A novel duck reovirus RT-LAMP-CRISPR / Cas SF01 detection kit, detection method and application

Abstract

The application discloses a novel duck reovirus RT-LAMP-CRISPR / CasSF01 detection kit, a detection method and application, and belongs to the technical field of animal epidemic disease molecular diagnosis and nucleic acid detection. The application provides an RT-LAMP-CRISPR / CasSF01 detection method and a matching kit for a conserved region of NDRV S1, and the specific LAMP primer amplification is matched with the secondary recognition of the crRNA guided CasSF01, the amplification is carried out at 65 DEG C for 30 min, the CRISPR endpoint detection is carried out at 37 DEG C, the visual endpoint fluorescence detection result is obtained, and the method does not depend on the conventional PCR thermal cycle process. Meanwhile, the LAMP system and the CRISPR system are prepositioned in different spaces of the same reaction tube and sequentially mixed, the opening and transfer operation after amplification is reduced, and the aerosol pollution risk caused by the operation is reduced.

Description

Technical Field

[0001] This invention relates to the field of molecular diagnostics and nucleic acid detection technology for animal diseases, and in particular to a novel duck reovirus RT-LAMP-CRISPR / CasSF01 detection kit, detection method and application. Background Technology

[0002] Novel duck reoviruses can cause disease in duck flocks and lead to production losses. Rapid and accurate nucleic acid testing of suspected samples is helpful for infection identification and control. Conventional PCR or real-time fluorescent PCR usually requires thermal cycling or quantitative fluorescence equipment; loop-mediated isothermal amplification can rapidly amplify nucleic acids under isothermal conditions, but when judging solely based on amplification signals, it may be affected by non-specific amplification or aerosol contamination.

[0003] CRISPR nucleic acid detection utilizes crRNA to re-identify target sequences and generates fluorescent signals through paracleavage activity activated by Cas protein. Combining RT-LAMP with CRISPR detection can achieve both isothermal rapid amplification and sequence-specific recognition. However, it is still necessary to screen suitable LAMP primers and crRNAs for the conserved target regions of NDRV, and optimize amplification conditions, Cas protein and crRNA concentrations, and reaction ligation methods. Summary of the Invention

[0004] The purpose of this invention is to provide a novel duck reovirus RT-LAMP-CRISPR / CasSF01 detection kit, detection method, and application to solve the problems existing in the prior art. By combining specific LAMP primer amplification with crRNA-guided CasSF01 secondary recognition, amplification is performed at 65℃ for 30 min, and CRISPR endpoint detection is performed at 37℃ to obtain visual endpoint fluorescence detection results.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a novel duck reovirus RT-LAMP-CRISPR / CasSF01 detection kit, the kit comprising an RT-LAMP reaction system and a CRISPR / CasSF01 system; The RT-LAMP reaction system contains LAMP primers composed of F3, B3, FIP, BIP and LF; The CRISPR / CasSF01 system contains crRNA, CasSF01 protein, and a fluorescent reporter probe; The nucleotide sequences of F3, B3, FIP, BIP and LF are shown in SEQ ID NO.2-SEQ ID NO.6, respectively; The sequence of the crRNA is shown in SEQ ID NO.8.

[0006] This invention also provides a novel method for detecting duck reovirus in the same tube sequence using RT-LAMP-CRISPR / CasSF01 for non-diagnostic and non-therapeutic purposes, comprising the following steps: The RT-LAMP reaction system was placed at the bottom of the reaction tube, and the CRISPR / Cas12a system was placed at the tube cap. LAMP amplification was performed on the nucleic acid of the test sample, the positive control, and the negative control using the RT-LAMP reaction system, respectively. After amplification, the LAMP amplification product was mixed with the CRISPR / Cas12a system by centrifugation and the reaction was continued. After the reaction was completed, the fluorescence signal was collected, and the presence or absence of the fluorescence signal was used to determine whether the sample was a novel duck reovirus. The RT-LAMP reaction system contains LAMP primers composed of F3, B3, FIP, BIP and LF; The CRISPR / CasSF01 system contains crRNA, CasSF01 protein, and a fluorescent reporter probe; The nucleotide sequences of F3, B3, FIP, BIP and LF are shown in SEQ ID NO.2-SEQ ID NO.6, respectively; The sequence of the crRNA is shown in SEQ ID NO.8.

