirements for the biochemical microenvironment (especially salt ion concentration). This embodiment is based on the optimal nucleic acid combination screened in Example 2, and systematically optimizes the concentration of the core components of the detection system in a gradient manner, while taking into account both detection rate and reagent economy.

[0052] 3.1 Optimization of the combination of Cas13a protein and crRNA concentration The ratio of effector protein to guide RNA in the system directly determines the assembly efficiency of the ribonucleoprotein (RNP) complex. This invention sets up three concentration gradients of Cas13a protein (100 nM, 200 nM, 400 nM) and three concentration gradients of crRNA (50 nM, 100 nM, 200 nM) for a 3×3 combination test.

[0053] The results are as follows Figure 4 As shown, the net fluorescence growth of the cleavage reaction exhibits a clear concentration dependence. When the concentrations of Cas13a and crRNA are low, the net fluorescence growth is also low, indicating that insufficient RNP assembly leads to low cleavage efficiency; as the concentrations of both increase, the fluorescence signal is significantly enhanced. Considering both detection sensitivity and reagent cost, this invention determines that 400 nM Cas13a protein and 200 nM crRNA are the optimal concentration combination.

[0054] 3.2 Optimization of T7 RNA polymerase concentration T7 RNA polymerase (purchased from Wuhan Aibotek Biotechnology Co., Ltd., catalog number: RK20582) is responsible for transcribing the amplification product into target RNA to activate Cas13a. To determine its optimal dosage, this invention tested concentration gradients from 1.25 U / μL to 7.5 U / μL.

[0055] The results are as follows Figure 5 As shown, when the T7 polymerase concentration is as low as 1.25 U / μL, insufficient substrate transcription leads to a decrease in fluorescence signal. When the concentration is increased to 3.75 U / μL, the fluorescence signal reaches a plateau, and further increases in the T7 polymerase concentration do not result in a significant increase in fluorescence signal. To avoid the introduction of impurities and non-specific reactions from excessive polymerase, this invention defines 3.75 U / μL as the optimal working concentration of T7 RNA polymerase.

[0056] 3.3 Optimization of magnesium ion concentration in the reaction system Given that T7 transcription requires a high magnesium environment while Cas13a cleavage is extremely sensitive to high salt, this invention optimizes the magnesium ion concentration in the system over a wide range of 10 mM to 60 mM to find the optimal balance between the activities of the two enzymes.

[0057] The results are as follows Figure 6 As shown, magnesium ion concentration has a significant impact on the cleavage efficiency of the system: under high salt conditions, the paraphyletic cleavage activity of Cas13a is severely inhibited, and the net fluorescence signal is suppressed to a low level; as the magnesium ion concentration decreases stepwise, the system signal increases in a stepwise manner. A magnesium ion concentration of 10 mM perfectly overcomes the biochemical environmental conflict of multiple enzymes in the single-tube coupling reaction, establishing the most critical buffer formulation parameter for this detection system.

[0058] 3.4 Final Optimized Reaction System Formulation Through the above system optimization, this invention establishes the optimal CRISPR-Cas13a reaction microenvironment for highly sensitive IBV detection. The final concentration formulation is limited to: Cas13a 400 nM, crRNA 200 nM, T7 RNA polymerase 3.75 U / μL, NTPs 1 mM, reporter probe 50 nM, and the magnesium ion concentration in the reaction buffer is limited to 10 mM.

[0059] Example 4: Evaluation of the Analytical Sensitivity (LOD) of the Detection System To accurately evaluate the detection capability of this detection system for trace amounts of viral nucleic acid, this embodiment prepared target RNA standards with known copy numbers and used a two-step method of "RT-RAA amplification-T7 / Cas13a transcriptional cleavage" for sensitivity testing.