[0007] Optionally, the LAMP amplification reaction conditions are 65℃ for 30 min; the LAMP amplification reaction system consists of 1.0 μL DNA polymerase, 10 U reverse transcriptase, 2.5 μL FIP, 2.5 μL BIP, 2.5 μL F3, 2.5 μL B3, 2.5 μL LF, 1.5 μL MgSO4, 2.5 μL buffer, 1.6 μL dNTP mixture, 5.0 μL nucleic acid of the sample to be tested, and nuclease-free water to a final volume of 25 μL.

[0008] Optionally, the CRISPR / CasSF01 system consists of 2 μL of 800 nM CasSF01 protein, 2.5 μL of 20x T7 terminator enzyme, 2 μL of 400 nM crRNA, 2.5 μL of 20x DTT, 1.25 μL of 40 μM fluorescent reporter probe, and 14.75 μL of nuclease-free water.

[0009] Optionally, the continued reaction is carried out at 37°C for 15 minutes.

[0010] Optionally, if there is a fluorescent signal and the fluorescence intensity is significantly higher than that of the negative control, the sample to be tested is determined to be / contains the novel duck reovirus; if there is no fluorescent signal, or the fluorescence intensity is not significantly different from that of the negative control, the sample to be tested is determined not to be / does not contain the novel duck reovirus.

[0011] The present invention discloses the following technical effects: This invention provides a method and kit for detecting RT-LAMP-CRISPR / CasSF01 in the conserved region of NDRV S1. The method combines specific LAMP primer amplification with crRNA-guided CasSF01 secondary recognition, amplification at 65℃ for 30 min, and CRISPR endpoint detection at 37℃ to obtain visualized endpoint fluorescence results, independent of conventional PCR thermal cycling. Furthermore, by pre-positioning the LAMP and CRISPR systems in different spaces within the same reaction tube and mixing them sequentially, the risk of aerosol contamination from opening the tube after amplification is reduced. This detection method exhibits excellent specificity and sensitivity. Additionally, it allows for selection of fluorescence method or blue light excitation for visual observation, making it suitable for on-site use. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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.

[0013] Figure 1 Amplification curves for four sets of NDRV LAMP candidate primers; Figure 2 The results show the optimized ratio of inner and outer primer concentrations for LAMP. Figure 3 The results show the optimized concentration ratio of outer primers to loop primers for LAMP. Figure 4 The final concentration of MgSO4 was optimized. Figure 5 The final dNTP concentration optimization results; Figure 6 The results of LAMP amplification at 63℃, 65℃, and 67℃ are shown. Figure 7 The results of LAMP amplification at 65℃, 67℃, and 69℃ are shown. Figure 8 Results of LAMP amplification time optimization; Figure 9 The results of CRISPR fluorescence curve screening for three crRNAs; Figure 10 The results of the endpoint fluorescence screening for three crRNAs; Figure 11 Optimize the curve for the combination of CasSF01 and crRNA concentrations; Figure 12 The endpoint fluorescence results are for the combination of CasSF01 and crRNA concentrations; Figure 13 The results are from the NDRV-positive plasmid template sensitivity test. Figure 14 Results of nucleic acid specificity assays for ARV, NDV, DPV, and H5 / H7 AIV; Figure 15 This is the result of repeatability verification. Detailed Implementation

[0014] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0015] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0016] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0017] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0018] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0019] Example 1 1. Target selection and positive plasmid preparation By comparing the S1 segment of NDRV 091 strain (GenBank: JX478256) with other NDRV S1 sequences, a relatively conserved region was selected, and the 489 nt fragment shown in SEQ ID NO.1 was synthesized as the target fragment. The positive plasmid was constructed by Beijing Qingke Biotechnology Co., Ltd.

[0020] SEQ ID NO.1: ATGTCGCTCCTGCCAACCCCGATCCTGTTCAAGTCGTTTAAAGTCCACTCGTGGCGTCTTCTGTCCCAATCCCCGTTCCACGTTCAATTCTGCGACGCAGGACTCCAATCCTACGACGTCCTATTCTCGTTTCCCTTCTGTGTGTGACCTTTCGCTCTGCTATTATCTTAATACGCCTTTCGAGTTTTGGAATTGCTGCTATTGAGGGTCGCCCCGGTGACTATTATTTGCTGTTCGCTGGCAAGTC CAGTGACTCTAACTCACGAGTATCTTTGTACGCTACGCGGCGAGCCGGTGACGATGGATCGCAACGAGGTGATACGCCTGATACTTTCCCTCCTCCCCTACCAGTCAAGCGACGTCGATCATTTGACGACACAGATCAAATCCCTCCAAAGCGCCGTCGACTCACTCAAAGAATCACAAGTGGTGGTGTTGAGACGCCTGACTACGATTACGTCGACGGTGGCAGATCTACAATCAACAACTGA.

[0021] 2. LAMP primer screening Four sets of LAMP primers were designed using PrimerExplorer, each set including F3, B3, FIP, BIP, and LF. Amplification was performed at 65℃ (amplification system: 40 U / μL Hieff). ® Bst Plus DNA Polymerase (Bst DNA polymerase 1μL, 200U / μL Hifair) ®III. Reverse transcriptase 0.05 μL, 20 μM FIP 2 μL, 20 μM BIP 2 μL, 20 μM F3 0.25 μL, 20 μM B3 0.25 μL, 20 μM LF 0.5 μL, 20x Eva green 1.25 μL, 100 mM MgSO4 1.5 μL, 10x resection buffer 2.5 μL, 25 mM dNTPs 1.4 μL, H2O to a final volume of 25 μL, template DNA added 5 μL) for 60 min and amplification curves compared. Figure 1 LAMP4 showed an earlier peak and better amplification performance, so LAMP4 was chosen as the primer set for subsequent reactions.

[0022] NDRV LAMP4 F3:CCCGATCCTGTTCAAGTCG, SEQ ID NO.2; NDRV LAMP4 B3: TGCCAGCGAACAGCAAATAATA, SEQ ID NO.3; NDRV LAMP4 FIP: GATTGGAGTCCTGCGTCGCAGAAAGTCCACTCGTGGCG, SEQ ID NO.4; NDRV LAMP4 BIP: TGTGTGTGACCTTTCGCTCTGCGGGCGACCCTCAATAGCA, SEQ ID NO.5; NDRV LAMP4 LF: GAACGTGGAACGGGGATTGG, SEQ ID NO.6; CrRNA-1: AGAGAAUGUGUGCAUAGUCACACAACGUGGAACGGGGAUUGGG, SEQ ID NO.7; CrRNA-2: AGAGAAUGUGUGCAUAGUCACACUCGUUUCCCUUCUGUGUGUG, SEQ ID NO.8; CrRNA-3: AGAGAAUGUGUGCAUAGUCACACCGCUCUGCUAUUAUCUUAA, SEQ ID NO. 9.

[0023] 3. Optimization of RT-LAMP reaction conditions (1) Optimization of the ratio of inner and outer primers Based on the previously determined optimal primer combination, the final concentration of the outer primer was fixed at 0.2 μM. The inner and outer primer ratios were optimized to 1:2, 1:4, 1:6, 1:8, 1:10, and 1:12. Amplification was performed at 65℃ on a fluorescence PCR instrument, with one cycle every 30 seconds. 120 cycles were performed for a total of 60 minutes. The optimal concentration and ratio of the inner and outer primers were determined based on the amplification curve.

[0024] (2) Optimization of circular primer concentration Based on the previously optimized reaction system, the final concentration of the outer primer was fixed at 0.2 μM. The ratio of outer primer to loop primer was optimized at 0:1, 1:1, 1:2, 1:4, 1:6, and 1:8. Amplification was performed at 65℃ on a fluorescence PCR instrument, with one cycle every 30 seconds. 120 cycles were performed, for a total of 60 minutes. The optimal concentration and ratio of outer primer to loop primer were determined based on the amplification curve.