[0060] 4.1 Preparation of target RNA standards (1) RNA was extracted from IBV-positive samples and reverse transcribed into cDNA. PCR amplification was performed using the optimal primer pair (cr3-F2R2 combination) selected in Example 2. To accommodate subsequent recognition of positive-strand crRNA, the T7 promoter sequence was specifically added to the 5' end of the downstream primer (R2). The amplified product was identified by agarose gel electrophoresis and then purified by gel extraction.

[0061] (2) In vitro transcription (IVT) to generate negative strand target: Using the purified PCR product as a template, in vitro transcription was performed using the T7 high-efficiency transcription kit (purchased from ABClonal, catalog number: RK20582). Since the T7 promoter is located downstream, this transcription process specifically synthesized negative strand RNA of the IBV target sequence.

[0062] (3) Purification and gradient dilution: The transcript was purified using RNA purification reagent. After determining the concentration, the initial copy number was calculated, and the target RNA standards were prepared by serial dilution with nuclease-free water at 10-fold concentrations.

[0063] 4.2 Two-step sensitivity test Step 1 (RT-RAA Amplification): Take 5 μL of the serially diluted RNA standards described above (with 0 copies of nuclease-free water as a negative control NTC) and add them to the RT-RAA reaction system (refer to Example 2.1). Incubate the reaction tubes at 42 degrees Celsius for 30 min to complete the reverse transcription and isothermal amplification of the target RNA.

[0064] Step 2 (T7 transcription coupled with Cas13a targeted cleavage): Take 3 μL of the RT-RAA amplification product obtained in Step 1 and add it to the optimized CRISPR-Cas13a single-tube reaction system of Example 3. Place it in a fluorescence quantitative PCR instrument and monitor at 37 degrees for 60 min.

[0065] 4.3 Sensitivity Results Analysis The results are as follows Figure 7 As shown, the limit of detection (LOD) for fluorescence detection is as low as 100 copies / reaction. This sensitivity fully meets the clinical need for accurate detection of trace viral loads in the early stages of infection.

[0066] Example 5: Evaluation of the broad spectrum and specificity of the detection system To verify the detection capability (broad spectrum) of the detection system of the present invention for various genotype strains and its anti-interference ability (specificity) in complex samples, this embodiment conducted specificity experiments on a variety of common avian pathogens.

[0067] 5.1 Selection of experimental strains and preparation of templates This embodiment selects two groups of representative pathogens for testing: (1) Broad spectrum test group (IBV variants): IBV strains or positive samples with wide clinical distribution and large evolutionary span were selected, covering the main GI lineages (GI-1, GI-13, GI-19, GI-22, GI-29) and GVI lineage (GVI-1).

[0068] (2) Specific test group (non-target pathogens): common avian pathogens that are easily confused in clinical practice are selected, including avian influenza virus (AIV), Newcastle disease virus (NDV), Marek's disease virus (MDV), infectious laryngotracheitis virus (ILTV) and infectious bursal disease virus (IBDV).

[0069] Total nucleic acid was extracted from each of the above pathogens and adjusted to the same concentration as the template for testing.

[0070] 5.2 Specificity and Broad-spectrum Testing Using equal amounts of the nucleic acids of the aforementioned pathogens as templates, RT-RAA amplification of cr3-F2R2 was performed using the primers screened in Example 2. Subsequently, 3 μL of each amplification product was added to the CRISPR-Cas13a reaction system finally optimized in Example 3, with a total volume of 20 μL for each CRISPR-Cas13a single-tube reaction system. The FAM fluorescence signal was continuously monitored at 37°C for 60 min.

[0071] 5.3 Results Analysis The results of the net fluorescence growth test after background subtraction at the reaction endpoint are as follows: Figure 8 As shown. Experimental data indicates: (1) Broad spectrum: All IBV genotype strains tested (GVI-1 and various branches of the GI lineage) effectively activated the cleavage reaction and produced significant positive fluorescence signals. This indicates that the target region of the nsp6-nsp7 region locked by the present invention based on large-scale bioinformatics comparison can effectively cover the prevalent IBV strains of different evolutionary branches and achieve broad-spectrum, non-discriminatory detection.