[0025] (3) Optimization of MgSO4 concentration Based on the previously optimized reaction system, six MgSO4 concentration gradients of 0 mM, 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM were designed for the reaction. Amplification was performed at 65℃ using a fluorescence PCR instrument, with one cycle every 30 seconds, for 120 cycles, totaling 60 minutes. The optimal MgSO4 concentration was determined based on the amplification curves.

[0026] (4) Optimization of dNTP concentration Based on the previously optimized reaction system, seven dNTP concentration gradients (0.6 mM, 0.8 mM, 1.0 mM, 1.2 mM, 1.4 mM, 1.6 mM, and 1.8 mM) were designed for the reaction. Amplification was performed at 65°C using a fluorescence PCR instrument, with one cycle every 30 seconds, for 120 cycles totaling 60 minutes. The optimal dNTP concentration was determined based on the amplification curves.

[0027] (5) Optimization of amplification temperature Based on the previously optimized reaction system, four temperature gradients (63℃, 65℃, 67℃, and 69℃) were designed for LAMP reactions. Isothermal amplification was performed on a fluorescence PCR instrument, with one cycle every 30 seconds, for 120 cycles totaling 60 minutes. The optimal amplification temperature was determined based on the amplification curves. Note: The instrument used in this invention for optimizing amplification temperature can only set three temperature gradients at a time. Therefore, there are two result graphs for the four temperature gradients (63℃, 65℃, 67℃, and 69℃). Slight differences in results at the same temperature may occur due to variations between replicates; this is normal.

[0028] (6) Optimization of amplification time Based on the previously optimized reaction system, templates of different concentrations, such as 10,000 copies / μL, 1,000 copies / μL, 100 copies / μL, and 10 copies / μL, were amplified at an isothermal temperature on a fluorescence PCR instrument. Each cycle lasted 30 seconds, and 120 cycles were performed for a total of 60 minutes. The amplification time was determined by the latest time to reach the plateau phase.

[0029] The ratio of inner to outer primers, the ratio of outer primers to loop primers, the concentration of MgSO4, the concentration of dNTPs, the reaction temperature, and the amplification time were optimized sequentially. Figures 2-8 The amplification effect was better when the ratio of inner to outer primers was 1:10 and the ratio of outer primers to loop primers was 1:6. The amplification conditions were 6 mM MgSO4, 1.6 mM dNTPs, and LAMP amplification at 65℃ for 30 min.

[0030] 4. Screening of crRNA and CasSF01 / crRNA combinations Within the LAMP amplification region, regions containing TTN PAM were identified, and 18–20 nt target regions were selected at their 3' ends to combine with the CasSF01 DR region to form three candidate crRNAs. Prepare the optimized LAMP reaction system and place it at the bottom of an 8-tube array. Add template and paraffin oil. Simultaneously, add the CRISPR digestion system (2 μL of 10 μM CasSF01 protein, 2.5 μL of 20x T7 terminator enzyme, 2.5 μL of 20x DTT, 2 μL of 10 μM CrRNA, 1.25 μL of 40 μM fluorescent reporter probe (FAM-TTATT-BHQ1), and 16 μL of water) to the cap of the 8-tube array. Incubate the reaction system in a constant temperature metal bath for 30 min. After the system temperature drops to room temperature, centrifuge the CRISPR digestion system from the PCR tube cap into the LAMP amplification system, vortex to mix, centrifuge again, and detect fluorescence changes at 37°C using a fluorescence PCR instrument. Repeat the cycle every 20 seconds for 90 cycles, for a total of 30 min. Compare the results ( Figures 9-10 The results showed a high CrRNA-2 signal and a rapid plateau phase.

[0031] Further comparison of CasSF01 / crRNA concentration combinations was conducted. Based on the previously optimized LAMP reaction system and the screened CrRNA, three CRISPR digestion systems were prepared (Combination 1: CasSF01 final concentration 400 nM, CrRNA final concentration 400 nM; Combination 2: CasSF01 final concentration 800 nM, CrRNA final concentration 400 nM; Combination 3: CasSF01 final concentration 800 nM, CrRNA final concentration 800 nM), to screen for the most suitable protein and CrRNA concentration combination. The reaction system was placed in a constant temperature metal bath for 30 min. After the system temperature cooled to room temperature, the CRISPR digestion system in the PCR tube cap was added to the LAMP amplification system by centrifugation, vortexed and centrifuged, and the fluorescence value was detected at 37℃ on a fluorescence PCR instrument. Each cycle lasted 20 s, and 90 cycles were performed for a total of 30 min. The results showed that the combination of CasSF01 800 nM and CrRNA-2 400 nM was optimal. Figures 11-12 ).

[0032] 5. Sequential RT-LAMP-CRISPR detection in the same tube Prepare a 25 μL RT-LAMP system according to Table 1 and place it at the bottom of an 8-tube reaction assembly. Add template and paraffin oil. Place the CRISPR / CasSF01 premix (2 μL 800 nM CasSF01, 2.5 μL 20x T7 terminator enzyme, 2 μL 400 nM CrRNA-2, 2.5 μL 20x DTT, 1.25 μL 40 μM fluorescent reporter probe, and 14.75 μL nuclease-free water) in the tube cap. Amplify at 65℃ for 30 min, then cool to room temperature and briefly centrifuge to mix the CRISPR system with the LAMP product in the tube cap. Incubate at 37℃ for 15 min, and finally interpret the results under blue light. Use an NDRV-positive plasmid as a positive control and water as a negative control. Note: If preparing fresh for immediate use, simply add the fluorescent reporter probe to the CRISPR / CasSF01 premix. For long-term storage, to prevent the Cas enzyme CasSF01 from cleaving the fluorescent reporter probe and generating fluorescence, which would affect the detection results, the fluorescent reporter probe needs to be added to the RT-LAMP reaction system.

[0033] Table 1 RT-LAMP reaction system The sequential RT-LAMP-CRISPR detection method includes the following steps: 1. Extract total nucleic acid from the sample to be tested, and add 5 μL to a 25 μL RT-LAMP system; 2. Place the RT-LAMP system at the bottom of the reaction tube and add enough paraffin oil to cover the surface of the reaction liquid; place the CRISPR / CasSF01 premixed system on the tube cap to keep the two systems spatially separated during the amplification stage; 3. The NDRV S1 target sequence was amplified by RT-LAMP by reacting at 65℃ for 30 min. 4. After the system cools to room temperature, perform a brief centrifugation to ensure that all the CRISPR premixed system in the tube cap enters the bottom of the tube and mixes with the LAMP amplification product; 5. Incubate at 37℃ for 15 min and observe the endpoint fluorescence under a blue light gel spectrometer.

[0034] Under the same blue light irradiation conditions, if the test tube shows a visible fluorescent signal and the fluorescence intensity is significantly higher than that of the negative control tube, it is judged as NDRV positive; if no visible fluorescent signal is shown, or the fluorescence intensity is not significantly different from that of the negative control tube, it is judged as NDRV negative.

[0035] 6. Sensitivity Test Positive plasmids were quantified using Qubit, and the copy number was calculated using the following formula: copies / μL = [6.02 × 10⁻⁶]. 23 ×DNA concentration (ng / μL) ×10 -9 [1] ÷ [intact plasmid length (bp) × 660]. The plasmid was diluted to 10000, 1000, 100, 50, 25, 12.5, and 6.25 copies / μL, with each concentration repeated 5 times. Results ( Figure 13 The results showed that, under the conditions of "5. Sequential RT-LAMP-CRISPR detection in the same tube", the lowest template concentration that could detect NDRV positive plasmid DNA in five replicates was 25 copies / μL; below 25 copies / μL, it could not be detected.

[0036] 7. Specificity test Detection was performed using nucleic acids related to avian reovirus (ARV), Newcastle disease virus (NDV), duck plague virus (DPV), and H5 / H7 subtype avian influenza virus (AIV) as templates. The detection method was performed according to "5. Sequential RT-LAMP-CRISPR Detection in the Same Tube," and the results were ( Figure 14 This indicates that, within the range of samples tested, no endpoint fluorescence signal significantly higher than that of the negative control was observed in non-NDRV nucleic acid samples, while the NDRV positive control showed a visible fluorescence signal.