[0072] (2) High specificity: In the test groups of non-target avian pathogens such as AIV, NDV, MDV, ILTV and IBDV, there was no net increase in fluorescence signal in the system, which was consistent with the results of the negative control (NTC).

[0073] In summary, this detection system demonstrates that while ensuring broad-spectrum coverage of IBV variants, it exhibits no cross-reactivity with common non-target avian pathogens, possessing extremely high specificity and meeting the needs for accurate identification of complex mixed infection samples in clinical settings.

[0074] Example 6: Comprehensive performance evaluation of the RT-RAA-Cas13a lateral flow chromatography system based on multiple types of real-world double-blind clinical samples To comprehensively evaluate the diagnostic efficacy, anti-interference ability, and detection physical boundaries of the detection system constructed in this invention in real and complex clinical samples, this embodiment conducted a double-blind comparative verification on 38 clinical samples suspected of IBV infection.

[0075] 6.1 Collection of clinical samples and nucleic acid extraction The 38 diseased poultry samples collected in this embodiment cover a wide range of viral shedding pathways, including respiratory and digestive tracts. The sample types are complex, specifically including throat swabs, cloacal swabs, lung tissue homogenates, tracheal tissue homogenates, and mixed tissue homogenates from various organs. Total RNA was extracted from all the above samples using the classic Trizol extraction method. The extracted total RNA was dissolved in RNase-free water and stored at -80°C for later use.

[0076] 6.2 Gold Standard Quantitative Detection and Standard Curve Establishment for Clinical Samples Using TaqMan quantitative PCR (qPCR) targeting the conserved regions of IBV as the gold standard, double-blind detection was performed on the above 38 RNA templates, and a system was simultaneously established for... Figure 9 The quantitative standard curve is shown. Based on the standard curve, the linear regression equation is Y = -3.60X + 42.52, the amplification efficiency is 89.7%, and the linear correlation coefficient R0 is [value missing]. 2 =0.994, indicating the accuracy of the quantitative system. Results showed that the 38 samples included 10 negative and 28 IBV positive samples. The viral load in the positive samples varied widely, covering the entire dynamic range from the clinically high viral load shedding phase to the extremely low viral load residual phase.

[0077] 6.3 Blind Clinical Trial and Result Analysis of the Lateral Flow Chromatography Test Strip System Take an equal amount of the above nucleic acid template and perform single-tube coupled detection using the RT-RAA-Cas13a side-flow chromatography system finally optimized in Example 3 of this invention. Observe the T / C line color development results within 10 minutes. The test strip structure is as follows: Figure 10 As shown, the results of the 38 samples were judged as follows: Figure 11 As shown in Table 2, the detection method of the present invention exhibits the following outstanding diagnostic efficacy, in conjunction with the detailed comparative data: (1) High clinical specificity and low background noise: Data shows that all 10 qPCR negative samples (sample numbers 29-38) were negative in the test strip system of this invention, and no non-specific T line color was induced, with a clinical negative concordance rate of 100%.

[0078] (2) Highly efficient and precise interception during the core viral shedding period: Statistics show that when the viral load of the sample is in the "infectious viral shedding period," which is of greatest significance for epidemiological control, the CRISPR test strip of this invention achieves 100% positive detection in 18 samples with Ct≤29.5. This means that in the clinical field, this invention can extremely sensitively screen out all positive live organisms or tissues with actual infectious risks, thus preventing the spread of the disease.

[0079] (3) System stability: In blind testing, a nonlinear response was observed between virus concentration and test strip color intensity in some samples. This phenomenon is highly consistent with the saturation kinetics of isothermal CRISPR cascade amplification as a nonlinear endpoint method. Even in extreme cases where there may be small sequence polymorphisms or high levels of biochemical inhibitor residues (such as sample 4), this invention still provides a stable positive result.