[0037] 8. Repeatability test Repeated detections were performed using positive plasmid templates at concentrations of 10000, 1000, and 100 copies / μL. The endpoint fluorescence performance at different concentrations was observed and recorded. Results are as follows: Figure 15 As shown, detection was performed using templates at three concentrations: high (10,000 copies / μL), medium (1,000 copies / μL), and low (100 copies / μL), demonstrating the good repeatability of the LAMP CRISPR detection method.

[0038] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A novel duck reovirus RT-LAMP-CRISPR / CasSF01 detection kit, characterized in that, The kit includes an RT-LAMP reaction system and a CRISPR / CasSF01 system; The RT-LAMP reaction system contains LAMP primers composed of F3, B3, FIP, BIP and LF; The CRISPR / CasSF01 system contains crRNA, CasSF01 protein, and a fluorescent reporter probe; The nucleotide sequences of F3, B3, FIP, BIP and LF are shown in SEQ ID NO.2-SEQ ID NO.6, respectively; The sequence of the crRNA is shown in SEQ ID NO.

8.

2. A novel RT-LAMP-CRISPR / CasSF01 detection method for duck reovirus in the same tube for non-diagnostic and non-therapeutic purposes, characterized in that, Includes the following steps: The RT-LAMP reaction system was placed at the bottom of the reaction tube, and the CRISPR / Cas12a system was placed at the tube cap. LAMP amplification was performed on the nucleic acid of the test sample, the positive control, and the negative control using the RT-LAMP reaction system, respectively. After amplification, the LAMP amplification product was mixed with the CRISPR / Cas12a system by centrifugation and the reaction was continued. After the reaction was completed, the fluorescence signal was collected, and the presence or absence of the fluorescence signal was used to determine whether the sample was a novel duck reovirus. The RT-LAMP reaction system contains LAMP primers composed of F3, B3, FIP, BIP and LF; The CRISPR / CasSF01 system contains crRNA, CasSF01 protein, and a fluorescent reporter probe; The nucleotide sequences of F3, B3, FIP, BIP and LF are shown in SEQ ID NO.2-SEQ ID NO.6, respectively; The sequence of the crRNA is shown in SEQ ID NO.

8.

3. The novel duck reovirus co-tube sequential RT-LAMP-CRISPR / CasSF01 detection method as described in claim 2, characterized in that, The LAMP amplification reaction conditions were 65℃ for 30 min; the LAMP amplification reaction system consisted of 1.0 μL DNA polymerase, 10 U reverse transcriptase, 2.5 μL FIP, 2.5 μL BIP, 2.5 μL F3, 2.5 μL B3, 2.5 μL LF, 1.5 μL MgSO4, 2.5 μL buffer, 1.6 μL dNTP mixture, 5.0 μL nucleic acid of the sample to be tested, and nuclease-free water to a final volume of 25 μL.

4. The novel duck reovirus co-tube sequential RT-LAMP-CRISPR / CasSF01 detection method as described in claim 2, characterized in that, The CRISPR / CasSF01 system consisted of 2 μL of 800 nM CasSF01 protein, 2.5 μL of 20x T7 terminator enzyme, 2 μL of 400 nM crRNA, 2.5 μL of 20x DTT, 1.25 μL of 40 μM fluorescent reporter probe, and 14.75 μL of nuclease-free water.

5. The novel duck reovirus co-tube sequential RT-LAMP-CRISPR / CasSF01 detection method as described in claim 2, characterized in that, The conditions for continuing the reaction were 37°C for 15 minutes.

6. The novel duck reovirus co-tube sequential RT-LAMP-CRISPR / CasSF01 detection method as described in claim 2, characterized in that, If there is a fluorescent signal and the fluorescence intensity is significantly higher than that of the negative control, the sample to be tested is determined to be / contains the novel duck reovirus; if there is no fluorescent signal, or the fluorescence intensity is not significantly different from that of the negative control, the sample to be tested is determined not to be / does not contain the novel duck reovirus.

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