[0080] (4) Scientific definition of physical detection limit (LOD) and grayscale boundary: For samples with extremely low viral load (latency period or residual recovery period), 10 samples had Ct>29.5. The test strips showed a boundary effect: Sample 9 (Ct=29.642) and Sample 24 (Ct=30.232) still showed weak positive color development, demonstrating the ability to impact extremely low concentrations, while the remaining 8 samples with Ct>29.5 did not show color development. Substituting this boundary Ct value into the quantitative equation for calculation, the detection limit (LOD) of the lateral flow chromatography test strip was found to be 10. 3 The magnitude of the copy / reaction.

[0081] Table 2 Clinical Pathological Material Detection Results

[0082] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A nucleic acid composition for detecting infectious bronchitis virus, characterized in that, This includes specific primer pairs for amplifying the conserved regions nsp6-nsp7 of infectious bronchitis virus and crRNA targeting the conserved region nsp7 of the infectious bronchitis virus. The downstream primer of the specific primer pair is connected to a T7 RNA polymerase promoter sequence at one end.

2. The nucleic acid composition according to claim 1, characterized in that, The specific primer pair includes an upstream primer with a nucleotide sequence as shown in SEQ ID No. 13 and a downstream primer with a nucleotide sequence as shown in SEQ ID No. 14 or SEQ ID No.

15.

3. The nucleic acid composition according to claim 1, characterized in that, The nucleotide sequence of the crRNA is shown in SEQ ID No.

11.

4. A kit for detecting infectious bronchitis virus, characterized in that, Includes the nucleic acid composition according to any one of claims 1 to 3, RT-RAA amplification reagent, T7 RNA polymerase, LwaCas13a protein, NTPs and reporter probe; The reporter probe is a single-stranded RNA consisting of 11 uracil ribonucleotides.

5. The reagent kit according to claim 4, characterized in that, When the kit is used for fluorescence detection, fluorescent groups and quenching groups are modified at both ends of the reporter probe, respectively; When the kit is used for lateral flow chromatography test strip detection, fluorescent groups and biotin are modified at both ends of the report probe, respectively.

6. A method for detecting infectious bronchitis virus for non-diagnostic and non-therapeutic purposes, characterized in that, Includes the following steps: (1) Using the nucleic acid of the sample to be tested as a template, RT-RAA isothermal amplification is performed using the specific primer pair in the nucleic acid composition of any one of claims 1 to 3 or the specific primer pair in the kit of claim 4 or 5 to obtain the amplification product; (2) The amplification product described in step (1) is mixed with T7 RNA polymerase, LwaCas13a protein, crRNA, NTPs and reporter probe and reacted to obtain the loading solution; (3) Visually detect the sample solution to determine whether infectious bronchitis virus exists.

7. The detection method according to claim 6, characterized in that, The reaction system described in step (2) comprises, in 20 μL, the following components at final concentrations: LwaCas13a protein 100-400 nM, crRNA 50-200 nM, T7 RNA polymerase 1.25-7.5 U / μL, reporter probe 50 nM, NTPs 1 mM, and magnesium ions 10-60 mM.

8. The detection method according to claim 6 or 7, characterized in that, The reaction in step (2) is carried out at a temperature of 37°C for 20 to 60 minutes.

9. The use of the nucleic acid composition according to any one of claims 1 to 3 or the kit according to claim 4 or 5 in the preparation of a product for rapid diagnosis of infectious bronchitis virus.

10. A rapid diagnostic kit for infectious bronchitis virus, characterized in that, The nucleic acid composition includes any one of claims 1 to 3, and further includes RT-RAA amplification reagent, T7 RNA polymerase, LwaCas13a protein, NTPs, reporter probe and lateral flow chromatography strip; The reporter probe is a single-stranded RNA composed of 11 uracil ribonucleotides, with one end modified with FAM or FITC and the other end modified with biotin.

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